Scand J Med Sci Sports 2007: 17: 595604
Printed in Singapore . All rights reserved
DOI: 10.1111/j.1600-0838.2006.00620.x
Effects of strength and endurance training on antioxidant enzyme
gene expression and activity in middle-aged men
D. Garc a-Lopez1, K. Hakkinen2, M. J. Cuevas1, E. Lima1, A. Kauhanen2, M. Mattila2, E. Sillanpaa2, J. P. Ahtiainen2,
L. Karavirta2, M. Almar1, J. Gonzalez-Gallego1
1
Institute of Biomedicine, University of Leon, Leon, Spain, 2Department of Biology of Physical Activity, University of Jyvaskyla,
Jyvaskyla, Finland
Corresponding author: Javier Gonzalez-Gallego, Institute of Biomedicine, University of Leon, 24071 Leon, Spain. Tel: 134
987 291258, Fax: 134 987 291267, E-mail: djgg@unileon.es
Accepted for publication 8 November 2006
This study was aimed at investigating the eects of a 21-
week period of progressive strength or endurance training on
peripheral blood mononuclear cells (PBMC) antioxidant
enzyme gene expression and activity in healthy middle-aged
untrained men. Strength (n 5 11) and endurance (n 5 12)
training were performed twice a week, including resistance
exercises to activate all the main muscle groups or cycle-
ergometer pedaling, respectively. mRNA levels of catalase,
glutathione peroxidase (GPx), mitochondrial superoxide
dismutase (MnSOD) and cytosolic superoxide dismutase
(CuZnSOD) were increased after 21 weeks of strength
training, while endurance training induced signicant
changes only in MnSOD and GPx mRNA levels. CuZn
It is well known that acute exhaustive exercise causes
signicant reactive oxygen species generation that
results in oxidative stress (Ji, 1993), which can induce
negative eects on health and well-being. In fact,
increased oxidative stress biomarkers (i.e., protein
carbonyls, MDA, and 8-hydroxyguanosine) have
been observed after supramaximal sprint exercises
(Cuevas et al., 2005), exhaustive long-distance cy-
cling or running (Almar et al., 2002; Aguilo et al.,
2005) as well as strength-type exercises (Avery et al.,
2003), both in trained and untrained humans. Anti-
oxidant enzyme activities such as superoxide dis-
mutase (SOD), catalase (CAT) and glutathione
peroxidase (GPx) provide the primary defense
against reactive oxygen species generated during
exercise, and the activities of these enzymes are
known to increase in response to exhaustive exercise
in both animal and human tissues (Ji, 1995). Cellular
defense against superoxide radicals is provided by
SOD, which dismutates them to form H2O2 and O2.
Two isozymes of SOD exist in mammalian cells,
diering in both cellular location and the metal
cofactor bound to its active site. The cytosolic super-
oxide dismutase (CuZnSOD) isoform is located pri-
Copyright & 2007 The Authors
Journal compilation & 2007 Blackwell Munksgaard
SOD protein content was signicantly increased only in
strength-trained subjects. The program of strength or
endurance exercise training had no signicant eects on
the activity of any of the antioxidant enzymes. In conclu-
sion, in a middle-aged population, 21 weeks of strength or
endurance training was a sucient stimulus to up-regulate
mRNA levels of PBMC antioxidant enzymes, the strength
training being a more optimal stimulus. However, the
discrepancies between enzyme protein and mRNA levels
suggest that the present systematic strength or endurance
training period had no benecial eects on enzymatic
antioxidant defense mechanisms in previously untrained
middle-aged men.
marily in the cytosol, whereas the mitochondrial
SOD (MnSOD) isoform is found in the mitochondria
(Ji, 1995). GPx is an enzyme responsible for reducing
H2O2 or organic hydroperoxides to water and alco-
hol, respectively, and it is located in both the cytosol
and the mitochondria. CAT catalyzes the breakdown
of H2O2 to form water and O2 (Ji, 1995). While there
is overlap between the function of CAT and GPx, the
two enzymes dier in their anity for H2O2. Thus,
when cellular levels of H2O2 are low, GPx is more
active than CAT in removing H2O2 from the cell
(Powers & Lennon, 1999).
Endurance training, which is characterized by
prolonged rhythmic, low-resistance, high-repetition
exercises (Fleck & Kraemer, 1988), has been postu-
lated as a potential muscles antioxidant defense
system up-regulator (Fatouros et al., 2004). In fact,
high maximal oxygen uptake (VO2max), which is a
consequence of systematic endurance training
(Tanaka & Swensen, 1998), has been correlated to
elevated antioxidant enzyme activity (Jenkins et al.,
1984). Furthermore, previous studies have reported
that high-intensity running training can elevate
antioxidant enzyme activities in erythrocytes and
595
Garc a-Lopez et al.
decrease neutrophil superoxide anion (O2
) produc- ities, none of them had any background in regular strength or
tion both at rest and in response to exhausting acute endurance training. They were not taking any medication
exercise (Miyazaki et al., 2001). However, both in known to aect hormonal or metabolic responses to exercise.
Before data collection, subjects were informed of the require-
treadmill-trained rats (Leeuwenburgh et al., 1994) ments and risks associated with participation and provided
and running-trained humans (Tiidus et al., 1996), written informed consent. All procedures were approved by
endurance training does not appear to aect antiox- the local Institutional Review Board for the protection of
idant defenses. Much less is known about the eects human subjects.
of chronic strength training on cellular antioxidant Participants were randomly assigned to one of three groups:
strength training group (S, n 5 12), endurance training group
capacity. Strength training, which typically consists (E, n 5 12) and non-training control group (C, n 5 8). One
of high-resistance and low-repetition exercises (Fleck subject from the S group dropped out during the study period,
& Kraemer, 1988), could prevent lipid peroxidation so at the end eleven subjects composed this group. Subjects
(Vincent et al., 2002; Ramel et al., 2004) or oxidative physical characteristics were obtained after the blood collec-
damage to DNA (Parise et al., 2005a), as well as tion, during the same laboratory session. Body fat content was
estimated by the InBody, bioelectric impedance technique
enhance the skeletal muscle cellular antioxidant ca- (InBody 720, Biospace, Seoul, Korea). Descriptive character-
pacity (Parise et al., 2005b), although there is no istics of the three groups are presented in Table 1.
sucient evidence proving these chronic adaptations.
Moreover, it should be noted, that to date, there
have been no reports documenting the behavior of
antioxidant enzymes in peripheral blood mononuc- Experimental design
lear cells (PBMC), following either strength or en- The study included a supervised 21-week training period,
durance training. Peripheral blood is an accessible which was performed by the S and E groups. The blood
source of cells that can be viewed as scouts, con- samples were collected and strength and aerobic performance
measurements were carried out before and after the training
tinuously maintaining a vigilant and comprehensive
period. Subjects from the S and E groups were also tested in
surveillance of the body for signs of infection or the middle (week 10.5) of the training period. For each
other threats (Whitney et al., 2003). PBMC are evaluation time (weeks 0, 10.5, and 21), blood collecting as
among the essential cell mediators of stress and well as strength and aerobic testing took place during three
inammation and produce cytokines, chemokines separated laboratory sessions. Moreover, for a given subject,
each one of the tests was carried out at the same time of the
and growth factors that can have benecial or
day throughout the experiment. A minimum of 48 h of rest
pathological eects on tissues (Connolly et al.,
2004). Therefore, exercise, depending on mode and
intensity, is believed to induce some level of local
Table 1. Subject physical and performance characteristics along the
muscle tissue disruption, resulting in a local inam- 21-week experimental training period
matory response that may include migration and
inltration of leukocytes into tissues. Leukocytes in C (n 5 8) S (n 5 11) E (n 5 12)
circulation may represent a population of cells on
Age (years) 53.3  2.5 54.9  1.9 53.6  2.4
their way toward participation in ongoing tissue Height (cm) 175.5  1.3 178.9  2.6 179.5  1.7
surveillance and adaptations, playing a role in re- Body mass (kg)
sponse to structural damage in skeletal muscle in- Pre-training 75.3  2.2 81.1  1.9 78.9  2.2
duced by exercise (Malm et al., 2000; Rowbottom & Mid-training 80.6  2.1 78.8  2.3
Post-training 75.0  2.3 80.2  2.0 77.2  2.1
Green, 2000). In fact, positive correlations between Body fat (%)
immunological variables in human blood and skele- Pre-training 17.5  1.7 22.0  2.7 20.9  2.0
tal muscle have been found in previous studies Mid-training 18.9  2.4* 18.8  2.0*
Post-training 17.3  1.1 19.22  2.2* 18.6  1.5*
(Malm et al., 2000). In any case, the study of the Leg press 1RM (kg)
PBMC genomic response to exercise may prove a Pre-training 151.4  4.4 161.8  8.2 144.6  7.7
useful area of research to deepen in the mechanisms Mid-training 177.9  9.2* 149.8  8.1
that link physical activity with health. Thus, our Post-training 161.2  7.2 197.5  9.3*#f$ 160.0  8.5*
VO2max (mL/min)
overall purpose was to investigate the eects of Pre-training 36.6  2.4 33.4  1.5 32.6  1.5
strength or endurance training on PBMC antioxi- Mid-training 34.7  1.6 34.6  1.7*
dant enzyme gene expression and activity in middle- Post-training 37.3  2.4 33.5  1.6 35.8  2.0*
aged untrained men.
Values are mean  SEM.
*Significant difference (Po0.05) from the corresponding pre-training
value,
Methods #
Significant difference (Po0.05) from the corresponding 10.5 week value,
Subjects f
Significant difference (Po0.05) from the corresponding C value,
$
Thirty-two healthy middle-aged men from the city of Jyvas- Significant difference (Po0.05) from the corresponding E value.
kyla took part in the study. Although subjects had been C, control group; S, strength training group; E, endurance training group;
previously involved with various recreational physical activ- SEM, standard error of the mean; 1RM, one repetition-maximum.

596

was allowed after the last training session before the tests were
conducted.
Strength and aerobic testing
Throughout the study, leg-press maximal dynamic force (one
repetition-maximum [1RM]) and maximal oxygen uptake
(VO2max) tests were performed on dierent lab sessions,
separated by a minimum of 48 h. A David 210 dynamometer
(David Fitness and Medical Ltd., Vantaa, Finland) was used
to measure maximal bilateral concentric force production of
the leg extensors (hip, knee and ankle extensors) (Hakkinen
et al., 1998). The subject was in a seated position so that the
hip angle was 1101. On verbal command, the subject per-
formed a concentric bilateral leg extension starting from a
knee-exed position of 701, trying to reach a full extension of
1801 against the resistance determined by the loads (kg)
chosen on the weight stack. In the testing of the maximal
load, separate 1RM contractions were performed. After each
repetition, the load was increased until the subject was unable
to reach a full-extended position. The last acceptable extension
with the highest possible load was determined as 1RM.
A VO2max test was carried out using a Monark Ergomedic
839E bicycle ergometer (Monark Exercise AB, Vansbro,
Sweden). The intensity was 50 W at the beginning of the test
and was increased by 20 W every second minute until exhaus-
tion. Heart rate was monitored continuously with a Polar
S810i hear rate monitor (Polar Electro Oy, Kempele, Finland),
and blood pressure once before and every second minute
during the test. The oxygen uptake (VO2) was measured
continuously using the Sensormedics Vmax229 (Sensormedics,
Yorba Linda, California, USA). Blood samples were taken
from ngertip every second minute to measure blood lactate
concentrations and determine aerobic and anaerobic thresh-
olds as described in detail previously (Aunola & Rusko, 1984).
Blood lactate was determined using a Lactate Pro LT-1710
lactate analyzer (Arkray, Kyoto, Japan).
Strength training
The 21-week whole-body strength training was carried out,
under supervision, twice a week, with a special emphasis on
the lower body. Therefore, each training session included two
exercises for the leg extensor muscles and one exercise for the
leg exors. The program included three to ve other exercises
for the other main muscle groups of the body. Mainly,
machine exercises were used throughout the training period.
All the exercises were performed using concentric muscle
actions, followed by eccentric actions. The loads were deter-
mined throughout the study during the training sessions every
34 weeks for the 21-week training period according to the
maximum-repetition method. The intensity ranged from 40%
to 80% of the 1RM. The number of sets in each exercise
increased and the number of repetitions decreased during the
training program. Thus, during the rst 7 weeks, subjects
trained with loads equivalent to 4070% of the 1RM, with 8
15 repetitions per set and two to four sets per exercise. During
weeks 814 loads were 6080% of the maximum, with 512
repetitions per set and two to ve sets per exercise. During the
last 7 weeks, loads were 6085% or the 1RM, with 510
repetitions per set and three to ve sets.
Endurance training
Endurance training was carried out by a bicycle ergometer
twice a week. All the training sessions were supervised and
heart rate monitoring was used. Thus, the heart rate levels of
Training and antioxidant enzymes
endurance training were determined on grounds of the aerobic
performance tests. During the rst 7 weeks, subjects trained
30 min/training session under the aerobic threshold. The rst
7 weeks also include a few training sessions during which the
subjects were accustomed to the intensity above the aerobic
threshold. During weeks 814, 1 of the 2 weekly sessions of
45 min was divided into four loading intervals: 15 min under
the level of aerobic threshold, 10 min between the aerobic
anaerobic thresholds, 5 min above the anaerobic threshold and
15 min again under the aerobic threshold. The other of the
2 weekly training sessions was 60 min under the aerobic
threshold. The focus of training during weeks 1521 was to
improve cycling speed and maximal endurance in a 60-min
session: 30 min under the aerobic threshold during the whole
session altogether, 2  10 min between the aerobic-anaerobic
thresholds and 2  5 min above the anaerobic threshold.
Every other training session included 90-min cycling under
the aerobic threshold.
Blood sample preparation and descriptive data collection
Venous blood samples (15 mL) were taken using EDTA as an
anticoagulant. Blood samples were obtained, using a catheter
closed by a stylet, from the braquiocephalic vein at rest, under
fasting conditions. Immediately after extraction, a measure of
3 mL of the whole blood was centrifuged at 1500 g for 10 min
at 4 1C, and plasma aliquots were stored at
85 1C until
s
further determination of Trolox -equivalent antioxidant ca-
pacity (TEAC) and reduced glutathione.
PBMC were separated from the whole blood by density
gradient centrifugation on Ficoll separating solution (Biochrom
AG, Berlin, Germany), as previously described (Cuevas et al.,
2005). For each sample, two 15-mL centrifuge tubes were used
to layer 6 mL of blood onto 4 mL of Ficoll. The suspension was
centrifuged for 30 min at 275 g and 20 1C. The mononuclear cell
layer was removed with manual pipetteing, washed one time in
Hanks solution and centrifuged for 10 min at 20 1C and 450 g
after the wash. Washed cells were resuspended in 1 mL of PBS.
Analyses were performed on frozen cells.
TEAC
The TEAC was determined according to the method based on
the oxidation of the 2,2 0 -azinobis-(3-ethylbenzothiazoline-6-
sulfonic acid) diammonium salt (ABTS) by potassium persulfte
to form a radical cation (ABTS1), as previously described (Re
et al., 1999). This intensely colored radical cation has a
maximum of absorption at 734 nm, and the direct scavenging
of the preformed radical by hydrogen-donating antioxidants
resulted in a decrease of the absorbance at 734 nm. The
absorbance at 734 nm was measured after 60 s from the start
of the reaction on a Hitachi Spectrophotometer (Hitachi High
Technologies America, Inc., Schaumburg (IL) USA). TEAC
values were calculated from the percentage of reduction of the
absorbance at 734 nm, indicating the ability of the compounds
to scavenge ABTS1, in relation to that induced by a Trolox
standard solution, under the ssame experimental conditions,
and are expressed as a Trolox equivalent.
Reverse transcription and quantitative real-time polymerase
chain reaction
Total RNA was isolated from PBMC by using a RiboPuret-
Blood Kit (Ambion, Inc., Austin (TX) USA) and quantied
by the uorescent method Ribogreen RNA Quantitation Kit
(Molecular Probes, Eugene (OR) USA). Residual genomic
597
Garc a-Lopez et al.
DNA was removed by incubating RNA with DNase I RNase-
free DNase (Ambion, Inc., Austin (TX) USA). First-standard
cDNA was synthesized using the High-Capacity cDNA Ar-
chive Kit (Applied Biosystems, Foster City (CA) USA). The
negative control (no transcriptase control) was performed in
parallel. cDNA was amplied using the TaqMan Universal
PCR Master Mix (Applied Biosystems) on an ABI 7000
(Applied Biosystems). TaqMan primers and probes for CAT
(Genbank X04076.1 and Hs00156308_m1), GPx (Genbank
Y00433.1 and Hs00829989_gH), CuZnSOD (Genbank
X02317.1 and Hs00533490_m1), MnSOD (Genbank M36693.1
and Hs00167309_m1) and (housekeeping gene) 18S rRNA
(Genbank X03205.1 and Hs99999901_s1) s
were derived from
the commercially available TaqMan Assays-on-Demand Gene
(Applied Biosystems). The relative changes in gene expression
levels were determined using the 2
DDCT method as described
previously (Livak & Schmittgen, 2001). The cycle number at
which the transcripts were detectable (CT) was normalized to the
cycle number of 18S detection, referred to as DCT.
Western blot analysis
For Western blot analysis PBMC were homogenized with
150 mL of 0.25 mM sucrose, 1 mM EDTA, 10 mM Tris and a
protease inhibitor cocktail (Tunon et al., 2003). Protein con-
centration was determinated as described previously (Lowry et
al., 1951). Samples containing 50 mg of protein were separated
by SDS-polyacrylamide gel electrophoresis (9% acrylamide)
and transferred to PVDF membranes. Non-specic binding
was blocked by preincubation of the PVDF membranes in PBS
containing 5% bovine serum albumin for 1 h. The membranes
were then incubated overnight at 4 1C with appropriate
antibodies. Antibodies against
s
CAT (6065 kDa) were pur-
chased from Calbiochem (Darmastadt, Germany), antibodies
against CuZnSOD (23 kDa) were purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz (CA) USA), and antibodies
against MnSOD (25 kDa) and GPx (23 kDa) were purchased
from Stressgen Biotechnologies (Ann Arbor (MI) USA).
Bound primary antibody was detected using a peroxidase-
conjugated secondary antibody (Dako, Glostrup, Denmark)
by chemiluminiscence using the ECL kit (Amersham, Piscat-
away (NJ) USA). The density of the specic bands was
quantitated with an imaging densitometer. The blots were
stripped in 6.25 mM Tris, pH 6.7, 2% SDS and 100 mM
mercaptoethanol at 50 1C for 15 min and probed again for
anti-beta-actin antibodies (Sigma-Aldrich, St. Louis (MO)
USA) (42 kDa) to verify equal protein loading in each lane.
Antioxidant enzyme activities
Immediately after obtaining the mononuclear cell extract,
quantitative determination of cytosolic CAT was performed.
Measurement of H2O2 concentration was recorded by using
UV absorbance at 240 nm as previously described (Aebi,
1984). Activity was expressed as U/mg protein. Cytosolic
GPx activity was quantied with continuous photometric
monitoring of oxidized glutathione (GSSG) at 37 1C. The
conversion of NADPH to NADP was evaluated using UV
absorbance at 340 nm (Flohe & Gunnzler, 1984). GPx activity
was calculated after substraction of the blank value, as U/mg
protein. Finally, total SOD was determined by using the
inhibition by SOD of the reaction of O2
with NBT following
a technique published previously (Oberley & Spitz, 1984).
NaCN was added 1 h before the measurement in order to
assay MnSOD activity. The MnSOD activity was subtracted
from the total SOD activity to calculate the CuZnSOD
activity. Both SOD activities were expressed as U/mg protein.
598
Statistical analysis
All data are reported as mean  standard error of the mean
(SEM). An analysis of variance (ANOVA) with repeated
measures for time (pre-training and 21 weeks) and group (S,
E, and C) was performed. A dierent ANOVA with repeated
measures for time (pre-training, 10.5 and 21 weeks) and
training group (S and E) was carried out to assess the mid-
term training-related eects. Bonferronis post-hoc analysis
was used where appropriate. Pearsons R was calculated to
assess correlations. A value of Po0.05 was regarded as
signicant. An SPSS1vrs. 13.0 statistical software (Chicago,
Illinois, USA) was used.
Results
Physical characteristics
Table 1 shows body mass and body fat percentage
from each group throughout the experimental per-
iod. The lack of a signicant time by training inter-
action points out that no between-group dierences
occurred over time. Regarding the S and E groups,
no signicant changes took place in the body mass,
although a signicant time main eect (F1,31 5 4.64,
Po0.05) was observed in body fat percentage, which
was signicantly reduced (Po0.05) in both experi-
mental groups after the rst 10.5 weeks of training.
The control group did not show any change regard-
ing body mass or body fat percentage.
Strength and endurance performance
Table 1 shows leg-press 1RM and VO2max values for
each group throughout the experimental period. The
1RM demonstrated a signicant time main eect
(Po0.001), as well as a training main eect
(Po0.05) and a time by training main eect
(Po0.01). In both S and E groups, 1RM was
signicantly increased (Po0.01) vs pre-training value
after the 21-week training period (22% and 10.6%
for S and E, respectively). In the S group, 1RM value
was signicantly increased across all time points: pre-
to mid-training (Po0.001) and mid- to post-training
(Po0.01). Moreover, the S group reached a signi-
cantly higher 1RM value than the E group after 10.5
weeks (Po0.05) and 21 weeks (Po0.01) of training.
The VO2max showed a signicant time main eect
(Po0.05), but no training main eect or time by
training main eect. In the E group, the VO2max
increased over the pre-training value after 10.5 weeks
(6.13%, Po0.05) and 21 weeks (9.88%, Po0.05).
The control group did not show any change regard-
ing 1RM or VO2max.
Antioxidant capacity
At pre-training, TEAC was 13.71  0.36,
13.49  0.35, and 13.21  0.26 nmol/mg prot for E,
S, and C group, respectively. Statistical analysis did
 Training and antioxidant enzymes
not show any signicant time, training or time by increased vs the pre-training value after 10.5 weeks
training main eects across the experimental period. (20%) and 21 weeks (40%) of endurance training.
Twenty-one weeks of strength or endurance ex- However, the mRNA level of GPx was only signi-
ercise training had no signicant eects on the cantly increased vs the pre-training value after 21
antioxidant enzyme activities (Table 2). Concerning weeks (31%) of training and no signicant eects on
protein content, there were no signicant time, train- the mRNA levels of CuZnSOD and CAT were found
ing or time by training main eects on CAT, GPx (Fig. 4). After the 21 weeks experimental period, S
and MnSOD. However, CuZnSOD protein content group values concerning CAT and CuZnSOD
showed a signicant time main eect (Po0.001), as mRNA were signicantly higher than E and C
well as a training main eect (Po0.001) and a time values. Again, the C group did not show any change
by training main eect (Po0.001; Fig. 1). In the S regarding gene expression of the antioxidant enzymes
group, CuZnSOD protein content was signicantly measured.
increased vs pre-training value after 10.5 weeks
(29.14%; Po0.001) and 21 weeks (49.67%;
Po0.001) of strength training. Although the baseline Discussion
value was similar among groups, the S group reached
a signicantly higher value than the E and C groups Strength and endurance training, when performed
after 10.5 weeks (Po0.05) and 21 weeks (Po0.01) of independently, induce dierent functional and struc-
training. No change regarding the protein content of tural adaptations. Strength training, which normally
the antioxidant enzymes measured was detected for aims to lead to increases in muscle mass and strength,
either endurance group (Fig. 2) or control group. can induce some other chronic adaptations such as a
Gene expression of all the antioxidant enzymes reduced blood pressure, body fat and mitochondrial
measured (MnSOD, CuZnSOD, CAT and GPx) density, as well as glycolytic enzyme activity (Tanaka
showed a signicant time main eect (Po0.05), as & Swensen, 1998; Kraemer et al., 2002). The results
well as a training main eect (Po0.05) and a time by of the present study showed that maximal dynamic
training main eect (Po0.05). Thus, in the S group, strength on leg-extension action was increased across
the mRNA level of all the antioxidant enzymes was all time points in the S group, while no increase took
increased after 21 weeks of training (32%, 26%, place in VO2max. A 22% increase in maximal strength
41%, and 38% for CAT, GPx, MnSOD and CuZn- was well in line with previous research results ob-
SOD, respectively). Moreover, increases in the tained in previously untrained middle-aged men with
mRNA level of CAT and CuZnSOD were already two training sessions a week (Hakkinen et al., 1998).
signicant after 10.5 weeks of regular strength train- On the other hand, endurance training, which nor-
ing (17% and 16%, respectively; Fig. 3). In the E mally aims to increase VO2max and endurance per-
group, MnSOD gene expression was signicantly formance capacity, decreases the activity of the
glycolytic enzymes, but increases intramuscular sub-
Table 2. Activity of antioxidant enzymes (U/mg protein) along the study strate stores, capillary and mitochondrial density
period (Tanaka & Swensen, 1998). Our results showed that
C (n 5 8) S (n 5 11) E (n 5 12) VO2max increased signicantly after 10.5 and 21
weeks of endurance training, indicating an improve-
CAT ment in aerobic capacity. Although the E group also
Pre-training 46.5  5.9 41.8  2.4 44.0  5.3
Mid-training 44.9  1.2 46.4  3.7
showed a signicant increase of 10% in maximal
Post-training 45.9  3.8 41.9  4.6 39.6  4.1 strength, this was signicantly lower than the gains
GPx reached by the S group. This result is also similar to
Pre-training 42.2  3.1 38.5  2.4 35.4  4.2 previous ndings showing that maximal strength can
Mid-training 39.8  0.6 37.2  2.4
Post-training 40.4  3.4 42.6  3.6 35.5  2.6
be increased to some extent also by endurance
MnSOD training (Izquierdo et al., 2005).
Pre-training 0.17  0.02 0.14  0.02 0.18  0.04 In addition to the well-established relationship
Mid-training 0.16  0.01 0.13  0.02 between endurance training and VO2max, several
Post-training 0.19  0.04 0.15  0.02 0.16  0.02
CuZnSOD studies have found a positive correlation between
Pre-training 2.7  0.3 2.2  0.4 2.8  0.7 aerobic training status and antioxidant enzyme ac-
Mid-training 2.4  0.2 2.1  0.4 tivity (Jenkins et al., 1984). Moreover, it has been
Post-training 3.0  0.7 2.4  0.4 2.5  0.3 shown that the ability to quench free radicals in
Values are mean  SEM. serum increases in relation to the maximum ability to
C, control group; S, strength training group; E, endurance training group; consume oxygen (Child et al., 1999). In contrast, we
SEM, standard error of the mean; GPx, glutathione peroxidase; MnSOD, did not detect signicant dierences in TEAC as a
mitochondrial superoxide dismutase; CuZnSOD, cytosolic superoxide function of either type or duration of training. No
dismutase. previous study has analyzed antioxidant capacity

599
Garc a-Lopez et al.
Pre-training Mid-training Post-training Pre-training Mid-training Post-training

Catalase GPx
-Actin -Actin

125 125
Relative Catalase proteinlevel

Relative GPx protein level

100 100

75 75

50 50

25 25 Fig. 1. Protein content of

antioxidant enzymes in per-
0 0 ipheral blood mononuclear
Pre-training Mid-training Post-training Pre-training Mid-training Post-training cells (PBMC) in response to
strength training. Upper pa-
nel: representative Western
Pre-training Mid-training Post-training Pre-training Mid-training Post-training blot photographs for each
MnSOD CuZnSOD time point. Lower panel:
densitometric analysis of
-Actin -Actin
Western blots, expressed as
percentage of pre-training
values (means  standard
125 200 error of the mean [SEM];
Relative CuZnSOD protein level
Relative MnSOD protein level

*#$ n 5 11). *Signicant dier-

100 ence (Po0.05) from the
150 *$ corresponding pre-training
75 value, #signicant dierence
100 (Po0.05) from the corre-
50 sponding mid-training value,
f
50 signicant dierence (Po
25 0.05) from the corresponding
C value, $signicant dier-
0 0 ence (Po0.05) from the cor-
Pre-training Mid-training Post-training Pre-training Mid-training Post-training responding E value.

adaptations induced by long-term strength/endur- from other age ranges are few and equivocal. For
ance training in middle-aged men. However, in example, there is an abundance of studies document-
healthy elderly subjects, it is not clear whether ing increases in SOD (Leeuwenburgh et al., 1997),
habitual physical activity favorably aects antioxi- CAT (Hollander et al., 2000) and GPx (Leeuwen-
dant potential (Vincent et al., 2002; Fatouros et al., burgh et al., 1997) activity after aerobic training in
2004), and conicting results, which may be partly young rodents and humans (Miyazaki et al., 2001;
due to a dierence in the type or training threshold Elosua et al., 2003). On the contrary, other authors
used, have been reported. have seen no changes or a decrease in antioxidant
To the best of our knowledge, the present study enzyme activity with endurance training. Thus, our
provides the rst evidence that systematic strength or data are consistent with previous studies that re-
endurance training increases mRNA levels of PBMC ported no signicant increases in antioxidant enzyme
antioxidant enzymes in untrained middle-aged men, activities after an aerobic cycling training (Tiidus
strength training being a more optimum stimulus in et al., 1996) or an aerobic walking/jogging training
this line. However, neither strength nor endurance (Fatouros et al., 2004), both in muscle (Tiidus et al.,
training programs induced changes in antioxidant 1996) and whole blood (Fatouros et al., 2004). We
enzyme activities. Moreover, 21 weeks of strength found only one study in which antioxidant enzyme
or endurance exercise training had no eect on the adaptation was measured in response to strength
protein content of MnSOD, CAT and GPx, and only exercise training, particularly in older subjects (Parise
an increase in CuZnSOD protein content was de- et al., 2005b). In contrast to our results, they demon-
tected in PBMC after a systematic strength training. strated improvements in antioxidant enzyme activ-
The eects of long-term exercise on the steady- ities after lower-limb unilateral strength training in
state dynamics of the enzymatic antioxidant defense older adults, reporting that 12 weeks of training
system are not clear, and there is an evident lack of resulted in an increased CAT and CuZnSOD activ-
studies focused on middle-aged men. Moreover, data ities, with no apparent changes in MnSOD activity in

600
 Training and antioxidant enzymes
Pre-training Mid-training Post-training Pre-training Mid-training Post-training

Catalase GPx
-Actin -Actin

Relative Catalase protein level

125 125

Relative GPx protein level

100 100

75 75

50 50

25 25

0 0
Pre-training Mid-training Post-training Pre-training Mid-training Post-training

Pre-training Mid-training Post-training Pre-training Mid-training Post-training

MnSOD CuZnSOD
-Actin -Actin
Fig. 2. Protein content of
antioxidant enzymes in per-
ipheral blood mononuclear

Relative CuZnSOD protein level

125 125
Relative MnSOD protein level

cells (PBMC) in response to

endurance training. Upper 100 100
panel: representative Wes-
tern blot photographs for 75 75
each time point. Lower pa-
nel: densitometric analysis 50 50
of Western blots, expressed
as percentage of pre-train- 25 25
ing values (means  stan-
0 0
dard error of the mean
[SEM]; n 5 12). Pre-training Mid-training Post-training Pre-training Mid-training Post-training

muscle tissue taken from the trained leg. The ndings
among previous studies are inconsistent, possibly
because of dierences in exercise mode, training
intensity and duration, as well as the dierent en-
zyme assay methods used and type of tissue analyzed.
For example, the relative sensitivity to detect SOD
activity varies between assays. Indeed, a 10-fold
dierence in sensitivity has been reported when seven
common methods to estimate SOD activity have
been compared (Oyanuagui, 1984). Hence, investiga-
tions using SOD procedures with low sensitivity
could fail to observe small-to-moderate training-
induced changes in tissue SOD activity due to a
limited sensitivity of the assay. Recently, it has also
been demonstrated that antioxidant enzyme activities
are aected by circannual rhythms, both in endur-
ance-trained and untrained young people (Balog et
al., 2006). All these facts could also explain the lack
of signicant changes in enzyme activity shown by
the present results.
In the present study, we have shown that 21 weeks
of whole-body strength training increased the protein
content of CuZnSOD, the magnitude of the training-
induced increase in PBMC being inuenced by the
duration of the training. It has been reported that 14
weeks of lower-limb strength training had no eects
on the protein content of CAT, CuZnSOD or
MnSOD in muscle tissue (Parise et al., 2005a). Based
on our observations, it is fair to assume that strength
exercise resulted in a cytosolic oxidative stress. Thus,
strength training exercise may have resulted in a
chronic elevation of cytosolic oxidant production.
In contrast, MnSOD is compartmentalized in the
mitochondrial inner membrane, and the lack of
adaptation in MnSOD protein after strength exercise
training may simply be related to the fact that
strength exercise is generally not associated with
mitochondrial stress to the same extent as endurance
exercise (Parise et al., 2005b). A second possibility
could be related to the fact that middle-aged adults
may be limited by a plateau eect with respect to
adaptation of MnSOD, as it has previously been
observed that older adults have signicantly higher
levels of MnSOD in skeletal muscle compared with
young adults, whereas there is no dierence between
young and old with respect to CuZnSOD (Gianni
et al., 2004). CuZnSOD may be more responsive to
the stresses of exercise and/or a greater exercise
intensity needed to elicit a response in MnSOD
over and above that induced by aging. In any case,

601
Garc a-Lopez et al.
150 *#$ 150

Relative Catalase mRNA level

*#

Relative GPx mRNA level

125 *$
125

100 100 Fig. 3. mRNA levels of antiox-

idant enzymes measured by
75 75 real-time PCR in peripheral
blood mononuclear cells
50 50
(PBMC) in response to
25 25 strength training. Levels of
antioxidant activities mRNA
0 0 were normalized against 18S
Pre-training Mid-training Post-training Pre-training Mid-training Post-training rRNA and presented as fold
change (  standard error of
*#
the mean [SEM]) of duplicate
*#$

Relative CuZnSOD mRNA level

150 150 samples from two separate ex-
Relative MnSOD mRNA level

*$ periments for each time point

125 125
(n 5 11). *signicant dierence
100 100 (Po0.05) from the correspond-
ing pre-training value, #signi-
75 75 cant dierence (Po0.05) from
the corresponding mid-training
50 50
value, fsignicant dierence
25 25 (Po0.05) from the correspond-
ing C value, $signicant dier-
0 0 ence (Po0.05) from the
Pre-training Mid-training Post-training Pre-training Mid-training Post-training corresponding E value.

150 150 *#
Relative Catalase mRNA level

Relative GPx mRNA level

125 125

100 100

75 75

50 50 Fig. 4. mRNA levels of antiox-

idant enzymes measured by
25 25 real-time PCR in peripheral
0 0
blood mononuclear cells
Pre-training Mid-training Post-training Pre-training Mid-training Post-training
(PBMC) in response to endur-
ance training. Levels of antiox-
idant activities mRNA were
*# normalized against 18S rRNA
150
Relative CuZnSOD mRNA level

150 and presented as fold change

Relative MnSOD mRNA level

* (  standard error of the mean

125 125
[SEM]) of duplicate samples
100 100 from two separate experiments
for each time point (n 5 12).
75 75 *signicant dierence (Po0.05)
from the corresponding pre-
50 50
training value, #signicant dif-
25 25 ference (Po0.05) from the cor-
responding mid-training value,
f
0 0 signicant dierence (Po0.05)
Pre-training Mid-training Post-training Pre-training Mid-training Post-training from the corresponding C value.

data obtained suggest that strength training could be (MnSOD, CuZnSOD, CAT and GPx). Furthermore,
per se a greater stressor than endurance training the present endurance training increased mRNA
because it activates mRNA expression of all antiox- levels of MnSOD and GPx after 21 weeks of training.
idant enzymes. Although the relative translation eciency for anti-
Changes in mRNA levels of antioxidant enzymes oxidant enzymes was not assessed in our study,
during the training process were not consistent with discrepancies between mRNA levels and activities
the changes in enzyme activities. The current study may be related to dierences in mRNA stability or
showed that strength training enhanced the gene translational eciency, similar to what has been
expression of all the antioxidant enzymes examined reported previously in the lung of old rats (Gomi &

602

Matsuo, 2001). It is possible that the expressions of
antioxidant enzymes mRNA were initially up-regu-
lated and then down-regulated. Thus, some factor
might act on individual mRNA to block their trans-
lation and thereby lead to their degradation. Alter-
natively, message degradation may be the primary
target of regulation. These ndings may also repre-
sent a mechanism of adaptation to regular training.
One can speculate that the training-stressed cells
provide high antioxidant enzyme transcript levels
for immediate translation whenever necessary. The
resulting accumulation of antioxidant enzymes them-
selves may regulate translation via a negative feed-
back loop if they are not used for an immediate
response. Individually, another possible explanation
for the elevation in MnSOD mRNA concentration
without changes in the level of MnSOD protein may
be in a kinase/phosphatase signal transduction path-
way that may exert a ne control over post-tran-
scripcional regulation of MnSOD expression
(Knirsch & Clerch, 2001). Another potential me-
chanism that cannot be excluded in our study is
that CAT may be inactivated by its substrate, hydro-
gen peroxide, due to formation of complex II or
complex III of CAT at high peroxide concentrations
(Lardinois & Rouxhet, 1996). Moreover, NO or NO-
derived products inhibit both CAT and GPx enzyme
activities (Nilakantan et al., 2005).
Training and antioxidant enzymes
idant enzyme activities in previously untrained
middle-aged men. These results conrm that dier-
ences mediated by factors such as tissue or charac-
teristics of the training program may determine the
presence or lack of antioxidant adaptations to train-
ing (Tiidus et al., 1996; Miyazaki et al., 2001;
Fatouros et al., 2004). Nevertheless, the period of
training in our study was a sucient stimulus to up-
regulate mRNA levels of PBMC antioxidant en-
zymes, the strength training being a more optimal
stimulus in this line, and further studies should be
carried out in order to investigate whether the lack of
adaptation of the PBMC antioxidant enzymes could
be due to loss in mRNA stability and eciency
translation, the type of tissue studied or the age of
the subjects. Our research underlines the necessity of
molecular studies, analyzing not only enzyme
activities but also protein contents and mRNA
levels, to identify adequately dierences in organ
adaptations to oxidative stress during physical train-
ing, the mechanisms involved and their potential
benets.
Key words: strength training, endurance training,
antioxidant enzymes, peripheral blood mononuclear
cells.

Perspectives Acknowledgements
The present data showed that 21 weeks of strength or This study was in part supported by a grant from the Ministry
endurance training had no eect on PBMC antiox- of Education, Finland.

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