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Department of Pharmacology and Institute of Public Health, The Panum Institute, University of Copenhagen. Department of Neuroanaesthesia, The Neuroscience Center, Copenhagen University Hospital, DK-2200 Copenhagen N, Denmark
1Correspondence: Institute of Public Health (c/o Department of Pharmacology), The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. E-mail: fipm{at}farmakol.ku.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: FPG • high-intensity exercise • ENDO III • reactive oxygen species
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In humans, the
available data suggest that immediately after exercise, the level of
oxidative damage to DNA is unchanged whereas DNA damage may appear
hours after the exercise (2)
. A similar situation has been
reported for DNA strand breaks, where DNA strand breaks first appeared
hours after an exhaustive running on a treadmill (3)
. It
has also been reported that a higher level of DNA strand breaks
appeared after strenuous bicycling (4)
. It appears that in
normal healthy subjects, a short bout of maximal exercise is usually
not sufficiently strenuous to cause significant effects on biomarkers
of oxidative stress immediately after exercise. This is probably
because the antioxidant response system can cope with an increased load
of ROS generated by a short bout of exhaustive exercise.
It is well known that exposure to low oxygen pressure leads to a
complex scenario of both metabolic and physiological changes. The
stress induced by hypoxia leads to an imbalance between antioxidant
capacity and oxidants, and can consequently be observed by many
biomarkers of oxidative stress. Rats exposed to hypobaric oxygen
pressure showed a higher level of oxidized glutathione 48 h after
exposure to hypoxia (5)
. Lipid peroxidation, detected by
an elevated malondialdehyde concentration in plasma and a lower level
of glutathione in blood, was observed in rats exposed intermittently to
hypoxia (equal to 7576 meters) for 6 h daily for 5 consecutive
days (6)
.
The effects of low oxygen pressure in humans have been investigated in
altitude studies for years. A stay at 5500 meters increased breath
pentane by 104% after 4 wk, and this effect was ameliorated by
pretreatment with vitamin E (7)
. Altitude studies
performed 1650–3048 meters above sea level for 1–2 wk including
exercise programs revealed increased lipid peroxidation and
urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) levels
compared with levels obtained at lower altitudes (8
9
10)
.
Although these data indicate that exercise in altitude is accompanied
by oxidative stress, they do not discriminate between the effect of
exercise and hypoxia.
The objective of this study was to investigate whether altitude hypoxia
makes normal healthy individuals more prone to generate DNA strand
breaks and oxidative DNA damage after a single bout of exhaustive
exercise. The oxidative DNA damage 8-oxodG was measured by HPLC;
DNA strand breaks, endonuclease III (ENDO III) -sensitive sites, and
formamidopyrimidine DNA glycosylase (FPG) -sensitive sites were
detected by the single cell gel electrophoresis (comet assay)
(11)
. Both ENDO III and FPG are DNA glycosylase enzymes
that hydrolyze the N-glycosylic bond between the deoxyribose and a
damaged base. The types of DNA damage detected by ENDO III are a broad
spectrum of oxidatively damaged pyrimidines; the FPG glycosylase
detects oxidative purine lesions (12)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Each symptom—headache,
gastrointestinal symptoms, fatigue, and/or weakness,
dizziness/lightheadedness, and difficulty of sleeping—was assessed on
a scale from 0 to 3 (maximal attainable score=15).
The exercise protocol started by a 5 min warm-up at 120 watt and 80 rpm
on a Monark 848 cycle ergometer (Monark, Sweden), followed by a
submaximal exercise test for 5 min, electing 
50% of the estimated
maximal oxygen uptake (VO2max) at sea level.
Immediately thereafter, the maximal exercise test was started. The
start load was estimated from the submaximal exercise and the workload
was increased by 40 watts every 90 s until exhaustion. The
VO2max and heart rate (HR) were recorded. The
VO2max situation was accepted if the oxygen
uptake reached a plateau, the plasma lactate concentration was above 8
mmol/l, or the respiratory exchange rate was above 1.15. The HR was
measured with a Polar Vantage XL (Oy, Finland); oxygen saturation was
recorded continuously by a Nellcor pulse oximeter (Haywar) with the
sensor attached to a fingertip. Gas exchange was recorded every 15 s with a MedGraphic CPX/d system (St. Paul, MN).
Twenty-four hour urine collections were obtained before and after the exercise protocol both at sea level and at high altitude. The first urine collection at high altitude was commenced immediately after arrival. The subjects were instructed to empty their bladder before start of the urine collection. In both environments, blood samples were obtained before, immediately after, 1 day (24 h), and 2 days (48 h) after the exercise session.
Isolation of lymphocytes
Ten milliliter venous blood were placed in
Vacutainer®CPTTM with sodium heparin (Becton Dickinson and Company,
Rutherford, NJ) and centrifuged 1650 g for 20 min at room
temperature. The plasma/lymphocyte layer was removed, washed in cold
RPMI 1640 media (Gibco, Grand Island, NY), and centrifuged at 400
g for 15 min at 4°C. Most of the supernatant was removed
and the pellet was resuspended in cold RPMI 1640 media supplemented
with 50% bovine calf serum and 10% dimethyl sulfoxide. At sea level,
the lymphocytes were stored at -70°C until analysis. At high
altitude, the lymphocytes were stored in liquid nitrogen and thereafter
transferred to -70°C at the return to Denmark. The possible
influence of storage in liquid nitrogen was investigated in a separate
experiment including blood samples from 5 donors.
Detection of DNA strand breaks and oxidative DNA damage
The procedure of the comet assay was the same as has been
described previously (14)
. The isolated lymphocytes were
mixed with 90 µl low melting point agarose and applied on a fully
frosted microscope slide that had been covered with 90 µl high
melting point agarose. The cells were lysed overnight in lysis solution
(1% Triton X-100, 2.5 mM NaCl, 0.1 Na2EDTA, 10
mM Tris, pH=10) and rinsed three times in buffer (40 mM HEPES, 0.1 M
KCl, 0.5 mM EDTA, 0.2 mg/ml BSA, pH=8). To detect oxidative DNA damage
and DNA strand breaks, the nucleoids were incubated in with either ENDO
III, FPG, or buffer for 45 min at 37°C (enzymes were a kind gift from
Serge Boiteaux, UMR217 Center National de la Recherche Scientifique et
Commissariat a l’Energie Atomique, France). Immediately after the
enzyme treatment, the nucleoids were immersed to an alkaline solution
(0.3 M NaOH, 1 mM Na2EDTA, pH>13) for 40 min and
electrophoresed for 20 min in the same alkaline solution (procedures
were carried out in a 4°C cold room). The electrophoresis solution
was recycled at a rate of 3.6 l/h. After electrophoresis, the
lymphocytes were washed twice in Tris buffer (0.4 M Tris-HCl, pH=7.5)
and stained with YOYO-1 in PBS (Molecular Probes, Eugene, OR) and
viewed in an Olympus fluorescence microscope. A detailed description of
the scoring of damage has been described previously (15)
.
Fifty images from each slide were scored visually as belonging to one
of five classes of DNA damage, from score 0 (round images) to score 4
(most of the DNA had migrated from the head to the tail). An average
level of DNA damage was calculated by multiplying the number of cells
in a class by the score number. A total of 100 cells were scored, thus,
the level of DNA damage range from score zero (undamaged) to
score 400 (highly damaged).
Analysis of urine samples
The 8-oxodG content in each of the urine collections was
determined as described previously (16)
. The volume of
urine was measured immediately after termination of urine collections
and 10 ml aliquots were frozen in liquid nitrogen for later analysis.
Due to the arrival time at high altitude, the collection of urine
amounted to 19 h before exercise. For the statistical analysis, we
estimated the 8-oxodG in these as 24 h urine samples by linear
extrapolation.
Statistics
The data were analyzed by a multifactorial ANOVA based on
repeated measurements with substitution of missing values. In a
two-factor ANOVA analysis, we used 85 degrees of freedom because the
original data material included 86 samples. Essentially similar results
were obtained by a one-way multifactorial ANOVA, with place and time as
the factors. The statistical analysis of one-way ANOVA was aided by
using Statistica® for Windows version 5.1 F,
StatSoft, 1997 (Tulsa, OK).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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depicts HR, VO2max, and arterial oxygen
saturation before and after exercise at sea level and high altitude.
Compared with sea level, resting HR increased and maximal HR decreased.
The VO2max at high altitude was lower compared
with sea level values. Arterial oxygen saturation at rest and after
exercise in hypoxia was lower compared with sea level. The AMS score
was 4.7 ± 2.7 (mean ±SD) on day 2 and gradually
decreased in the ensuing days of hypoxia (day 3: 2.9±1.6, day 4:
2.5±2.1). There were no significant correlations between the AMS score
and 8-oxodG, levels of DNA strand breaks, ENDO III-sensitive sites, or
FPG-sensitive sites (r2<0.04, data not shown).
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Figure 1
depicts the level of DNA strand breaks, ENDO III-sensitive sites, and
FPG-sensitive sites. Hypoxia increased the level of DNA strand breaks
on all the days at altitude. ENDO III-sensitive sites were increased on
day 3 at altitude compared with sea level and pre-exercise level.
FPG-sensitive sites remained unchanged. Urine samples collected during
the first day of hypoxia had a higher 8-oxodG content compared with the
sea level values (Fig. 2
).
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At high altitude, exercise further aggravated the induction of strand
breaks compared with pre-exercise levels (Fig. 1)
. In contrast,
normoxic exercise had no significant effect on DNA strand breaks. A
significantly decreased level of FPG-sensitive sites was seen 24 h
after the exercise at sea level, but not after hypoxic exercise.
At sea level the lymphocytes were stored at -70°C, whereas they were stored in liquid nitrogen at the mountain and then transferred to -70°C after the return to Denmark. The freezing media were the same. We set up an experiment to test the effect of the different storage conditions on DNA strand breaks and oxidative DNA damage in lymphocytes isolated in normoxic conditions from five normal subjects that were not included in the experimental protocol. Scores of the DNA damage (means ±SD) after storage in -70°C (DNA strand breaks: 23±17, endonuclease III-sensitive sites: 1±7, FPG-sensitive sites: 117±41) were not different from scores of samples stored in liquid nitrogen for a week and transferred to -70°C before analysis (DNA strand breaks: 24±14, endonuclease III-sensitive sites 6±13, FPG-sensitive sites 131±19).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We did
not detect any change in the level of urinary 8-oxodG after exercise.
This agrees with reports showing that 8-oxodG and FPG-sensitive sites
were the same before and after normoxic exercise (18)
. It
may reflect that the present exercise protocol was not strenuous
enough. High urinary concentration of 8-oxodG is probably detected only
after exercise at intensities accompanied by muscle soreness.
Previously we had found that vigorous exercise 10 h daily for 30
days resulted in higher urinary 8-oxodG level (19)
. Also,
elevated content of 8-oxodG in the quadriceps femoris muscle was
detected in subjects that performed 200 eccentric muscle actions at an
intensity being 60% of the maximum isometric contraction
(20)
.
Our data indicate that acute hypoxia is associated with oxidative
stress. Increased levels of DNA strand breaks and oxidative DNA damage
are normally regarded as a signal of an ongoing pathological process,
although we cannot distinguish between early or late phases of the
process (21)
. A common source to the production of
oxidative DNA damage and DNA strand breaks is by generation of excess
amounts of ROS. Enhanced production of ROS has been shown in vitro in
chicken cardiomyocytes, and cultured Hep3B cells exposed to acute
hypoxia (22
, 23)
. In rats exposed to 10% oxygen, there
was an increased production of ROS in the cells of the mesenteric
microcirculation (24)
. Moreover, evidence from in vitro
experiments shows that hypoxia evokes an inflammatory response in
vascular tissue and human peripheral blood mononuclear cells,
determined by augmentation of the expression of inflammatory mediators
such as tumor necrosis factor 
(TNF-), interleukins (IL-1, IL-6,
IL-8), inducible nitric oxide synthase, and adhesion molecules
(25
26
27
28)
. Other in vitro experiments have shown that
hypoxia increases lipopolysaccharide-induced activity of human alveolar
macrophages (29
, 30)
. The inflammatory processes are
likely to be involved in the pathogenesis of hypoxia-related diseases
such as high-altitude pulmonary edema (HAPE) and high-altitude cerebral
edema. In subjects with HAPE, very high levels of IL-6 and C-reactive
protein (CRP) have been measured in serum (31)
and of IL-6
in bronchoalveolar fluid (32)
. In subjects without HAPE,
altitude hypoxia for 1–3 days caused moderate increases in serum
levels of IL-6 (33
34
35)
, IL-1 receptor antagonist, and CRP
(34)
. The human cellular immune system seems to react
promptly in response to hypoxia: hypobaric hypoxia (380 Torr) for 20
min markedly increased circulating levels of lymphocytes, CD16+ natural
killer cells, and CD14+ monocytes (36)
.
Several investigations indicate that prolonged exercise and
high-intensity exercise in normoxic conditions evoke inflammatory
processes similar to hypoxia. A short bout of maximal exercise for 5
min had no effect on serum cytokines, whereas sustained exercise at
60–65% of VO2max increased serum levels of IL-6
and TNF- (37)
. Other studies of exercise with a
duration less than 20 min showed unaltered levels of IL-6 and TNF-
in blood after running (38
, 39)
and swimming
(40)
. This suggests that only exercise with a propensity
of forcing damage to the muscle fibers activates an inflammatory
response, as evidenced by secretion of cytokines in the blood. The
parallel activation of the inflammation system by exhaustive exercise
and hypoxia has been shown to exert a higher effect when subjects
exercise in hypoxia: hypoxic exercise enhanced the concentration of
CD16+ and CD56+ natural killer cells compared with normoxic exercise
(41)
. Data obtained after 4 days at 4350 meters suggested
that exercise exacerbated hypoxia-induced increases in IL-6
(33)
.
The mechanism by which hypoxic exercise exacerbates the inflammatory
response possible lies in the reduced oxygen delivery to the muscle
tissue. In subjects enrolled in our study, maximal workloads at sea
level decreased the arterial oxygen saturation. This kind of arterial
hypoxemia is widely known to occur during maximal exercise in
endurance-trained subjects (42)
, and may be secondary to a
reduced pulmonary arterial mean transit time of red blood cells because
of a high maximal cardiac output, leading to insufficient oxygen
diffusion time. The decrease in arterial oxygen saturation was further
aggravated during hypoxic exercise, as has been described
(43)
. Exercise induces a reduction in intracellular muscle
PO2 that is aggravated during hypoxic exercise
(44)
. Thus, further studies with control of oxygenation
during exercise are necessary to separate the effect of exercise on the
inflammatory response in relation to the greater hypoxemia.
Nonetheless, our findings of enhanced levels of DNA strand breaks after
hypoxic exercise demonstrate the presence of a factor that is likely to
enforce a proinflammatory response to hypoxia. Because single-stranded
DNA breaks are a strong and probably the only activator of
poly-ADP-ribose polymerase (45)
, increased levels of DNA
strand breaks may constitute an important link in hypoxia-induced
inflammation cascades. The mechanism of poly-ADP-ribose
polymerase-mediated toxicity possibly involves an inhibition of
mitochondrial function, which alters the cellular energy metabolism and
may lead to apoptosis (45)
. Because both apoptosis and
altered energy metabolism is associated with formation of ROS,
poly-ADP-ribose polymerase enzymes may further contribute to the higher
level of DNA strand breaks detected during hypoxia.
In conclusion, the present report demonstrates that hypoxia generates DNA strand breaks and oxidative DNA damage in humans. Exercise performed in normoxic conditions had no effect on the generation of DNA strand breaks, whereas hypoxic exercise produced more DNA strand breaks. This implies that during a condition of hypoxic stress, the protection by the antioxidant system is insufficient to avoid generation of DNA strand breaks after exhaustive exercise. By activation of poly-ADP-ribose polymerase, induction of DNA strand breaks by ROS may play an important role in hypoxia-induced inflammation.
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ACKNOWLEDGMENTS |
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Received for publication October 12, 2000. Accepted for publication December 13, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. J. Clin. Invest. 90,2333-2339
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P < 0.05 compared
with sea level and pre-exercise value.