Aging Clin Exp Res DOI 10.1007/s40520-015-0366-8

ORIGINAL ARTICLE

Organ and tissue-dependent effect of resveratrol and exercise on antioxidant defenses of old mice Bui Thanh Tung1 • Elisabet Rodriguez-Bies2 • Hai Nguyen Thanh1 • Huong Le-Thi-Thu1 • Pla´cido Navas2 • Virginia Motilva Sanchez3 • Guillermo Lo´pez-Lluch2

Received: 28 October 2014 / Accepted: 20 April 2015 Ó Springer International Publishing Switzerland 2015

Abstract Background Oxidative stress has been considered one of the causes of aging. For this reason, treatments based on antioxidants or those capable of increasing endogenous antioxidant activity have been taken into consideration to delay aging or age-related disease progression. Aim In this paper, we determine if resveratrol and exercise have similar effect on the antioxidant capacity of different organs in old mice. Methods Resveratrol (6 months) and/or exercise (1.5 months) was administered to old mice. Markers of oxidative stress (lipid peroxidation and glutathione) and activities and levels of antioxidant enzymes (SOD, catalase, glutathione peroxidase, glutathione reductase and transferase and thioredoxin reductases, NADH cytochrome B5-reductase and NAD(P)H-quinone acceptor oxidoreductase) were determined by spectrophotometry and Western blotting in different organs: liver, kidney, skeletal muscle, heart and brain. Results Both interventions improved antioxidant activity in the major organs of the mice. This induction was accompanied by a decrease in the level of lipid peroxidation in the liver, heart and muscle of mice. Both resveratrol and

& Bui Thanh Tung [email protected] 1

School of Medicine and Pharmacy, Vietnam National University, Hanoi, Floor 5 Building Y1, 144 Xuan Thuy, Cau Giay, Hanoi, Vietnam

2

Centro Andaluz de Biologı´a del Desarrollo, Universidad Pablo de Olavide-CSIC, CIBERER, Instituto de Salud Carlos III, Carretera de Utrera Km. 1, 41013 Seville, Spain

3

Departmento de Farmacologı´a, Facultad de Farmacia, Universidad de Sevilla, 41012 Seville, Spain

exercise modulated several antioxidant activities and protein levels. However, the effect of resveratrol, exercise or their combination was organ dependent, indicating that different organs respond in different ways to the same stimulus. Conclusions Our data suggest that physical activity and resveratrol may be of great importance for the prevention of age-related diseases, but that their organ-dependent effect must be taken into consideration to design a better intervention. Keywords Antiaging  Antioxidant  Resveratrol  Exercise  Old mice

Introduction Imbalance in the activity of antioxidant enzymes and the production of free radicals by metabolic activities, mainly associated with mitochondria, have been associated with the aging process by the free radical theory of aging [1, 2]. The main radicals in cells are derived from reactive oxygen species (ROS) and have been considered active factors in aging and aging research because of their potential involvement in many degenerative diseases. These ROS are highly reactive and damage many biological macromolecules such as DNA, RNA, protein and lipids [3]. For this, antioxidant enzymes constitute an important defense system to clear up the harmful ROS in vivo and to prevent oxidative damage of macromolecules. Resveratrol (trans-3,40 ,5-trihydroxystilbene) (RSV) is a naturally occurring phytoalexin found in red wine, berries and peanuts. RSV has shown many positive effects on biological systems ranging from cancer chemoprevention [4], prevention of inflammation [5] and antioxidant

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capacity [6]. Although its effect has been studied for more than a decade, the molecular mechanisms of RSV remain elusive [7]. Although a direct activation seems to be unlikely, the modulation of sirtuins by regulation of NAD?/ NADH ratio and the activation of the AMPK-dependent pathway seem to be the main mechanisms involved in the RSV effect on cells and organisms affecting longevity, metabolism regulation, cancer, inflammation, etc. [7, 8]. During the last years, the activation of the DNA damagedependent pathway [9, 10] by activating ATM and the regulation of the different pathways affected by this kinase probably indicate a common mechanism of action for all the effects of RSV on the cell and organisms. Furthermore, several investigations have demonstrated the role and protective effect of RSV against certain forms of oxidant damage, through a hydrogen-electron donation from its hydroxyl groups [11] or by increasing the expression of antioxidant enzymes [12]. Therefore, RSV may be an important dietary factor to improve health and prolong the average lifespan in animals [13, 14]. Physical activity is associated with better health mainly by its effect on muscle strength and the cardiovascular system. On the other hand, physical activity can also positively affect physiological endogenous antioxidant defenses in old subjects. It reduces the production of oxidants and oxidative damage, improves antioxidant defense system and increases the resistance of organs and tissues against the deleterious action of free radicals [15]. Furthermore, the physical activity level correlates closely with antioxidant enzymatic activities, especially related to the glutathione-dependent system in the liver and brain [16]. Furthermore, recently we have described the positive relationship of coenzyme Q10-dependent prevention of LDL oxidation and physical activity in elderly people [17, 18]. The aim of the present study was to evaluate the possible antiaging properties of RSV and/or physical activity in old mice by modulating endogenous antioxidant activities and enzyme levels in different organs. Thus, the content of glutathione, sulfhydryl group and lipid damage through malondialdehyde (MDA) and the activity and levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), NAD(P)H-quinone acceptor oxidoreductase (NQO1), NADH cytochrome b5 reductase (CytB5Rase) and thioredoxin reductase (TrxR) in different mice organs were determined. Furthermore, the antioxidant ratio (R) indicated as the activity of SOD related to the sum of the activities of CAT and GPx, which has been related to cell senescence [19, 20] was also determined. The different responses of organs to the same stimulus and their relationship with the induction of endogenous antioxidant systems are discussed.

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Materials and methods Animals and feeding regimen Male mice (C57BL/6 J) at the age of 18 months were used for these experiments. The experiments were of duration 6 months until their killing. Thus, at the end of the study, the mice were 24 months of age (old mice). A total of 16 mice were used, divided into four groups: Control no trained (Control-NT), Control trained (Control-T), Resveratrol no trained (RSV-NT) and Resveratrol trained (RSV-T). The animals were fed with a basal diet (Teklad Global Diet chow 2014S, Harlan) and kept in a thermostatically controlled cage holder at 22 °C with a 12 h lighting cycle. All animals were maintained according to a protocol approved by the Ethical Committee of the University Pablo de Olavide and following the international rules for animal research. Training consisted of running at a speed of 20 m/min, 20 min/day, 5 days/week, for all the time. The group Control was fed a liquid containing ethanol in water (180 lL ethanol/100 mL H2O) and the group RSV was fed a liquid containing resveratrol [180 lL of a dilution of 55 mg/mL trans-resveratrol in ethanol in100 mL H2O, reaching a concentration of 100 mg/L (0.01 % RSV)] in opaque bottles to avoid light-dependent decomposition. Drinking water was changed twice a week for both groups. Taken into consideration an average drinking of 4–5 mL/day and the weight of the animals, the calculated dose of RSV was around 500 lg/animal/day (16.67 mg/ kg/day). Animals were killed by cervical dislocation and dissected. The brain, kidney heart, muscle and liver were frozen in liquid nitrogen and stored at -80 °C until the analysis. All animals were maintained according to a protocol approved by the Ethical Committee of the University Pablo de Olavide of resolution 03/09 and following the international rules for animal research. Body weight Mice’s body weight was measured every 2 weeks to check for a possible influence of physical performance and RSV. Treatment of sample Frozen tissue from the brain, kidney heart, muscle and liver was homogenized in nine volumes of ice-cold tissue lysis buffer containing 150 mM sodium chloride, 1.0 % NP-40, 50 mM Tris, pH 8.0, and 1 mM PMSF (phenylmethylsulfonyl fluoride) with protease inhibitors (Sigma). Homogenates were centrifuged at 10009g for 10 min at 4 °C.

Aging Clin Exp Res

Single-use aliquots of the homogenates were stored at -80 °C before measurements. The protein concentration was determined by the Bradford’s method. Measurement of antioxidant activities and oxidative damage The SOD activity was spectrophotometrically measured using the method developed by Marklund and Marklund [21]. Briefly, SOD was detected on the basis of its ability to inhibit superoxide-mediated oxidation of pyrogallol. One unit was determined as the amount of enzyme that inhibited oxidation of pyrogallol by 50 %. CAT activity was measured by following the rate of disappearance of H2O2 at 240 nm [22]. One unit of CAT activity is defined as the amount of enzyme catalyzing the degradation of 1 lmol H2O2 per min and specific activity corresponding to transformation of H2O2 (in nmol) per min per mg protein. The whole amount of glutathione, reduced (GSH) plus oxidized (GSSG) forms, was determined by the method suggested by Anderson [23]. The amount of glutathione was expressed as nmol per mg total protein. The GPx activity was determined in a coupled assay with glutathione reductase by measuring the rate of NADPH oxidation at 340 nm using H2O2 as the substrate [24]. GR activity was determined by following the oxidation of NADPH at 340 nm as described by Carlberg and Mannervik [25]. GT activity was determined by Habig’s methods [26] based on the conjugation of 1-chloro-2,4dinitrobenzene (CDNB) with reduced glutathione. Enzymatic activity was calculated by using the extinction coefficient of 9.6 mM-1 cm-1 for CDNB and expressed as nmol/min/mg protein. Total CytB5Rase activity was assayed by measuring the rate of potassium ferricyanide reduction spectrophotometrically, according to the method of Strittmatter and Velick [27]. The enzyme activity was calculated using the extinction coefficient of 6.22 mM-1 cm-1 for NADH and expressed as nmol/min/mg protein. NQO1 activity was determined spectrophotometrically by monitoring the reduction of the standard electron acceptor, 2,6-dichlorophenol-indophenol (DCPIP) at 600 nm as described by Benson et al. [28] in the absence or presence of dicoumarol. The dicoumarol-inhibitable part of DCPIP’s reduction was calculated as NQO1 activity using the extinction coefficient of 21.0 mM-1 cm-1 and expressed as nmol DCPIP reduced/min/mg protein. TrxR activity was determined by the method of Hillet et al. [29] and based on the reduction of 5,50 -dithiobis

(2-nitrobenzoic acid) (DTNB) determined by the increase in absorbance at 412 nm. A unit of activity was defined as 1.0 nmol 5-thio-2-nitrobenzoic acid (TNB) formed/min/mg protein. Protein thiol (SH) groups were estimated by Ellman’s method [30]. Briefly, 0.5 ml of sample homogenate was added to a cuvette containing 0.5 ml phosphate buffer (0.1 M, pH 7.4); 0.2 ml of 3 mM 5,5-dithiobis (2-nitrobenzoic acid) was then added to start the reaction. After 10 min, absorbance was measured at 412 nm. The amount of SH groups was calculated according to the formula: mol SH/ml = [(Dsample/14,150)/dilution factor]/ml. Lipid peroxidation assay was performed by determining the reaction of malondialehyde with two molecules of 1-methyl-2-phenylindole at 45 °C as described by Ge´rardMonniern et al. [31]. Peroxidized lipids are expressed as nmol MDA equivalents/mg protein. All the biochemical analyses and enzyme activities were determined in triplicate per sample. Activity or determination per sample was considered as the mean of these three determinations. Data are the result of the mean of the samples from four animals per treatment (n = 4). Immunoblotting analysis Homogenates of samples were separated by 10 % (v/v) SDS-PAGE and then transferred to nitrocellulose membranes and subjected to immunoblot analyses using the primary antibodies anti-Cu, Zn-SOD (SOD1), anti-CAT, (Santa Cruz Biotechnology), anti-CytB5Rase (rabbit polyclonal antibody kindly provided by Dr. J. M. Villalba, Universidad de Co´rdoba, Spain), anti-GPx1, anti-TrxR1, anti-TrxR2 (Acris Antibodies, Germany) or anti-NQO1 (Abcam, Cambridge, UK) and secondary antibodies horseradish peroxidase-conjugated goat anti-rabbit or antisheep antibodies (Calbiochem, Germany). Protein expression levels were corrected for whole protein loading determined by staining membrane with Ponceau S. Protein expressions were visualized by the ChemiDocTM XRS? System and compiled with Image LabTM 4.0.1 Software (Bio-Rad Laboratories). Statistical analysis The results were analyzed by two-way ANOVA using SigmaPlot 10.0 program (Systat Software Inc.). All values were expressed as mean ± SE. The critical significance level a was established at 0.05 and, then, statistical significance was defined as P \ 0.05.

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Results RSV and exercise decreased oxidative damage in old mice To evaluate the levels of some oxidative damage markers, we determined the level of glutathione and MDA in different tissues. The results are shown in Table 1. The highest levels of glutathione were found in the liver, whereas other tissues showed similar lower levels around 6 nmol/mg proteins. In this case, exercise increased the level in the heart, muscle and liver without any effect on the brain and the kidney. On the other hand, RSV increased the level in the brain, muscle and liver without any effect on the kidney and the heart. Combined RSV with exercise only had an effect on the liver and heart. The lLevels of MDA, indicating lipid peroxidation, were higher in the brain and lower in the heart, kidney, and liver. Exercise decreased the level MDA in the liver, whereas RSV decreased MDA only in muscles. A combination of both produced a clear decrease in the heart, muscle and liver. Antioxidant activities are improved by RSV and exercise in old mice We determined the activity of several antioxidant enzymes in the different tissue (Tables 2, 3 and 4). Kidney and liver showed the highest CAT activity, whereas heart showed the lowest levels (Table 2). Interestingly, in those organs showing the highest activity, kidney and liver, it was further increased by exercise. RSV only induced activity in kidney, whereas the combination of both increased the activity in both organs. A trend toward increase in muscle was found, but without reaching statistical significance. Table 1 Oxidative stress markers in old mice

Control-NT

In the case of SOD, the response was different. Liver showed the highest activity, but exercise or RSV decreased it whereas their combination increased it. Heart activity was also induced by both as well as its combination. Exercise combined with RSV slightly increased its activity in brain. A trend toward an increase was also found in muscle, but without significance. Other important enzymatic activity involved in eliminating H2O2, GPx, was higher in kidney, muscle and liver and lower in heart and brain (Table 3). Interestingly, GPx activity increased in all organs after RSV treatment or training. This increase was significant in the liver, heart and kidney in the case of training, whereas it was significant in the brain, heart and liver in the case of RSV. The combination of both increased significantly the activity only in the kidney and liver. Interestingly, GR and GST activities did not respond to exercise or RSV as GPx. GR was not affected, whereas GST only increased in the heart and liver with exercise, RSV and their combination. Other interesting antioxidant activities linked to antioxidant protection in cell membranes were also affected by exercise or RSV depending on the organ (Table 4). CytB5Rase was induced by exercise in kidney and by the combination of exercise and RSV in kidney and liver. No effect was found with RSV alone. On the other hand, NQO1 activity was strongly induced in liver by both exercise and RSV, whereas in the heart exercise induced while RSV decreased it. Thioredoxin reductase activity was only determined in the kidney and liver. Exercise, RSV and its combination increased significantly TrxR activity in kidney. However, the contrary effect was found in the liver which showed a trend toward a decrease with exercise or RSV that was significant when both were combined.

Control-T

RSV-NT

RSV-T

Glutathione Brain

5.67 ± 0.14

5.60 ± 0.46

6.97 ± 0.50*

6.16 ± 0.54

Heart

6.19 ± 0.22

8.44 ± 0.91*

7.49 ± 0.65

7.69 ± 0.39*

Kidney

5.85 ± 0.69

6.45 ± 0.43

6.34 ± 0.69

6.52 ± 0.55

Muscle

6.52 ± 0.44

10.27 ± 0.52*

8.70 ± 0.65*

7.75 ± 0.72

Liver

21.4 ± 5.8

37.9 ± 0.1*

35.3 ± 0.3*

38.5 ± 2.3*

Brain

4.66 ± 0.24

4.68 ± 0.53

4.82 ± 0.38

4.71 ± 0.29

Heart

0.47 ± 0.06

0.31 ± 0.11

0.34 ± 0.05

0.29 ± 0.06*

Kidney

0.51 ± 0.12

0.55 ± 0.03

0.50 ± 0.07

0.52 ± 0.03

Muscle

1.07 ± 0.05

1.14 ± 0.02

0.69 ± 0.06*

0.85 ± 0.05*

Liver

0.45 ± 0.11

0.32 ± 0.06*

0.38 ± 0.07

0.32 ± 0.07*

MDA

Values are the mean ± SE. * Significant difference vs. Control-NT levels, P \ 0.05. Glutathione (sum of oxidized and reduced form) and MDA levels are indicated as nmol/mg protein

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Aging Clin Exp Res Table 2 Antioxidant CAT and SOD activities in old mice

Control-NT

Control-T

RSV-NT

RSV-T

Brain

2.07 ± 0.09

2.04 ± 0.05

2.11 ± 0.02

2.21 ± 0.07

Heart

3.37 ± 0.43

3.14 ± 0.39

3.35 ± 0.29

2.88 ± 0.34

Kidney

195 ± 22

236 ± 21*

250 ± 13*

276 ± 18*

Muscle

44.4 ± 5.4

57.9 ± 5.2

56.3 ± 5.1

58.1 ± 2.6

77 ± 3

110 ± 13*

85 ± 9

104 ± 7*

Brain

2.34 ± 0.12

2.39 ± 0.08

2.55 ± 0.1

2.77 ± 0.09*

Heart

7.51 ± 0.35

9.25 ± 0.34*

8.62 ± 0.21*

Kidney

2.21 ± 0.25

2.41 ± 0.14

2.50 ± 0.08

CAT

Liver SOD

10 ± 0.35* 2.35 ± 0.21

Muscle

19.4 ± 4.8

26.7 ± 4.2

27.7 ± 1.8

32.5 ± 1.3*

Liver

1542 ± 386

1298 ± 260

1470 ± 246

1761 ± 370

Values are the mean ± SE. * Significant difference vs. Control-NT levels, P \ 0.05. Activities are indicated as nmol/min/mg protein

Table 3 Antioxidant GPx, GR and GST activities in old mice

Control-NT

Control-T

RSV-NT

RSV-T

GPx Brain

17.4 ± 0.5

19.7 ± 0.6

21.5 ± 1.5*

19.7 ± 0.6

Heart

13.6 ± 2.4

16.1 ± 2.1*

16.1 ± 1.4*

14.7 ± 0.7

Kidney

30.3 ± 1.5

39.8 ± 3.8*

36.5 ± 1.4

39.5 ± 1.8*

Muscle

28.7 ± 0.5

30.5 ± 0.8

30.1 ± 1.3

28.5 ± 1.1

Liver

35.1 ± 12.8

79.2 ± 4.3*

69.9 ± 4.3*

85.7 ± 13.1*

GR Brain

2.30 ± 0.18

2.09 ± 0.08

2.19 ± 0.09

1.99 ± 0.14

Heart

4.84 ± 1.09

5.39 ± 0.61

5.30 ± 0.28

4.09 ± 0.55

Kidney

24.1 ± 0.9

22.5 ± 0.3

22.5 ± 1.2

26.6 ± 1.4

Muscle

8.36 ± 1.12

5.84 ± 1.10

9.42 ± 1.15

8.15 ± 0.89

Liver GST

28.2 ± 1.3

28.9 ± 1.6

27.0 ± 2.1

24.2 ± 0.5

Brain

90.7 ± 2.4

92.4 ± 1.4

97.4 ± 2.7

99.9 ± 4.7

Heart

18.4 ± 0.6

12.4 ± 0.6*

13.6 ± 0.5*

12.8 ± 0.8*

Kidney

35.1 ± 7.2

28.4 ± 1.0

31.5 ± 1.8

32.9 ± 1.9

Muscle

50.4 ± 7.6

54.2 ± 1.6

51.9 ± 3.7

54.5 ± 2.0

Liver

751 ± 212

1094 ± 106*

1130 ± 118*

897 ± 119

Values are the mean ± SE. * Significant difference vs. Control-NT levels, P \ 0.05. Activities are indicated as nmol/min/mg protein

Antioxidant protein levels are differentially affected by RSV and/or exercise in old mice Our experience shows that in many cases, increase of enzymatic activity is not accompanied by higher protein levels and vice versa [12, 32, 33]. For this reason, we also determined changes in the protein levels of antioxidant enzymes as indicated in the tables (Fig. 1). CAT levels were only induced by exercise and/or RSV in muscle, whereas other organs did not respond to these stimuli. However, SOD1 did show modifications at the protein level in all the organs studied, with kidney and

muscle being the most affected. Interestingly, in brain and liver, exercise induced SOD1 expression, but this effect was avoided when combined with RSV. In the case of GPx1, only the liver showed response to all the stimuli, whereas the brain only showed higher levels when both exercise and RSV were combined. In the case of GR, the response varied from lower levels in the brain to higher levels in the heart after exercise and RSV combination. In the case of the NAD(P)H-depending enzymes, CytB5Rase and NQO1, induction of activity found in kidney was accompanied by higher levels of the protein in this organ, whereas in the heart this increase was found when

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Aging Clin Exp Res Table 4 Antioxidant CytB5Rase, NQO1 and TrxR activities in old mice Control-NT

Control-T

RSV-NT

RSV-T

CytB5Rase Brain

245 ± 2

254 ± 5

258 ± 6

228 ± 1

Heart

772 ± 91

730 ± 72

715 ± 63

890 ± 69* 197 ± 21*

Kidney

148 ± 15

165 ± 13*

150 ± 8

Muscle

4.1 ± 0.5

5.3 ± 0.2

4.4 ± 1.0

5.7 ± 0.8

Liver

328 ± 33

351 ± 28

336 ± 26

381 ± 42*

NQO1 Brain Heart

5.3 ± 0.1

5.7 ± 0.3

16.3 ± 3.5

19.0 ± 2.9*

7.4 ± 0.4*

6.8 ± 0.7*

12.3 ± 2.7*

13.5 ± 2.5*

Kidney

1.1 ± 0.2

2.4 ± 0.2*

1.6 ± 0.4

3.1 ± 0.9*

Muscle

0.5 ± 0.0

0.5 ± 0.0

0.7 ± 0.1

0.6 ± 0.1

Liver

5.4 ± 0.9

11.7 ± 0.5*

7.7 ± 1.5*

9.8 ± 1.7*

TrxR Brain

Not determined

Heart

Not determined

Muscle

Not determined

Kidney

1.6 ± 0.4

2.3 ± 0.3*

3.0 ± 0.5*

3.0 ± 0.3*

Liver

5.2 ± 0.7

4.6 ± 0.3

4.4 ± 0.5

2.8 ± 1.0*

Values are the mean ± SE. * Significant difference vs. Control-NT levels, P \ 0.05. Activities are expressed as nmol/min/mg protein

RSV and exercise were combined and was accompanied by lower levels of protein. However, in the case of NQO1, the decrease in the activity found in heart when RSV and exercise were combined, was accompanied by lower levels of protein. However, the contrary was found in liver, higher activity accompanied by lower amount of protein. TRxR proteins showed a complex response to RSV and/ or exercise. Remarkably, in kidney, RSV or exercise seems to decrease the levels of TRxR1, but when combined these levels increased. In liver, this protein responded with higher presence induced by exercise but not by RSV, although the activity was inhibited by both interventions.

Discussion It is widely considered that the deleterious and irreversible changes produced by free radicals throughout the life of the organism are one of the main factors involved in aging [1]. Thus, free oxygen radicals have been proposed as important causative agents of aging. For this reason, in the theory of aging coined by Harman [34] in 1956, it is postulated that aging is produced by oxidative reactions caused by a higher production of free radicals or a lower capacity to eliminate them or to repair their oxidative effect. Abundant evidences show that a variety of ROS and other free radical are truly involved in the occurrence of molecular damage,

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which can lead to structural and functional disorders, diseases and death. RSV has been shown to have potent antiaging and health-promoting activities by modulating antioxidant activities in cells among other effects [12]. The same antiaging effect by modulating endogenous antioxidant capacity has been also associated with caloric restriction that is considered to be mimicked by RSV [32, 35]. On the other hand, physical activity ameliorates age-related impairments by reducing the oxidative damage and also improving antioxidant defense systems. Many antioxidants and antioxidant activities are involved in the protection against oxidative damage. These endogenous enzymatic antioxidant defenses include CAT, SOD, CytB5Rase, NQO1, glutathione, GPx, GR, GST and TrxR. Lipid peroxidation is one of the main events induced by oxidative stress and is particularly active in biomembranes like mitochondria. Polyunsaturated fatty acids (PUFAs) are one family of the most important components of cell membranes in living systems. Free radicals attack PUFAs leading to the formation of highly reactive electrophilic aldehydes, including MDA, 4-hydroxy-2-nonenal (HNE), and the most abundant products. Nohl’s study has reported accumulation of lipid peroxidation products during aging [36]. Furthermore, we have found that the antiaging effect of CR is more effective when the source of fat is rich in monounsaturated and saturated fatty acids than when rich in PUFAs [37–39]. In accordance with these studies, we have found that MDA levels increase with aging in mice liver. In this study, we also found that RSV and/or exercise can protect these membranes in the muscle, heart and liver in old mice indicating an induction of the endogenous antioxidant systems in these organs by mainly affecting SOD and GPx activities and levels of glutathione (Table 1). In this study, we show that RSV and/or exercise can affect endogenous antioxidant activities in different ways depending on the organ. Taking into consideration the different roles and locations of each enzyme, this effect can reflect the adaptive mechanisms of these organs against a mild oxidative stress induced by exercise or regulated by RSV. Our results agree with previous work by Wong et al. [40] which showed that long-term RSV intake attenuates oxidative damage in tissues specially affected during aging such as liver, heart or kidney. Moreover, the previous study of Thirunavukkarasu and coworkers [41] showed that exercise increases glutathione-dependent activities. Similarly, in the present study, administration of RSV and exercise improved the activity of GPx in old mice. However, the complex relationship between antioxidant activities can induce wrong conclusions from activity or protein levels. We show that in many cases, activity is not accompanied by similar changes at the protein levels.

Aging Clin Exp Res Fig. 1 Protein expression of antioxidant enzymes in different organs in old mice. Indicated organs were processed as indicated in ‘‘Materials and Methods’’ and the presence of CAT, SOD1, GPX1, GR, CytB5Rase, NQO1, TRxR1 and TRxR2 proteins determined by Western blotting. Quantification was performed considering Ponceau Red staining as loading control. Results refer to the levels found in each organ of the control group. *Significant differences vs. control levels in each organ. P \ 0.05

Furthermore, the relationship between antioxidant activities must also be taken into consideration. In fact, it was proposed that the imbalance in the ratio of SOD to CAT and GPx results in the accumulation of H2O2 that through the Fenton reaction results in the formation of hydroxyl radicals which are highly reactive and damage

macromolecules such as DNA, protein and lipids. For this reason, the balance in the activity of these enzymes has been directly related to cell senescence [19, 42]. Interestingly, a previous work demonstrated that the R ratio [R = SOD/(CAT ? GPx) in activities] increases in liver along with age [12]. The results shown in this manuscript

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demonstrate that both exercise and RSV decrease this ratio, indicating a higher protective effect in the liver. However, R was not affected in the other organs. In conclusion, our results indicate that both RSV and exercise improve in different manner the activities of endogenous antioxidant enzymes such as CAT, SOD1, GPx, GR, GST, NQO1 in old mice, and in some cases preventing the decrease of these activities associated with aging. Consequently, RSV supplementation and a higher physical activity should be strongly encouraged in older people, not only to improve physical function, avoid sarcopenia and maintain higher independence, but also to attenuate oxidative damage caused by aging. However, we cannot extrapolate the effects of these interventions in one or few organs to the whole organism. A deeper study of the regulation of antioxidant enzymatic activities and expression in relationship with aging is needed. Acknowledgments We thank Almudena Velazquez Dorado and Ana Sanchez Cuesta for their technical support. The group was financed by the Andalusian Government as the BIO177 Group through FEDER funds (European Commission). The research was financed by the Spanish Government Grant DEP2012-39985 (Spanish Ministry of Economy and Competitiveness). Tung Bui Thanh received a fellowship from the AECID program (Spanish Ministry of Foreing Affair). ERB, PN and GLL are also members of the Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras (CIBERER), Instituto Carlos III. Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest. Human and Animal Rights All animals were maintained according to a protocol approved by the Ethical Committee of the University Pablo de Olavide of resolution 03/09 and following the international rules for animal research. This article does not contain any studies with humans performed by any of the authors.

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