Download changes in the activity of selected adaptive enzymes in chicken liver

Bull. Vet. Inst. Pulawy 48, 503-506, 2004
CHANGES IN THE ACTIVITY OF SELECTED ADAPTIVE
ENZYMES IN CHICKEN LIVER AFTER SINGLE GAMMA
IRRADIATION
MARCEL FALIS, KATARÍNA BEŇOVÁ, MICHAL TOROPILA,
EDINA SESZTÁKOVÁ AND JAROSLAV LEGÁTH
University of Veterinary Medicine, 041 81 Košice, Slovakia
e-mail: [email protected]
Received for publication April 21, 2004.
Abstract
Material and Methods
Chickens were used as experimental animals to
investigate the influence of gamma radiation on changes in the
activity of selected liver adaptive enzymes and serum
corticosterone. Analyses were carried out on days 1, 5, 14, and
30 after irradiation. The activity of tyrosine aminotransferase
increased significantly on day 5 after irradiation. The activity
of tryptophane-2,3-dioxygenase increased significantly on
days 1 and 5 after irradiation. Serum corticosterone levels
showed an increase on days 1 and 5 and decrease on days 14
and 30 after irradiation. Total liver proteins decreased
significantly on day 1 and increased insignificantly on days 5,
14, and 30 after irradiation.
Animals. The experiment was carried out on
21-day-old broiler crossbreds. From hatching up to day
21 they were reared in previously disinfected (11)
experimental facilities (12) and were supplied with feed
and water ad libitum. The rations consisted of BR I and
BR II commercial granulated feed. The broilers were
irradiated at the Faculty of Natural Sciences of UPJS in
Košice, directly in adjusted plexiglas cages, using an
apparatus CHISOSTAT 60Co-CHIRANA.
Experimental procedure. The experimental
broilers were exposed to a single-dose whole-body
gamma radiation of 4.5 Gy (output 0.295 Gy/min).
Analyses were carried out on days 1, 5, 14, and 30 after
the irradiation. Control broilers were exposed to sham
radiation, i.e. they were handled in the same way except
for irradiation with gamma rays. The birds were killed
by decapitation (2, 3). The pooled blood samples were
collected in Petri dishes which were kept on ice and the
blood serum,
obtained after centrifugation, was
analysed. Liver samples were also taken and were stored
frozen in liquid nitrogen. The corticosterone
concentration was determined by fluorimetric analysis
(17). Total protein concentration in the liver was
determined by Bio-La sets (Lachema-Brno). The activity
of liver tryptophane-2,3-dioxygenase (TO) was
determined by the method described by Knox and
Auerbach (9), and the activity of liver tyrosine
aminotransferase (TAT) by the method of
Diamondstone (4). Measurements were done using a
spectrophotometer Spekol 11 and fluorimeter
(Spektrofluorimeter FP-550, JASCO). Six birds from
each group were analysed on average. The significance
of differences between experimental broilers and the
controls was evaluated by the unpaired t-test (Prism 3.0,
Software). The experiment was conducted in summer.
Key words: chickens, corticosterone, gamma
irradiation, tryptophane-2,3-dioxygenase, liver proteins,
tyrosine aminotransferase.
In the history, organisms have been always
exposed to the effect of ionizing radiation of various
intensity. During evolution adaptive mechanisms have
developed in several animal species and helped them to
eliminate harmful influence of ionizing radiation.
The sensitivity to the radiation depends on the level of
phylogenesis. Therefore, as the object of our
experiments we chose Gallus domesticus that belongs to
the most resistant domestic animals. The response of the
organism to radiation depends not only on the species of
animals but also on their age, sex, nutrition and health
status. In the present study we observed the reaction of
21-day-old chickens at the beginning of irradiation.
Irradiation with lethal or sublethal doses causes
significant metabolic changes in the exposed organisms.
Ionizing radiation causes changes in the metabolism of
lipids, saccharides, proteins and enzymes and induces
acute phase mechanisms. Corticosterone plays an
important role in this process.
The aim of the study was to observe the
changes in corticosterone level in relation to the changes
in the activity of “adaptive enzymes” in the liver of
fattening chickens after single whole-body irradiation.
504
Results
The serum concentration of corticosterone (Fig.
1) increased insignificantly on days 1and 5 and
decreased on days 14 and 30 after irradiation.
When compared to the control, the
concentration of total proteins in the liver (Fig. 2) was
decreased significantly on day 1, increased
insignificantly on days 5 and 14 and did not differ on
day 30 of the experiment.
The activity of tryptophane-2,3-dioxygenasis
(Fig. 3) was not significantly decreased on day 30 after
irradiation. As for other periods of our observation, it
showed a significant increase on day 5 after irradiation.
The liver activity of tyrosine aminotransferase
(Fig. 4) decreased significantly on day 1. As for other
periods, it was increased significantly on day 5
compared to the controls.
60
pmol/ml
50
40
control
irradiated
30
20
10
0
0
5
10
15
20
25
30
Fig. 1. Changes in chicken serum corticosterone. The values are given as means ± S.E.M.
120
100
g/l
80
control
irradiated
60
40
20 **
0
0
5
10
15
20
25
30
Fig. 2. Changes in total liver proteins. The values are given as means ± S.E.M. ** P<0.01.
6
***
mmol/hod/g
5
4
control
irradiated
3
2
1
0
0
5
10
15
20
25
30
Fig. 3. The changes of tryptophan-2,3-dioxygenase activity in the liver. The values are given as means ± S.E.M. *** P<0.001.
nmol/min/g
505
9
8
7
6
5
4
3
2
1
0
**
control
irradiated
*
0
5
10
15
20
25
30
Fig. 4. Changes in tyrosine aminotransferase activity in the liver. The values are given as means ± S.E.M. * P< 0.05, ** P<0.01.
Disscussion
Metabolic changes in animal organisms under
physiological conditions as well as after the action of
some negative factors (10), such as ionizing radiation,
have been the subject of many studies.
It is well known that the activity of some
adaptive enzymes, e.g. tyrosine aminotransferase and
tryptophane-2,3-dioxygenase increases rapidly under the
action of stress factors or hormones. Even non-lethal
doses of ionizing radiation can activate the
hypothalamo-hypophyseal-adrenal axis (15). This
activation is accompanied by an increase in secretion
of adrenal cortex glucocorticoids that may induce the
activity of the studied enzymes. Changes in the serum
corticosterone were also observed.
After enzyme activation by steroid hormones or
other stress factors (e.g. ionizing radiation), the de novo
synthesis of proteins occurs through increasing the
amount of m-RNA available for translation.
The different sensitivity of inductive processes
may also be explained by a specific way of irradiation
damage to DNA, which may occur in some genes only
while other genes remain undamaged, or by irradiationinterference with biosynthesis of certain proteins on the
translation level (13). Similar to glucocorticoids,
irradiation alone stimulates the transfer of amino acids
into hepatocytes (7). The excessive amino acids,
released from radiosensitive tissues after their
accumulation in the liver, induce the activity of the
enzymes which control the metabolism of amino acids
in the liver. It is known that in vivo concentration of
amino acids is a limiting factor in the process of their
transformation to glucose, and glucagon or c.AMP are
the main activators of amino acid transport to
hepatocytes and the concentrations of these activators
are increased after irradiation (8). Our results showed
marked changes in the serum enzyme activities in the
irradiated chickens. The initial increase in corticosterone
concentrations observed after the irradiation indicated
stressful action of ionizing radiation on the organism.
This change was observed in all groups tested.
Corticosterone induces activity of many enzymes and
therefore the changes in the activity of adaptive enzymes
in relation to the changes in corticosterone
concentrations were studied.
Neither decrease nor significant increase in the
enzyme activities is observed after irradiation of pure
isolated enzymes, contrary to irradiated live systems
(14).
The enzymes in irradiated organisms may be
damaged due to changes in the
“active centre of
proteins”, prosthetic compartments of enzymes, or
damages to enzyme bonds. An increase in the activity
of these enzymes indicates a prevalence of catabolic
processes in irradiated bodies, associated not only with
changes in cellular permeability (1), development of
serious histological changes in hepatocytes during the
first three days after irradiation, but also with
stimulation of synthesis of de novo transamination
enzymes induced by increased secretion of adrenal
cortex after irradiation. In addition to direct damage
caused by ionizing radiation and to other factors, the
activity of adaptive enzymes may also be influenced by
intake of food and water. Toropila et al. (1996)
observed an increase in tryptophane pyrolase and
alanine aminotransferase and a decrease in aspartate
aminotransferase activity in the liver during 12-day
starvation of rats.
According to the literature data, an organism in
the stage of rapid growth is more sensitive to ionizing
radiation than that in adulthood. During the postnatal
growth, the irradiation causes growth retardation or
growth disorders because of the damage to
differentiation of cell processes in the growth zones (5).
References
1. Albaum H. G.: Serum enzymes following whole-body
radiation in the rabbit. Radiation Res 1960, 12, 186─194.
506
2. Bugarský A., Takáčová D., Korim P.: A ban on cruelty to
animals – current legislation in SR, Slov Vet čas 1995, 20,
43.
3. Decree No. 231 of the Code, (1998) of the Ministry of
Agriculture of the Slovak Republic about keeping pet
animals, wild animals, and dangerous animals and about
the protection of laboratory animals.
4. Diamondstone T. I.: Anal Biochem 1966, 16, 395-401.
5. Dostál M.: Military radiobiology, part 1., textbook,
VLVDÚ JEP, Hradec Králové, 1975, p. 234.
6. Dostál M.: Military radiobiology, part 2., textbook,
VLVDÚ JEP, Hradec Králové, 1975, p. 562.
7. Flory W., Neuhaus O. W.: Induced transport of amino
acids in rat liver after whole-body irradiation. Radiat Res
1976, 68, 138-147.
8. Kilberg M. S., Neuhaus O. W.: Hormonal control of
amino-acid transport in the liver of rats exposed to wholebody irradiation. Radiat Res 1978, 73, 360-372.
9. Knox E., Auerbach V. H.: The hormonal control of
tryptophane peroxidase in the rat. J Biol Chem 1955, 214,
307-313.
10. Kottferová J.,Korenéková B., Jacková A., Siklenka P.,
Hurná E.: Distribution of cadmium in layers after vitamin
D3 supplementation. Health and diseases of animals,
Košice 1999, 48-50.
11. Kubíček K., Novák P., Kočišová A., Rodl P.: Disinfection,
disinsection and rat control in Schemes, Tables and
Figures, Veterinary and Pharmaceutical University Brno,
2000, p. 100.
12. Ondrašovič M., Ondrašovičová O., Para Ľ Kočišová A.:
Veterinary Care about the Environment – Practical
Lessons. UVM Košice. MAGNUS Košice, 1993, p. 153.
13. Streffer Ch., Scharfferus S.: The induction of liver
enzymes by cortisol after combined treatment of mice with
x-irradiation and inhibitors of protein biosynthesis. Int J
Radiat Biol 1971, 20, 301-313.
14. Toropila M.: Practical lessons in radiobiology.
Magnus,1993, Košice, p.75.
15. Toropila M., Ahlers I., Ahlersová E., Ďatelinka I., Beňová
K.: Effects of low doses of continuing gamma radiation on
changes in activity of aspartate aminotransferase and
alanine aminotransferase in the liver and serum of
laboratory rats. Folia Vet 1995, 39, 29-32.
16. Toropila M., Ahlers I., Ahlersová E. Ondrašovič M.,
Beňová K.: The influence of prolonged fasting on changes
in the activity of selected adaptive enzymes in rat liver.
Vet Med-Czech 1996, 41, 41-44.
17. Van der Vies J., Bakker R.F.M., de Wied, D.: Correlated
studies on plasma free corticosterone and on adrenal
steroid formation rate in vitro. Acta Endocrinol 1960, 34,
513 –523.