G Model TOX-50439; No.

of Pages 11

ARTICLE IN PRESS
Toxicology xxx (2009) xxxxxx

Contents lists available at ScienceDirect

Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study
a , Ana I. Galan a , Encarna Palacios a , Maria A. Diez a , Begona Muguerza a , Cesar Cobaleda a , Maria E. Munoz b c d Jose I. Calvo , Okezie I. Aruoma , Isidro Sanchez-Garcia , Rafael Jimenez a,
a

Department of Physiology & Pharmacology, Campus M. Unamuno, University of Salamanca, E-37007 Salamanca, Spain Department of Physiotherapy, School of Physiotherapy & Nursing, Campus M. Unamuno, University of Salamanca, E-37007 Salamanca, Spain Department of Pharmaceutical and Biomedical Sciences, Touro College of Pharmacy New York, 230 West 125 Street, New York 10027, USA d Centre for Cancer Research, CSIC/University of Salamanca, Campus M. Unamuno, E-37007 Salamanca, Spain
b c

a r t i c l e

i n f o

a b s t r a c t
The efcacy of long-term intake of a novel functional food supplement FuncionaTM containing vitamins and juiced fruits was evaluated in order to assess the net effect of physical activity and antioxidant potentials in healthy older adult population. The long-term (2 years) and large-scale (400 older adult subjects) interventional study was based on both moderate-intensity exercise practice and concurrent supplementation. Sustained exercise-induced oxidative stress as reected in signicantly increased blood thiobarbituric acid-reactive substances (TBARS) (+15%), protein carbonyl groups (PC) (+18%) and oxidized glutathione (GSSG) (+112%) concentrations, and leukocyte 8-OHdG contents (23%). Exercise decreased the reduced/oxidized glutathione (GSH/GSSG) molar ratio (43%) and plasma vitamin C levels (22%). Supplementation with FuncionaTM was signicant in preventing oxidative damage to lipid, protein and DNA, and normalizing blood GSSG, GSH/GSSG and vitamin C levels. Thus daily intake of the antioxidant functional beverage counteracts the exercise-induced oxidative stress in free-living older subjects, and might be necessary to restore impaired antioxidant balance due long-term regular exercise. 2009 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 14 July 2009 Received in revised form 12 September 2009 Accepted 14 October 2009 Available online xxx Keywords: Antioxidant functional food supplementation Antioxidant vitamins Elderly Clinical study Oxidative stress Regular exercise

1. Introduction Physical exercise is accepted as a vital component in the maintenance of good health as a strategy that mitigates the functional decline associated with ageing (ACSM, 2004; Ferrari, 2007; FisherWellman and Bloomer, 2009; Stewart, 2005). Booth and Roberts (2008) have reported that higher aerobic capacity and often higher skeletal muscle strength are associated with a lower prevalence of most chronic diseases and that maintenance of aerobic capacity and skeletal muscle strength by lifelong physical activity delays the biological ageing in most organ systems, therefore delaying premature death. The benets of exercise seem paradoxical, considering that exercise also increases oxygen consumption, which in turn leads to an increased mitochondrial production of presumably harmful reactive oxygen species (ROS), and it can also lead to oxidative stress and cellular injury (Urso and Clarkson, 2003; Fisher-Wellman and Bloomer, 2009; Goldfarb et al., 2005; Ristow et al., 2009; Rousseau et al., 2006; Stewart, 2005).

Corresponding author. Tel.: +34 923 294 672; fax: +34 923 294 669. E-mail address: rajim@usal.es (R. Jimenez). 0300-483X/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2009.10.015

Moderate to high intensity aerobic and endurance exercise causes signicant ROS generation in several tissues and increases oxidative stress biomarkers (Goldfarb et al., 2005, Aruoma, 1994). The majority of studies report signicant increases in oxidative stress biomarkers, and decreases in antioxidant vitamins and reduced glutathione (GSH) after short- and long-term exercise training in both young and aged persons (Bloomer et al., 2006; Fisher-Wellman and Bloomer, 2009; Jessup et al., 2003; Ji and Leeuwenburgh, 1996; Viitala and Newhouse, 2004). Although the efcacy of biological antioxidant network may be dependent on the efcient genetic up-regulation of the antioxidant enzymatic defence system and nutritional intake of vitamins and polyphenols (Banerjee et al., 2003; Bloomer et al., 2006; Fisher-Wellman and Bloomer, 2009; Rebrin et al., 2005; Soobrattee et al., 2005; Urso and Clarkson, 2003) their activities seem to decline with the aging process, exercise and deciencies in dietary intakes (Kostka et al., 2000; Bloomer et al., 2006; Harman, 2001; Rousseau et al., 2006). Systematic exercise training may also result in lower levels of oxidative stress mainly due to adaptations induced from the cumulative effect of repeated exercise bouts and exposure to ROS (Banerjee et al., 2003; Bloomer et al., 2006; Fisher-Wellman and Bloomer, 2009; Hofer et al., 2008; Ji et al., 2006; Radak et al., 2005; Ristow et al., 2009). Thus, the exercise-induced oxidative stress may

Please cite this article in press as: Munoz, M.E., et al., Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study. Toxicology (2009), doi:10.1016/j.tox.2009.10.015

G Model TOX-50439; No. of Pages 11 2

ARTICLE IN PRESS
M.E. Mu noz et al. / Toxicology xxx (2009) xxxxxx 2.2. Subjects and participant grouping Participation in the study was offered to 720 eligible members, and a cohort of 400 elderly free-living persons with a sedentary lifestyle was selected (306 women and 94 men; 6086 years old when selected) (Fig. 1). None of the subjects had performed any kind of programmed physical activity nor had devoted more than 1 h per week to programmed physical activity along the 4 months prior to the study. All volunteers completed a medical history, diet and supplementation history, and physical activity questionnaire to determine eligibility. Thirty exclusion criteria were dened that included alcohol consumption, regular use of antioxidant supplements, abnormal values in clinical parameters, and pathologies such as cancer, diabetes mellitus, respiratory and cardiovascular insufciency, etc. The participants had normal dietary habits and were able to safely carry out aerobic exercises of moderate intensity. The subjects were fully informed about all the experimental procedures and the risks and benets associated with the study, and those that agreed to participate provided written consent. The subjects selected were randomly assigned to one of three groups: Exercise (EXER), Exercise plus Antioxidant Treatment (EXAT) and Control (CO). For different reasons (not death) 23 individuals abandoned the study (10, 9 and 4 subjects from the CO, EXER and EXAT groups, respectively). 2.3. Experimental procedures Subjects with a sedentary lifestyle formed the CO group; they were not subjected to any intervention (no training and no antioxidant treatment) and participated only in the measurement procedures. The EXER and EXAT groups followed a supervised class-based exercise programme for 2 years, delivered in two cycles (10 months/cycle, 3 sessions/week, 50 min/session) separated by a 2-month interval of resting conditions (this interruption was imposed by the GEREPRO manager and was justied by the high temperatures prevailing during the Spanish summer). The exercise programme included the use of multiple components, according to the key practices recommended by the ACSM for elderly subjects (2004). Only the subjects in the EXAT group received a nutritional treatment along each cycle, consisting of the daily intake of 330 mL (one tetrapak) of a functional antioxidant beverage, BiofrutasTM (now FuncionaTM ) (Grupo Leche Pascual, Madrid, Spain). The energetic and nutritional values, and the major components and their concentrations in FuncionaTM are shown in Table 1. So, by drinking FuncionaTM , the subjects of the EXAT group received as supplement a total of 5.3 mg of vitamin E, 36.3 mg of vitamin C and 413 g of vitamin A per day, representing between 60% and 100% of the European RDA for elderly people, depending on the vitamin and sex considered (Garca-Gabarra, 2006a,b). The subjects of the EXER group were given a placebo, consisting of an aqueous solution of sucrose (9%), dextrose (3%) and anhydrous citric acid (0.0825%), pH 3.8. The exercise programme was performed in groups comprising of 2030 participants, under the supervision of a trained physiotherapist. The exercise regime (which was the same for all participants irrespective of their age and gender) was designed to maintain tness, cardiorespiratory capacity, muscular strength and endurance, and the exibility of the major muscle groups (Galn et al., 2006). Each workout session consisted of aerobic exercises, beginning with a 10 min warm-up period (stretching and walking) and nishing with a 5-min cool down and relaxation period. The main part of each session, 3540 min, consisted of multiple-set regimens of strength and endurance, and of static and dynamic techniques of exibility training. The intensity of the exercises was gradually increased and was regulated by the number of repetitions of particular exercises and series, as recommended by the ACSM (2004). Maximal effort never surpassed 70% of the maximum heart rate (100% of maximum load of effort during exercises = 200 minus age, according to Worms rule). In the middle and at the end of each session, participants drank water ad libitum for adequate hydration, as well as the placebo (EXER group) or the corresponding daily dose of FuncionaTM (EXAT group); these liquids were also taken under supervision to control intake on the resting days. The degree of compliance to the experimental protocol (attending the exercise sessions and complying with the daily intake of FuncionaTM ) was assessed as the percentage of exercise sessions attended by the participant (or the percentage of tetrapaks drunk) with respect to the total number of sessions (or doses) programmed per cycle. 2.4. Dietary records All participants were instructed to keep to their normal diets and to not consume any supplemental antioxidants during the study period. Dietary intake was assessed over the week prior to each of the four assessments performed along the study; data were obtained through the 24-h dietary recall and food record method (National Center for Health Statistics, U.S. Guides for Dietary Recall Interview, 19992001). The diet records were analyzed for total calories, protein, carbohydrate, and fat and antioxidant vitamins. 2.5. Assessment schedule, sampling and evaluations Participants samples were collected at weeks 0 (baseline measurements) and 40 of each cycle, performing 4 assessments along the study: 1st, 2nd, 3rd and 4th (Fig. 1). A 7-day diet description was obtained from each participant during the

not always be negative and could act (possibly through hormetic mechanisms) as an activator in redox-regulated processes involved in the adaptive responses to exercise (Goon et al., 2009; Ji et al., 2006; Radak et al., 2005; Ristow et al., 2009). The adaptive processes may decline during ageing, particularly following prolonged exercise, and fail in some disease states prevalent in the elderly (Yu and Chung, 2006). These facts suggest that the balance between the benecial and potentially harmful effects of exercise might be of particular importance in the older adults, whose low mitochondrial efciency, failure to adapt in response to exercise, and nutritional deciency of antioxidant vitamins are commonly associated with low plasma levels of antioxidants and low antioxidant enzyme activity (Fisher-Wellman and Bloomer, 2009; Harman, 2001; Urso and Clarkson, 2003; Yu and Chung, 2006). Although antioxidants are widely used as supplements supposedly to augment the healthpromoting effects of exercise, the net effect of regular physical activity on oxidative stress and antioxidant potentials in healthy older adult subjects, and the requirements for antioxidant nutrients of exercising elderly subjects need to be fully dened. Antioxidant supplementation may provide protection against the negative health consequences of ROS caused by exercise (Goldfarb et al., 2005; Jessup et al., 2003; Ristow et al., 2009). However, contradictory results have been reported (mostly in sportspersons, young or adult) where antioxidant effects have not been observed after supplementation with vitamin E (Huang et al., 2000; Viitala and Newhouse, 2004), C (Chao et al., 1999; Huang et al., 2000), E plus C (Huang et al., 2000) or E in combination with other antioxidants (Chao et al., 1999; Urso and Clarkson, 2003). Whilst some reports suggest a clear benet (Fisher-Wellman and Bloomer, 2009), particularly after high doses of antioxidant vitamin supplements (Goldfarb et al., 2005; Hartmann et al., 1995; Jessup et al., 2003), certain vitamins, when used alone or in high dosages, may in fact act as prooxidants (Bloomer et al., 2006; Urso and Clarkson, 2003). Data concerning long-term and large-scale intervention studies in elderly people based on controlled exercise and low dose antioxidant supplementation are lacking. The few results obtained from adult and older adult subjects derive from investigations carried out either after a bout of acute exercise, with only a few individuals and/or using high doses of antioxidant vitamins, or for a short period of antioxidant treatment (Bloomer et al., 2006; Fisher-Wellman and Bloomer, 2009; Huang et al., 2000; Jessup et al., 2003; Senturk et al., 2005). There thus remains an urgent need to evaluate the effects of long-term treatment with antioxidant food supplements in exercising elderly subjects. The aim of this study was (1) to verify the occurrence of oxidative stress after a long-term controlled programme of moderate exercise in healthy older adult persons and, (2) to evaluate whether concurrent daily supplement intake of a functional antioxidant beverage containing antioxidant phytochemicals and vitamins E, C and A at approximately nutritional doses counteracts or attenuates exercise-induced oxidative stress.
2. Subjects and experimental procedures 2.1. Study design The present study was carried out in non-institutionalised older subjects enrolled in a Geriatric Revitalization Program (GEREPRO) run by the Social Network of Community Centres for the Elderly in Salamanca (Spain). This Program, a long-term and large-scale intervention study, was performed as a randomized and controlled investigation based on the evaluation of both exercise and nutritional antioxidant treatment. The class-based exercises were selected, programmed, supervised, monitored, and evaluated by physiotherapists specialized in older adult subjects. The antioxidant treatment consisted of a long-term controlled supplementary intake of an antioxidant beverage, enriched with vitamins E, C and A. The investigation protocol conformed to the principles outlined in the Declaration of Helsinki (1996) for research on human beings. Approval for all experimental procedures was obtained from the Ethics Committee of the University of Salamanca and the Bioethics Board of the University Hospital of Salamanca.

Please cite this article in press as: Munoz, M.E., et al., Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study. Toxicology (2009), doi:10.1016/j.tox.2009.10.015

G Model TOX-50439; No. of Pages 11

ARTICLE IN PRESS
M.E. Mu noz et al. / Toxicology xxx (2009) xxxxxx 3

Fig. 1. Recruitment activity, groups, cycles of the trial, stages and number of participants in each group and stage of the study.

week before each assessment for comparing nutrient intakes among the groups. Blood pressure and resting heart rate, and anthropometric parameters were also evaluated. Blood samples were collected from all subjects at week 0 (1st and 3rd assessments of the study) and at week 40 (2nd and 4th assessments) of each cycle, that is, before and after interventions. The samples were collected by venipuncture after an overnight fasting period, and 24 h after the last session of exercise and/or the last dose of the antioxidant beverage. Complete blood cell determinations were performed in blood samples using an automated Coulter Counter at a clinical laboratory. Another blood sample was centrifuged and the main plasma biochemical markers (glucose, total cholesterol, HDL cholesterol, triglycerides, urea, uric acid, creatinine, total protein, albumin and thyroxine-binding prealbumin) and enzymic markers (AST, ALT, ALP and -GT) for the diagnosis of clinically healthy subjects were evaluated by routine methods with an automated auto sampler (Hitachi, mod. 717, Boehringer-Ingelheim, Germany). The rest of the plasma was stored in aliquots in liquid nitrogen until use for the determination of thiobarbituric acid-reactive substances (TBARS), carbonyl derivatives (PC), total glutathione (TGSH) and vitamins E, C and A concentrations. After blood centrifugation, leukocytes were immediately collected and frozen in liquid nitrogen until use for the determination of 8-hydroxy2 -deoxyguanosine (8-OHdG). 2.6. Laboratory measurements Plasma TBARS and PC concentrations were determined spectrophotometrically (Palomero et al., 2001). Total DNA from peripheral leukocytes was extracted by the pronase/ethanol method, and the amount of 8-OHdG was measured essentially by means of an HPLC system using deoxyguanosine (dG) and 8-OHdG as standards (Loft and Poulsen, 1999). The 8-OHdG levels were expressed as the number of 8-OHdG molecules per 105 dG. Plasma and whole blood TGSH were evaluated by the enzymatic procedure using glutathione reductase and 5,5 -dithio-bis (2-nitrobenzoic acid) (DTNB). For analysis of GSSG samples, 2M-vinylpyridine was used to derivatize GSH (Adams et al., 1983). Plasma antioxidant vitamins were determined using HPLC methods. Plasma ascorbate concentrations were estimated with electrochemical detection interfaced with a Kromasil 100NH2 column (250 mm 4.6 mm ID) (Kutnink et al. (1987). Vitamins E (-tocopherol) and A (all-trans-retinol) were

determined using a Phenomenex luna C18 column (250 mm 4.6 mm ID). The eluate was monitored by UV detection at 290 and 325 m, respectively (Talwar et al., 1998). 2.7. Statistical analysis Data are presented in terms of means SEM. Statistics and graphics were computed with a software package for Macintosh computers. Data were processed using JMPTM version 5.0 statistical software (SAS Institute, Cary, NC), and are presented as means SEM. Initially, one-way (grouping was the only factor) ANOVA was conducted to examine whether there were differences between groups in the pre-intervention values of each dependent variable. Then, owing to the intercycle inactivity period (no training or nutritional treatment) and to the number of drop-outs when the rst cycle of the study had concluded (see Fig. 1), the statis-

Table 1 Energetic and nutritional values and major components of the functional beverage FuncionaTM . (Values per 100 g of product) Energetic value Nutritional value Protein Carbohydrates Lipid Composition Vitamin E (d--tocopherol) (1.5 mg added) Vitamin C (ascorbic acid) (9.0 mg added) Vitamin A (retinol) (120 g added) Skimmed milk Concentrated juice from tropical fruits Water, sugar, dextrose, stabilizer 248 kJ (58 kcal) 0.36 g 14.2 g 0.05 g 1.6 mg 11.0 mg 125 g 10.0% 33.7% 56.1%

Please cite this article in press as: Munoz, M.E., et al., Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study. Toxicology (2009), doi:10.1016/j.tox.2009.10.015

G Model TOX-50439; No. of Pages 11 4

ARTICLE IN PRESS
M.E. Mu noz et al. / Toxicology xxx (2009) xxxxxx Table 2 Main anthropometric, functional, biochemical and hematological variables determined at the beginning of the study. Groups Parameters Age (years) Height (m) Weight (kg) Women Men BMI (kg/m2 ) Women Men Control 68.8 0.7 1.54 0.09 68.2 1.2 66.9 1.3 74.6 2.2 29.1 0.5 29.2 0.5 28.2 1.1 EXER 67.3 0.4 1.53 0.06 68.8 0.7 67.5 0.7 77.3 2.2 29.2 0.3 29.3 0.3 28.7 1.2 EXAT 69.1 0.6 1.54 0.08 70.1 0.9 69.6 1.0 78.2 1.5 29.6 0.3 29.1 0.4 28.3 0.8

tical analyses of the data obtained in each cycle were examined in two separate parts (participants who completed the rst cycle were included in a comparison between the 1st and 2nd tests, and participants who completed the second cycle were included in a comparison between the 3rd and 4th tests). In addition, statistical analyses were performed with a 3 2 repeated-measures ANOVA model to detect differences within and between groups for each time point. The 3 2 repeatedmeasures ANOVA model was performed on the dependent variables as a function of intervention (three groups: CO, EXER, EXAT) and time (two levels: pre- and postintervention). The pairs (CO vs EXER) and (CON vs EXAT) and (EXER vs EXAT) were compared using post hoc within the 3 2 repeated-measures ANOVA model. If a signicant (group treatment) interaction was found that is, when the F-ratios were signicant a post hoc Scheff test was conducted (on the between and/or the within subject factors) to discriminate between means when ANOVA yielded signicant results. An analysis of covariance was performed on the outcome variables at the end of each cycle in order to examine the possible inuence of dietary intake, even though no statistical differences were observed between groups at study entry. The covariate used was the baseline value for each subject for the particular outcome variable being analyzed. When the ANCOVA revealed that the covariate contributed to the outcome signicantly, then the predicted means generated by the ANCOVA were analyzed with a post hoc Scheff test. Statistical signicance was accepted at p < 0.05.

Blood pressure (mmHg; max/min) Women 138 2/74 2 Men 132 5/73 3 Glucose (mg/dL) Women Men Total cholesterol (mg/dL) Women Men Triglycerides (mg/dL) Women Men HDL cholesterol (mg/dL) Total protein (g/dL) Albumin (g/dL) Prealbumin (mg/dL) Urea (mg/dL) Uric acid (mg/dL) Calcium (mg/dL) Phosphorus (mg/dL) Erythrocytes (106 /L) Women Men Hemoglobin (g/dL) Hematocrit (%) Women Men Leukocytes (103 /L) Platelets (103 /L) 100 4.2 99 4.7 100 6 208 4 208 5 210 9 90.9 6.4 91.5 8.6 89.5 8.5 45.4 1.1 7.19 0.04 3.77 0.04 20.8 1.2 44.1 1.3 4.13 0.15 9.45 0.06 3.62 0.05 4.62 0.01 4.58 0.01 4.84 0.05 14.4 0.1 42.7 0.1 42.0 0.1 46.1 0.3 6.00 0.02 217 1

141 2/78 2 140 4/77 2 97.1 1.6 97.0 1.8 97.7 2.1 210 2 210 3 208 9 98.7 5.9 100 6 94 10 44.4 0.6 7.18 0.03 3.80 0.02 19.8 0.8 43.0 0.8 4.05 0.08 9.46 0.03 3.60 0.03 4.62 0.01 4.59 0.003 4.84 0.01 14.4 0.1 42.6 0.1 42.2 0.1 45.6 0.1 5.98 0.01 218 1

140 2/76 1 139 3/76 1 99.0 2.6 98.6 3.2 100 4 209 3 212 3 199 6 90.9 5.3 92.7 3.8 85.4 6.3 44.7 0.7 7.08 0.03 3.76 0.02 20.1 0.6 42.2 0.9 4.32 0.11 9.51 0.04 3.66 0.04 4.66 0.01 4.56 0.006 4.94 0.02 14.4 0.1 42.7 0.1 41.7 0.1 45.4 0.1 6.15 0.01 218 1

3. Results 3.1. Participant characteristics and compliance to treatment Three hundred and seventy-seven of the original 400 subjects completed the study. The anthropometric, biochemical and haematological data are within the normal ranges for the population in this study (Table 2). The subjects diets were consistent with the RDA measured by the 24-h dietary recall, and dietary intake did not differ signicantly between groups at any time point as regards average calorie intake (between 1800 and 2200 kcal/day) and the daily consumption of macronutrients, minerals and vitamins, except for the intake of antioxidant vitamins in the supplemented subjects, which were signicantly higher than in the EXER and Control subjects. The subjects of the EXER and EXAT groups who performed the rst cycle of the study had average attendances/compliances of 80% and 87%, respectively. These values were slightly higher in the second cycle. The data in Tables 24 as well as Figs. 2 and 3 show data from the subjects whose attendance at the exercise sessions and compliance with the antioxidant treatment was 75%. Data in Figs. 4 and 5 are based on the subjects whose attendance and compliance were 75% and 90% separately. 3.2. Effects of exercise and concurrent daily intake of FuncionaTM on systemic oxidative stress biomarkers Values for plasma TBARS and PC concentrations in Controls, EXER and EXAT subjects are shown in Table 3. In the CO group, no signicant differences were observed upon comparing the data obtained at the beginning and at the end of each of the cycles. This absence of changes in the levels of oxidation of lipid and protein in controls was also detected after separating the subjects by sex or age, in all of the four categories dened for age (65, 6670, 7175 and 76 years old). Regarding the EXER data, signicant interactions (group treatment) were found when a 3 2 repeated-measures ANOVA was performed for plasma TBARS and PC, the TBARS and PC values in the EXER group being signicantly higher at post-training than at pre-training. The mean post-training values of TBARS and PC were also signicantly higher than those of the controls at the same time point. In addition, TBARS levels at the beginning of the second cycle were lower than at the beginning of the rst cycle; however, the effect of exercise on TBARS in each individual cycle seems to be genuine since, although they start from different basal values, the relative increases in TBARS were similar after the rst (+15%) and the second (+14%) cycle. On the other hand, these effects were

Mean values (SEM) corresponding to the rst assessment in Controls (n = 80), exercise (EXER; n = 180) and exercise plus antioxidant-supplemented (EXAT, n = 140) groups.

related to the degree of attendance at the programmed sessions, in such a way that the increases in TBARS values in subjects who attended 90% of the sessions of the 1st cycle were signicantly higher (+24%, p < 0.05) than in those who attended 75% of the sessions (+15%). Values for plasma PC concentrations were similar among groups at the beginning of each the two cycles of the study, and exercise signicantly increased protein oxidation with respect to pre-training, in both the rst (+18%) and the second cycle (+11%). As in the case of the TBARS, the effects of exercise on PC were linked to the degree of attendance. For the subjects attending the rst cycle of exercise, the plasma TBARS levels were slightly higher in the men than in the women (Fig. 2A), but these differences were not signicant. Although exercise-induced signicant increases in TBARS levels in the four categories dened, the effects of exercise were greater in subjects between 71 and 75 years old than in the other age groups (Fig. 2C). Similar changes were observed after separating the PC data. In the EXAT subjects, concurrent daily intake of the functional beverage completely prevented the prooxidant effects of training on lipid and protein, detecting a signicant treatment effect with the

Please cite this article in press as: Munoz, M.E., et al., Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study. Toxicology (2009), doi:10.1016/j.tox.2009.10.015

G Model TOX-50439; No. of Pages 11

ARTICLE IN PRESS
M.E. Mu noz et al. / Toxicology xxx (2009) xxxxxx 5 1st cycle Before After 3.13 0.10 (+1%) (2.943.32) 3.39 0.07b,c (+15%) (3.303.48) 2.60 0.12b,c,e (19%) (2.522.68) 2nd cycle Before 2.75 0.06 (2.642.86) 2.66 0.06 (2.432.63) 2.79 0.06 (2.722.92) After 2.79 0.08 (+1%) (2.672.91) 3.03 0.07b,c (+14%) (2.893.09) 2.42 0.05b,c,e (13%) (2.342.50)

Table 3 Effects of exercise and concurrent dietary antioxidant supplementation on the plasma concentrations of TBARS and protein carbonyl groups in elderly persons. Groups

TBARS (M) Control

3.10 0.11 (2.923.28) 2.95 0.06 (2.853.05) 3.19 0.07 (3.073.31)

EXER EXAT Protein carbonyl groups (M) Control

29.7 1.4 (27.931.5) 30.5 0.8 (29.731.3) 31.1 0.7e (30.232.0)

29.4 1.5 (1%) (27.631.3) 36.1 0.9b,c (+18%) (35.237.0) 28.9 0.9e (7%) (28.129.7)

30.2 0.9 (28.831.6) 31.5 0.9 (30.632.4) 32.6 0.5 (31.833.4)

30.5 0.07 (+1%) (29.331.7) 34.9 0.5b,c (+11%) (34.335.6) 30.2 0.4e (7%) (29.630.9)

EXER EXAT

Mean values (SEM) in Controls (n = 7080) and in subjects performing the 1st and 2nd cycle of exercise (EXER group, n = 171180) or exercise plus antioxidant treatment (EXAT group, n = 136140), before and after the interventions (1st and 2nd, and 3rd and 4th assessments, respectively). (%): % of change with respect to before; ( ): 95% condence intervals; b, c, e: p < 0.05, signicantly different from before and to the Controls and Exercise groups, respectively.

repeated-measures ANOVA test (Table 3). Thus, a signicant intragroup difference was observed on comparing the data obtained before and after the interventions (training and supplement) (Fig. 2), effects that were statistically signicant in both the rst and the second cycle of the study. A similar degree of protection was observed after antioxidant supplementation for the PC levels (Table 3) since the exercise-induced increases in PC were completely abolished (with the mean values being similar to those of the Controls and signicantly lower than those observed before intervention in the non-supplemented subjects in both intervention cycles). 3.3. 8-Oxo-deoxyguanosine The data for the oxidation of peripheral leukocyte DNA corresponding to the rst cycle of interventions are shown in Table 4. No signicant differences among groups were observed in the baseline 8-OHdG levels. The baseline values of 8-OHdG in the Controls remained unchanged along the study, even after stratifying the subjects by sex or age. Regular exercise signicantly increased leukocyte 8-OHdG contents in the EXER subjects. At the end of the rst cycle the mean values of 8-OHdG were signicantly higher than the pre-exercise values (+23%, Table 4). A similar prooxidant effect was observed in the subjects who had completed the second cycle. The prooxidant effect of training on DNA was completely prevented
Table 4 Effects of exercise and concurrent dietary antioxidant supplementation on the leukocyte contents of 8-OHdG in elderly persons who performed the rst cycle of interventions. Groups Control EXER EXAT Before 30.5 2.61 (14.844.4) 29.5 2.29 (16.440.1) 29.9 2.83 (19.840.3) After 30.6 2.30 (+0.3%) (16.443.8) 36.2 1.91b,c (+23%) (27.056.4) 22.0 3.42b,c,e (26%) (10.837.9)

when the subjects were supplemented daily with the antioxidant beverage (EXAT group) with the mean post-treatment 8-OHdG values being signicantly lower than the basal ones in the EXER group. This antioxidant effect was also observed in the subjects performed the second cycle. 3.4. Plasma glutathione and glutathione redox status The values of the TGSH plasma concentrations (Fig. 3) were similar among groups at the beginning of the rst (Fig. 3A) and the second (Fig. 3B) cycles of the study. TGSH levels in the Controls remained stable along the study. By contrast, training reduced TGSH concentrations in the EXER subjects, inducing signicant reductions with respect to pre-training values in both the rst (29%, Fig. 3A) and the second (34%, Fig. 3B) cycle of the study. In the EXAT group, concurrent daily intake of the functional beverage did not prevent the exercise-induced TGSH depletion neither in the 1st nor in the 2nd cycle (Fig. 3). Regarding whole blood glutathione and glutathione redox status (Fig. 4), no signicant differences were observed in the baseline values of GSH, GSSG or the GSH/GSSG ratio among the Control, EXER and EXAT subjects. Interestingly, there were no signicant differences in these biomarkers in the Control group along the study. Signicant interactions (group treatment) were found when a 3 2 repeated-measures ANOVA was performed for blood GSSG, GSH and the GSH/GSSG ratio in the EXER and EXAT groups. These ndings clearly indicate that training enhanced the blood GSSG levels and reduced those of GSH and the GSH/GSSG molar ratio; these changes were statistically signicant with respect to the pretraining and control values, and were observed in both cycles of the study. These changes in glutathione status were related to the degree of compliance. Fig. 4 shows the data corresponding to the second post-training test (4th assessment data); the data depicted were obtained from subjects whose compliance was 75% and 90%. The mean blood GSSG concentrations in the EXER subjects who attended 75% and 90% of the exercise sessions were signicantly higher than the pre-training values (117% and 144%, respectively), and than in the Controls (112% and 137%, respectively) (Fig. 4A and B). By contrast, the GSH/GSSG molar ratio values in the EXER subjects were signicantly lower than at pre-training (45% and 62%, respectively), and were also signicantly lower than the Control values (43% and 52%, respectively) (Fig. 4C and D).

Mean values (SEM) in Controls (n = 34) and in subjects performing the exercise programme (EXER group, n = 44) or exercise plus antioxidant treatment (EXAT group, n = 45), before and after the interventions (1st and 2nd assessments, respectively). The 8-OHdG levels were expressed as the number of 8-OHdG molecules per 105 dG; (%): % of change with respect to before; ( ): 95% condence intervals;. b, c, e: p < 0.05, signicantly different from before and to the Control and Exercise groups, respectively.

Please cite this article in press as: Munoz, M.E., et al., Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study. Toxicology (2009), doi:10.1016/j.tox.2009.10.015

G Model TOX-50439; No. of Pages 11 6

ARTICLE IN PRESS
M.E. Mu noz et al. / Toxicology xxx (2009) xxxxxx

Fig. 2. Effects of exercise and of concurrent dietary antioxidant supplementation on the plasma contents of TBARS in elderly people following the rst cycle of the study. Inuence of sex (panels A and B) and age (panel C). Mean values (SEM). The results correspond to data of TBARS obtained before and after interventions (1st and 2nd assessments, respectively) and are depicted separately by sex (women = W, n = 8795; men = M, n = 5459) and age (65 years old, n = 31; 6670 years old, n = 47; 7175 years old, n = 38, and 76 years old, n = 30); b and e: a signicant difference (p < 0.05) with respect to before and to the EXER group, respectively.

The exercise-induced changes in the glutathione redox status were prevented in the subjects attending the exercise sessions and concurrently taking the antioxidant beverage. Thus, blood GSSG levels in the EXAT subjects who drank 75% and 90% of the intended doses of the beverage were signicantly lower than those observed in the exercising subjects treated with placebo (31% and 46%, respectively). The GSH/GSSG molar ratio in the EXAT subjects was also almost normalized at the end of the antioxidant treatment (Fig. 4C and D). The mean value of this ratio was higher than that observed in the non-supplemented subjects whose compliance was 75% (p < 0.053) and 90% (p < 0.001), but was similar to that of the controls at the same time point, and also similar to the pre-treatment values. 3.5. Effects of exercise and concurrent daily intake of FuncionaTM on plasma levels of antioxidant vitamins The variations in the plasma levels of retinol (vitamin A), tocopherol and vitamin C are shown in Fig. 5. No signicant

differences were observed in the baseline concentrations of the three vitamins among groups, in both the rst and the second cycle. At the end of the rst cycle of training, vitamin A (9%, Fig. 5A) and -tocopherol (8%, Fig. 5B) levels were slightly but not signicantly reduced with respect to their baseline values, whereas the concurrent antioxidant treatment slightly increased the levels of these vitamins (+14% and +16%, respectively), although none of these changes were statistically signicant. Similar changes were observed in the subjects who performed the second cycle of interventions. Regarding the vitamin C plasma levels, signicant differences were observed when comparing the results from the subjects that had completed one and two cycles of training (EXER group) with the data from the subjects under antioxidant treatment (EXAT group). Thus, in the EXER group, the post-training levels of vitamin C were signicantly lower than baseline and control values in the subjects who completed the rst cycle and, particularly, the second cycle of sessions (Fig. 5C and D), clearly indicating a training-induced

Please cite this article in press as: Munoz, M.E., et al., Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study. Toxicology (2009), doi:10.1016/j.tox.2009.10.015

G Model TOX-50439; No. of Pages 11

ARTICLE IN PRESS
M.E. Mu noz et al. / Toxicology xxx (2009) xxxxxx 7

Fig. 3. Effects of exercise and of concurrent dietary antioxidant supplementation on the plasma contents of total glutathione in elderly people following the rst (panel A) and the second (panel B) cycle of the study. Mean values (SEM). Results correspond to the data on total glutathione obtained before and after the interventions in each cycle from the Control ( , n = 70), EXER ( , n = 139) and EXAT ( , n = 118) subjects; b and c: signicant difference (p < 0.05) with respect to before interventions (or baseline) and to the Control group.

reduction in the plasma levels of this vitamin. The effects of exercise depended on the degree of attendance of the subjects at the exercise sessions: vitamin C levels were 14% and 22% lower (p < 0.05) than the baseline in the subjects whose attendances were 75% and 90%, respectively (Fig. 5C). Moreover, the training-induced decreases in vitamin C depended on sex, the reductions being higher in the men than in the women (Fig. 5D). In the EXAT group, the training-induced reductions in vitamin C were totally abolished by the concurrent antioxidant treatment, the mean values being signicantly higher than those observed in the EXER group at the same time point. These effects of the functional beverage in the EXAT subjects depended on the degree of compliance with the experimental protocol. When compliance was 75%, vitamin C levels were 13% higher (p < 0.05) than those observed in the EXER subjects (Fig. 5C) (the level was 28% when compliance was 90%). The antioxidant supplement-induced normalization was observed in both the men and the women (Fig. 5D) and in the four categories dened for age. Finally, after supplementation none of the members of the EXAT group had serum vitamin concentrations below the estimated desirable plasma concentrations of >50 mol/L vitamin C; in the EXER group, vitamin C was below this desirable level in 34% of the subjects. 4. Discussion Chronic exercise may both increase oxidative stress and augment antioxidant defences (Giacomo et al., 2009; Ristow et al., 2009). Concurrent dietary antioxidant supplementation prevented or palliated the exercise-induced increases in TBARS, PC, GSSG and 8-OHdG concentrations and the reductions in the GSH/GSSG molar

Fig. 4. Effects of exercise and of concurrent dietary antioxidant supplementation on the whole blood glutathione redox status in elderly people after separating the subjects by the degree of compliance to the experimental protocol of the study (compliance 75% and 90%). Mean values (SEM). Results correspond to data obtained after ending the 2nd cycle (4th assessment) and are expressed as absolute values for GSSG concentrations (panels A and B) and for the GSH/GSSG molar ratio (panels C and D). Controls ( ), EXER ( ) and EXAT ( ); b, c and e: signicant difference (p < 0.05) with respect to before interventions (or baseline) and to the Control and EXER groups, respectively. Subjects number: 70, 119 and 107 for compliance 75% in CO, EXER and EXAT groups, respectively; 70, 73 and 67 subjects for compliance 90% in CO, EXER and EXAT groups, respectively.

Please cite this article in press as: Munoz, M.E., et al., Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study. Toxicology (2009), doi:10.1016/j.tox.2009.10.015

G Model TOX-50439; No. of Pages 11 8

ARTICLE IN PRESS
M.E. Mu noz et al. / Toxicology xxx (2009) xxxxxx

Fig. 5. Effects of exercise and of concurrent dietary antioxidant supplementation on the plasma levels of retinol, -tocopherol and vitamin C in elderly people following one or both intervention cycles in the study. Controls ( , n = 68), EXER ( , n = 133) and EXAT ( , n = 112) groups. Mean plasma concentrations (SEM) of retinol (panel A) and -tocopherol (panel B) before (data from 1st and 3rd assessments) and after (2nd and 4th assessments) interventions in the subjects attending the rst and second cycles of the study. The results in panels C and D correspond to the data on vitamin C levels obtained after ending the 2nd cycle (data from 4th assessment), and are depicted separating by the degree of compliance to the experimental protocol (panel C) and by sex (panel D); n = 68123; b, c, e and m: signicant difference (p < 0.05) with respect to before (baseline) and to the Control and EXER groups and to men, respectively.

ratio and plasma levels of vitamin C. The exercise-induced increases in the plasma levels of TBARS and PC reported here are consistent with those previously reported in studies carried out in sedentary and trained healthy elderly subjects (Finaud et al., 2006; Jessup et al., 2003; Ji, 1999; Mergener et al., 2009; Meydani and Evans, 1993) but also differ from some other data from the literature in which reductions or absence of changes in serum TBARS levels in older subjects have been reported after a long-term period of resistance and aerobic exercise training (Fatouros et al., 2004; Parise et al., 2005b; Shin et al., 2008; Vincent et al., 2002). Regarding the GSH depletion observed in the EXER group, it has been reported that regular exercise either increases, decreases or does not change the levels of TGSH, GSH or GSSG in sedentary and trained subjects (Ji and Leeuwenburgh, 1996; Rousseau et al., 2006; Vincent et al., 2002). The baseline leukocyte 8-OHdG contents from the subjects of the EXER group were signicantly enhanced by exercise, in agree-

ment with the data reported by Goon et al. (2009), Mergener et al. (2009) and Evans et al. (2004) in aged persons, but not with the data from some other references (Hofer et al., 2008; Parise et al., 2005a). Thus, Mergener et al. (2009) have recently reported signicant increases (by about 8090%) in DNA oxidative damage in older adults that exercised regularly; in addition, Goon et al. (2009) have reported that Tai Chi practice during 6 months decreased normal DNA and increased mildly damaged DNA in volunteers aged 45 years and above; after 12 months of Tai Chi, the subjects had decreased normal DNA and increased severely damaged DNA. GSH depletion under stressing exercise and reductions of other antioxidants may increase the hydroxyl radical formation, which in turn leads to DNA damage. Sustained exercise, even at moderate intensities can cause macromolecular oxidation in elderly people, indicating that the rate of ROS generation under the exercise programme overwhelms

Please cite this article in press as: Munoz, M.E., et al., Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study. Toxicology (2009), doi:10.1016/j.tox.2009.10.015

G Model TOX-50439; No. of Pages 11

ARTICLE IN PRESS
M.E. Mu noz et al. / Toxicology xxx (2009) xxxxxx 9

efciency of the antioxidant defence systems, possibly due to the age-related attenuation (Kostka et al., 2000; Yu and Chung, 2006) of the training-induced adaptations of some antioxidant enzymes and/or to the inadequate levels of non-enzymatic antioxidants (Johnson, 2002; Parise et al., 2005b; Rousseau et al., 2006). Emerging evidence suggests that antioxidant-rich diet or supplementation with antioxidant vitamins or antioxidant mixtures reduce basal and exercise-induced oxidative stress by scavenging ROS and up-regulating the activities of antioxidant enzymes (Bloomer et al., 2006; Viitala and Newhouse, 2004). The ROS generated during exercise may also inuence the redox-regulated processes that control some of the adaptive responses to exercise, including gene expression, the induction of antioxidant enzymes, etc. (Fisher-Wellman and Bloomer, 2009; Goon et al., 2009; Parise et al., 2005b; Radak et al., 2005; Ristow et al., 2009). This raises the possibility that antioxidant supplementation could suppress some adaptive responses to exercise and ultimately counteract the benecial effects linked to these adaptations. Previous reports have indicated that VO2 max can be signicantly enhanced (and blood pressure was signicantly reduced) in both the exercising but nonsupplemented subjects and in the exercising and supplemented subjects (some indices of cardiovascular risk were also improved in a similar way in both groups) (Galn et al., 2006). Exercise can reduce the pool of vital antioxidant molecules in the elderly. Thus, supplementation with antioxidant vitamins in ratios and quantities close to the amounts consumed through a balanced whole-food diet (Bloomer et al., 2006; Jessup et al., 2003; Ji, 1999; Viitala and Newhouse, 2004) has been suggested. Most of the reported antioxidant intervention studies have been carried out in young people and/or sportspersons, generally using high or very high dose of antioxidants vitamins (Chao et al., 1999; Goldfarb et al., 2005; Huang et al., 2000; Senturk et al., 2005) and have been performed on low numbers of subjects or in short intervention periods (Huang et al., 2000; Senturk et al., 2005). However, only one interventional study had explored up to now the effect of antioxidant supplements in elderly persons doing regular exercise (Jessup et al., 2003). This underscores the uniqueness of the study reported here: essentially, this was a controlled long-term study on the daily intake of a functional antioxidant beverage at near nutritional doses to a large-scale population. The dietary supplementation with the antioxidant beverage led to the normalization of the redox status of glutathione, totally prevented the exercise-induced increases in TBARS, PC and 8-OHdG levels and also prevented the reductions normally observed in the levels of antioxidant vitamins, particularly vitamin C. These results are in complete agreement with reports of previous studies showing that antioxidant vitamin supplements (or a mixture of these) either prevents, attenuates, or totally antagonizes the exercise-induced oxidative injury to macromolecules in subjects of different ages, sex and training levels (Bloomer et al., 2006; Davison et al., 2007; Fisher-Wellman and Bloomer, 2009; Goldfarb et al., 2005; Jessup et al., 2003; Mastaloudis et al., 2004a; McAnulty et al., 2007; Senturk et al., 2005; Sureda et al., 2008; Traber, 2006). Other studies have not shown antioxidant effects either in trained or untrained subjects supplemented daily with vitamin E (Huang et al., 2000; Viitala and Newhouse, 2004), C (Chao et al., 1999; Huang et al., 2000), A (Chao et al., 1999), E plus C (Huang et al., 2000) or antioxidant mixtures (Chao et al., 1999; Fisher-Wellman and Bloomer, 2009). The reasons for the disparities in the various results reported could be related to a variety of factors, including the antioxidant(s) tested, the daily dose of vitamins administered (either singly or in combination), the nature, amount and timing of the exercise, the methodology used for assessing oxidative stress (for example, timing for blood sampling after the last exercise test/session, number and type of biomarkers evaluated, etc.)

and, especially, to the number, age and tness of the subjects evaluated. Studies in the elderly and in the young utilizing very high doses of vitamin E (800 UI -tocopherol/day) over 48 days have shown that vitamin E-supplement signicantly prevents lipid peroxidation induced by eccentric exercise (Meydani et al., 1993). Endurance exercises in combination with high doses of vitamin E for 16 weeks reduced oxidative stress and TBARS in both sedentary and exercisetrained older subjects (Jessup et al., 2003), similar to what has been observed in adult persons under antioxidant vitamin treatment for 8 weeks (Senturk et al., 2005). Evidence of the effect of exercise and antioxidant supplementation on oxidative DNA damage has not been denitively established, and the role of physiological concentrations of antioxidant vitamins in preventing DNA damage in humans remains to be conrmed (Barzilai and Yamamoto, 2004; Loft and Poulsen, 1999). Antioxidant vitamins and other antioxidants evaluated in vitro and administered to animals (Barzilai and Yamamoto, 2004) or humans (Barzilai and Yamamoto, 2004; Hartmann et al., 1995) can prevent DNA damage and increase DNA repair capacity in leukocytes under oxidative stress. A 14-day supplementation with vitamin E (Hartmann et al., 1995) and a long-term supplementation with vitamin C plus vitamin E (Mastaloudis et al., 2004b) have both reported prevention of the exercise-induced oxidative injury to DNA measured as signicant decreases in the blood (Barzilai and Yamamoto, 2004) and urinary (Huang et al., 2000) levels of 8-OHdG after exercise. Our present study, based on a long-term supplementation with a functional beverage, provided protection against the oxidative damage generated during regular exercise in the elderly. By contrast, other studies have failed to observe any protective effect after the intake of vegetable juice powder concentrate for 2 weeks (Bloomer et al., 2006) or after high-dose vitamin E or C supplementation for 2 months (Huang et al., 2000). The antioxidant effects observed on biomarkers of oxidative stress in the EXAT subjects might be due to the antioxidant vitamins and phytochemicals contributed daily through intake of the antioxidant beverage. Thus, whereas exercise-induced signicant decreases in plasma vitamin C levels, similar to those observed by others (Rousseau et al., 2006), these were totally prevented by the antioxidant drink when compliance with treatment was 75% and, particularly 95%. In addition, plasma vitamins A and E decreased slightly in the EXER subjects, but increased by about 15% with respect to the pre-intervention values in the supplemented individuals. It is well known that antioxidant vitamins and some phytochemicals may directly scavenge ROS and up-regulate the activities of antioxidant enzymes (Masella et al., 2005; Soobrattee et al., 2005; Urso and Clarkson, 2003), and it is possible that a long-term synergy between vitamins C and E could have taken place in the EXAT subjects since supplementation with the three antioxidant vitamins together has been shown to be more effective that either vitamin alone (Banerjee et al., 2003; Urso and Clarkson, 2003). Indeed, as reported in this study, vitamins C and E supplementation can restore the exercise-induced alterations in the GSH/GSSG ratio, an outcome consistent with the report of Urso and Clarkson (2003). Besides antioxidant vitamins, the food beverage used in this study contained 33.7% of a variety of tropical fruit juices known to contain signicant quantities of antioxidant phytonutrients, whose antioxidant actions are similar to, or even greater than, those of carotene (Masella et al., 2005; Soobrattee et al., 2005). Some of these phytochemicals (mainly polyphenols, bioavonoids and carotenoids) have excellent bioavailability (Rebrin et al., 2005; Soobrattee et al., 2005), conceivably act synergistically with antioxidant vitamins (Bloomer et al., 2006; Soobrattee et al., 2005) and, together with antioxidant vitamins (Clarkson and Thompson, 2000), are able to induce phase-2 enzymes, thus increasing the

Please cite this article in press as: Munoz, M.E., et al., Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study. Toxicology (2009), doi:10.1016/j.tox.2009.10.015

G Model TOX-50439; No. of Pages 11 10

ARTICLE IN PRESS
M.E. Mu noz et al. / Toxicology xxx (2009) xxxxxx Clarkson, P.M., Thompson, H.S., 2000. Antioxidants: what role do they play in physical activity and health? Am. J. Clin. Nutr. 72, 637646. Davison, G., Gleeson, M., Phillips, S., 2007. Antioxidant supplementation and immunoendocrine responses to prolonged exercise. Med. Sci. Sports Exerc. 39, 645652. Evans, M.D., Dizdaroglu, M., Cooke, M.S., 2004. Oxidative DNA damage and disease: induction, repair and signicance. Mutat. Res. 567, 161. Fatouros, I.G., Jamurtas, A.Z., Villiotou, V., Pouliopoulou, S., Fotinakis, P., Taxildaris, K., Diliconstantinos, G., 2004. Oxidative stress responses in older men during endurance training and detraining. Med. Sci. Sports Exerc. 36, 20652072. Ferrari, C.K., 2007. Functional foods and physical activities in health promotion of aging people. Maturitas 58, 327339. Finaud, J., Lac, G., Filaire, E., 2006. Oxidative stress: relationship with exercise and training. Sports Med. 36, 327358. Fisher-Wellman, K., Bloomer, R.J., 2009. Acute exercise and oxidative stress: a 30year history. Dyn. Med. 8, 125. Galn, A.I., Palacios, E., Ruiz, F., Dez, A., Arji, M., Almar, M., Moreno, C., Calvo, J.I., Munoz, M.E., Delgado, M.A., Jimnez, R., 2006. Exercise, oxidative stress and risk of cardiovascular disease in the elderly. Protective role of antioxidant functional foods. Biofactors 27, 167183. Garca-Gabarra, A., 2006a. Ingesta de nutrientes: conceptos y recomendaciones internacionales (1a parte). Nutr. Hosp. 21, 291299. Garca-Gabarra, A., 2006b. Ingesta de nutrientes: conceptos y recomendaciones internacionales (2a parte). Nutr. Hosp. 21, 437447. Giacomo, C., Acquaviva, R., Sorrenti, V., Vanella, A., Grasso, S., Barcellona, M.L., Galvano, F., Vanella, L., Renis, M., 2009. Oxidative and antioxidant status in plasma of runners: effect of oral supplementation with natural antioxidants. J. Med. Food 12, 145150. Goldfarb, A.H., Bloomer, R.J., McKenzie, M.J., 2005. Combined antioxidant treatment effects on blood oxidative stress after eccentric exercise. Med. Sci. Sports Exerc. 37, 234239. Goon, J.A., Aini, A.H., Musalmah, M., Anum, M.Y., Nazaimoon, W.M., Ngah, W.Z., 2009. Effect of Tai Chi exercise on DNA damage, antioxidant enzymes, and oxidative stress in middle-age adults. J. Phys. Act. Health 6, 4354. Harman, D., 2001. Aging: overview. Ann. NY Acad. Sci. 928, 121. Hartmann, A., Niess, A.M., Grunert-Fuchs, M., Poch, B., Speit, G., 1995. Vitamin E prevents exercise-induced DNA damage. Mutat. Res. 346, 195202. Hofer, T., Fontana, L., Anton, S.D., Weiss, E.P., Villareal, D., Malayappan, B., Leeuwenburgh, C., 2008. Long-term effects of caloric restriction or exercise on DNA and RNA oxidation levels in white blood cells and urine in humans. Rejuvenat. Res. 11, 793799. Huang, H., Helzlsouer, K., Appel, L., 2000. The effects of vitamin C and vitamin E on oxidative DNA damage: results from a randomized controlled trial. Cancer Epidemiol. Biomarkers Prev. 9, 647652. Jessup, J.V., Horne, C., Yarandi, H., Quindry, J., 2003. The effects of endurance exercise and vitamin E on oxidative stress in the elderly. Biol. Res. Nurs. 5, 4755. Ji, L.L., Leeuwenburgh, C., 1996. Glutathione and exercise. In: Somani, S. (Ed.), Pharmacology in Exercise and Sports. CRC Press, New York, pp. 97123. Ji, L.L., 1999. Antioxidant and oxidative stress in exercise. Proc. Soc. Exp. Biol. Med. 222, 283292. J., 2006. Exercise and hormesis: activation of Ji, L.L., Gomez-Cabrera, M.C., Vina, cellular antioxidant signaling pathway. Ann. NY Acad. Sci. 1067, 425435. Johnson, P., 2002. Antioxidant enzyme expression in health and disease: effects of exercise and hypertension. Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 133, 493505. Johnston, C.S., Dancho, C.L., Strong, G.M., 2003. Orange juice ingestion and supplemental vitamin C are equally effective at reducing plasma lipid peroxidation in healthy adult women. J. Am. Coll. Nutr. 22, 519523. Kostka, T., Drai, J., Berthouze, S.E., Lacour, J.R., Bonnefoy, M., 2000. Physical activity, aerobic capacity and selected markers of oxidative stress and the anti-oxidant defence system in healthy active elderly men. Clin. Physiol. 20, 185190. Kutnink, M.A., Hawkes, W.C., Schaus, E.E., Omaye, S.T., 1987. An internal standard method for the unattended high-performance liquid chromatographic analysis of ascorbic acid in blood components. Anal. Biochem. 166, 424430. Loft, S., Poulsen, H.E., 1999. Markers of oxidative damage to DNA: antioxidant and molecular damage. Methods Enzymol. 300, 166184. Masella, R., Di Benedetto, R., Vari, R., Filesi, C., Giovannini, C., 2005. Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione-related enzymes. J. Nutr. Biochem. 16, 577586. Mastaloudis, A., Morrow, J.D., Hopkins, D.W., Devaraj, S., Traber, M.G., 2004a. Antioxidant supplementation prevents exercise-induced lipid peroxidation, but not inammation, in ultramarathon runners. Free Radic. Biol. Med. 36, 13291341. Mastaloudis, A., Yu, T.W., ODonnell, R.P., Frei, B., Dashwood, R.H., Traber, M.G., 2004b. Endurance exercise results in DNA damage as detected by the comet assay. Free Radic. Biol. Med. 36, 966975. McAnulty, S., McAnulty, L., Nieman, D., Morrow, J., Dumke, C., Utter, A., 2007. Carbohydrate effect: hormone and oxidative changes. Int. J. Sports Med. 28, 921927. Mergener, M., Martins, M.R., Antunes, M.V., da Silva, C.C., Lazzaretti, C., Fontanive, T.O., Suyenaga, E.S., Ardenghi, P.G., Maluf, S.W., Gamaro, G.D., 2009. Oxidative stress and DNA damage in older adults that do exercises regularly. Clin. Biochem. 42, 16481653. Meydani, M., Evans, W.J., 1993. Free radicals, exercise and aging. In: Yu, B.P. (Ed.), Free Radicals in Aging. CRC Press, Boca Raton, FL, pp. 183204. Meydani, M., Evans, W.J., Handelman, G., Biddle, L., Fielding, R.A., Meydani, S.N., Burrill, J., Fiatarone, M.A., Blumberg, J.B., Cannon, J.G., 1993. Protective effect of

biosynthesis of cellular antioxidants and counteracting alterations in the glutathione redox status (Masella et al., 2005). Therefore, it is possible that additional antioxidants provided by the functional beverage, other than vitamins E, C and A, might have contributed to the protection against the exercise-induced oxidative stress. Plasma and tissue levels of some antioxidant vitamins have been shown to decrease in aged persons after exercising (Bloomer et al., 2006; Clarkson and Thompson, 2000; Rousseau et al., 2006). The results in the EXER and EXAT subjects are in agreement with what has been observed in sportspersons and elderly subjects enrolled in short-time placebo-controlled studies (Hartmann et al., 1995; Jessup et al., 2003), with the results observed in older adult women supplemented with physiological doses of vitamins E and A (Wolters et al., 2004), and with those obtained in middle-aged men after a combined antioxidant supplementation for 12 weeks at a moderate dose (Volkovova et al., 2005). In addition, in some studies carried out with antioxidant functional foods, increases in the plasma antioxidant activity and vitamin C levels (Snchez-Moreno et al., 2003), reductions of about 45% in TBARS levels (Johnston et al., 2003), and increases in plasma vitamin C and lymphocyte DNA resistance to oxidative stress (Riso et al., 2005) have been observed. 5. Conclusions Regular moderate-intensity training over a long-term period in the elderly induces oxidative stress, leading to partial depletion of vitamin C and GSH, and the oxidation of lipids, proteins, GSH and DNA. The results on supplemented subjects provide experimental evidence that the daily supplement of approximately nutritional dose of antioxidant vitamins over a long-term period was able to avoid oxidative damage to macromolecules. For elderly people, regular and prolonged exercise, even though of moderate intensity, should perhaps be accompanied by a daily consumption of antioxidant food supplements in order to prevent exercise-induced oxidative stress, to reach an adequate level of antioxidant defence, and to optimize the control of the cellular redox status. Conict of interest statement The authors declare that there are no conicts of interest. Acknowledgments We are especially grateful to the study participants for their extraordinary cooperation. This study was supported by grants from the Salamanca University (grant ref. LCI2) and Grupo Leche Pascual (Madrid, Spain). References
ACSM American College of Sports Medicine, 2004. Physical activity programs and behaviour counselling in older adult populations. Med. Sci. Sports Exerc. 36, 19972003. Adams, J.D., Lauterburg, B.H., Mitchell, J.R., 1983. Plasma glutathione and glutathione disulde in the rat: regulation and response to oxidative stress. J. Pharmacol. Exp. Ther. 227, 749754. Aruoma, O.I., 1994. Free radicals and antioxidant strategies in sports. J. Nutr. Biochem. 5, 370381. Banerjee, A.K., Mandal, A., Chanda, D., Chakraborti, S., 2003. Oxidant, antioxidant and physical exercise. Mol. Cell. Biochem. 253, 307312. Barzilai, A., Yamamoto, K., 2004. DNA damage responses to oxidative stress. DNA Repair 3, 11091115. Bloomer, R.J., Goldfarb, A.H., McKenzie, M.J., 2006. Oxidative stress response to aerobic exercise: comparison of antioxidant supplements. Med. Sci. Sports Exerc. 38, 10981105. Booth, F.W., Roberts, C.K., 2008. Linking performance and chronic disease risk: indices of physical performance are surrogates for health. Br. J. Sports Med. 42, 950952. Chao, W.H., Askew, E.W., Roberts, D.E., Wood, S.M., Perkins, J.B., 1999. Oxidative stress in humans during work at moderate altitude. J. Nutr. 129, 20092012.

Please cite this article in press as: Munoz, M.E., et al., Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study. Toxicology (2009), doi:10.1016/j.tox.2009.10.015

G Model TOX-50439; No. of Pages 11

ARTICLE IN PRESS
M.E. Mu noz et al. / Toxicology xxx (2009) xxxxxx 11 Shin, Y.A., Lee, J.H., Song, W., Jun, T.W., 2008. Exercise training improves the antioxidant enzyme activity with no changes of telomere length. Mech. Ageing Dev. 129, 254260. Soobrattee, M.A., Neergheen, V.S., Luximon-Ramma, A., Aruoma, O.I., Bahorun, T., 2005. Phenolics as potential antioxidant therapeutic agents: mechanism and actions. Mutat. Res. 579, 200213. Stewart, K.J., 2005. Physical activity and aging. Ann. NY Acad. Sci. 1055, 193206. Sureda, A., Tauler, P., Aguil, A., Cases, N., Llompart, I., Tur, J.A., Pons, A., 2008. Inuence of an antioxidant vitamin-enriched drink on pre- and post-exercise lymphocyte antioxidant system. Ann. Nutr. Metab. 52, 233240. Talwar, D., Ha, T.K., Cooney, J., Brownlee, C., OReilly, D.S., 1998. A routine method for the simultaneous measurement of retinol, alpha-tocopherol and ve carotenoids in human plasma by reverse phase HPLC. Clin. Chim. Acta 270, 85100. Traber, M.G., 2006. Relationship of vitamin E metabolism and oxidation in exercising human subjects. Br. J. Nutr. 96 (Suppl. 1), S34S37. Urso, M.L., Clarkson, P.M., 2003. Oxidative stress, exercise, and antioxidant supplementation. Toxicology 189, 4154. Viitala, P., Newhouse, I.J., 2004. Vitamin E supplementation, exercise and lipid peroxidation in human participants. Eur. J. Appl. Physiol. 93, 108115. Vincent, K.R., Vincent, H.K., Braith, R.W., Lennon, S.L., Lowenthal, D.T., 2002. Resistance exercise training attenuates exercise-induced lipid peroxidation in the elderly. Eur. J. Appl. Physiol. 87, 416423. Volkovova, K., Barancokova, M., Kazimirova, A., Collins, A., Raslova, K., Smolkova, B., Horska, A., Wsolova, L., Dusinska, M., 2005. Antioxidant supplementation reduces inter-individual variation in markers of oxidative damage. Free Radic. Res. 39, 659666. Wolters, M., Hermann, S., Hahn, A., 2004. Effects of 6-month multivitamin supplementation on serum concentrations of alpha-tocopherol, beta-carotene, and vitamin C in healthy elderly women. Int. J. Vitam. Nutr. Res. 74, 161168. Yu, B.P., Chung, H.Y., 2006. Adaptive mechanisms to oxidative stress during aging. Mech. Ageing Dev. 127, 436443.

vitamin E on exercise-induced oxidative damage in young and older adults. Am. J. Physiol. 264, 992998. Palomero, J., Galn, A.I., Munoz, M.E., Tunn, M.J., Gonzlez-Gallego, J., Jimnez, R., 2001. Effects of ageing on the susceptibility to the toxic effects of cyclosporin A in rats. Changes in liver glutathione and antioxidant enzymes. Free Radic. Biol. Med. 30, 836845. Parise, G., Brose, A.N., Tarnopolsky, M.A., 2005a. Resistance exercise training decreases oxidative damage to DNA and increases cytochrome oxidase activity in older adults. Exp. Gerontol. 40, 173180. Parise, G., Phillips, S.M., Kaczor, J.J., Tarnopolsky, M.A., 2005b. Antioxidant enzyme activity is up-regulated after unilateral resistance exercise training in older adults. Free Radic. Biol. Med. 39, 289295. Radak, Z., Chung, H.Y., Goto, S., 2005. Exercise and hormesis: oxidative stress-related adaptation for successful aging. Biogerontology 6, 7175. Rebrin, I., Zicker, S., Wedekind, K.J., Paetau-Robinson, I., Packer, L., Sohal, R.S., 2005. Effect of antioxidant-enriched diets on glutathione redox status in tissue homogenates and mitochondria of the senescence-accelerated mouse. Free Radic. Biol. Med. 39, 549557. Riso, P., Visioli, F., Gardana, C., Grande, S., Brusamolino, A., Galvano, F., Porrini, M., 2005. Effects of blood orange juice intake on antioxidant bioavailability and on different markers related to oxidative stress. J. Agric. Food Chem. 53, 941947. Ristow, M., Zarse, K., Oberbach, A., Klting, N., Birringer, M., Kiehntopf, M., Stumvoll, M., Kahn, C.R., Blher, M., 2009. Antioxidants prevent health-promoting effects of physical exercise in humans. Proc. Natl. Acad. Sci. U.S.A. 106, 86658670. Rousseau, A.S., Margaritis, I., Arnaud, J., Faure, H., Roussel, A.M., 2006. Physical activity alters antioxidant status in exercising elderly subjects. J. Nutr. Biochem. 17, 463470. Snchez-Moreno, C., Cano, M.P., de Ancos, B., Plaza, L., Olmedilla, B., Granado, F., Martin, A., 2003. Effect of orange juice intake on vitamin C concentrations and biomarkers of antioxidant status in humans. Am. J. Clin. Nutr. 78, 454460. Senturk, U.K., Yalcin, O., Gunduz, F., Kuru, O., Meiselman, H.J., Baskurt, O.K., 2005. Effect of antioxidant vitamin treatment on the time course of hematological and hemorheological alterations after an exhausting exercise episode in human subjects. J. Appl. Physiol. 98, 12721279.

Please cite this article in press as: Munoz, M.E., et al., Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study. Toxicology (2009), doi:10.1016/j.tox.2009.10.015