Oxidative Stress

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Relationship with Exercise and Training ..... Myoglobin can also be oxidised by auto-oxidation aract, cancers, Alzheimer's or .... and base repair damage.[10,51 ...

REVIEW ARTICLE

Sports Med 2006; 36 (4): 327-358 0112-1642/06/0004-0327/$39.95/0  2006 Adis Data Information BV. All rights reserved.

Oxidative Stress Relationship with Exercise and Training Julien Finaud, G´erard Lac and Edith Filaire Laboratoire Biologie Interuniversitaire des Activit´es Physiques et Sportives, Universit´e Blaise Pascal de Clermont-Ferrand, Aubi`ere, France

Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 1. Free Radicals (FR) and Activated Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 1.1 Biochemistry of Reactive Oxygen Species (ROS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 1.1.1 Dioxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 1.1.2 Superoxide Ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 1.1.3 Hydrogen Peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 1.1.4 Hydroxyl Radical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 1.2 Programmed Formation of ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 1.3 Unprogrammed Formation of ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 1.3.1 ROS Formation During Oxygen Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 1.3.2 ROS Formation During Ischaemia Reperfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 1.3.3 ROS Formation During Haemoglobin and Myoglobin Oxidation . . . . . . . . . . . . . . . . . . . . . 332 1.3.4 Other Ways of ROS Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 1.4 Biological Effects of ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 1.4.1 Positive Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 1.4.2 Negative Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 2. Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 2.1 Enzymatic Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 2.1.1 Superoxide Dismutase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 2.1.2 Catalase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 2.1.3 Glutathione Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 2.1.4 Relationship with Exercise and Training . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 2.2 Non-Enzymatic Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 2.2.1 Vitamin E (Tocopherol) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 2.2.2 Vitamin C (Ascorbic Acid) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 2.2.3 β-Carotene and Vitamin A (Retinol) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 2.2.4 Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 2.2.5 Thiols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 2.2.6 Coenzyme Q10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 2.2.7 Uric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 2.2.8 Heat Shock Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 2.2.9 Ferritin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 2.2.10 Albumin, Caeruloplasmin and Bilirubin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 2.3 Antioxidant Supplementation in Athletes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 2.3.1 Beneficial Effects of Antioxidant Supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 2.3.2 Pro-Oxidant Effects of Overloaded Antioxidant Supplementation . . . . . . . . . . . . . . . . . . . 340 2.4 Summary: Exercise and the Antioxidant System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 3. Methods to Assess Oxidative Stress in Biological Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 3.1 Direct Detection of FR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 3.2 Measurement of Oxidative Damage to Lipids, Proteins and DNA Molecules . . . . . . . . . . . . . . . 341

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3.2.1 Lipid Peroxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 3.2.2 Protein Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 3.2.3 DNA Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 3.2.4 Other Indirect Oxidative Stress Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 3.3 Antioxidant Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 3.3.1 Enzymatic Antioxidant Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 3.3.2 Antioxidant Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 3.3.3 Other Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 3.3.4 Total Antioxidant Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 3.4 Summary: is There an Ideal Method? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 4. Oxidative Stress and Physical Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 4.1 Oxidative Stress and Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 4.1.1 Aerobic Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 4.1.2 Anaerobic Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 4.1.3 Mixed Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 4.2 Training Effects on Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 4.2.1 Aerobic Training . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 4.2.2 Anaerobic Training . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 4.2.3 Mixed Training . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 4.2.4 Relationship Between Training Load and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . 350 4.2.5 Oxidative Stress and Overtraining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 4.3 Summary: Oxidative Stress, Training and Overtraining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

Abstract

Free radicals are reactive compounds that are naturally produced in the human body. They can exert positive effects (e.g. on the immune system) or negative effects (e.g. lipids, proteins or DNA oxidation). To limit these harmful effects, an organism requires complex protection – the antioxidant system. This system consists of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase) and non-enzymatic antioxidants (e.g. vitamin E [tocopherol], vitamin A [retinol], vitamin C [ascorbic acid], glutathione and uric acid). An imbalance between free radical production and antioxidant defence leads to an oxidative stress state, which may be involved in aging processes and even in some pathology (e.g. cancer and Parkinson’s disease). Physical exercise also increases oxidative stress and causes disruptions of the homeostasis. Training can have positive or negative effects on oxidative stress depending on training load, training specificity and the basal level of training. Moreover, oxidative stress seems to be involved in muscular fatigue and may lead to overtraining.

Regular physical activity, associated with a balanced diet, is known as an important factor for health.[1] However, exhaustive and/or intense physical activity can induce diseases, injuries and chronic fatigue, which can lead to the overtraining syndrome, partially because of the toxicity of free radicals (FR). FR, which are highly produced during physical exercise,[1] are involved in muscular fa 2006 Adis Data Information BV. All rights reserved.

tigue, many diseases and aging.[2,3] However, they exert positive effects on the immune system and essential metabolic functions.[4] Antioxidants are components that suppress FR and their harmful effects. If the production of FR is larger than antioxidant activity, there is an oxidative stress state with cell damages.[5] Sports Med 2006; 36 (4)

Oxidative Stress and Physical Activity

Physical activity increases the FR production and the antioxidant utilisation. Nutrition provides an important part of the antioxidant; however, insufficient micronutrient supply is often reported in athletes.[6,7] It has also been shown that oxidative stress can increase during periods of intensive training. Therefore, oxidative stress may be one of the actors of the overtraining syndrome.[8,9] This article presents the basis of oxidative stress and determines the relationship between oxidative stress, exercise, training and overtraining.

329

Among FR, reactive oxygen species (ROS) are derived from oxygen. ROS contains FR and reactive forms of oxygen (table I). This article will focus on ROS, which are involved in essential physiological phenomenon such as immunity or oxidative stress. Other FR families exist, such as reactive nitrogen species (RNS) and reactive sulphur species (RSS) [table I]. These species could be formed by reactions with ROS or could increase ROS production.[14] 1.1 Biochemistry of Reactive Oxygen Species (ROS)

1. Free Radicals (FR) and Activated Species FR are molecules or molecule fragments with one or more unpaired electrons in the valence shells.[10-12] FR are very unstable and very reactive because they tend to catch an electron to other molecules (oxidation).[5,13] Their lifetime is very short (from milliseconds to nanoseconds [table I]). FR are produced by an electron transfer that requires a high energy input.[11] When reacting with other radicals or molecules, a FR can form new radicals.[5]

1.1.1 Dioxygen

Aerobic organisms require dioxygen (O2) because this molecule acts as an electron acceptor during the oxidation of energetic substrates. Paradoxically, dioxygen is a permanent threat.[10] Indeed, ROS are continually produced from exogenous origins (radiation exposure, air pollutants, intoxication by oxygen, smoke, alcohol) or from endogenous origin (oxygen metabolism).[4,11] During oxygen metabolism, dioxygen receives two elec-

Table I. Classification and main effects of free radicals Free Radical

Contraction

Reactive oxygen species

ROS

Half-life

Main effects

Superoxide ion

O2•–

10–5 sec

Lipid oxidation and peroxidation Protein oxidation DNA damage

Ozone

O3

Stable

Singlet oxygen

1O2

1 µsec

Hydroxyl radical

OH•

Hydrogen peroxide

H2O2

Stable

Hypochlorous acid

HOCl

Stable

10–9 sec

Alkoxyl radical

RO•

10–6 sec

Peroxyl radical

ROO•

7 sec

Hydroperoxyl radical

ROOH•

Reactive nitrogen species

RNS

Nitric oxide

NO•

Nitric dioxide

NO2•

Peroxynitrite

ONOO•–

Reactive sulphur species

RSS

Thyil radical

RS•

 2006 Adis Data Information BV. All rights reserved.

Lipid peroxidation DNA damage Proteins oxidation 1–10 sec 0.05–1 sec Proteins oxidation DNA damage ROS production

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trons. Dioxygen prefers to receive one electron at a time and converts it into a superoxide ion (O2•–).[5] Following this process, 2–5% of oxygen consump˙ 2) is converted to O2•–. tion (VO

tive and very toxic ROS and there is no specific antioxidant against this FR. This FR causes lipid peroxidation and protein oxidation.[16] 1.2 Programmed Formation of ROS

1.1.2 Superoxide Ion O2•– is created with

the addition of one electron on dioxygen (equation 1) and becomes highly reactive. O 2 + e-® O 2 •(Eq. 1) Fenton’s reaction is an iron-salt-dependent decomposition of dihydrogen peroxide, generating the highly reactive hydroxyl radical. It occurs in the presence of ferrous ions (Fe2+) and O2•–. Iron is mainly present in tissues in a ferric ion state (Fe3+). The reaction (equation 2d) is called the Haber-Weiss reaction. a

O 2•- + H + → O 2• H

b

O 2•H + O 2•-+ H + → H 2O 2 + O 2

c

Fe 3+ + O 2•- → Fe 2+ + O 2

d

Fe 2+ + H 2O 2 → Fe 3+ + OH + OH -



(Eq. 2)

In the immune system, neutrophils and macrophages are in charge of destroying foreign substances (antigens). Those immunity cells produce O2•– with the reduced nicotinamide-adenine dinucleotide phosphate (NADPH)-oxidase system, which is present in leukocytes.[17] During this process (equation 4), two O2 molecules are needed so this reaction is called ‘oxidative burst’. NADPH-oxidase

2 O 2 + NADPH →

2 O 2•- + NADP + + H +

(Eq. 4) As shown in section 1.1.3, O2•– can be converted to H2O2 by SOD in Fenton’s reaction. After that, H2O2 can be transformed into HOCL, which is very active for antigen degradation.[18] Thus, an important quantity of ROS can be formed during the immunity process and plays an essential biological role for homeostasis control.[15,17] 1.3 Unprogrammed Formation of ROS

1.1.3 Hydrogen Peroxide

Equation 3 summarises the first and the second stages of Fenton’s reaction (equations 2a and 2b). This reaction forms hydrogen peroxide (H2O2) in an acid environment and is catalysed by the superoxide dismutase (SOD) enzyme. SOD

2 O 2•- + 2 H + → H 2O 2 + O 2

(Eq. 3) H2O2 is not a FR because it has no unpaired electron, but it is considered a ROS because of its toxicity and its capacity to cause ROS formation. In leukocytes, myeloperoxydase (MPO) transform H2O2 in hypochlorous acid (HOCL), one of the strongest physiological oxidants and a powerful antimicrobial agent.[15] 1.1.4 Hydroxyl Radical

Hydroxyl radical (OH•) is the end product of Fenton’s reaction (equation 2). OH• is a very reac 2006 Adis Data Information BV. All rights reserved.

1.3.1 ROS Formation During Oxygen Metabolism

It is generally considered that the oxygen metabolism, which occurs into mitochondria, is associated with the generation of ROS.[19] Oxidative phosphorylations result in adenosine triphosphate (ATP) formation. Substrate oxidation occurs in the Krebs’ cycle and in the electron transport chain with oxygen as the electron acceptor. In the respiratory chain, 95–99% of oxygen consumed is reduced into water (H2O) by a tetravalent reduction (equation 5) catalysed by coenzyme Q (CoQ).[17,20] CoQ

O 2 + 4 e- + 4 H + → 2 H 2 O

(Eq. 5) However, 1–5% of O2 will form O2•–.[21-23] The ROS formation is proportional to the respiratory chain activity, but the latter is not always propor˙ 2.[19] Two major sites of production of tional to VO ROS have been localised in the respiratory chain: Sports Med 2006; 36 (4)

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331

Mitochondria

Electron chain transport NADH

NAD+

Complex I

Succinate

Fumarate

Retonone

Complex III

Complex II Reversed CoQH2 e- flow

CoQ CoQ·-

O2 ® O2·-

-

Antimycin A

b cyt. FeSR Myxothiazol

Fig. 1. Possible locations of mitochondrial reactive oxygen species formation inside electron chain transport (reproduced from Nohl et al.,[26] with permission.  the Biochemical Society). b cyt. = b cytochrome; CoQ = coenzyme Q; CoQH2 = reduced coenzyme Q10; CoQ•– = oxidised coenzyme Q10; FeSR = Rieske iron-sulphur protein; NAD+ = oxidised nicotinamide-adenine dinucleotide; NADH = reduced nicotinamide-adenine dinucleotide; O2•– = superoxide ion.

complex I and complex III (see figure 1).[20,23] Distribution and quantity of ROS production inside these complexes fluctuate according to the needs of ˙ 2, central temperature and other parameATP, VO ters that vary with physical exercise.[19] Inside complexes I and III, reduced coenzyme Q10 (CoQH2) contribute to ROS formation (equation 6).[24] CoQ may be transformed into a superoxide generator when the ubisemiquinone anion, arising from oneelectron oxidation of ubiquinol, becomes accessible to protons.[25] CoQH 2 + O 2 ® CoQH • + O 2 ••

CoQH + O 2 ® CoQ + H + + O 2•-

(Eq. 6) There is a synergistic action of CoQH2 and cytochrome b566 in complex III.[24] However, this hypothesis is still controversial because CoQ, in its reduced form, may act as an antioxidant.[19] It was recently shown that ROS are not spontaneously released from mitochondria, but appear when the mitochondrial membrane potential reaches a maximum (state IV).[26] This fact is confirmed by other studies.[27] The site of single-electron deviation to dioxygen seems to be ubiquinol interacting with the Rieske iron-sulphur protein and low-potential cytochrome b of the complex III.[26] Another study re 2006 Adis Data Information BV. All rights reserved.

vealed that about 50% of O2•– production arises from reduced nicotinamide-adenine dinucleotide (NADH)-dehydrogenase inside complex I, between a mercurial-sensitive and a retonone-sensitive component, most likely a nonhaeme iron-sulphur function. This hypothesis is still controversial.[26] The possible locations of ROS formation inside the mitochondrial respiratory chain are represented in figure 1. The assumption that mitochondria are the major intracellular source of ROS was essentially based on in vitro experiments with isolated mitochondria.[19,20,26] Artifacts due to the preparation procedure or inadequate measurement of ROS may lead to methodological mistakes.[26] In vivo study provides direct evidence that mitochondria (in heart muscle) produce ROS during exercise.[16] So, both in vitro and in vivo studies tended to affirm that the mitochondrial respiratory chain can not only be a major source of ROS at rest and during exercise in the working muscle, but also in tissues such as liver, kidneys and non-working muscles that undergo partial ischaemia during physical exercise.[19] At the same time, mitochondria are particularly susceptible to the ROS-induced oxidative damage on lipids, proteins and DNA. In particular, damage to mitoSports Med 2006; 36 (4)

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Intracellular space Mitochondria: Mn-SOD GPX Electron chain transport Membranes: Vit E, carotenoids, flavonoids

ROS NADPH Nucleus XO

Cytosol: CAT, GPX, Cu-Zn-SOD, Vit C, GSH

Extracellular space Blood Circulating antioxidants: Vit C, lipoic acid, GSH

ROS

Membranes, circulating cells and lipoproteins: Vit C, carotenoids, flavonoids

Fig. 2. Potential sources of reactive oxygen species (ROS) in skeletal muscle and locations of the major intracellular and extracellular antioxidants. CAT = catalase; GPX = glutathione peroxidase; GSH = glutathione; NADPH = reduced nicotinamide-adenine dinucleotide phosphate; SOD = superoxide dismutase; Vit = vitamin; XO = xanthine oxidase.

chondrial DNA (mtDNA) induces alterations to the polypeptides in the respiratory complexes, with consequent decrease of electron transfer, leading to further production of ROS. Thus, a vicious circle of oxidative stress and energetic decline is established.[28,29] However, training does not seem to modify ROS release from mitochondria.[27] Nevertheless, there is a lack of knowledge about the exact mechanisms of ROS production inside the mitochondria, and further studies are needed. 1.3.2 ROS Formation During Ischaemia Reperfusion

The second major source of ROS is ischaemia reperfusion phenomenon, which occurs after surgical interventions, after shocks or during physical exercise (figure 2).[17,30-32] During exhaustive or anaerobic exercise, blood flow is brought to active territories such as skeletal muscles while other tissues can be in an hypoxic situation.[1,19] After exercise, these tissues receive a great quantity of oxygen. This phenomenon is described as ‘ischaemia reperfusion’. Xanthine dehydrogenase (XDH) has an important role in the formation of uric acid from purins degradation (ATP, adenosine diphosphate  2006 Adis Data Information BV. All rights reserved.

[ADP] and adenosine monophosphate [AMP]). Inside hypoxic tissues, XDH can be converted into xanthine oxidase (XO).[20,32] During reperfusion, O2•– can then be formed by a reaction catalysed by XO between oxygen, hypoxanthine and xanthine.[1,33,34] Nevertheless, the role of XO in muscles is discussed because there is a poor amount of XO inside them.[19,32] Other alternative explanations seem to be possible to explain the increased production of FR during ischaemia reperfusion. Some studies have shown that ischaemia reperfusion increased mitochondrial FR production.[19] Other studies pointed out that phagocyte infiltration, catecholamine, myoglobin and methmyoglobin auto-oxidation take part in FR production during ischaemia reperfusion.[35] 1.3.3 ROS Formation During Haemoglobin and Myoglobin Oxidation

Oxidation of haemoglobin can cause ROS formation.[4,17,36] In the human body, 3% of the total haemoglobin (about 750g) is transformed by autooxidation. This reaction, which increases during exercise, produces methaemoglobin and O2•–.[1,37-40] Sports Med 2006; 36 (4)

Oxidative Stress and Physical Activity

Myoglobin can also be oxidised by auto-oxidation or by FR during ischaemia reperfusion with the production of H2O2.[35,38,40] Myoglobin can then interact with H2O2 and produce other radicals such as ferryl radicals or peroxyl radicals.[41-43] 1.3.4 Other Ways of ROS Production

Other processes involved in ROS production during exercise are increased central temperature, catecholamine and lactic acid, which has the ability to convert O2•– into OH•.[1,44] 1.4 Biological Effects of ROS 1.4.1 Positive Effects

ROS are involved in the immunity phenomenon, in particular by acting against antigens during phagocytosis.[10,12,17] This role increases during inflammation. Inflammation can be caused by physical exercise, particularly by intense and traumatising exercises such as eccentric exercises.[45] Although most studies have concentrated on the harmful effects of FR, ROS play an important role in cellular signals or in biogenesis of cells because they can serve as cell messengers or modify oxidation-reduction (redox) status.[5,12,46-48] ROS are also known to be involved in enzyme activation, in drug detoxification or in facilitating glycogen repletion.[10] ROS also play an essential role in muscular contraction.[47-50] This role has been pointed out because inhibition of ROS production leads to a loss of muscular fibres contractile force. Conversely, increasing ROS leads to an increased strength contraction.[47,49,50] However, an important amount of ROS in muscular tissue is implicated in muscular fatigue and can represent one of the negative effects of ROS. 1.4.2 Negative Effects

Despite some helpful effects, ROS have possible harmful effects because they can alter the size and shape of the compounds they interact with.[1,10,51,52] Consequently, ROS can induce apoptosis into healthy cells and can provoke inflammation or altered cellular functions. All those deteriorations take part in some degenerative pathology such as cat 2006 Adis Data Information BV. All rights reserved.

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aract, cancers, Alzheimer’s or Parkinson’s diseases or in cell aging.[3] Lipid Oxidation

Lipoprotein oxidation is an important factor in the pathogenesis of atherosclerosis.[53,54] In fact, ROS initiate lipoprotein oxidation, in particular lowdensity lipoprotein (LDL) oxidation.[55] This oxidation is dependent on blood antioxidant capacity[56,57] and can increase with oxidative stress linked to physical exercise.[58,59] However, those effects are partially or totally compensated in athletes because exercise decreases cardiovascular accident risk.[59] ROS also have the ability to oxide polyunsaturated free-fatty acids (PUFFA), which take part in cell membrane constitution.[11,21,51] This reaction initiates lipid peroxidation, a chain reaction that produces other FR such as ROO• or ROOH• and substances such as conjugated dienes or malondialdehyde (MDA).[54] Lipid peroxidation changes the fluidity of cell membranes, reduces the capacity to maintain an equilibrated gradient of concentration, and also increases membrane permeability and inflammation.[60] Consequently, it is possible to detect a loss of intracellular liquids, a diminution of calcium transport in the endoplasmic reticulum, alterations of mitochondrial functions and cell alterations, together with loss of cryptozoic proteins and enzymes.[10,31] Every type of cell can be damaged by ROS, including muscular cells and erythrocytes.[61] Protein Oxidation

ROS can also oxidise blood and structural proteins and inhibit the proteolytic system.[62] During oxidation, proteins can lose amino acids or can be fragmented. Those reactions lead to alteration of structural proteins or alteration of enzyme functions.[60] Protein and amino acid oxidation is accompanied by overall increases in the relative level of protein carbonyl groups[11,63-65] and of oxidised amino acids,[16,66] which are used as general indexes for the occurrence of oxidative damage.[16,60,67] Protein oxidation can be the consequence of inflammation, physical exercise or ischaemia reperfusion.[65,66] Oxidised proteins are catabolised in order to reform amino acids but carbonyl by-products cannot enter this process. Therefore, they induce a Sports Med 2006; 36 (4)

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Exercise

ROS-RNS

Alteration of mitochondrial functions

Increased anaerobic pathways utilisation

Decreased electron transfer Decreased ATP formation Increased ROS formation

Increased Pi Acidosis

Alteration of the action potential

Alteration of the redox status

Increased Pi Acidosis

Muscular fatigue Decreased force

Overtraining? Fig. 3. The different hypothesis about the effects of reactive oxygen species (ROS) on muscular fatigue. ATP = adenosine triphosphate; redox = oxidation-reduction; RNS = reactive nitrogen species; Pi = inorganic phosphate.

proteolysis blocking and an accumulation of oxidised proteins.[64,65] Consequently, protein turnover, genetic transcription and cell integrity are reduced under ROS actions. ROS also have the ability to alter the lysosomal system and the proteasomes, two major pathways by which proteins are degraded.[62] DNA Oxidation

ROS are also known to cause DNA strand breaks and base repair damage.[10,51,63,68] Every part of DNA is susceptible to attack by ROS.[69] The DNA repair system is continual, but its capacity can be overreached or the repair processes can be altered.[68,70] As a consequence, DNA oxidation can provoke mutagenesis and is a major contributor to human cancer and cell aging.[17,60,68,70,71] Different major sources of DNA damages have been found as a result of: smoking, chronic inflammation and leakage from mitochondria, which increased with physical exercise.[51,70,71] Implication of FR in Muscular Fatigue

A minimal amount of ROS is necessary for muscular contraction.[47,49,50] Nevertheless, oxidative stress, which results in muscle-increased ROS concentration, is associated with muscular fatigue dur 2006 Adis Data Information BV. All rights reserved.

ing contraction and in post-exercise muscular damage and suffering.[1,7,31,72-76] The different hypothesis about the effects of ROS on muscular fatigue is summarised in figure 3. Precisely, when ROS concentration is too important or too prolonged, a timeand dose-dependent muscular force decrease and a time- and dose-dependent muscular fatigue increase can be observed.[47,72,77] Numerous factors seem to be implicated in FR-induced muscular fatigue (figure 3). The alteration of the mitochondrial functions with exposure to ROS is considered a major factor of muscular fatigue.[72,77] Indeed, mitochondria are particularly susceptible to the ROS-induced oxidative damage on lipids, proteins and DNA, and damages to mtDNA can induce alterations to the respiratory complexes, with a consequent decrease of electron transfer and ATP formation. Thus, aerobic pathways become less efficient. Consequently, it seems that this phenomenon can induce an increase of anaerobic pathways utilisation. This can have negative effects in muscle because anaerobic pathways induce both an increased inorganic phosphate (Pi) level and acidosis, which are two major factors of muscular fatigue.[72] Sports Med 2006; 36 (4)

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Contractile proteins (actin and myosin) and calcium pump are muscular compounds that are sensitive to redox status. Redox status is directly linked and modified by ROS concentration. Thus, when ROS production is important, redox status can be altered. Consequently, muscular contraction (contractile proteins) and muscular contraction control (calcium pump) may be altered.[33] ROS can induce an intracellular calcium increase and intracellular enzymes inactivation (particularly enzymes implicated in aerobic and anaerobic pathways) into muscular cells, which could participate to muscular fatigue.[78] Moreover, during certain forms of exercise, such as eccentric exercise, an important iron release (from ferritin or haemoglobin) can be observed. Iron release can aggravate exercise and post-exercise oxidative stress and muscular fatigue and damages.[79] Moreover, data tend to show that the action potential for muscle contraction can be modified by ROS.[80] Indeed, ROS causes remarkable perturbation of the inward potassium transport system in muscle. It has been shown that muscle soreness-induced decrease in maximal force generation is a result of an increase in muscular nitric oxide (NO).[81] Indeed, NO was reported to decrease contractile force by inhibition of Ca21-ATPase activity in the sarcoplasmic reticulum. Moreover, NO induces hyperpolarisation of membrane potential (thereby leading to reduced force generation) and also may directly inhibit the force-generating proteins in skeletal muscle. In summary, it seems possible that ROS- and RNS-induced decrease in maximal force generation can be a part of a protective mechanism by which skeletal muscle protects itself from further peak force-generated damage.[81] Moreover, repetitive muscular ROS-induced fatigue associated with inadequate recovery is supposed to induce overtraining syndrome.[73,82,83] 2. Antioxidants An antioxidant can be defined as a substance that helps to reduce the severity of oxidative stress either by forming a less active radical or by quenching the damaging FR chain reaction on substrates such as proteins, lipids, carbohydrates or DNA.[84] A range of antioxidants are active in the body including  2006 Adis Data Information BV. All rights reserved.

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enzymatic (endogenous) and non-enzymatic (mainly brought by food) antioxidants.[74] All of them can be intracellular or extracellular antioxidants (figure 2). Antioxidant enzymes include SOD, catalase (CAT) and glutathione peroxidase (GPX). Non-enzymatic antioxidants include a variety of FR quenchers such as vitamin A (retinol), vitamin C (ascorbic acid), vitamin E (tocopherol), flavonoids, thiols (including glutathione [GSH], ubidecarenone (ubiquinone Q10), uric acid, bilirubin, ferritin) and micronutrients (iron, copper, zinc, selenium, manganese), which act as enzymatic cofactors. The antioxidant system efficiency depends on nutritional intakes (vitamins and micronutrients) and on endogenous antioxidant enzyme production, which can be modified by exercise, training, nutrition and aging.[84] Moreover, the antioxidant system efficiency is important in sport physiology because exercise increases the production of FR. 2.1 Enzymatic Antioxidants 2.1.1 Superoxide Dismutase

SOD is the major defence upon superoxide radicals and is the first defence line against oxidative stress. SOD represents a group of enzymes that catalyse the dismutation of O2•– and the formation of H2O2 (equation 7). SOD

2 O 2•- + 2 H + → H 2O 2 + O 2

(Eq. 7) In all cells, at rest, the major part of mitochondrial-produced O2•– is reduced by mitochondrial SOD and the other part diffuse into the cytosol.[85] In muscular cells, 65–85% of SOD activity is done in the cytosol.[74] Different forms of SOD are present in the body (see table II and figure 2). Manganese is a cofactor of the Mn-SOD form, which is present in mitochondria as well as copper and zinc, which are cofactors of Cu-Zn-SOD, present in cytosol. 2.1.2 Catalase

CAT is present in every cell and in particular in peroxysomes, cell structures that use oxygen in order to detoxify toxic substances and produce Sports Med 2006; 36 (4)

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Table II. Localisation and actions of antioxidant enzymes Antioxidants

Cofactors

Cellular localisation

Targets

Mn-SOD

Manganese

Mitochondria

Anion superoxide Peroxynitrite

Cu-Zn-SOD

Copper Zinc

Cytosol – mitochondria (membrane)

Anion superoxide Peroxynitrite

CAT

Iron

Peroxysome, cytosol and mitochondria

Hydrogen peroxide

GPX

Selenium

Cytosol and mitochondria

Hydrogen peroxide Peroxynitrite

CAT = catalase; GPX = glutathione peroxide; SOD = superoxide dismutase.

H2O2.[86] Catalase converts H2O2 into water and oxygen (equation 8).

®

2.2 Non-Enzymatic Antioxidants

CAT

2 H 2O

2 H2O + O 2

2.2.1 Vitamin E (Tocopherol)

(Eq. 8) Catalase can also use H2O2 in order to detoxify some toxic substances via a peroxidase reaction (equation 9). This reaction needs a substrate such as phenol, alcohol (ethanol; A) or formic acid. CAT

H 2O 2 + H 2A (substrate) → 2 H 2O + A

(Eq. 9) 2.1.3 Glutathione Peroxidase

The GPX present in cell cytosol and mitochondria has the ability to transform H2O2 into water (equation 10). This reaction uses GSH and transforms it into oxidised glutathione (GSSG).

®

GPX

H 2O 2 + 2 GSH

GSSG + 2 H2O

(Eq. 10) GPX and CAT have the same action upon H2O2, but GPX is more efficient with high ROS concentration and CAT has an important action with lower H2O2 concentration.[21,86] 2.1.4 Relationship with Exercise and Training

Antioxidant enzymes are endogenous (table II). However, their production can be modulated by certain factors. Exercise and training are well known to be potential factors of SOD, CAT and GPX increase as shown by numerous studies (see section 3 for more details).[16,22,74,87]

 2006 Adis Data Information BV. All rights reserved.

Vitamin E is a fat-soluble vitamin made up of several isoforms known as tocopherols. α-Tocopherol is the more active and abundant form.[88] Vitamin E has been called the most important chainbreaking antioxidant because of its abundance in cells and mitochondrial membranes and its ability to act directly on ROS.[78] Vitamin E interacts with numerous antioxidants such as vitamin C, GSH, βcarotene or lipoic acid. Those antioxidants have the capacity to regenerate vitamin E from its oxidised form.[50] Vitamin E plays an important role in cell membranes because it stops lipid peroxidation (table III). The molecular structure of vitamin E enables ROS inactivation in a lipid environment, particularly for peroxyl radicals, which come from LDL oxidation into membranes or into blood.[53,89,90] A vitamin E deficiency is frequent in healthy occidental populations.[90,91] Such deficiency could increase oxidative stress and fatigue associated with decreased oxidative capacity and endurance.[77,78,92,93] Athletes often use vitamin E supplementation in order to prevent exercise-induced ROS muscular damages and fatigue.[7,33,78,87,94,95] However, the results are often contradictory probably because of methodological differences such as vitamin status of subjects before studies, vitamin E supplementation quantity, frequency and duration, or training level.[6,76] Indeed, trained subjects present a higher vitamin E status, whereas overreaching seems to decrease it.[33,76]

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Table III. Localisation and actions of the main non-enzymatic antioxidants Antioxidant

Localisation

Actions

Targets

Vitamin E (tocopherol)

Lipids Cell/mitochondria membranes

Lipid peroxidation inhibition Membrane stabilisation

ROOH – 1O2

Vitamin A (retinol)

Lipids Cell membranes

Lipid peroxidation reduction

1O2

Vitamin C (ascorbic acid)

Aqueous middle Cytosol Extracellular liquids

Vitamin E regeneration LDL protection

Glutathione

Aqueous middle

Substrate for GPX Vitamins E and C regeneration

Direct antioxidants

Cysteine

Intracellular middle

Glutathione precursor

Lipoic acid

Aqueous middle

Lipid peroxidation inhibition Vitamins C and E and cysteine regeneration

Thioredoxin

Aqueous middle

Mn-SOD synthesis Vitamin C regeneration

– ROOH

OH• – O2•–

1O2

– OH•

H2O2 – ROOH H2O2

Glutaredoxin

Aqueous middle

Flavonoids

Linked with glucids

Pro-oxidant enzymes inhibition Pro-oxidant ions (Fe2+, Fe3+, Cu2+) trapping LDL protection

Coenzyme Q10

Internal membrane of mitochondria

Vitamin C and E regeneration LDL protection

Uric acid

Aqueous middle

O2•– – OH• – ROOH – RO•

ROO•

ROOH – OH• – O3 – HOCL Pro-oxidant ions (Fe2+, Fe3+, Cu2+) ONOOH trapping Erythrocytes, haemoglobin, DNA, lipids protection

Indirect antioxidants HSP

Aqueous middle

Protection of proteins (cells)

Iron

Aqueous middle

Catalase cofactor

Ferritin

Aqueous middle

Free iron trapping

Zinc

Aqueous middle

SOD cofactor (Cu-Zn-SOD) LDL and thiols protection FR production inhibition

Copper

Aqueous middle

SOD cofactor (Cu-Zn-SOD)

Selenium

Aqueous middle

GPX cofactor

Manganese

Aqueous middle

SOD cofactor (Mn-SOD)

Albumin

Aqueous middle

Give electron to FR Cu2+ trapping

Caeruloplasmin

Aqueous middle

Give electron to FR Cu2+, Fe2+ and Fe3+ trapping

Bilirubin

Aqueous middle

Give electron to FR Lipid peroxidation inhibition Erythrocyte protection

1O2

= singlet oxygen; FR = free radicals; GPX = glutathione peroxidase; H2O2 = hydrogen peroxide; HOCL = hypochlorous acid; HSP = heat shock proteins; LDL = low-density lipoprotein; O2•– = superoxide ion; ONOOH = peroxynitrous acid; O3 = ozone; OH• = hydroxyl radical; RO• = alkoxyl radical; ROO• = alkylperoxyl radical; ROOH = hydroperoxyl radical; SOD = superoxide dismutase.

 2006 Adis Data Information BV. All rights reserved.

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2.2.2 Vitamin C (Ascorbic Acid)

Vitamin C is a water-soluble vitamin and is probably the most important antioxidant in extracellular fluids, but is also effective in cytosol.[82,96] Vitamin C is more abundant in tissues in which ROS production is more important. This phenomenon is defined as an adaptation against oxidative stress.[96] In fluids, vitamin C has the ability to neutralise ROS (OH•, O2•–, fatty acid peroxyl radical (LOO•), alkoxyl radical [RO•]).[82] Inside cells, vitamin C reinforces the action of vitamin E and GSH by regenerating their active form after they have reacted with ROS.[56,78,97] Vitamin C also has the ability to trap copper ions, which have a powerful oxidant action. Thus, vitamin C supplementation has often been studied. In athletes, its preventive effects against oxidative stress are discussed.[32,95,98,99] A deficiency in vitamin C has negative effects on performance and vitamin C supplementation (especially in combination with other antioxidants such as vitamin E) helps to maintain an adequate vitamin C level in tissues.[7] 2.2.3 β-Carotene and Vitamin A (Retinol)

Vitamin A is a fat-soluble vitamin present in many lipid substances. β-carotene, present in cell membranes, is converted into vitamin A when the body needs it. Although the mechanism of its in vivo action is unclear, β-carotene is suggested to deactivate ROS (in particular singlet oxygen and lipid radicals) and to reduce lipid peroxidation.[74,100] Although less important than vitamin E inside the antioxidant system, β-carotene and vitamin A act in tandem with vitamin C and vitamin E in order to protect cells against ROS.[101] β-carotene supplementation seems to have beneficial effects against exercise-induced oxidative stress without enhancing physical performance.[102,103] 2.2.4 Flavonoids

Flavonoids (Fl-OH) are phenolic substances formed in plants from amino acids phenylalanine, tyrosine and malonate.[93,104] In vitro studies pointed out the antioxidant effects of flavonoids that have the ability to inhibit pro-oxidant enzymes or to form complexes with pro-oxidant ions such as Fe2+, Fe3+ or Cu2+. Flavonoids also have direct trapping action  2006 Adis Data Information BV. All rights reserved.

upon some ROS by direct hydrogen atom donation. Despite increasing evidence for the in vitro antioxidant effects of flavonoids, there is a lack of knowledge on their in vivo actions.[52,104,105] However, some studies tend to confirm the in vivo antioxidant properties of flavonoids.[106] Moreover, flavonoids seem to have a sparing effect on vitamin E and βcarotene.[52,106] The in vivo effects of flavonoids are discussed because some of them can have pro-oxidant effects and because flavonoids are present in the body as metabolite forms that have poor antioxidant effects.[105,107,108] 2.2.5 Thiols

Thiols are a class of molecules characterised by the presence of sulfhydryl residues (–SH) at their active site.[109] Thiols are synthesised from sulphur amino acids: cysteine or methionin, which is a cysteine precursor. They have numerous functions in biological systems, e.g. protein synthesis, redox, cell biogenesis and immunity. They also play a major role in the antioxidant defence network.[109] GSH is the major thiol present in an organism. It acts like a substrate for GPX in peroxidase ROS inhibition. GSH can also directly detoxify ROS and enhances the functional antioxidant capacity of vitamins C and E.[110,111] In the presence of oxidative stress, it is possible to observe a decrease of the GSH/GSSG ratio and of the total thiol quantity.[109,112,113] These phenomena seem to be involved in the aetiology of some neurodegenerative diseases such as Parkinson’s or Alzheimer’s disease.[114] They are also observed in aging or after physical exercise.[112,113] A low GSH concentration in cells may be associated with cellular damage and decreased immunity efficiency, which can be compensated with a vitamin C and E supplementation.[115] Such results tend to show that these antioxidants have the same targets and work together against them. Lipoic acid is a thiol that inhibits lipid peroxidation and helps to reduce vitamins C and E from their oxidised form.[50,116,117] It can also reduce cystin (the oxidised form of cysteine) into cysteine in order to promote thiol genesis.[109,114,118] Therefore, lipoic acid supplementation helps to increase antioxidant protection and may have some therapeutic effects.[50,109] Sports Med 2006; 36 (4)

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2.2.6 Coenzyme Q10

Coenzyme Q10 (CoQ10) is an endogenous molecule that is essential for ATP synthesis and is especially present in mitochondrial membrane.[48,119] CoQ10 is known to act as an antioxidant with a direct action upon peroxyl radicals or with an indirect action by regenerating vitamins C and E.[120,121] CoQ10 also has beneficial effects, such as protection against cardiovascular diseases, cancer and cell aging or apoptosis.[48,119,122,123] However, CoQ10 acts as a mediator for gene expression and protein synthesis in muscle.[48] In this case, CoQ10 acts as a prooxidant by giving rise to O2•–, which is converted to H2O2 by SOD. H2O2 then acts as a second messenger for genetic expression. CoQ10 supplementation has been tested in sporting groups with limited results on oxidative stress reduction and physical performance.[124,125] 2.2.7 Uric Acid

Uric acid is an end-product of purine metabolism in humans.[113,126,127] Intense physical exercise is known to increase plasmatic concentrations of uric acid.[90,128] Plasmatic uric acid can then diffuse into muscles in order to protect them from FR oxidation.[129] Indeed, uric acid, in plasma and in muscle, is also one of the more important antioxidants with direct effects on singlet oxygen, HOCL, peroxyl radical, peroxynitrite or ozone.[36,126,130-134] Some studies demonstrated that uric acid represents a great part (>50%) of the plasmatic antioxidant capacity.[131] Thus, uric acid helps to protect erythrocytes, cell membranes, hyaluronic acid and DNA from FR oxidation. Another important antioxidant property of uric acid is the ability to form stable complexes with iron ions. This process inhibits Fe3+, catalysed vitamin C oxidation and lipid peroxidation.[135,136] Therefore, uric acid is a vitamin C protector but is also a vitamin E protector.[56] In vivo, it is possible to detect and measure its FR-induced oxidation product (allantoin) in body fluids after episodes of oxidative stress, such as physical exercise.[113,127,137,138] 2.2.8 Heat Shock Proteins

Heat shock proteins (HSP) are a variety of proteins known to have protective effects against various stresses. HSP increase with exercise, particular 2006 Adis Data Information BV. All rights reserved.

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ly with body temperature variations, inflammation and oxidative stress.[139-141] HSP are considered antioxidants because they protect cells and intracellular proteins against FR-induced damage.[17,139,140] Physical training and antioxidant supplementation promote a lower HSP basal level but the ability to have a quick HSP liberation under stressful situations remains unchanged.[140] Given the potential of ROS to damage intracellular proteins during subsequent bouts of muscle contractions, data suggested that, under oxidative stress conditions, the pre-existing antioxidant pathways may be complemented by the synthesis of HSP.[141] Thus, HSP could represent an important protection mechanism against exerciseinduced damage to muscle.[139-141] 2.2.9 Ferritin

Iron is required for normal cell growth and proliferation and can have antioxidant effects as a cofactor of catalase. However, iron ions can have pro-oxidant effects in Fenton’s reaction or can oxidise vitamin C and reduce antioxidant protection against FR.[136,142] Therefore, excess iron is potentially harmful and ferritin, one of the major proteins of iron metabolism, plays an important part in the maintenance of iron balance.[143] Several studies support a protective role of ferritin against FRmediated damage because ferritin minimises FR formation by sequestering iron in blood or in cells.[142,144,145] In addition, an increase of ferritin synthesis is observed in response to physical exercise, cellular damage and inflammation, which promote oxidative stress.[144-148] Indirect and direct links between FR and genetic expression of ferritin were shown in some studies.[142-145] 2.2.10 Albumin, Caeruloplasmin and Bilirubin

Albumin, caeruloplasmin and bilirubin act as nonspecific chain-breaking antioxidants by giving electrons to FR.[13] Albumin (a thiol protein) and caeruloplasmin are implicated in copper transport and so they reduce FR generation by Fenton’s reaction.[12,149] Bilirubin, a biliary protein coming from haemoglobin, increases with oxidative stress and has antioxidant effects in body fluids.[147,150,151] However, these proteins have a limited antioxidant action because their action is indirect and is effecSports Med 2006; 36 (4)

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tive in body fluids such as blood, far from the major FR production localisation, especially during physical exercise. 2.3 Antioxidant Supplementation in Athletes 2.3.1 Beneficial Effects of Antioxidant Supplementation

Antioxidant supplementation among athletes is well documented. The results of these studies are often contradictory because of antioxidant compounds and quantity. Indeed, some studies showed that the association of several antioxidants has better effects than single-compound supplementation.[152-154] Moreover, the subjects’ profiles (age, nutritional status, training level and physical activity category) can influence the results.[33,44] In a large majority of studies, antioxidant supplementation does not enhance exercise performance or physical capacity in non-deficient athletes.[7,44,78,103] In return, antioxidant supplementation provides protection against the negative health consequences of FR caused by exercise. Thus, antioxidant supplementation helps athletes to maintain an optimal health, which is a key condition to attaining the best performance. This is particularly important during periods of intensive training and/or competition, which cause greater needs of antioxidants.[152-155] In this case, a normal diet is not always sufficient.[6,33,156] Some antioxidants (vitamins A, E and C) can protect subjects from FR-induced muscle damage during exercise or can have anabolic effects.[76,102] Results tend to show that antioxidant supplementation can have beneficial effects in athletes by preventing against antioxidant deficiencies and FR harmful effects on tissues, particularly muscular tissues.[152,154] 2.3.2 Pro-Oxidant Effects of Overloaded Antioxidant Supplementation

Every antioxidant is a redox agent that can protect against FR in some circumstances and promote FR production in others.[157] Therefore, some precautions must be taken because antioxidants can have pro-oxidant effects, especially when megadoses are used.[44,79] Such findings were demonstrated for vitamin C,[79] β-carotene[6,7] and CoQ10.[158] It was also shown that quercetin can have pro-oxidant  2006 Adis Data Information BV. All rights reserved.

effects because long-term administration of quercetin significantly decreases glutathione concentration and glutathione reductase activity in rats.[159] Moreover, antioxidants present in food are in a balanced biochemistry state compared with supplement antioxidant compounds.[157] 2.4 Summary: Exercise and the Antioxidant System

Both enzymatic and non-enzymatic antioxidants play a vital role in protecting tissues from excessive oxidative damages.[44,84] The respective actions of the antioxidants are summarised in table III and figure 2. This role is especially important during exercise, which is associated with FR production, in relation to intensity, duration and training status.[87] Thus, because of low antioxidant dietary intakes and exercise and training modifications on the antioxidant system, some antioxidant supplementation in certain antioxidant nutrients seems to be justified.[22] However, the theoretical basis for which antioxidants should enhance performance is not clear. Studies have generally found that antioxidant supplements do not improve performance but improve antioxidant status.[152,154] In return, large amounts of antioxidant in nutrition could have negative effects.[44,79] Therefore, it seems that antioxidant supplementation must be extremely controlled for composition, duration and dose (depending on nutritional intakes) in order to be efficient for athletes’ health and performance. 3. Methods to Assess Oxidative Stress in Biological Systems Oxidative stress can be estimated according to the measurement of: (i) FR; (ii) radical-mediated damages on lipids, proteins or DNA molecules; and (iii) antioxidant enzymatic activity or concentrations. Results must be interpreted with caution because of possible contradictions.[44,160,161] 3.1 Direct Detection of FR

The production of ROS can be revealed according to direct methods. The electron spin resonance technique is a direct spectroscopic method that alSports Med 2006; 36 (4)

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lows the direct measurement of ROS from their paramagnetic properties.[12,97,162] Measurements can be made in vitro, in vivo or ex vivo. However, the most precise measurements (in vivo) are not practicable in humans because of the toxicity of the products used for such methods.[12,44] Blood samples can be collected in tubes containing a solution with spin trappers, which are ROS stabilisers. After centrifugation, the serum is analysed by a spectroscopic method. However, the results are to be interpreted with caution because of the short half-life of the ROS, their strong ability to react and their weak concentration.[1,97,161] This direct method allows a better understanding of the ROS reactions, and reagents used can also quantify ROS.[12] Realised ex vivo, it raises the problem of the short ROS half-life, these being stabilised in the serum after blood samples. 3.2 Measurement of Oxidative Damage to Lipids, Proteins and DNA Molecules 3.2.1 Lipid Peroxidation

A basic approach to study oxidative stress would be to measure the rate of peroxidation of membrane lipids or fatty acids. Lipid peroxidation leads to the breakdown of lipids and to the formation of a wide array of primary oxidation products such as conjugated dienes or lipid hydroperoxides, and secondary oxidation products including MDA, F2-isoprostane or expired pentane, ethane or hexane. Measurement of conjugated dienes is interesting because it detects molecular reorganisation of polyunsaturated fatty acids during the initial phase of lipid peroxidation. Because of this specificity, this method is often used to assess oxidative stress.[18,44] Lipid hydroperoxide is another marker of the initial reaction of FR and is a specific marker of cellular membrane damage.[12,97] Other products are often used to measure oxidative stress but have the disadvantage of being secondary oxidation products. One of them, MDA, is produced during fatty acid auto-oxidation. This substance is most commonly measured by its reaction with thiobarbituric acid, which generates thiobarbituric acid reactive substances (TBARS). Although  2006 Adis Data Information BV. All rights reserved.

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TBARS assay is not specific to MDA (this induces MDA overestimation), this method is accepted as a general marker of lipid peroxidation but results are subject to caution.[12,44,111,161] In addition, some studies tend to show that MDA is not an adapted method to assess oxidative stress after exercise.[111] A further technique for the measurement of lipid peroxidation is the analysis of volatile hydrocarbon end products such as pentane, hexane and ethane in expired air. Such a method is non-invasive but is not precise because these gases can be formed by other ways than FR oxidation.[12] More recently, it was found that F2-isoprostanes are produced by FR catalysed peroxidation of arachidonic acid.[18] Numerous recent studies have shown that quantification of F2-isoprostane compounds could be a reliable method for endogenous lipid peroxidation and oxidant injury assessment as well as some other recent markers such as blood oxidised LDL or antibodies against oxidised LDL.[59,93,163] 3.2.2 Protein Modification

FR-induced modification of proteins causes the formation of carbonyl groups into amino acid side chains. An increase of carbonyl is present in every phenomenon linked to oxidative stress.[66] Thus, the measurement of carbonyl formation is the most usual method for determination of FR-damaged proteins.[65,66] Total proteins are often measured in order to use the carbonyl/protein ratio, which is a more precise index of protein oxidation.[164] This method is particularly interesting because the half-life of carbonyl is long. Thus, a high amount of carbonyl can show cumulative effects of oxidative stress, which is essential in some studies (e.g. longitudinal following of athletes). Oxidised amino-acid (e.g. o-o′-dityrosine) quantification is an alternative method for oxidised protein measurement.[16] This method can be non-invasive (with the possibility of using urine samples) and presents a methodological interest (high concentrations and stability of measured compounds). Nevertheless, there is a lack of knowledge in oxidised amino acid kinetics, which limits the interpretation of the results.[12,16] Sports Med 2006; 36 (4)

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3.2.3 DNA Modification

ROS induce several types of DNA damage including strand breaks, DNA-protein cross-links and base modifications. Nowadays, numerous methods are used for DNA modification quantification.[69] The most used marker is the nucleotide 8-hydroxy-2′-deoxyguanosine (8-OHdG), which is produced by FR-induced guanine oxidation. The oxidised DNA is continually repaired, and oxidised nucleotides such as 8-OHdG are excreted via blood and urine. Thus, invasive or non-invasive methods are possible.[12] However, there have been doubts about the precision of this method because of the possible artefactural production of 8-OHdG by autooxidation during and after sample extraction.[69,165] 3.2.4 Other Indirect Oxidative Stress Markers

Creatine kinase (CK) and myoglobin are markers of muscular cellular damage.[9,166] These parameters can also be considered indirect markers of oxidative stress because lipid peroxidation induces damage of cellular membranes.[9,12] Thus, they are more permeable and allow the release of these intracellular substances.[76] However, CK and myoglobin are not specific markers of oxidative stress, especially in athletes who can have high plasmatic CK and myoglobin values because of sports characteristics (shocks, contacts), which induce cellular damage.[9] Moreover, trained athletes have higher basal values of CK and myoglobin.[76] Another effect on the cellular membrane is an alteration of its pliability, inducing a modification of blood rheology, which has been used to evaluate states of fitness in athletes.[167] 3.3 Antioxidant Measures 3.3.1 Enzymatic Antioxidant Activity

Enzymatic antioxidant activity (SOD, CAT and GPX) is quantified in a large majority of studies. This method can evaluate the quality of antioxidant protection at rest but can also show the importance of oxidative stress, especially after physical activity. After exercise, their evolution represents an adaptation upon FR production.[168,169] Antioxidant enzyme activity can be modified differently: increase  2006 Adis Data Information BV. All rights reserved.

at first (adaptation) or decrease if oxidative stress is important or long (utilisation). 3.3.2 Antioxidant Vitamins

Plasmatic quantification of antioxidant vitamins (vitamin A, C and E) is a common method to assess antioxidant protection and to detect vitamin deficiency.[12,13] As well as antioxidant enzymes, antioxidant vitamin concentrations are modified in the presence of oxidative stress and can be indirect markers of oxidative stress.[12] However, some precautions must be taken, especially for vitamin C. A stabiliser must be added in samples in order to stabilise this vitamin.[98] Vitamin E, in blood, is transported linked to blood lipids. Therefore, cholesterol is often measured in order to obtain the vitamin E/cholesterol ratio, which is a good marker of vitamin E status.[59] Caution is recommended when interpreting plasma antioxidant concentrations because variations, during exercise or training, may represent a redistribution between tissue and plasma.[87] 3.3.3 Other Antioxidants

A further technique for the measurement of antioxidant capacity of the body and oxidative stress is the measurement of thiol proteins. Like other antioxidants, a loss of thiol proteins can appear during a long period of oxidative stress. However, the quantification of GSH, the most important thiol in the human body, and GSSG (the oxidised form of GSH) is a popular technique to assess oxidative stress. The GSH/GSSG ratio is an interesting marker of oxidative stress because FR oxidise GSH into GSSG.[112,113] Uric acid is an important plasmatic and muscular antioxidant.[131,137] However, uric acid concentration can vary because of oxidative stress, purins cycle and renal excretions. Uric acid alone, therefore, can not represent a reliable marker of antioxidant capacity and oxidative stress; however, allantoin, a uric acid oxidation product, may be a valuable endogenous marker of oxidative stress. Thus, it could be considered as a good parameter to quantify oxidative stress because allantoin is theoretically absent from human body fluids under normal circumstances. Nevertheless, some results suggest that alSports Med 2006; 36 (4)

Oxidative Stress and Physical Activity

lantoin alone may have limited value as a marker of oxidative stress because allantoin can be oxidised and degraded by FR in blood samples. Thus, allantoin quantity can underestimate oxidative stress.[137] 3.3.4 Total Antioxidant Capacity

The large number of antioxidants in human fluids or in tissue makes it difficult to measure each antioxidant separately. Therefore, several methods have been developed to measure the total antioxidant capacity (TAC) of a biological sample.[162] The use of a pro-oxidant in order to quantify the oxygen radical absorbance capacity is one of the most used techniques.[13,170] The TAC gave an overall value corresponding to the sum of all antioxidants.[13,170] However, the interpretation of the changes in the antioxidant capacity is difficult because it can increase as a result of nutritional effects or because of an adaptation of oxidative stress. Furthermore, some antioxidant concentrations can be modified without any evolution of the TAC.[171] 3.4 Summary: is There an Ideal Method?

In general, every method has its interests and its limits, and because of this complexity, no single measurement of oxidative stress or of antioxidant status is going to be sufficient. Indeed, the interpretation of the values coming from a single marker could be a source of error. Therefore, a battery of measurements, including TAC, isolated antioxidant and markers of FR-induced damage on lipids, proteins and DNA seems to be a reliable method to assess oxidative stress.[13] 4. Oxidative Stress and Physical Activity 4.1 Oxidative Stress and Exercise 4.1.1 Aerobic Exercise Effects on FR Production

In 1982, Davies et al.[172] were the first to show that exercise increases FR production. Since then, a lot of studies have investigated the effects of exercise on oxidative stress. Most of them were carried  2006 Adis Data Information BV. All rights reserved.

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out with methods including aerobic exercise (running, cycling and swimming [see table IV]).[51,53,58,90,96,162,173-175] Aerobic exercise is accom˙ 2, which may increase FR panied by an increased VO ˙ 2, which, in activity. Aerobic exercise increases VO turn, may increase FR production. Therefore, many studies suggested that such physical activity enhanced FR production both in animals and in humans.[51,53,58,90,96,173-176] However, this phenomenon cannot occur with low exercise intensity (5km – 6 × wk – 10wk

Accominotti et al.[192] (1991)

Cycling (follow up)

Tessier et al.[112] (1995)

˙ 2max – 3 × wk – 10wk Running