Egypt. J. Exp. Biol. (Zool.), 11(1): 71 – 78 (2015)
© The E gypt ian S oc iet y of E xper im ent al B iology
RESEARCH ARTICLE
Z ei na b I . At ti a
Mon a M. H ega zi *
Moh am ed H. Mou r ad **
O m eym a A. A sh our **
AN T IOX ID AN T EN ZY MES ST AT U S IN L IVER AN D WH IT E MU SC LE OF N IL E
T IL AP I A EX POSED T O DIFFER EN T H YPOX IC LEV ELS AN D D U R AT ION S
ABSTRACT:
The effect of hypoxi a on Nile tilapi a,
Oreochromis niloticus for one day (trial 1) and
for 30 days (trial 2) was studied. The hypoxi c
oxygen concent rations used in the two trial s
were 2, 1, and 0.5 mg O 2 L - 1 in comparison to
control normoxic group 7 mg O 2 L - 1 . The
assayed enzymes were: superoxi de dismutase
(SO D),
catalase
(C AT),
glutathione
peroxi dase (G Px), glutathione S-transferase
(GST), glutathione reductase (G R), γ-glutamyl
cysteinyl synthetase (γ- G CS), and γglutamyl transpepti dase (γ-G T). The activities
of the enzymes assayed were s ignificantl y
increased in liver and white muscle of fish
exposed to both short term and long term of
hypoxia exposures, except in white muscle of
fish expos ed to 2 and 1 m g O 2 L - 1 . The
changes in these param eters were intensified
at l ow levels and of long hypoxia exposure.
The si gnificance of these alterati ons in
enzym e activities is discussed.
KEY WO RDS :
A n ti oxi d ant E nzym es, Ni l e T il api a , Li v er ,
W hit e Mus cl e, H y poxi a
CO RRES PO NDENCE:
Z ei na b I . At ti a
Zool . Dep., Facult y of Science,
University, Tanta, Egypt
E-mail: [email protected]
Tanta
Mon a M. H ega zi *
Moh am ed H. Mou r ad **
O m eym a A. A sh our **
*Zool. Dep., Faculty of Science, Tanta
University, Tanta, Egypt
**National Institute of Oceanography and
Fi sheri es, Al exandri a, Egypt
ARTICLE C OD E: 07.01.15
ISSN: 2090 - 0511
INTRO DU CTIO N:
Many species of tilapia have been
cultured in developing countries where animal
protein is lacking. Nile tilapia is by far the most
important farmed tilapia species in the world.
Tilapia is the most familiar and popular fish in
Egypt, as well as, in the Middle East and warm
climate countries (Philippart and Ruwet, 1982;
El-Sayed, 2006). Fish production should be
increased in Egypt to meet the demand of the
increasing population. Several problems face
fish production. Among these problems are the
low dissolved oxygen concentrations (hypoxia).
Environm ental hypoxia occurs commonly
in
aquatic
environments.
Despite
the
significant physiological challenge many fishes
have evolved the ability to survive extended
periods of exposure to hypoxia (i.e. low lev els
of oxygen) and even anoxia (i.e. total lack of
oxygen) (Nikinmaa and Rees, 2005). Hypoxia
tolerant fishes, successfully surviving low
oxygen conditions, undergo a new danger after
oxygen resumption. The el ectron transport
chain being reduced under hypoxic state can
produce elevated levels of reactive oxygen
species (ROS) during reoxygenation that may
cause oxidative stress (Rahal et al., 2014).
Therefore, these speci es must evolve welldeveloped antioxidant systems.
Fish exposed to hypoxia may result in
oxidative stress although it is not directly
evidenced yet. Some clues could be given by
an increase in activities of antioxidant
enzym es under hypoxia conditions (Lushchak
et al., 2001). Usually antioxidant enzymes are
upregulat ed by an increase in i ntracellular
ROS l evels. Once produced, ROS may damage
cellular components and tissues particularly
targeting proteins, lipids, and nucleic acids,
often leading to cumulative organ injury
(Livingstone, 2001). O xidative stress arises if
ROS generation prevail their degradation
(Lushchak and Bagnyukova, 2006).
In order to protect against oxidative
stress, organisms have devel oped antioxidant
system s consi sting of low-molecular weight
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Attia et al., Antioxidant Enzymes in Liver and White Muscle of Nile Tilapia Exposed to Different Hypoxic Levels and Durations
compounds including glutat hione, ascorbic
and uric acid, tocopherols and proteins.
Superoxide dismutase (SOD) and catal ase
decom pose O 2 and H 2 O 2 , respectivel y.
Glutathione peroxidase (G Px) detoxifies both
H 2 O 2 and organic hydroperoxi des, while
glutathione-S-transferase (G ST) detoxifies
various compounds by conj ugating them with
glutathione. Glutathi one reductase (G R)
reduces oxidized gl utathione using NADPH,
and associated en zym es like glucose-6phosphate dehydrogenase (G 6P DH) which
supplies reduced equival ents for GR (Herm esLima, 2004).
Fish found both in the tropics and
tem perate waters have i ncipient limiting
oxygen levels on fish growth i n aquaculture
which may occur in hypoxia. Incipient limiting
levels generally average at 73 mmHg (2.29
mg L -1 at 28ºC for warm water fish). In
reference to Nile tilapia, studies have shown
that the incipient oxygen requirement s are
between 1.39 m g L - 1 to 2.92 mg L - 1 (Mallya,
2007). Thereby, i n the present study, 2, 1,
and 0.5 mg O 2 L - 1 concentrations were
selected to mimic t he average possible
hypoxic conditions faced by the fish in their
natural habitat.
Since Nile tilapia are cultured for human
consumpti on the effects of hypoxia in their
muscle and liver are obviously of great
interest. Thus, the present investigation
aimed t o st udy the potential oxidative stress
adverse ef fects of l ong and short term
exposure to different concentrations of
hypoxia on Nil e tilapia fi ngerli ngs.
M ATER IAL AND METHO DS:
Fish husbandry:
Nile tilapia juveniles were obtained from
the fish hatching pond i n Fowa city (K afer ElShei kh
Governorate).
The
fish
were
transported in oxygenated cell ophane bags at
mid-November when the am bient water
tem perature was 15 ± 2ºC. Fish of nearl y
equal size (10 ± 1. 2 g) w ere distributed in 48
L gl ass aquaria. Four fish were placed in
every aquarium giving a rearing density of 1.2
g L − 1 . The fish rearing density was reduced at
the beginning of the experim ental tim e to
prevent the final densities from exceeding the
recommended threshold. The upper rearing
density lim it of the fish for toxicity testing was
2.5 g L − 1 (APHA, 1998). The maximum final
rearing density at the end of the exp erim ent
was 2.36 g L − 1 . Each aquarium was equipped
with a continuous air fl ow system. Di ssol ved
oxygen l evel in the aquaria was mai ntained at
80–85% air saturati on (8 mgL − 1 ). The dail y
ration w as 3% of fish wei ght, and the fish
were fed comm erci ally available pellets
(25.2% protei n, lipids, carbohydrate and fi bers
with total energy, 2505 kcal kg −1 ) once dail y
at 10:00 a.m.
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71
Thermal acclimation of fish:
The fish were acclimated to the
laboratory conditions for one week. Each
aquarium was equipped with high precision
thermostat controlled heater. The temperature
was steadily raised from 15–16ºC during 10
days by 2ºC every two days to reach the
temperature 26 ± 0.5ºC; the optimum
temperature giving maximal growth of Nil e
tilapia (Mishrigi and Kubo, 1978). The fish
were acclimat ed to this temperature for four
weeks. Fifty percent of the aquarium water was
daily replaced by dechlorinated water, which
was previously adjusted to 26ºC.
Experimental hypoxia design:
Following laboratory thermal acclimation,
the aquaria were classified into three groups.
The first left at normal level (8.0 mg O 2 L -1 )
oxygen saturation. The second hypoxic group
(60 fish) was divided into three sub-groups
exposed to the hypoxic concentrations 2, 1 and
0. 5 mg O 2 L - 1 , in five-covered aquaria for each
sub-group for only one day (the first trial). The
third hypoxic group (60 fish) was divided into
three sub-groups exposed to t he hypoxic
concentrations 2, 1 and 0.5 mg O 2 L -1 , in fivecovered aquaria for each sub-group for 30
days (the second trial).
During the experiments, the oxygen
concentration was slowly and gradually
decreased to the desired dissolved oxygen
(DO). Nitrogen gas was pumped into water to
lower the DO level in order to create the
hypoxic conditions of the experiment (Lehmann
et al., 2005). Final oxygen l evels were
obtained by bubbling N 2 into the polystyrene
container (20% decrease per hour), and the
temperature was kept constant at 26 ± 0.5 ºC.
The loss of equilibrium indicated the lowest
survivable oxygen level. To ensure that the
fish were not gasping at the surface t o
artificially increase oxygen uptake, the treated
aquaria were covered with a suitabl e glass
plates during the exposure time. Dissolved
oxygen levels were monitored continuously
using a dissolved oxygen meter, Hanna
(model, HI 9142, Italy), by placing the
el ectrode i nto the treated aquaria. Electrodes
were calibrated to zero oxygen with a freshly
prepared 2% sodium persulfate solution.
Calibration to 100% was done with air.
Tissue sampling:
Fish were killed by a sharp blow on the
head and weighed. A small pi ece of whit e
muscle and right liver lobe were carefully
excised on ice, avoiding squeezi ng the tissue,
washed in ice-cold isotonic NaCl saline and
blott ed dry and weighed. The tissue was
immediately
homogenized
in
ice-cold
phosphate buffer (50 mM, pH 7.4) nearly 10%
(w/v) using Omni international homogenizer
(USA) at 22,000 rpm for 20 s each with 10 s
intervals. The homogenat e was centrifuged at
2000 ×g in cooling centrifuge (Hettich,
G ermany) at 4ºC for 15 min and the
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Egypt. J. Exp. Biol. (Zool.), 11(1): 71 – 78 (2015)
supernatant was saved. The supernatant was
freeze-thawed thrice to completely disrupt
mitochondria (Salach,
1978).
Then the
supernatant was again centrifuged at 6000×g
in cooling centrifuge at 4 ºC for 15 min and the
yielded supernatant which contains the
cytosolic and mitochondrial enzymes was
saved for immediate enzym e assays. The
UV/vis Spectrophotom eter (JENWAY 6505,
UK) was used for the m easurements of enzym e
activities at 25ºC.
Enzyme activity assays:
Total superoxide dismutase (SOD, EC
1.15.1.1) activity was assayed by the m ethod
of Paoletti and Mocali (1990). Samples were
assayed by measuring inhibition of NADH
oxi dation
by
β-mercaptoethanol
in
the
presence of EDTA and Mn. NADH solution was
made fresh daily, the assays were run by
adding sequentially to the cuvett e: 0.80 ml of
50 mM phosphate buffer (pH 7.4), 55 µL EDTAMn solution (EDTA/MnCl 2 100/50 mM), 40 µL
NADH solution (7.5 mM), and different volum e
from sample tissue extract. The reaction was
then
initiated
by
adding
100
µl
βmercaptoethanol solution (10 mM). The
changes in Δ E of NADH were followed per
minute for 15 min at 340 nm (ε = 6.22 mM -1 cm 1
). One unit of SOD activity is defined as the
amount of cell extract (mg protein) required to
inhibit the rate of NADH oxidation of the
control by 50%.
Catalase (CAT, EC 1.11.1.6) activity was
assayed according to the method of Cohen et
al. (1970). One milliliter of 50 mM phosphate
buffer (pH 7.4) and 10 γ L of tissue extract was
added to the cuvette. The reaction was then
initiated by the addition of 300 γL of 30 mM
H 2 O 2 prepared by diluting 0.34 mL of 30%
H 2 O 2 to 100 mL of 50 mM phosphate buffer (pH
7.4).
Specific
catalase
activiti es
were
determined following the changes in the
absorbance of H 2 O 2 at 240 nm (ε = 0.0394 1
mM −1 1cm −1 ).
Glutathione
peroxidase
(GPx,
EC
1.11.1.9) activity was assayed according to the
method of Paglia and Valentine (1967). One
milliliter of 50 mM sodium phosphate (pH 7.4)
containing 2 mM EDTA and 0.15 mM NADPH
and 10 γ L of tissue extract was added to the
cuvette. The reaction was then initiated by the
addition of 10 γL of 1mM GSSG. Specific GP x
activities were determined following the
changes in the absorbance of NADPH per min
at 340 nm (ε = 6.22 mM −1 1 cm −1 ).
Glutathione-S-transferase
(GST,
EC
2.5.1.18) activity was assayed according to the
method of Habig et al. (1974). The final
reaction mixture contained 1 mM CDNB, 1 mM
GSH in 50 mM phosphate buffer pH 7.4 and
the reaction was initiated by the addition of 50
γL tissue extract. Specific GST activities were
determined following the changes in the
absorbance of CDNB per min at 340 nm (ε =
9.6.00 mM−1 1 cm −1 ).
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Glutathione reductase (GR, EC 1.6.4.2)
activity was assayed according to the m ethod
Smith et al. (1988). The final reaction mixture
contained 0.44 mM GSSG, 0.30 M EDTA, in
0. 1 M phosphate buffer pH 7.0 10 µL tissue
extract and 0.036 mM NADPH was added just
before the enzymatic determination as the
starting reagent. Specific GR activities were
determined following the changes in the
absorbance of NADPH per min at 340 nm (ε =
6. 22 mM −1 1 cm −1 ).
Gama-Glutamylcysteine synthetase (γGCS, EC 6.3.2.2) activity was assayed
according to the m ethod of Huseby and
Stromme (1974). The enzym e activity was
assayed in 1 mL reaction mixture contained
100 mM Tris–HCl buffer (pH 8.0), 150 mM KCl,
5 mM ATP, 2 mM PEP, 10 mM glutamate,
10mM γ-amino butyrate, 20 mM MgCl, 2 mM
Na EDTA, 0.2 mM NADH, 17 g PK, and 17 mg
LDH. The reaction was then initiat ed by the
addition of 10 γL tissue extract. Specific γGCS activities were determined following the
changes in the absorbance of NADH per min at
340 nm (ε = 6.22 mM −1 1 cm −1 ).
Gama-Glutamyl transferase (γ-GT, EC
2. 3.2.2) activity was assayed according to the
method of Silber et al. (1986). The rate of the
substrate
analogue
glutamyl -p-nitroanilide
cleavage to form p-nitroaniline (pNA) by
transfer of a glutamyl moiety to glycylglycine
was monitored for at least 10 min. One
milliliter of 100 mM and 250 γ L of 20 mM γglutamyl -3-carboxy-4-nitroanilide were added
to the cuvette. The reaction was then initiated
by the addition of 10 γL tissue extract. Specific
γ-GT activiti es were determined following the
changes in the absorbance per min at 405 nm
(ε = 9.2 mM −1 1 cm −1 ).
Chemicals:
All chemicals used in this study were
purchased from Sigma Chemical Co. (St.
Louis, MO, USA) and were of analytical grade.
Statistical analyses:
Results are presented as mean ±
standard deviation. The statistical evaluation
of all data was done using one-way analysis of
variance (ANOVA) followed by Dennett's t est.
P value ≤ 0.05 were regarded as statistically
significant. The p is normally considered
significant at the value p ≤ 0.05.
RESULTS:
The obtained results of t he specific
en zym e activities of liver and w hite muscle of
Nile tilapia reared at 26ºC in the t hree
different level s of hypoxi a after one day (the
first tri al) and thirty days (the second trial) of
exposure were tabulated i n the tables 1-7.
The en zym e activities are express ed as µM
min - 1 gram - 1 wet weight tissue except SO D
calcul ated as mg tissue that results in 50%
SO D activity inhibition. The en zym es activity
in liver of fish reared in t he three different
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hypoxia concentrations (2, 1, and 0.5 mg O 2
L - 1 ) in the first and second trial was
significantly
(P
≤
0.05)
increased
in
comparison with their respective control s.
However, the enzym es activity in white
muscle w as significantly (P ≤ 0.05) increased
in comparison with their respective control s
except in fish reared in 0.5 mg O 2 L - 1 in the
first trial and the three different hypoxia
concentrations i n the second tri al.
73
The increase i n SOD activity (Table 1)
after one day of hypoxia, in liver w as 48%,
78% and 2.19 fold, respectivel y whil e, in whit e
muscl e was 13%, 21% and 66%, respectively.
After 30 days of hypoxia, the increase in liver
en zym e activity w as 56%, 88% and 2.25 fol d
respectivel y while, the increase in whit e
muscl e was 36%, 52% and 82%, respectively.
Table 1. Effect of hypoxia concentrations on superoxide dimutase activity (U/ g wet weight tissue) in the liver and white
muscle of Nile tilapia acclimated at 26ºC
DO mg
Liver
One day
30 days
White muscle
One day
30 days
7 Controls
2
1
0.5
232.4 ± 34.8
341.6 ± 49.6
345.0 ± 36.2*
535.2 ± 58.2*
413.3 ± 54.8*
643.3 ± 71.5*
510.3 ± 77.9*
771.7 ± 97.8*
152.2 ± 19.5
223.3 ± 32.1
173.3 ± 30.8
305.3 ± 36.2*
185.5 ± 24.2
341.7 ± 43.6*
253.7 ± 33.6*
408.3 ± 60.6*
- Each reading represents Mean ± SD of 10 fish.
-The data were subjected to One Way ANOVA with replication, (significance at P ≤ 0.05).
- The significance of difference between treated fish and control was checked by Dunnet test (*significance at P ≤0.05).
increase in liver enzym e acti vity was 19%,
40%, and 74%, respectively while, the
increase i n white muscle was 43%, 51%, and
87%, respectivel y.
The i ncrease i n CAT acti vity (Tabl e 2)
after one day of hypoxia, was 18%, 81% and
2.26 fold respecti vely while, the i ncrease in
white m uscle was 46%, 36%, and 73%,
respectively. After 30 days of hypoxi a the
Table 2. Effect of hypoxia concentrations on catalase activity (µM/min/g wet weight tissue) in the liver and white muscle of
Nile tilapia acclimated at 26ºC
DO mg
Liver
One day
30 days
White muscle
One day
30 days
7 Controls
2
1
0.5
130. 1 ± 17.8
228.9 ± 28.7
197.5 ± 30.2*
274.5 ± 43.1*
236.5 ± 38.1*
320.9 ± 53.2*
295.2 ± 42.6*
398.3 ± 63.2*
64.7 ± 10.7
135.4 ± 20.4
94.8 ± 315.5
194.1 ± 23.5*
88.3 ± 13.3
205.5 ± 35.2*
112.1 ± 25*
254.4 ± 44.4*
- Each reading represents Mean ± SD of 10 fish.
-The data were subjected to One Way ANOVA with replication, (significance at P ≤ 0.05).
- The significance of difference between treated fish and control was checked by Dunnet test (*significance at P ≤ 0.05).
increase in liver enzym e acti vity was 35%,
61%, and 89%, respectively while; the
increase in whit e m uscle was 67%, 2 and 2.43
folds respectively.
The i ncrease in GST activity (Tabl e 3)
after one day of hypoxia, was 51%, 85% and
2.30 fold respecti vely while, the i ncrease in
white m uscle was 37%, 44%, and 77%,
respectively. After 30 days of hypoxia, the
Table 3. Effect of hypoxia concentrations on glutathione-S-transferase (GST) activity (µM/min/g wet weight tissue) in the
liver and white muscle of Nile tilapia acclimated at 26ºC
DO mg
Liver
One day
30 days
White muscle
One day
30 days
7 Controls
2
1
0.5
1.2 ± 0.13
1.78 ± 0.20
1.8 ± 0.25*
2.40 ± 0.3*
2.27 ± 0.3*
2.87 ± 0.4*
2.81 ± 0.46*
3.67 ± 0.56*
0.47 ± 0.07
0.92 ± 0.19
0.65 ± 0.08
1.54 ± 0.16*
0.68 ± 0.08
1.84 ± 0.30*
0.84 ± 0.3*
2.24 ± 0.4*
- Each reading represents Mean ± SD of 10 fish.
-The data were subjected to One Way ANOVA with replication, (significance at P ≤ 0.05).
- The significance of difference between treated fish and control was checked by Dunnet test (*significance at P ≤ 0.05).
The i ncrease i n GPx acti vity (Tabl e 4)
after one day of hypoxi a, was 56%, 876% and
2.23 fold, respectively whil e the i ncrease in
white m uscle was 17%, 39%, and 60%,
respectively. After 30 days of hypoxia, the
ISSN: 2090 - 0511
increase in liver enzym e acti vity was 36%,
52% and, 76%, respectivel y while the
increase i n white m uscle w as 40%, 85% and
2.24 fol d, respecti vely.
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Egypt. J. Exp. Biol. (Zool.), 11(1): 71 – 78 (2015)
Table 4. Effect of hypoxia concentrations on glutathione peroxidase (GPx) activity (µM/min/g wet weight tissue) in the liver
and white muscle of Nile tilapia acclimated at 26ºC
DO mg
Liver
One day
30 days
White muscle
One day
30 days
7 Controls
2
1
0.5
1.39 ± 0.19
2.30 ± 0.21
2.17 ± 0.3*
3.14 ± 0.40*
2.59 ± 0.3*
3.51 ± 0.6*
3.10 ± 0.5*
4.057 ± 0.4*
0.78 ± 0.21
1.07 ± 0.15
0.92 ± 0.13
1.50 ± 0.27*
1.09 ± 0.16
1.97 ± 0.31*
1.25 ± 0.21*
2.39 ± 0.42*
- Each reading represents Mean ± SD of 10 fish.
-The data were subjected to One Way ANOVA with replication, (significance at P ≤ 0.05).
- The significance of difference between treated fish and control was checked by Dunnet test (*significance at P ≤ 0.05).
30 days of hypoxia, the increase in liver enzyme
activity was 56%, 86% and 2.23 fold,
respectively while, the increase in white muscle
was 76%, 2. 05 and 2.69 folds, respectively.
The increase in GR activity (Table 5) after
one day of hypoxia, was 41%, 66% and 86%
respectively while; the increase in white muscle
was 40 %, 49% and 2 fold, respectively. After
Table 5. Effect of hypoxia concentrations on glutathione reductase (GR) activity (µM/min/g wet weight tissue) in the liver and
white muscles of Nile tilapia acclimated at 26ºC
DO mg
Liver
One day
30 days
White muscle
One day
30 days
7 Controls
2
1
0.5
1.69 ± 0.20
2.16 ± 0.25
2.38 ± 0.23*
3.37 ± 0.42*
2.82 ± 0.43*
4.03 ± 0.34*
3.15 ± 0.37*
4.82 ± 0.57*
0.81 ± 0.21
1.20 ± 0.10
1.14 ± 0.20
2.13 ± 0.28*
1.21 ± 0.21
2.47 ± 0.45*
1.63 ± 0.30*
3.25 ± 0.51*
- Each reading represents Mean ± SD of 10 fish.
-The data were subjected to One Way ANOVA with replication, (significance at P ≤ 0.05).
- The significance of difference between treated fish and control was checked by Dunnet test (*significance at P ≤ 0.05).
increase in liver enzym e acti vity was 42%,
92% and 2.34 f old, respecti vely while, the
increase i n white muscle was 2.12, 2. 73, and
3.60 fol ds, respectively.
The increase in γGCS activity ( Table 6)
after one day of hypoxia was 44%, 61% and
2.02 fold, respectively whil e the i ncrease in
white muscle was 0.9%, 0.93%, and 8%,
respectively. After 30 days of hypoxia, the
Table 6. Effect of hypoxia concentrations on γ-Glutamyl cysteinyl synthetase (γ-GCS) activity (µM/min/g wet weight tissue)
in the liver and white muscle of Nile tilapia acclimated at 26ºC
DO mg
Liver
One day
30 days
White muscle
One day
30 days
7 Controls
2
1
0.5
2.20 ± 0.25
2.76 ± 0.30
3.18 ± 0.28*
3.93 ± 0.65*
3.56 ± 0.34*
5.39 ± 0.77*
4.45 ± 0.46*
6.46 ± 0.86*
0.98 ± 0.12
1.38 ± 0.25
0.92 ± 0.20
2.51 ± 0.34
0.93 ± 0.13
3.23 ± 0.37*
0.99 ± 0.16
4.30 ± 0.43*
- Each reading represents Mean ± SD of 10 fish.
-The data were subjected to One Way ANOVA with replication, (significance at P ≤ 0.05).
- The significance of difference between treated fish and control was checked by Dunnet test (*significance at P ≤ 0.05).
After 30 days of hypoxia, the increase in liver
en zym e activity was 74%, 93% and 2. 38 fold,
respectivel y while the increase i n white was
36%, 52%, and 2.04 f old, respecti vely.
The i ncrease in γ-G T activity (Tabl e 7)
after one day of hypoxia, was 2.14, 2.47, and
3.11 folds, respectivel y while; the increase in
white w as 37%, 39%, and 41%, respectivel y.
Table 7. Effect of hypoxia concentrations on γ-glutamyl transpeptidase (γ-GT) activity (µM/min/g wet weight tissue) in the
liver and white muscle of Nile tilapia acclimated at 26ºC
DO mg
Liver
One day
30 days
White muscle
One day
30 days
7 Controls
2
1
0.5
0.80 ± 0.11
1.51 ± 0.24
1.72 ± 0.17*
2.63 ± 0.27*
1.98 ± 0.24*
2.92 ± 0.43*
2.49 ± 0.39*
3.59 ± 0.66*
0.418 ± 0.06
0.73 ± 0.12
0.59 ± 0.08
0.99 ± 0.09*
0.61 ± 0.07
1.10 ± 0.16*
0.62 ± 0.09*
1.48 ± 0.26*
- Each reading represents Mean ± SD of 10 fish.
-The data were subjected to One Way ANOVA with replication, (significance at P ≤ 0.05).
- The significance of difference between treated fish and control was checked by Dunnet test (*significance at P ≤ 0.05).
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Attia et al., Antioxidant Enzymes in Liver and White Muscle of Nile Tilapia Exposed to Different Hypoxic Levels and Durations
DISC USSIO N:
The present data has shown readily
m easurable amounts of oxidative stress
biomarkers are present in liver a nd white
muscle tissues of Nile tilapia. Therefore, we
assessed them t o give wide picture of
oxidative stress and antioxidant defense in
hypoxia tolerant Nile tilapia. Antioxi dant
enzym es such as SOD, CAT, and GP x provide
the first line of cellul ar defense against toxi c
free radicals resulting oxidative stress. These
enzym es react directly with oxygen free
radicals to yi eld non-radical product s. CAT
and GPx, on the other hand, convert hydrogen
peroxi de into water, thereby neutralizing its
toxi city (O 'Bri en et al., 2000; Kirkman and
G aetani, 2007). SOD m etaboli zes free
radicals and dismutat es superoxide ani ons
(O 2 ٠) to H 2 O 2 and protects the cell against
O 2 ٠ m ediated lipid peroxi dation (Maier and
Chan, 2002). In sever oxidative stress, it has
been reported that superoxide radicals inhibit
CAT activity and H 2 O 2 suppresses SO D
activity in t he cell (Hassan, 1978). A strong
positive correlation was found between SO D
and CA T activiti es that may be explained by
the protective role of SO D for CA T (Lushchak
et al., 2005). Reduction i n CAT activity might
be du e to increased endogenous production
of the superoxide anion or increased nitri c
oxide end products, or decreased activity of
GPx and SO D or all of these factors.
However, the present pattern of antioxi dant
enzym es acti vity did not showed any
inhibition. This may reflect a high ability of
Nile tilapia to cope with ROS production
produced by the present hypoxia exposure
levels as an adaptive response. In fish that
survived, there were increases i n antioxi dant
defenses. It can be concluded that ROS
produced as a m ediator of hypoxia toxicity in
fish. R esistance developm ent is related with
increased activities of antioxidant enzym es,
which were important in the protecti on to
hypoxia exposure.
In the present experim ent the activity of
the enzym es SOD, CA T, G ST, GPx, and G R
of short and long term exposure, in liver and
white m uscl e of Nil e tilapia showed significant
increase, except i n white muscle of fish
exposed to 2 and 1 m g O 2 L - 1 showed no
significant alt erations. The i ncrease was more
prominent at the low oxygen concentration.
Antioxi dant enzymes act in concert to rem ove
various ROS produced by free radical
reactions. The increase in t he activity of these
antioxidant enzym es due t o hypoxia exposure
may be related to: 1) increased RO S
production, 2) increased activity of antioxi dant
defense system of Nil e tilapia. The increase in
the activity of these enzym es m ay decrease
the imbalance bet ween the antioxi dant
defenses and ROS producti on to lessen the
severity of oxidative stress.
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75
In other studi es, goldfish exposed to
hypoxia conditions increased CAT activity i n
the liver, GPx and G 6P DH activities in the
brain (Lushchak et al., 2001), and hypoxi a
increased SO D activity i n the liver, brain and
gills in common carp (Víg and Nemcsók,
1989). In anot her study on common carp,
hypoxia enhanced CAT and GPx activities i n
brain (Lushchak et al., 2005). In other cases,
antioxi dant
enzym es
show ed
a
m ore
conventi onal response to hypoxic stress,
rem aining unchanged or being lowered over
hypoxia.
In the present study we observed that
the activiti es of CAT and GR were
significantly i ncreased in liver during hypoxia.
The i ncrease in CAT activity and the
maintenance of hi gh constit utive levels of
other antioxidant enzym es (SOD, GPX, GS T,
G 6PDH, and GS H) may have controlled the
extent of oxyradical -induced lipid peroxi dation
to a level that is physiologically tolerable f or
the organ. Hydroxyl radicals (·O H) are
involved in the i nitiation and propagation of
lipid peroxidation in v ivo (Halliwell and
G utt eridge,
1999).
By
cat alyzing
the
dismutati on of O 2 into H 2 O 2 , SOD helps t o
prevent the reduction of Fe (III) to Fe (II)
[reaction 1: O 2 + Fe (III) Fe (II) + O 2 ], a
substrate for the ·OH-generating Fenton
reaction [reaction 2: Fe (II) + H 2 O 2 Fe
(III) + OH + ·OH] (Halliwell and G utteridge,
1999). C atalase and GPx also have pivotal
rol es in the removal of H 2 O 2 . Moreover, GSH
must be recycled t o support continuous i n
vivo activity of GPx (Storey, 1996; H erm esLima et al., 1998). Thus high levels of GR and
G 6PDH in Nil e til apia liver should be critical
for the cellular decomposition of H 2 O 2 .
Furthermore, GST and GPx are important i n
minimizing t he accumulation of the toxic
products of li pid peroxidation, including
malodial dehyde, hydroxynonenal, and lipi d
hydroperoxides (H ermes-Lima et al., 1998).
The sites responsible for the formation of
oxyradicals
in
postanoxi c
liver,
and
consequently
the
inducti on
of
lipi d
peroxidation, could be mitochondria (Shlaf er
et al. , 1987; Ruuge et al., 1991), endoplasmic
reticulum, through a cytochrom e P-450
system (Halliw ell and G utteridge, 1999), and
num erous oxidases such as xanthine oxidase
(Hermes-Lima and Storey, 1995; Halliwell and
G utt eridge, 1999).
It was dem onstrated an increase i n
activities of antioxidant enzymes of goldfish,
Carassius auratus under hypoxic conditions
(Lushchak et al., 2001 & 2005) that supports
the hypothesis t hat hypoxia prepares the
organism for oxidative stress during hypoxi a
as proposed by Herm es-Lima et al. (1998).
The liver and m uscle also dem onstrated
changes in activities of studied enzym es. For
example, liver SOD increased within the first
hours of hypoxia. This response fit s the
hypothesis of preparative adapt ation t o
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76
Egypt. J. Exp. Biol. (Zool.), 11(1): 71 – 78 (2015)
oxidative stress initially
proposed
and
devel oped by Hermes-Lima et al. (1998).
The activity of γGCS and γG T was
significantly increased in t he liver and white
muscle of Nile tilapia expos ed to hypoxia
except in white muscle of fish reared at 2 an d
1 mg L-1 concentrations in trial1. Glutathione
is de novo synt hesized i n both eukaryot es and
prokaryot es by the sequential action of γGCS.
The enzyme γGCS is rate-limiting and i s
feedback-inhibit ed by GSH (Kelly et al.,
2002). Studies on γ-GCS in mammal s
indicated that increase in ROS and/or
decreases in i ntracellular G SH would activate
the gen e expression of γ-GCS (Mulcahy et al.,
1997; Sekhar et al., 1997; Tomonari et al.,
1997). The enzym e γG CS is the only enzym e
of the cycle l ocated at the outer surfa ce of
plasma m embrane. Accordingly, the increase
in γGCS activity may indicate i ncrease in ROS
and/or decreases in intracellular GSH. Thi s
conception may be supported by: 1) the
predicted RO S formation as indicted by the
attendant
increase
in
the
activity
of
antioxidant enzymes, 2) the increase in the
activity of antioxidant enzymes share in
diminishing glutat hione level.
The enzym e γG T involved in the transfer
of amino acids across the cellular m embrane
and also involved in glutathione m etabolism
by transferring the glutamyl moiety to a
variety of acceptor mol ecul es incl uding water.
Glutathione biosynthesis by way of the
gamma-glutamyl
cycle is important
for
maintaining G SH hom eost asis and normal
redox status (Zhang et al., 2005). γGT pl ays
key roles in GSH hom eostasis by breaking
down
ext racellular
GSH
and
provi ding
cysteine, the rate-limiting substrate, for
intracellular de novo synthesis of GSH,
certain L-amino acids and peptides leaving
the cysteine product to preserve intracellular
hom eostasis of oxidative stress. During
oxidative stress, γG T gene expressi on i s
increased, and this is believed to constitute
an adaptation to stress. The i ncrease in γG T
activity indicates increased ROS production in
liver and white muscl e of Nil e tilapia exposed
to the hypoxia exposure. In accordance with
W hitfield (2001) and Lee et al. (2004),
increased γG T activity m ay be a response to
oxidative stress, one whi ch can increase the
transport of glutathione precursors into cell.
Therefore,
glutathione
status
is
important to the resistance of oxidati ve stress
in fish. Under stressful conditions, glutathione
synthesis de novo occurs (Winston and Di
Giulio, 1991). The glutathione levels are
highest in anoxia-t olerant turtles and freezetolerant frogs, and the maintenance of the
ratio GSH/GSSG i n the cell is critical to
organisms t hat periodically undergo oxidative
stress (Storey, 1996). GSH is reported to be
involved in the protection of membranes
against li pid peroxidation (Ritola et al., 2002;
ISSN: 2090 - 0511
Vladimirov, 2002), supported by a negative
correlation
between
lipid
peroxi dation
products and G SH l evel s and a positive
correlation between t he first param eter and
GSSG content.
Arguably, reproductive success is the
most important factor in determining species
fitness and survival. The effects of hypoxia on
reproduction and development of marine
animals was studi ed. Diez and Davenport
(1990) showed that embryos of dogfish,
Scyliorhinus canicula exposed to 20% oxygen
saturation suffered from 100% mortality after
three weeks, while those exposed to 50%
oxygen
saturation
survived
the
entire
experimental period. Increased larval mortality
and reduced hatching success were found for
nese, Chondrostoma nasus embryos w hen
exposed to 10% air saturation (Keckeis et al.,
1996). In contrast, the study of Berntsen et al.
(1990) showed that natural hatching in mature
salmon eggs was induced by hypoxia. High
mortality
and
detrimental
effects
on
development and growth w ere found, w hen
oyster, Crassostrea virginica larvae were
exposed to < 0.07 mg O 2 L - 1 for more than 24 h
(Baker and Mann, 1992). Delayed developm ent
of
mussel, Mytilus
edulis
embryos t o
prodissoconch larvae was found when embryos
were exposed to 0.6–1.3 mg O 2 L -1 for 60 h
(Wang and Widdows, 1991). The paucity of
data on reproductive impairm ent caused by
hypoxia makes it difficult to decipher the cause
of the declines in natural fish populations in
hypoxic environm ents. Zhou (2001) showed
that hypoxia (1 mg O 2 L -1 ) significantly
retarded gonad development, reduced growth,
fertilization success, reproductive output and
larval hatching and larval success of the
common carp Cyprinus carpio. Reduction i n
growth and fecundity impli es a reduction i n
individual fitness, which may subsequently
lead to population decline. Research relating
hypoxia to growth and reproductive impairment
is therefore instructive, for predicting effects of
hypoxia on natural fish populations.
In conclusi on, the results of the present
st udy and compilation of data from earli er
st udies revised by Leveelahti et al. (2014)
suggest that: 1) hypoxia does not always
result in unchecked oxidative stress that
culminat es in tissue damage and 2) not all
fish show a general increase in key markers
of redox-activated antioxidant defense that is
usuall y found i n response to elevated
hypoxia-i nduced
oxi dative
stress.
Furthermore, the results suggest that there is
no clear association between the oxi dative
stress response and hypoxia tolerance of the
species. Rather, in several of the species
st udied,
the
observed
redox
activated
antioxi dant defenses were ti ssue-specific and
may be more aligned to phylogenetic
rel ationships than to the species-specific
oxygen requirements.
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Attia et al., Antioxidant Enzymes in Liver and White Muscle of Nile Tilapia Exposed to Different Hypoxic Levels and Durations
77
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‫ﺣﺎﻟﺔ اﻹﻧﺰﻳﻤﺎت اﻟﻤﻀﺎده ﻟﻸﻛﺴﺪه ﻓﻰ اﻟﻜﺒﺪ و اﻟﻌﻀﻼت اﻟﺒﯿﻀﺎء ﻟﺴﻤﻚ اﻟﺒﻠﻄﻰ اﻟﻨﯿﻠﻰ اﻟﻤﻌﺮض‬
‫ﻟﺪرﺟﺎت وﻓﺘﺮات ﻣﺨﺘﻠﻔﻪ ﻣﻦ ﻧﻘﺺ ﻣﺴﺘﻮي اﻻﻛﺴﺠﯿﻦ‬
**‫ اﻣﯿﻤﻪ ﻋﺒﺪاﻟﻌﻈﯿﻢ ﻋﺎﺷﻮر‬،*‫ ﻣﻨﻰ ﻣﺤﻤﺪ ﺣﺠﺎزى‬،**‫ ﻣﺤﻤﺪ ﺣﺴﻦ ﻣﺮاد‬،*‫زﻳﻨﺐ اﺑﺮاھﯿﻢ ﻋﻄﯿﻪ‬
‫ ﻣﺼﺮ‬،‫ ﺟﺎﻣﻌﺔ طﻨﻄﺎ‬،‫ ﻛﻠﯿﺔ اﻟﻌﻠﻮم‬،‫* ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﯿﻮان‬
‫ ﻣﺼﺮ‬،‫ اﻷﺳﻜﻨﺪرﻳﺔ‬،‫** اﻟﻤﻌﮫﺪ اﻟﻘﻮﻣﻲ ﻟﻌﻠﻮم اﻟﺒﺤﺎر واﻟﻤﺼﺎﻳﺪ‬
‫وﺟﺪ ت زﻳﺎده ذات أھﻤﯿﻪ إﺣﺼﺎﺋﯿﻪ ﻓﻰ ﻧﺸﺎط اﻻﻧﺰﻳﻤﺎت ﻓﻰ‬
‫اﻟﻜﺒﺪ واﻟﻌﻀﻼت اﻟﺒﯿﻀﺎء ﻟﻠﺴﻤﻚ اﻟﻤﻌﺮض ﻟﻔﺘﺮات طﻮﻳﻠﻪ وﻗﺼﯿﺮه‬
1 ,2 ‫ﻣﻦ ﻧﻘﺺ اﻻﻛﺴﺠﯿﻦ ﻣﺎﻋﺪا ﻧﺸﺎط اﻻﻧﺰﻳﻤﺎت ﻓﻲ اﻟﺘﺮﻛﯿﺰ‬
‫ﻟﺘﺮ ﻓﻰ اﻟﻌﻀﻼت اﻟﺒﻀﺎء ﻓﺈﻧﻪ ﻟﻢ ﻳﺤﺪث‬/‫ﻣﻠﻠﻰ ﺟﺮام اﻛﺴﺠﯿﻦ‬
‫ ﻟﻮﺣﻈﺖ ھﺬه اﻟﺘﻐﯿﯿﺮا اﻟﻘﯿﺎﺳﯿﻪ‬.‫ﺑﻪ ﺗﻐﯿﺮات ذات أھﻤﯿﻪ إﺣﺼﺎﺋﯿﻪ‬
‫وﺗﻢ ﻣﻨﺎﻗﺸﺔ أھﻤﯿﺔ‬. ‫ﻋﻨﺪ اﻟﺘﻌﺮض اﻟﻄﻮﻳﻞ ﻟﻨﻘﺺ اﻷﻛﺴﺠﯿﻦ‬
.‫ھﺬه اﻟﺘﻐﯿﺮات ﻓﻲ اﻷﻧﺸﻄﺔ اﻹﻧﺰﻳﻤﯿﺔ‬
: ‫اﻟﻤﺤﻜﻤﻮن‬
‫ ﻋﻠﻮم طﻨﻄﺎ‬،‫ ﻣﺤﻤﺪ ﻋﺒﺪ اﻟﻤﻨﻌﻢ ﺣﺠﺎزي ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﯿﻮان‬.‫د‬.‫أ‬
‫ ﻋﻠﻮم اﻟﻤﻨﺼﻮره‬،‫ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﯿﻮان‬
ISSN: 2090 - 0511
‫ ﻋﺰه إﺳﻤﺎﻋﯿﻞ ﻋﺜﻤﺎن‬.‫د‬.‫أ‬
‫ﺗﻢ ﻗﯿﺎس اﻹﻧﺰﻳﻤﺎت اﻟﻤﻀﺎده ﻟﻸﻛﺴﺪه ﻓﻲ اﻟﻜﺒﺪ‬
‫واﻟﻌﻀﻼت اﻟﺒﯿﻀﺎء ﻟﺴﻤﻚ اﻟﺒﻠﻄﻲ اﻟﻨﯿﻠﻲ اﻟﻤﻌﺮض ﻟﻨﻘﺺ‬
‫اﻷﻛﺴﺠﯿﻦ ﻟﻤﺪة ﻳﻮم )اﻟﺘﺠﺮﺑﻪ اﻻوﻟﻲ( وﻟﻤﺪة ﺛﻼﺛﻮن ﻳﻮﻣﺎ‬
‫ ﺗﻢ ﻓﻰ اﻟﺘﺠﺮﺑﺘﯿﻦ ﺗﻌﺮﻳﺾ اﻟﺴﻤﻚ ﻟﺘﺮﻛﯿﺰات‬.(‫)اﻟﺘﺠﺮﺑﻪ اﻟﺜﺎﻧﯿﻪ‬
‫ﻟﺘﺮ )ﻧﻘﺺ‬/‫ ﻣﻠﻠﻰ ﺟﺮام أﻛﺴﺠﯿﻦ‬0.5 ,1 ,2 ‫أوﻛﺴﯿﺠﯿﻦ ھﻰ‬
‫ ﻣﻠﻠﻰ ﺟﺮام‬7 ‫اﻻﻛﺴﺠﯿﻦ( ﻣﻘﺎرﻧﻪ ﺗﻌﺮض ﻣﺠﻤﻮﻋﻪ اﻟﻰ ﺗﺮﻛﯿﺰ‬
‫ﻧﺸﺎط‬
‫ﻗﯿﺎس‬
‫ﺗﻢ‬
.(‫)اﻟﻜﻨﺘﺮول‬
‫ﻟﺘﺮ‬/‫اﻛﺴﺠﯿﻦ‬
superoxide dismutase (SOD), catalase (CAT), ‫اﻹﻧﺰﻳﻤﺎت‬
glutathione peroxidase (GPx), glutathione S-transferase
(GST), glutathione reductase (GR),
γ-glutamyl
cysteinyl synthetase (γ- GCS), and γ- glutamyl
‫ وﻗﺪ‬.‫ ﻓﻰ اﻟﻜﺒﺪ واﻟﻌﻀﻼت اﻟﺒﯿﻀﺎء‬transpeptidase (γ- GT)
On Line ISSN: 2090 - 0503
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