Arch.Geflügelk., 74 (4). S. 274–278, 2010, ISSN 0003-9098. © Verlag Eugen Ulmer, Stuttgart
Effect of low dose gamma-radiation upon antioxidant enzymes in
chick embryo liver
Einfluss einer geringen Dosis an Gamma-Strahlung auf die antioxidativen Enzyme in der
Leber von Hühnerembryonen
M. Vilic1, Jasna Aladrovic1, Blanka Beer Ljubic1, Saveta Miljanic2 and P. Kraljevic1
Manuskript eingegangen am 5. Oktober 2009, angenommen am 9. Januar 2010
Introduction
It has been shown that low doses and low dose rates of
ionizing radiation can stimulate induction of antioxidant
defence systems, such as glutathione (GSH), glutathione
peroxidase (GSH-Px), superoxide dismutase (SOD) and
catalase (CAT) (KOJIMA et al., 1997; YAMAOKA, 2006). Most
of these studies of low dose radiation effects were carried
out in various tissues of rodents. In the avian there are
results of antioxidant status mostly in liver, brain, yolk and
yolk sac membrane of bird embryo under physiological
conditions (GAAL et al., 1995; SURAI, 1999). Thus, chick
embryo liver on the 19th day of incubation was found to be
higher in SOD, CAT and GSH-Px activity as well as GSH
level when compared with brain and yolk sac membrane.
It is also known that the liver in birds comprises the major
body storage of antioxidants (SURAI, 2003).
In literature there is no investigation available which
shows that low dose gamma radiation has effects upon
antioxidant status in chick embryonic organs after chick
embryo irradiation. Investigations, though a few, of low
dose ionizing radiation in the range from 0.05 to more
than 1 Gray (Gy) are primarily directed on fertility, hatchability and body weight of chickens (MRAZ, 1971; TODOROV
et al., 1986; ZAKARIA, 1989; JILO and LÖHLE, 1991; ZAKARIA,
1991; GERRITS and DIJK, 1992). Although these previous
studies showed contradictory data, it is reported that dose
of gamma radiation between 0.1 to 1 Gy, especially the
dose between 0.1 and 0.5 Gy (JILO and LÖHLE, 1991) or
between 0.06 to 0.3 Gy, (TODOROV et al., 1986), could
increase chicken egg hatchability and chicken body weight
after egg irradiation before incubation.
In a previous study (unpublished results) effects of doses
of 0.15 Gy and 0.3 Gy of gamma irradiation on enzyme
activity in chicken blood plasma and antibody titer of Newcastle disease virus in blood serum of chickens hatched
from eggs irradiated on the 19th day of incubation were observed. The dose of 0.3 Gy showed a more significant effect
when compared to the dose of 0.15 Gy. Therefore, if our
investigations (this one and those in the future) show that
low dose of gamma radiation stimulate antioxidant status
in chickens, as is a case in mammals, our results could have
real practical application. Consequently, the aim of this
study was to investigate lipid peroxidation and antioxidant
status in liver of chick embryo after acute low dose of
0.3 Gy gamma irradiation on the 19th day of incubation.
Materials and Methods
Experimental protocol
Fertilized eggs produced by a commercial flock of COBB
500 broiler breeds were irradiated with gamma rays
(source 60Co) on the 19th day of incubation. In the same
experiment, there were included the same number of eggs
unexposed to gamma-radiation and served as controls.
Non-irradiated eggs were retained in the same place for a
period of time equal to that required for irradiated eggs.
Control eggs were subjected to a sham irradiation. All eggs
were placed in the same commercial incubator Victoria
(Pavia, Italy), capacity of 22100 eggs for 18 days. Incubator had automatic control of temperature (37.8°C), humidity (60–62% relative humidity), and incubation rack turning. On the 19th day of incubation the eggs were irradiated
and transferred to hatching trays located in the same
incubator. After the hatch, chicks were housed on the floor
at a temperature of 33°C. Two-day-old chicks used in the
studies did not have access to feed but had access to water
ad libitum. Both groups of chicks hatched from irradiated
eggs and chicks hatched from non-irradiated eggs were
kept under the same conditions. The study was reviewed
and approved by the Ethics Committee of the Faculty of
Veterinary Medicine University of Zagreb. (Class: 640-01/
06-17/30; Record Number: 61-01/139-06-60).
Irradiation and dosimetry
The eggs (chick embryos) were irradiated with the dose of
0.3 Gy gamma radiation from panoramic 60Co source
(activity about 3 PBq) of the Ruder Bos kovic Institute,
Zagreb, Croatia (MILJANIC and RANOGAJEC-KOMOR, 1997).
The dose rate was about 23.84 mGy/s, and a source
axis-to-egg axis distance was 3.06 m. Dosimetric measurements were performed with an ionization chamber type
2581 and a Farmer Dosimeter type 2570 (NE Technology
Limited). The dose is specified as absorbed dose to water
(measured free in air).
Sample preparation
1 Department of Physiology and Radiobiology, Faculty of Veterinary Medicine,
University of Zagreb, Croatia
2 Division of Materials Chemistry, Ruder Boskovic Institute, Croatia
Chick embryos and newly hatched chicks were sacrificed
on 6, 24, 48, 72 and 96 h after irradiation. The liver was
immediately removed, washed in cold saline, weighed,
Arch.Geflügelk. 4/2010
Vilic et al.: Low dose gamma-radiation and antioxidants
275
quick frozen in liquid nitrogen and placed in deep-freezer
at –80°C until analysis for antioxidant parameters. Seven
chick embryos or chicks were used at each time intervals in
both groups. For the measurement (three months liver
storage ago) liver tissue was homogenized on ice in
0.14 mol/l KCl using a Teflon-glass Schüthomogenplus homogenizer (Schütt Labortechnik, Germany) at 2,800 rpm
during 0.5 min. Ration of tissue mass to volume of buffer
was 1:5. Liver homogenate was centrifuged at 20,000 × g
for 30 min. to prepare the supernatant using Sigma 3K15
centrifuge (Germany). The supernatant was used for lipid
peroxide and GSH level, and activity of GSH-Px, SOD and
CAT assay.
method of LOWRY et al. (1951) using bovine serum albumin
as standard.
Assay of antioxidant parameters
Results
Lipid peroxide concentration. The concentrations of thiobarbituric acid reactive substances (TBARS) were determined
by the method of OHKAWA et al. (1979). It detects malondialdehyde and other thiobarbituric acid-reactive substances generated by free radical-mediated peroxidation of
unsaturated lipids (expecting cholesterol and monoenoic
or dienoic phospholipids). Absorption peak was measured
at 532 nm and concentration of lipid peroxide was expressed in terms of nanomoles of TBARS/g protein.
The results of TBARS and GSH level as well as antioxidant
enzymes GSH-Px and SOD in the liver of chick embryo
irradiated with 0.3 Gy gamma-rays on the 19th day of
incubation are presented on Figure 1. The result of CAT activity in the liver of chick embryo irradiated with 0.3 Gy
gamma-rays on the 19th day of incubation are presented on
Figure 2.
Irradiation resulted in significantly higher GSH, TBARS
and GSH-Px levels 6 h after irradiation and in significantly
lower GSH, TBARS and GSH-Px levels at 48 h after irradiation. Thereafter, no significant differences could be observed. SOD was significantly lower at 48 h, but no effects
could be observed at other times. CAT was not significantly
different during the experiment.
Gluthatione level. The concentration of GSH was determined by the method of BEUTLER et al. (1963). This method
is based upon development of a relatively stable yellow
colour when 5,5'- dithiobis-(2-nitrobenzoic acid) is added
to sulfhydril compounds. The absorbance was read at
412 nm. GSH level was calculated as µmol/g protein.
Statistical analysis
Obtained results of antioxidant parameters were expressed
as mean ± standard deviation (SD) and the significance of
differences between the control and irradiated groups was
analysed in STATISTICA (STATSOFT, 2005). After testing
for normal distribution (Kolmogorov–Smirnov test of normality), the results were tested by Student’s t-test whereas
a p value < 0.05 was selected to indicate significance.
Discussion
Glutathione peroxidase activity. The GSH-Px activity was
measured using commercial kit RANSEL (Randox Laboratories Ltd., Ardmore, Diamond Road, Crumlin, Co. Antrim,
United Kingdom). The GSH-Px activity assay method is
based on the ability of GSH-Px to catalyze the oxidation of
glutathione by cumene hydroperoxide. In the presence of
glutathione reductase and NADPH, oxidized glutathione is
immediately converted to reduced form with concomitant
oxidation of NADPH to NADP+ (PAGLIA and VALENTINE,
1967). The decrease in absorbance at 340 nm was measured.
GSH-Px activity was expressed in terms of U/g protein.
Superoxide dismutase activity. The SOD activity was measured by the method of FLOHE and OTTING (1984). The SOD
activity was determined on an SABA autoanalyzer with
RANSOD reagent (Randox Laboratories Ltd., Ardmore,
Diamond Road, Crumlin, Co. Antrim, United Kingdom).
The method used on determination of SOD activity is
based on the formation of superoxide radicals from
xanthine by the action of xanthine oxidase, which react
with 2- (4-iodophenyl)3-(4-nitrophenyl)5-phenyltetrazole
chloride to produce formazan red stain. Activity of SOD
was measured as the grade of inhibition of this reaction
and expressed in terms of U/mg protein.
Catalase activity. The CAT activity was measured by the
method of JOHANSSON and BORG (1988). The method was
based on the reaction of the enzyme with methanol in the
presence of an optimal concentration of hydrogen peroxide. The produced formaldehide was measured spectrophotometricaly with 4-amino-3-hydrazino-5-mercapto.1,
2,4-triazole (Purpald) as a chromogen at 470 nm. CAT
activity was expressed in terms of U/g protein.
Estimation of protein. All parameters were expressed per g
of protein. Protein concentration was measured by the
Arch.Geflügelk. 4/2010
The results of our study indicate that chick egg irradiation
on the 19th day of incubation with low dose gamma radiation induces alteration of antioxidant status in the embryo
liver during the first 48 h after egg exposure. The level of
TBARS and GSH-Px activity increased significantly on the
6th hour after egg irradiation while the level of GSH
increased on the 6th and on the 24th hour after egg irradiation. On the other side all investigated antioxidant
parameters, except CAT activity, significantly decreased on
the 48th hour after egg irradiation. After that all antioxidant parameters tended to increase and they were almost
on the same level as control parameters.
At the moment we do not know the actual reason of
changes of these parameters in chick embryo liver. However, we suppose that the increased level of TBARS on the
6th h after egg irradiation could be correlated with the lipid
content of the chick embryo liver. Namely, it is known that
the lipid metabolism in chick embryo is predominating
energy source during the last week of the development
(NOBLE, 1986) and that chick embryo accumulated a large
amount of lipids in the liver and extrahepatic tissues,
during which the total lipid content increased toward
hatching (MOORE and DORAN, 1962). In addition, chick
embryo liver on the 19th day of incubation contains very
high content of unsaturated fatty acids (NOBLE, 1986;
NOBLE and COCCHI, 1990; SPEAKE et al., 1998). Furthermore,
SATO et al. (2006) showed that the hepatic function may
already differ between broilers and layers at the embryonic
development where the lipid content in liver of broiler
chick on the 18th day of incubation and newly hatched
chicks was higher than the lipid content in liver of layer
chick embryo. Consequently, this described lipid contents
especially in broiler chick embryo liver at the end of
embryonic development could be probably explained by
the increase level of TBARS in our study.
276
Vilic et al.: Low dose gamma-radiation and antioxidants
* Star marks represent significant differences (P < 0.05)
** (P < 0.01)
*** (P < 0.001) between means of irradiated group and control group
Figure 2. Catalase (CAT) activity in liver of chick embryo and
newly hatched chicks after gamma-irradiation with the dose
of 0.3 Gy and dose rate of 23.8 mGy/s (mean ± SD of 7 liver
samples)
Katalase (CAT) Aktivität in der Leber von Hühnerembryonen und
frisch geschlüpften Küken nach einer Bestrahlung mit GammaStrahlen in einer Dosierung von 0,3 Gy und einer Dosierungsrate
von 23,8 mGy/s (Mittelwerte ± SD von 7 Leberproben)
Figure 1. Level of lipid peroxidation (TBARS) and
gluthatione (GSH) as well
as activity of glutathione
peroxidase (GSH-Px) and
superoxide
dismutase
(SOD) in liver of chick
embryo and newly hatched
chicks after gamma-irradiation with the dose of
0.3 Gy and dose rate of
23.8 mGy/s (mean ± SD of
7 liver samples)
Gehalte an Oxidationsprodukten (TBARS) und Glutathion (GSH) sowie Aktivitäten von Glutathionperoxidase (GSH-Px) und Superoxid-Dismutase (SOD) in der
Leber von Hühnerembryonen und frisch geschlüpften
Küken nach einer Bestrahlung mit Gamma-Strahlen
in einer Dosierung von 0,3
Gy und einer Dosierungsrate
von 23,8 mGy/s (Mittelwerte
± SD von 7 Leberproben)
Our results concerning this increase TBARS level in
broiler chick embryo liver after egg exposure to low dose
gamma radiation could hardly be compared with results in
literature. Namely, in literature the contradictory results
have been published on the level of lipid peroxidation in
different organs of mammals after low dose irradiation;
the TBARS level in different organs of mammals is mostly
decreased after low dose gamma irradiation (YAMAOKA et
al., 1991; YAMAOKA, 2006), while PATHAK et al. (2007)
reported the increase of TBARS level in kidney of mice
after whole body irradiation.
The increase of GSH level and GSH-Px activity in the
chick embryo liver on the 6th h after egg irradiation could
be possible due to its function in cell defence from oxidative stress in earlier hours after irradiation. Namely,
GSH-Px plays a primary role in antioxidant defence in
chick embryo liver from hydrogen peroxide and lipid
hydroperoxide particularly at the end of embryonic development (SURAI, 1999). On the other side GSH, the most
abundant non-enzymatic antioxidant present in the cell,
directly reacts with reactive oxygen species (MEISTER and
ANDERSON, 1983) thus playing an important role in protecting tissues from oxidative damage.
Our results are consistent with the reports of KOJIMA et
al. (1997) and PATHAK et al. (2007) who have demonstrated
that low dose gamma-radiation enhances GSH content in
mice liver and kidney after whole body exposure. Further,
PATHAK et al. (2007) also showed increase of TBARS level in
kidney of mice after whole body irradiation and reported
that this increase in the lipid peroxidation might cause an inArch.Geflügelk. 4/2010
Vilic et al.: Low dose gamma-radiation and antioxidants
crease in the antioxidant defence status to regulate the cellular homeostasis. Therefore, it can not be excluded that the increase of TBARS in our study is also responsible for increased
GSH level and GSH-Px activity in the chick embryo liver.
On the other side the decreased levels of GSH and investigated antioxidant enzymes (GSH-Px and SOD) in chick embryo liver on the 48 h after egg irradiation are not consistent
with the literature data on the same antioxidant parameters
which were increased in different organs of mammals after
exposure to low dose of x or gamma radiation (KOJIMA et al.,
1998; KOJIMA et al., 1999; YAMAOKA, 2006). One of the reasons of these differences could be explained by specific biochemical processes such as lipid metabolism at the end of
chick embryo development previously described in our discussion. The other reason of decreased level of GSH, GSH-Px
and SOD could be physiologic processes such as lung ventilation which differs from those in mammals. Namely during
the first 18 days of the embryonic development the oxygen
transfer and carbon dioxide occurs at the chorioallantoic
membrane. After that, on the 19th day of incubation the embryo internally pips the air cell inner membrane and begins
lung ventilation on air cell gas with an increase in oxygen
consumption between day 20 and post-hatching day 1
(MENNA and MORTOLA, 2002; MORTOLA and LABBÈ, 2005).
Therefore, it could be possible that the decrease of the antioxidant parameters recorded in our experiment on the 48th
h after irradiation i.e. on the 21st day of incubation is probably due to a higher susceptibility of the chick embryo on
oxidative stress which is supposedly caused by low dose irradiation on the 19th day of the embryonic development.
277
cells from oxidative stress. However, there is a lack of
results on antioxidant status in organs of chicken after low
dose eggs or whole-body irradiation. This study was performed to investigate the effect of low-dose gamma-irradiation upon activity of glutathione peroxidase (GSH-Px),
superoxide dismutase (SOD), catalase (CAT) and level of
glutathione (GSH) and lipid peroxidation (TBARS) in liver
of chick embryo and newly hatched chicks. Fertilized
chicken eggs were irradiated with the dose of 0.3 Gy
gamma radiation (source 60Co) on the 19th day of incubation. The liver samples were taken on 6, 24, 48, 72 and
96 h after egg irradiation. The antioxidant parameters
were measured spectrophotometrically.
Level of GSH and lipid peroxidation as well GSH-Px
activity increased in the liver of chick embryos on the 6th
hour after irradiation. GSH-Px and SOD activity significantly decreased in the liver of chick embryo on the 48 h
after egg exposure. Lipid peroxidation level and GSH level
also significantly decreased in the liver of chick embryo on
the 48 h after egg exposure. CAT activity during the experiment was not statistically different when it compared to
controls. These results suggest that chick embryo exposure
to dose of 0.3 Gy gamma-radiation on the 19th day of
incubation causes alteration of antioxidant status in chick
embryo liver. It also seems, as is the case in mammals, that
GSH and GSH-Px could be the first choice in antioxidant
defence in chicken embryo liver at early time after low
dose irradiation.
Key words
Conclusions
In conclusion, the obtained results show that the chicken
eggs exposure to the dose of 0.3 Gy gamma-radiation with
dose-rate of 23.8 mGy/s on the 19th day of incubation
causes an oxidative stress in early hours after irradiation as
well as a decrease in non-enzymatic and enzymatic antioxidant defence system in the hatching chicks i.e. 48 hours
after low dose irradiation. Furthermore, it also seems, as is
the case in mammals, that GSH and GSH-Px could be the
first choice in antioxidant defence in chicken embryo liver
at an early time after low dose irradiation. Irradiation of
the chicken eggs on the 19th day of incubation with low
dose of 0.3 Gy gammaradiation and dose rate of
23.84 mGy/s do not have an effect on the antioxidant status in liver of chickens hatched from irradiated eggs.
Our study on the effect of low dose gamma radiation on
the antioxidant status in chick embryo liver is, actually, one
of the basic scientific investigations, and we hope that other
low doses and dose rates of gamma irradiation could increase antioxidant status and resistance of chickens hatched
from irradiated eggs against stress, which is very often the
cause of many disturbances in the production of chicken
meat or eggs, as well as the cause of some deseases.
Acknowledgements
This investigation was financed by the Ministry of Science,
High Educations and Sport of the Republic of Croatia. The authors wish to thank the Ministry for their financial assistance.
Summary
Exposure of rodents to low-dose whole-body irradiation
could cause various antioxidant mechanisms that defend
Arch.Geflügelk. 4/2010
Chick embryo, gamma radiation, antioxidative enzymes,
liver
Zusammenfassung
Einfluss einer geringen Dosis an Gamma-Strahlung
auf die antioxidativen Enzyme in der Leber von
Hühnerembryonen
Das Aussetzen von Nagetieren einer geringen Dosis von
Strahlung löst antioxidative Mechanismen im Organismus
aus, die die Zellen vor oxidativem Stress schützen. Die
Kenntnisse über die Auswirkung einer Bestrahlung von
Bruteiern oder das ganzen Körpers von Hühnern mit geringen Dosen an Strahlung auf den antioxidativen Status der
Organe sind dagegen begrenzt. Die vorliegende Studie
wurde daher durchgeführt, um die Effekte einer geringen
Dosis von Gamma-Strahlung auf die Aktivitäten der Glutathion-Peroxidase (GSH-Px), Superoxid-Dismutase (SOD),
Katalase (CAT) und den Gehalten an Glutathion (GSH)
sowie Oxidationsprodukten (TBARS) in der Leber von
Hühnerembryonen und frisch geschlüpften Küken zu
untersuchen. Hierzu wurden befruchtete Hühnereier am
19. Bruttag einer Dosis von 0,3 Gy Gamma-Strahlung
(60Co) ausgesetzt. Die Leberproben wurden 6, 24, 48, 72
und 96 h nach der Bestrahlung gesammelt. Die Parameter
der Regulation der Oxidationsprozesse wurden spektrophotometrisch bestimmt.
Die Gehalte an GSH und TBARS sowie die GSH-Px-Aktivität nahm in der Leber der Embryonen 6 h nach der
Bestrahlung zu. Dagegen nahmen die Aktivitäten von
GSH-Px und SOD sowie die Gehalte an TBARS und GSH in
der Leber 48 h nach der Bestrahlung signifikant ab. Im
Vergleich zur Kontrolle waren keine signifikanten Veränderungen der CAT-Aktivität fest zu stellen. Die Ergebnisse
lassen den Schluss zu, dass die Bestrahlung von Hühner-
278
Vilic et al.: Low dose gamma-radiation and antioxidants
embryonen am 19. Bruttag mit 0,3 Gy Gamma-Strahlung
zu Veränderungen im antioxidativen Status in der Leber
des Embryos führt. Wie auch bei Säugetieren scheinen
GSH und GSH-Px die erste Stufe einer antioxidativen
Bekämpfungsstrategie in der Leber von Hühnerembryonen
unmittelbar nach einer Bestrahlung mit geringer Dosierung darzustellen.
Stichworte
Hühnerembryo, Gamma-Strahlung, antioxidative
Enzyme, Leber
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Correspondence: Marinko Vilic, DVM, PhD, Department of Physiology and
Radiobiology, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova
55, HR-10000 Zagreb, Croatia ; E-mail: [email protected]
Arch.Geflügelk. 4/2010