Central European Journal of BiologyEffects of intermittent hypoxia different regimes on mitochondrial lipid peroxidation and glutathione-redox balance in stressed rats * E-mail: ogonch
Trang 1Central European Journal of Biology
Effects of intermittent hypoxia different
regimes on mitochondrial lipid peroxidation
and glutathione-redox balance in stressed rats
* E-mail: ogonchar@yandex.ru
Received 15 November 2007; Accepted 22 February 2008
Abstract: The purpose of this study was to compare the influence of two regimes of intermittent hypoxia (IH) [repetitive 5 cycles of 5 min
hypoxia (7% O2 or 12% O2 in N2) followed by 15 min normoxia, daily for three weeks] on oxidative stress protective systems in liver
mitochondria To estimate the effectiveness of hypoxia adaptation at the early and late preconditioning period, we exposed rats to acute
6-h immobilization at the 1st and 45th days after cessation of IH We showed that severity of hypoxic episodes during IH might initiate
different adaptive programs Moderate hypoxia during IH prevents mitochondrial glutathione pool depletion induced by immobilization
stress, maintains GSH-redox cycle via activation of glutathione peroxidase, glutathione-S-transferase, glutathione reductase, NADP+
-dependent isocitrate dehydrogenase, and increases Mn-SOD activity Such regimen of hypoxic preconditioning caused the decrease
of mitochondrial superoxide anion generation as well as of basal and stimulated in vitro lipid peroxidation and this protective effect
remained for 45 days under renormoxic conditions Hypoxic adaptation in a more severe regimen exerted beneficial effects on the
mitochondrial antioxidant defense system only at its later phase
© Versita Warsaw and Springer-Verlag Berlin Heidelberg
Keywords: Intermittent hypoxia • Adaptation • Mitochondria • Lipid peroxidation • Glutathione • Glutathione enzymes • Antioxidant defense
Department of Hypoxic States,
Bogomoletz Institute of Physiology,
National Academy of Sciences of Ukraine,
01024 Kyiv, Ukraine
Research Article
Abbreviations
GPx - glutathione peroxidase
GR - glutathione reductase
GSH - reduced glutathione
GSSG - oxidized glutathione
GST - glutathione-S-transferase
IDPm - mitochondrial NADP+-dependent isocitrate
dehydrogenase
IH - intermittent hypoxia
IHT - intermittent hypoxic training
LPO - lipid peroxidation
Mn-SOD - manganese superoxide dismutase
O2.- - superoxide anion
ROS - reactive oxygen species
TBARS - thiobarbituric acid-reactive substances
1 Introduction
For many years scientists focused on the study of the adaptive processes as a biological phenomenon that involves cells reacting at a molecular level to achieve greater cellular resistance against damaging factors, including oxidative stress [1]
Intermittent hypoxia might effectively stimulate various metabolic processes and this phenomenon
is widely used in sport and medicine practice [2] The multiple brief exposures of hypoxia/reoxygenation in an intermittent hypoxia (IH) are comparable with ischemia/
reperfusion and exert protective effects similar to those observed in ischemic preconditioning [3-6] It was demonstrated that IH adaptation helps to prevent mitochondrial DNA deletion and preserve mitochondrial ultrastructure [3], as well as to improve energy production
in metabolic processes by increasing formation of
Trang 2mitochondria in the brain and liver, activating electron
flux through respiratory complex I, and increasing
efficiency of oxidative phosphorylation [1] However,
the precise mechanisms underlying the antioxidant
protective effects of IH on mitochondria are not clear
Investigations in our and other research laboratories
showed that the adaptation to intermittent hypoxia can
reduce damage caused by other stresses, including
ischemia [6], physical exercise [7], and more severe and
sustained hypoxia [8] The duration of the protective effect
of hypoxic preconditioning has not been determined yet
Some researchers consider it to be no more than 7 days
[5,8
A recent study showed that the duration,
frequency, and severity of hypoxic episodes of hypoxic
preconditioning are also important in achieving
adequate protective effects against stress-induced
injury Experimental data on the degree and duration
of hypoxic exposure as critical factors determining
whether hypoxia is a beneficial or a noxious agent are
contradictory [9,10]
Hypoxia/reoxygenation can induce excessive ROS
generation [11-13] Impaired electron flux through the
mitochondrial respiratory chain is an important reason
for oxidative stress during hypoxia and reoxygenation
[14,15] The accumulation and direct transfer of reducing
equivalents within the mitochondrial respiratory chain to
molecular oxygen can give rise to superoxide anion, singlet
molecular oxygen, hydroxyl radical, and peroxynitrite
[11] The matrix glutathione redox cycle in coordination
with the Mn-SOD-mediated scavenging of superoxide is
crucial for preventing excessive ROS accumulation in
mitochondria [16] The glutathione system is considered
to be one of the main regulators of the intracellular
redox environment, executing control over the
mitochondrial redox state and antioxidant defense, and
governing the redox regulation of metabolic processes
[17] Some authors emphasize that the GSH play an
important role in the formation of adaptive responses
as redox-sensitive transcription factors are commonly
controlled by the thiol redox status of the cells [18]
The function of GSH and GSH-related enzymes
depend on the redox status of NADP(H), a reducing
cofactor used for the regeneration of glutathione from
glutathione disulfides [17] It is well established that
three enzymes are candidates for mitochondrial NADPH
generation, isocitrate dehydrogenase, malic enzyme,
and the nicotinamide nucleotide transhydrogenase [19]
Recently, it has been demonstrated that mitochondrial
NADP+-dependent isocitrate dehydrogenase (IDPm) is
a major NADPH producer in the mitochondria and, thus,
plays a key role in cellular defense against oxidative
stress-induced damage [20]
Although the mechanisms underlying regulation
of intracellular GSH levels by hypoxia is known [21], specific role of mitochondrial GSH, GSH-related, and NADP+-dependent enzymes in the survival of the cells during repeated hypoxia/reoxygenation has not been reported yet
The aim of this study is to compare the effects of two regimes of IH (with severe and moderate short-term hypoxia exposures) on the intensity of lipid peroxidation processes and glutathione – redox balance
in liver mitochondria To estimate the effectiveness of hypoxic adaptation within the early and late periods, we investigated mitochondrial oxidative stress- protective responses after acute immobilization at the 1st and 45th day after cessation of IH
2 Experimental Procedures
2.1 Materials
5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), GSH, GSSG, glutathione reductase (from bakers yeast), thiobarbituric acid (TBA), EGTA, defatted bovine serum albumin (BSA), cytochrome C, N-ethylmaleimide, adrenaline, 1-chloro-2,4-dinitrobenzene (CDNB), NADPH, NADP+, threo-DS-isocitrate, 2-vinilpyridine, and hydrogen peroxide were obtained from Sigma, Fluka, and Merck All other reagents were of analytical grade
2.2 Animals and study design
Wistar rats weighing 220-260 g were used They were housed in Plexiglas cages (4 rats per cage) and maintained in an air-filtered and temperature controlled (20-22°C) room Rats received a standard pellet diet and water ad libitum and were kept under artificial light-dark cycle of 12 h The present study was approved by the Institutional Animal Ethics Committee, Bogomoletz Institute of Physiology, Kyiv (Ukraine) The rats were randomly divided into 8 groups of six rats each: 1 - control (C) Rats were sedentary and under normoxic condition
2 - acute stress (AS) Rats were exposed to a single 6-h immobilization in the animal homeroom The immobilization was performed using a plastic rodent restrainer that allowed for a close fit to rats 3 - intermittent hypoxia in regimen I (IHT (I)) Animals were subjected to intermittent hypoxic training during
21 days Hypoxic episodes were created by breathing hypoxic gas mixtures (7% O2 in N2) under normobaric conditions in a special chamber We used experimentally repeated short-term hypoxia (5 min) with normoxic intervals (15 min) Rats were subjected to five such sessions daily 4 - acute stress (6-h immobilization)
Trang 3at the 1st day after cessation of intermittent hypoxia
in regimen I (IHT (I) + AS1) 5 - acute stress
(6-h immobilization) at the 45th day after cessation
of intermittent hypoxia in regimen I (IHT (I)+ AS45).
6 - intermittent hypoxia in regimen II (IHT (II)) These
animals were subjected to intermittent hypoxic
training by breathing hypoxic gas mixtures (12%
O2 in N2) under normobaric conditions in a special
chamber Short-term hypoxia was repeated 5 min with
15 min-long normoxic intervals Rats had such five
sessions daily for 21 days 7 - acute stress (6-h
immobilization) at the 1st day after cessation of
intermittent hypoxia in regimen II (IHT (II) + AS1) 8 -
acute stress (6-h immobilization) at the 45th day after
cessation of intermittent hypoxia in regimen II (IHT (II)+
AS45)
Ambient O2 levels in the chamber were continuously
monitored using of a Beckman O2 analyzer (model
OM-11) by sampling the air in the chamber The duration of
the gas flows during each hypoxic and normoxic episode
was regulated by timed solenoid valves
Animals of groups 2, 4, 5, 7, and 8 were killed
immediately after the experiment by decapitation In
other groups, animals were sacrificed 24 h after the last
hypoxic training session At the time of sacrifice, the
animals were lightly anaesthetized with ether
2.3 Mitochondria isolation
Rat liver mitochondria were isolated by differential
centrifugation as described by Jonson and Lardy [22],
with some modifications Liver was collected in isolation
medium A (250 mM sucrose, 10 mM Tris/HCl, pH 7.6),
1 mM EGTA and 0.5% defatted bovine serum albumin)
and homogenized After centrifugation of the homogenate
at 1000 g for 5 min, the supernatant was strained on
gauze and recentrifuged at 12 000 g for 15 min The
resulting pellet was resuspended in ice-cold isolation
medium B (250 mM sucrose, 10 mM Tris/HCl, pH 7.6)
and 0.1 mM EGTA) and a new series of centrifugation
was performed The final washing and resuspension of
mitochondria was in the medium B without EGTA and
BSA Protein concentration was determined by the
Lowry method, using BSA as a standard
2.4 Lipid peroxidation assay
Lipid peroxidation (LPO) in isolated mitochondria was
measured from the formation of thiobarbituric acid - reactive
substances (TBARS) [23] The mitochondrial suspension
(200 µL) was mixed with solution containing 0.8% TBA
(dissolved in 50 mM NaOH), 20% TCA, 0.25 N HCl The
mixture was heated at 90oC for 15 min The amount of
TBARS formed was detected at 532 nm Sensitivity to
in vitro LPO was estimated by incubation of identical
mitochondria samples with 10 µM FeSO4 and 0.1 mM ascorbic acid at 37oC for 30 min The reaction was stopped by addition of 20% TCA
2.5 Determination of superoxide radical production
Endogenous O2 ∙− production was assessed as an index
of the mitochondrial capacity for production of ROS by spectrophotometry during the reduction of ferricyto-chrome c [24] Each sample was placed in a test tube containing Krebs buffer Then 15 µM cytochrome c was added to the sample and was incubated for 15 min at 37oC
At the end of this period, the buffer was removed and the absorbance read at 550 nm after the addition of 3 mM N-ethylmaleimide to inhibit further reduction of cytochrome c A mixture containing the same reagents, except the cytochrome c addition, was used as a blank for the same sample The amount of O2 ∙− produced was calculated as the change between ferricytochrome c and ferrocytochrome c (ε 550 = 21000 M-1 cm-1) and the results were expressed in nmol of O2∙− per min per mg mitochondrial protein
2.6 Enzymatic assays
Enzymatic activity in the mitochondrial preparations was determined upon solubilization in 0.5% deoxycholate
Superoxide dismutase (EC 1.15.1.1) activity was measured by the method of Misra and Fridovich [25], which is based on the inhibition of autooxidation of adrenaline to adrenochrome by SOD contained in the examined samples The samples were preincubated for
60 min at 0oC with 6 mM KCN, which produces total inhibition of Cu, Zn-SOD The results were expressed
as specific activity of the enzyme in units per mg protein
One unit of SOD activity is defined as the amount of protein causing 50% inhibition in conversion of adrenaline
to adrenochrome under specified conditions
Selenium-dependent glutathione peroxidase (EC 1.11.1.9) (GPx) activity was measured by the
method of Rotruck et al [26] with some modifications
Briefly, the reaction mixture contained Tris-HCl buffer (100 mM, pH 7.4), sodium azide (10 mM), EDTA (2 mM), reduced glutathione (2.5 mM), mitochondrial suspension (200 µL) and H2O2 (2 mM) The contents were incubated at 37oC for 3 min and reaction was arrested by 15% TCA The supernatant was assayed for glutathione content by using Ellmans reagent
Glutathione -S- transferase (EC 2.5.1.18) (GST) activity was determined by assaying
GHS, as described by Warholm et al [27]
Trang 4The working solution contained 100 µL of
20 mM GSH, 1 mM EDTA in 200 mM phosphate buffer
Conjugated product was recorded continuously for 5
min at 30oC, at 340 nm (ε 340 =9.6x10-3M-1cm-1)
Glutathione reductase (EC 1.6.4.2) (GR) reaction
mixture contained phosphate buffer (200 mM, pH
7.0), EDTA (2 mM), NADPH (2 mM), and GSSG (20
mM) The reaction is initiated by the addition of 100 µL
mitochondrial suspension and decrease in absorbance
at 340 nm is followed at 30oC [28]
The NADP+ − dependent isocitrate dehydrogenase
(EC 1.1.1.42) (IDPm) activity was measured by the
production of NADPH at 340 nm The reaction mixture
contained 50 mM Tris/HCl buffer (pH 7.4), 10 mM MgCl2,
5 mM threo-DS-isocitrate, and 2 mM NADP+ [29]
2.7 Glutathione content
Total glutathione – the sum of reduced glutathione and
oxidized glutathione (GSH and GSSG) − was determined
by a method where glutathione is extracted from the liver
mitochondria with 5% ice-cold-sulfosalicytic acid and
after neutralization with triethanolamine sequentially
oxidized by DTNB (0.6 mM) and reduced by NADPH
(0.3 mM) in the presence of glutathione reductase
(2 U/ ml) [30] For determination of the GSSG alone, the
GSH presented in solutions was converted by incubation
with 2 µL of 2-vinilpyridine at 4oC for 1 h The rate of
2-nitro-5-thiobenzoic acid formation was monitored at
412 nm and compared to standard curves made with
GSH and GSSG, respectively The GSH concentration
is calculated as total glutathione – 2 x [GSSG]
2.8 Statistical analysis
Data are expressed as mean ± SEM for each group The
differences among experimental groups were detected
by one-way analysis of variance (ANOVA) with post hoc
multiple comparisons using Bonferroni’s test
3 Results
3.1 Lipid peroxidation
The results indicate that basal level of LPO was the
highest in liver mitochondria after 6-h immobilization
(Figure 1A) Intermittent hypoxia in regimen I caused
an increase in basal TBARS content by 23% (P<0.01),
while LPO level after IH in regimen II was unchanged
in comparison with the control Stimulation LPO
demonstrated a similar tendency in the changes
of TBARS indexes at these experimental groups
(Figure 1B) Groups 5, 7, 8 displayed a reduction of
15-19% (P<0.01) in basal LPO level in mitochondrial fraction as well as in LPO following an addition of exogenous inducers in comparison with the group 2 In contrast, the basal TBARS content in mitochondria of group 4 remained approximately at acute immobilization level (group 2) and Fe2+/ascorbate-stimulated in vitro
LPO was the highest compared to other experimental groups
A
0 0,5 1 1,5 2 2,5
1 2 3 4 5 6 7 8
a a
b * ^^ # a * + b *
B
0 2 4 6 8 10 12 14 16 18 20
1 2 3 4 5 6 7 8
a a a*
a * ^
#
* +
a *
Figure 1 Effect of different regimes of intermittent hypoxia (IH)
and acute immobilization on mitochondrial lipid peroxi-dation – basal LPO (A) and Fe 2+ /ascorbate - induced LPO (B) Values are mean ± SEM n=6 in each group The data were analyzed for statistical significance using
ANOVA followed by Bonferroni post hoc test a -P<0.001;
b - P<0.01 vs control; * - P<0.001 vs immobilization (group 2); # -P<0.001 statistically significant differences between groups 3 and 6; + -P<0.001 statistically sig-nificant differences between groups 4 and 7 as well as between groups 5 and 8; ^- P<0.001; ^^- P< 0.05 statistically significant differences between groups 4 and 5 as well as between groups 7 and 8 Groups: 1- control; 2- a single 6h immobilization; 3- IH in regimen I; 4- 6h immobilization at the first day after cessation of IH
in regimen I; 5- 6h immobilization at the 45 th day after cessation of IH in regimen I; 6- IH in regimen II; 7- 6h immobilization at the first day after cessation of IH in regimen II; 8- 6h immobilization at the 45 th day after ces-sation of IH in regimen II.
Trang 53.2 Superoxide radical production
The data for superoxide anion production is shown at
Figure 2 A The generation of O2∙− was significantly lower
in rats exposed to IH (II) than in rats exposed to IH(I)
Liver mitochondrial samples had a higher production of
O2∙− after immobilization (P<0.001) In all cases using
immobilization after IH (I) and IH (II), O2 ∙− generation
were lower than in unadapted rats
3.3 Superoxide dismutase activity
Figure 2B illustrates that after sessions of IH (II), the activity of Mn-SOD was increased by 29% compared
to normoxic rats (P<0.001) A similar tendency in Mn-SOD activity was observed in rats after immobilization
at the 1st day after cessation of IH (II) (group 7) The reduction in Mn-SOD activity was observed in rats exposed to immobilization as well as to IH (I) compared
to the control Mitochondrial Mn-SOD activity in rats of group 3 was 46% lower than group 6 rats (P<0.001)
Changes in the Mn-SOD activity in the rats exposed to 6h immobilization at the 45th day after cessation of IH (I)
as well as of IH (II) were similar
3.4 Glutathione pool
Table 1 presents the changes in the glutathione pool in liver mitochondria Immobilization caused an increase
in GSSG level by 69% (P<0.001), a decrease in GSH content by 18% (P<0.001), and the ratio of reduced
to oxidized form was 2.06 times less than the control average (P<0.001) The IH (I)-exposed animals resulted
in significant enhancement of mitochondrial GSSG concentration by 39% (P<0.001) For these animals the total GSH and reduced GSH content were depleted by
16 and 21%, respectively (P<0.001) In contrast, the IH (II) pretreatment unchanged these indexes compared
to the normoxic rats After immobilization at the 1st and 45th day, rats that had IH (I) and IH (II) exhibited
a decrease of GSSG content (10-28%, P<0.001) and
an elevation of GSH/GSSG ratio compared to the group 2 (besides the group 4) The concentration of mitochondrial reduced GSH in animal groups 4 and 5 remained low, which is nearly identical to that observed
in rats of group 2
3.5 Glutathione-related enzymes and NADP+ -dependent isocitrate dehydrogenase activities
A 6-h acute immobilization induced a decrease in GPx activity by 22% (P<0.001), an enhance in GST and IDPm activities by 16% and 12%, respectively (P<0.01)
However, the activity of GR had a tendency to decrease compared to the control (Table 2) IH (II) increased mitochondrial activities of GR and IDPm and decreased the GST activity The activity of GPx tended to increase
in comparison with normoxia In opposition, IH (I) caused diminution in GR and IDPm activities by 22% and 15%
(P<0.001), and increased GPx and GST activities by 25% (P<0.001) and 20% (P<0.01), respectively In the rats that were immobilized on the 1st day after cessation
of IH (I) the activities of GR, IDPm were lower and the activity of GST was higher than in the stressed rats after
A
0 0,2
0,4
0,6
0,8
1 1,2
1,4
1,6
1,8
2
1 2 3 4 5 6 7 8
a
c b** b **
###
c * ++ c * ++
0
1
2
3
4
5
6
1 2 3 4 5 6 7 8
b
*
*
a # a+
* c
*
*
B
Figure 2 Effect of different regimes of intermittent hypoxia (IH) and
immobilization on mitochondrial superoxide anion pro-duction (A) and Mn-SOD activity (B) Values are mean
± SEM n=6 in each group The data were analyzed for statistical significance using ANOVA followed by
Bonfer-roni post hoc test a -P<0.001; b - P<0.01; c - P<0.05 vs control; * - P<0.001; ** - P<0.01 vs immobilization (group 2); # -P<0.001; ### -P<0.05 statistically significant differ-ences between groups 3 and 6; + -P<0.001; ++ -P<0.01 statistically significant differences between groups 4 and
7 as well as between groups 5 and 8 ; ^- P<0.001 sta-tistically significant differences between groups 4 and 5
as well as between groups 7 and 8 Groups: 1- control;
2- a single 6h immobilization; 3- IH in regimen I; 4- 6h immobilization at the first day after cessation of IH in regi-men I; 5- 6h immobilization at the 45 th day after cessation
of IH in regimen I; 6- IH in regimen II; 7- 6h immobilization
at the first day after cessation of IH in regimen II;8- 6h immobilization at the 45 th day after cessation of IH in regi-men II.
Trang 6IH (II) Changes in GR, GPx, GST, and IDPm activities in
the rats exposed to immobilization on the 45th day after
cessation of both IH (I) and IH (II) were similar
3.6 Correlation between oxidative stress
marker and GSH content, activities of GSH-related and NADP+-dependent enzymes in mitochondria
The kinetics of TBARS content changes in liver
mitochondria during IH in the two regimes before
and after immobilization were compared with GSH
concentration, GR, GPx, GST and NADP+-dependent
isocitrate dehydrogenase activities For stressed rats
at the 1st and 45th day after IH in regimen I, a positive
correlation was observed between IDPm and GR
activities (r=0.98) as well as between IDPm activity
and the GSH content (r=0.90) There were negative
correlations between the IDPm activity and TBARS
content (r=−0.54), IDPm and GPx activities (r=−0.18), IDPm and GST activities (r=−0.87) as well as between levels of GSH and TBARS (r=−0.98) In mitochondria of rats that had immobilization in early and later periods after cessation of IH (II), IDPm activity was positively correlated with GR (r=0.73) and GPx (r = 0.92) activities,
as well as with the GSH content (r= 0.27) We found
a negative correlation between contents of TBARS and GSH (r=−0.92) and IDPm and GST activities (r =−0.98) No significant correlation between the TBARS content and the IDPm activity (r =−0.09) was observed in these groups of rats
4 Discussion
In the present study the severe hypoxia used in sessions of intermittent hypoxia causes a significant increase in the mitochondrial O2 ∙− production, as well
Groups Experimental
conditions Total glutathionenmol/ mg protein GSSGnmol/mg protein GSHnmol/mg protein GSH/GSSG
1 Control 5.27±0.37 0.23±0.12 4.82±0.29 20.92±0.67
2 AS 4.73±0.20 0.39±0.16 a 3.95±0.19 a 10.14±0.61 a
3 IH (I) 4.44±0.38 b 0.32±0.02 a 3.80±0.36 a 11.86±0.93 a
4 IH (I) + AS 1 4.34±0.24 a 0.35±0.02 a* 3.65±0.25 a 10.65±1.24 a
5 IH (I) + AS 45 4.75±0.21 0.29±0.01 a**^ 4.18±0.19 c 14.71±0.38 a*^
6 IH (II) 5.14±0.47 ### 0.26±0.01 # 4.63±0.46 ## 17.86±0.92 a#
7 IH (II)+ AS 1 4.97±0.24 0.31±0.02 a*++ 4.35±0.22 +++ 14.05±0.58 a*+
8 IH (II) + AS 45 4.84±0.24 0.28±0.02 a* 4.28±0.24 15.30±1.11 a*
Table 1 Changes in liver mitochondria glutathione pool after intermittent hypoxia of different regimes and acute immobilization.
Values are means ± SEM n=6 in each group The data were analyzed for statistical significance using ANOVA followed by Bonferroni post hoc test
a -P<0.001; b - P<0.01; c - P<0.05 vs control; * - P<0.001; ** - P<0.01 vs immobilization (group 2); # -P<0.001; ## - P<0.01; ### - P<0.05 statistically significant differences between groups 3 and 6; + -P<0.001; ++ - P<0.01; +++ - P<0.05 statistically significant differences between groups 4 and
7 as well as between groups 5 and 8; ^- P<0.001 statistically significant differences between groups 4 and 5 as well as between groups 7 and 8.
Table 2 Changes in activities of mitochondrial GSH-related enzymes and NADP + dependent isocitrate dehydrogenase after different regimes of
intermittent hypoxia and acute immobilization.
Values are means ± SEM n=6 in each group The data were analyzed for statistical significance using ANOVA followed by Bonferroni post hoc test a -P<0.001; b - P<0.01; c - P<0.05 vs control; * - P<0.001; ** - P<0.01 vs immobilization (group 2); # -P<0.001 statistically significant differences between groups 3 and 6; + -P<0.001 statistically significant differences between groups 4 and 7 as well as between groups 5 and 8; ^- P<0.001;
^^ - P<0.01 statistically significant differences between groups 4 and 5 as well as between groups 7 and 8
Groups Experimental
conditions
GR µmol NADPH/
min/ mg protein
GPx µmol GSH/
min/ mg protein
GST µmol conjugated product/
min/ mg protein
IDPm µmol NADPH/
min/ mg protein
1 Control 20.27±0.47 3.11±0.13 11.50±0.64 19.90±0.61
2 AS 19.16±1.07 2.42±0.15 a 13.28±0.57 b 18.00±0.56 b
3 IH (I) 15.85±0.53 a 3.88±0.27 a 13.76±0.72 b 16.96±0.85 a
4 IH (I) + AS 1 14.96±0.34 a* 2.70±0.25 c 12.75±0.71 17.03±0.55 a
5 IH (I) + AS 45 17.96±0.58 a * ^ 2.47±0.33 a 12.07±0.28 18.70 ±0.59 ^^
6 IH (II) 21.58±0.42 c # 3.42±0.14 # 10.12±0.81 c 23.51±0.78 a #
7 IH (II) + AS 1 19.33±0.55 + 3.06±0.12 * 10.70±1.01 *+ 21.47±1.04 c * +
8 IH (II) + AS 45 19.21±0.48 2.72±0.20 11.61±0.50 ** 18.67±0.49 ^
Trang 7as in TBARS level The intensification of oxidative
process in mitochondria is accompanied by an increase
in GSSG content and a decrease in GSH/GSSG ratio,
which are essential indicators of oxidative stress in
cell compartments [31] Our results also suggest that
mitochondria of rats exposed to IH in regimen I are
more susceptible to stimulated in vitro LPO than the
mitochondria of rats exposed to IH in regimen II
It is known that hypoxia, as well as reoxygenation,
can induce excessive ROS generation, resulting from
the univalent reduction of molecular oxygen to O2 ∙−
by electrons that leak from the mitochondrial electron
transport chain, mainly from complexes I and III
[13,32] The repetitive situation of short-term severe
hypoxia followed by short-term reoxygenation causes
a disturbance of intracellular pro-oxidant/antioxidant
homeostasis, which is manifested in the intensification
of lipid peroxidation [11,14,33] The enhanced LPO in
mitochondria leads to the loss of mitochondrial membrane
fluidity, membrane ionic permeability, including proton
permeability, which uncouples oxidative phosphorylation,
as well as activity of membrane – bound enzymes [12]
This is in agreement with the findings of Joyeux-Faure et
al [10] where repetitive cycles of 40 sec of hypoxia (5%
O2) for 35 days increased ischemia-induced infarction of
rat hearts An increase in superoxide anion and hydrogen
peroxide levels in mitochondria were observed after 60
cycles of intermittent hypoxia (each cycle consisting of
15 sec of 1.5% O2 followed by 4 min of normoxia) [33], a
severe high-altitude hypoxia exposure, as well as after
an anoxia/reoxygenation [11,12]
Most of the O2 ∙− generated by mitochondria is
released into the mitochondrial matrix, where it is
converted to H2O2 by a specific intramembrane Mn-SOD
[32] Mn-SOD is particularly responsive to oxidative
stress and may be activated in a variety of stressful
conditions, including oxygen deprivation [34] In our
study we demonstrated a decline in the Mn-SOD activity
after severe hypoxia in IH (I) that is in accordance with an
enhancement of superoxide production in mitochondria
The present results are in good consent with the findings
of Jackson et al [35] that hypoxia decreases expression
of Mn-SOD mRNA in epithelial cells and in lung
fibroblasts At the same time, reoxygenation increases
expression, but decreases the activity of Mn-SOD in
rat neurons Yamashita et al [8] had shown that during
preconditioning (1% O2 for 1 h) the activity of Mn-SOD
in culture myocytes slightly decreases, but increases
after a 24 h reoxygenation Moreover,
mitochondria-generated RNS and ROS can mediate posttranslational
modifications of mitochondrial proteins that result in their
inactivation (Mn-SOD, adenine nucleotide translocator)
or altered function (cytochrome C, aconitase) [34,36]
Some authors explain this observation by high susceptibility of Mn-SOD to oxidative inactivation
by ONOO- [36] It had been shown that increased
3-nitrotyrosine levels of Mn-SOD in vivo are associated
with decreased specific activity of the enzyme [34] It
is known that NO competes with SOD for the removal
of O2 ∙− by forming ONOO-, when NO concentration is
in range of mitochondrial SOD level [37] This process
is enhanced during hypoxia when mitochondrial nitric-oxide synthase is functionally upregulated [38]
Our experimental data showed that sessions of IH (I) cause a decrease in the total and reduced glutathione
It was reported earlier that, in rats, severe hypoxia depletes GSH stores in liver mitochondria [37,39] and leads to short-term falls in intracellular GSH levels in
endothelial and alveolar cells in vitro and also in vivo
[21]
The depletion of glutathione probably occurred by
conjugation reactions via the GST or by GSSG formation
through increased H2O2 production and GPx activity [17]
A possible involvement of GSH in the metabolism of aldehydes and peroxides in our study was demonstrated
by maintenance of adequate Se-GPx and GST activities
in rats exposed to IH (I) Because catalaseis absent in the mitochondria of most animal cells, GPx plays a key role in metabolizing H2O2 in mitochondria [16,17]. Recent studies had shown that the high levels of oxidative and nitrosative stress lead subsequently to induction of GPx mRNA transcription and protein expression in various cell lines [40]
In contrast, slight changes in superoxide anion production and the TBARS level, which we registered in rats exposed to moderate hypoxia in regimen II, are in general consent with high activity of Mn-SOD (by 29% in comparison with normoxia) This statement is based on the assumption that higher SOD activity means faster rate of conversion and, hence, disappearance of O2 ∙− [31] As was reported by Wispe [41], Mn-SOD activity
shows a biphasic increase after preconditioning in vivo
that might be due to a conversion from pro Mn-SOD to Mn-SOD or to a change in its oligomer structure
In this study, it was demonstrated that GPx activity
in rats adapted to regimen II had a tendency to increase
in comparison with the control An increase in the activity of Se-GPx is in accordance with the increased content of H2O2, which serves as the substrate for this enzyme and influences its activity [16] We suppose that the development of H2O2 generation in this situation does not translate into proportional mitochondrial damage, and this is confirmed by the TBARS index, perhaps, due to the increased antioxidative defense found in mitochondria and/or ability of H2O2 to diffuse out the mitochondrial matrix [17] An increase in ROS
Trang 8scavenges, such as SOD and GSH, after sessions of
moderate hypoxia in IH (II) might represent an important
physiological adaptation that counteracts an increase in
oxidative stress under hypoxic conditions
Since the mitochondrial GSSG cannot be exported
into cytosol for reconversion into GSH, mitochondrial
NADPH is an essential reducing equivalent for the
regeneration of GSH from GSSG by the activity of
mitochondrial GR [16,17] Recently, it was demonstrated
that the control of mitochondrial redox balance and
oxidative damage is one of the primary functions of
IDPm [20] Lee et al [42] have shown that decreased
expression of IDPm markedly elevates the mitochondrial
ROS generation, DNA fragmentation, lipid peroxidation,
and a significant reduction in ATP level However,
the role of IDPm, which catalyzes decarboxylation of
isocitrate into α-ketoglutarate with concurrent product of
NADPH in the mitochondria during intermittent hypoxia,
is insufficiently known
In the present study, we showed that increases in
GR and IDPm activities promote coordinated action of
GSH redox-cycle enzymes during IH in regimen II In
contrast, IH (I)- exposed rats had decreased activity
of IDPm in parallel with a decrease in the GR activity
These results are in accordance with the fact that
severe hypoxic stimulus (PO2 below 20 Torr) results
in a general loss of Krebs cycle enzyme activity In
contrast, more modest levels of hypoxia (PO2 80-100
Torr) under IH or acclimatization to high altitude hypoxia
shift the anaerobic glycolysis to aerobic metabolism and
stimulate oxidative enzyme activities [2] Based on the
positive correlation between IDPm, GR, GPx activities
and indexes of oxidative stress we hypothesize that
ICDm is involved in oxidative stress protection and in
mitochondrial GSH-redox regulation during adaptation
to IH
For the estimation of hypoxic adaptation efficiency on
mitochondrial functions we used acute immobilization
Rats exposed to immobilization demonstrated
several indications of oxidative stress, an alteration of
glutathione metabolism, a decrease of GPx activity, and
a decreased GSH/GSSG ratio These data suggest
that immobilization is a stressful and physiologically
sensitive period for mitochondria and can cause dramatic
biochemical changes in GSH-redox balance
Acute immobilization on the 1st day after cessation
IH in strong hypoxic regimen caused a clearly
pronounced increase in mitochondrial TBARS content
in vivo, decrease in GSH/GSSG ratio, which -in addition
to depletion of reduced GSH and inhibition of GPx,
GR and IDPm activities- are indicative of enhanced
oxidative processes in mitochondria At the same time in
animals of the above group, the superoxide production
was lower and the Mn-SOD activity was higher than in the corresponding indices in the acutely stressed rats Disturbance in pro-/antioxidant balance in these rats made the mitochondria more sensitive to stimulated
in vitro LPO whose index was found to be significantly
higher in comparison with unadapted rats
Increased activities of GSH-related and NADP+ -dependent enzymes, as well as the Mn-SOD activity
in rats adapted in regimen II, may be the reason for significant attenuation of superoxide anion generation and TBARS levels after immobilization The strengthening of mitochondrial antioxidant protection during sessions of IH (II) may serve as an adaptive mechanism of tolerance to new oxygen injury It is possible that such cross adaptation, when resistance
to one factor induces resistance to other factors, is a polygenic phenomenon that requires the simultaneous activation of multiple stress-responsive genes It is known that during hypoxia mitochondria function as
O2 sensors due to increasing of ROS release, which may act as signaling molecules [15] The mild oxidative stress during hypoxia/reoxygenation could activate O2 -sensing-related transcriptional factors, such as HIF-1, AP-1, NF-ĸB, that are associated with adaptive changes
in mammalian cell metabolism, including the expression
of proteins and enzymes that respond to hypoxic stress [18,43] It had been proposed that mitochondrial ROS could also be a triggering factor for the stabilization of HIF-1α during hypoxia [15]
Immobilization at the 45th day after cessation of IH in regimen II induced changes in LPO index, GSH/GSSG ratio, and activities of GSH-related enzymes similar to those observed in liver mitochondria of rats subjected
to immobilization in the early period These data confirm long-lasting activation of mitochondrial antioxidative system by IH that persists 45 days after termination of the hypoxia/reoxygenation stimulus
An interesting observation made in the present study is that the intensity of oxidative processes in mitochondria of rats exposed to immobilization within the later period after cessation of IH (I) (group 5) was less than in rats exposed to immobilization at ones after cessation of IH in regimen I (group 4) At the same time, the TBARS content and activities of GR, GPx, NADP+- dependent IDPm, and Mn-SOD in mitochondria of rats that had acute stress on the 45th day after cessation of IH
in regimen I, as well as in regimen II, were analogous
It is possible that the hypoxic adaptation in regimen I when we used stronger hypoxia was more effective at its later phase This is in agreement with the hypothesis of “a second window of protection” of hypoxic preconditioning when the oxygen radicals generated during the initial preconditioning period activate endogenous antioxidant
Trang 9defense at late phase [44] The mechanism of the late
phase effect of preconditioning is still not clear It was
reported that the late phase effect of sublethal ischemia
observed 24 h after preconditioning highly correlated
with the induction of mitochondrial Mn-SOD or heart
shock proteins [8]
In our study, we demonstrated that mitochondria
adapted to moderate hypoxia enhance production and
activity of ROS scavenges and that play an important
role in the development of oxidative tolerance to new
damaging factors
Acknowlegements
I would like to thank N Petrova and T Dubovaya for technical assistance
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