Download effect of paracetamol injection in ovo in the course of hatching and

Bull Vet Inst Pulawy 50, 537-542, 2006
EFFECT OF PARACETAMOL INJECTION IN OVO IN THE
COURSE OF HATCHING AND THYROID HORMONE LEVELS
IN CHICKEN EMBRYOS
MARCIN W. LIS, ANDRZEJ SECHMAN1, JERZY W. NIEDZIÓŁKA AND JANUSZ RZĄSA1
Department of Animal Hygiene and Breeding Environment,
1
Department of Animal Physiology,
University of Agriculture, al. Mickiewicza 24/28, 30-059 Kraków, Poland
[email protected]
Received for publication July 25, 2006.
Abstract
The aim of the study was to determine the effect of
paracetamol (acetaminophen, P) on the course of
embryogenesis and thyroid hormone (TH) levels in chicken
embryos. Eggs from a broiler breeder flock were incubated
under standard conditions. They were candled on the 5th d of
incubation in order to discard unfertilized eggs and those with
dead embryos. At the 6th d of incubation the eggs with living
embryos were divided into 3 groups (n=120 in each); two of
them were in ovo injected with P (4 mg/egg in 100 µl of aqua
pro injectione) or with 100 µl of aqua pro injectione (control
C1), while one group was non-injected (control C0).
Thyroxine (T4), triiodothyronine (T3), and reverse-T3 (rT3)
levels were measured radioimmunologically in samples of
blood plasma collected from chick embryos on days: 11, 14,
16, 18, 20 (external piping), and on day 21 of incubation, i.e.
just after hatching. The rest of the embryos were used for the
investigation of hatchability parameters. In the P-treated group,
a significant increase in the mortality of the embryos was
found. It was mainly caused by the elevation of dead embryo
number on the 6th and 7th d of incubation, i.e. just after P
injection. In this group, much more damage to the liver and
hyperplasia of the stomach were also observed. The time of
hatching in P-treated chicks was significantly delayed. In
comparison with both control groups, in P-treated embryos T4
was significantly higher on days 14, 16, and 18 of incubation,
while it was considerably lower on day 20 and just after
hatching. T3 concentrations in P-exposed embryos were
significantly lower on day 11 and higher on day 14 and just
after hatching. In P-injected embryos, rT3 levels were higher
only on day 14. The results obtained indicate that paracetamol
exerts unfavourable effects on chick embryogenesis; it
decreases hatchability and delays hatching. This effect is
probably associated with thyroid hormone alterations
following P-treatment in late embryogenesis.
Key words: chicken embryo, paracetamol,
thyroid hormones, hatching.
Paracetamol (acetaminophen; P) is a popular
antipyretic and analgesic drug in human medicine. The
antipyretic and analgesic effects of paracetamol are
associated with a selective inhibition of the COX-2
cyclooxygenase and thereby synthesis of prostaglandin
in the brain. P is absorbed completely from the digestive
tract and the maximum concentration in blood is reached
after 30-60 min following treatment. P is mainly
metabolised in the hepatocytes and its half time in blood
is about 2 h (16). As early as in 1966, Dikstein et al.
(12) tried to use paracetamol in poultry breeding as the
antihypertermic preparation and stimulator of the growth
process in broiler chickens.
P had been considered as a non-toxic medicine
until 1966, when its hepatotoxicity was described (6, 10,
and 31). The hepatotoxic effect is caused by one of P
metabolites: N-acetyl-p-benzoquinone imine (NAPQI),
which is able to evoke necrosis of hepatocytes (16). It
has been established that paradoxically P is less harmful
for children than adults are, because children have less
intensive metabolism of hepatocytes (16). The
hepatotoxic effect of P was also described in the case of
cultured chick hepatocytes (19); however, the
mechanism of this effect is not known.
Laub et al. (17), using mouse embryos, showed
that large doses of P did not affect the development of
the preimplanted embryo. However, Lum and Wells (20)
established that P administered to pregnant mice could
be potentially teratogenic. Moreover, Stark et al. (28)
observed that the addition of P to the medium of
cultured rat embryos produced an increased incidence of
abnormally open anterior neuropores. Abnormalities in
cultured embryos, following P exposure, were also
shown by Weeks et al. (34). However, they found that
the N-acetylcysteine, a glutathione precursor, protects
embryos from P toxicity. These results indicate that
paracetamol
affects
mammalian
embryonic
development; however, in the literature there is no data
concerning the effect of P on chicken embryogenesis.
Therefore, the aim of the present study was to examine
the consequence of paracetamol in ovo injection on
chicken embryogenesis. Since thyroid hormones play a
crucial role during this process and affect the time of
538
hatching, and the length of incubation (8, 14, 24-26),
their concentrations following P injection in ovo were
also investigated.
Material and Methods
Eggs (weighing on the average 60.2 ± 8.4 g)
from the same broiler breeder flock of ISA 215 line
were put to the incubator (Masalles 65 DIGIT) and
incubated under standard conditions (1-18 d of
incubation: t=37.8°C, RH=55%, 19-21 d of incubation;
t=37.2°C, RH=70%). The eggs were candled on the 5th d
of the incubation, and unfertilised eggs and those with
dead embryos were discarded. On the 6th d of the
incubation, the eggs with living embryos (n=360) were
randomly divided into three equal groups. The
experimental group was in ovo injected with 4 mg of
paracetamol (POLFA-Kutno, Poland; Lot. No. 0255011)
dissolved in 100 µl of aqua pro injectione (Polpharma,
Poland). The administered dose of paracetamol was
calculated based on the human acceptable daily dose (4
g P/60 kg b.w.). The eggs of the two control groups
were injected with 100 µl of aqua pro injectione
(control C1) or were not treated (control C0). The
injections were given at the small end of the egg, deeply
into albumin according to Borzemska et al. (4).
During the incubation, the pathological analysis
of dead embryos of each group was performed. The age,
malformations, and malpositions of the embryo were
estimated at the moment of death (5).
Blood samples from each group were collected
on days 11, 14, 16, 18, 20 (external piping), and on the
21st d of incubation, i.e. just after hatching. They were
centrifuged 10 min (2 000 x g) and then plasma pools
obtained from two to three embryos (days 11-20) and
individual plasma samples from hatched chicks were
kept in –20°C until hormone determination. Thyroid
hormones (TH): thyroxine (T4), triiodothyronine (T3),
and reverse-T3 (rT3) concentrations in plasma samples
were measured radioimmunologically according to the
method earlier described (1, 27). Standard solutions of
T4, T3, and rT3 were prepared with chicken plasma free
of endogenous thyroid hormones. Antibodies against T4
and T3 were obtained from Sigma (USA), while the
antibody against rT3 from Professor Jerzy Kosowicz
(University of Medical Sciences, Poznań, Poland).
These antibodies have no or very weak cross reactivities
with
other
iodothyronines.
The
radioactive
iodothyronines were purchased from NEN (Belgium).
The lowest limit of sensitivity for T4 and T3 was 1.25
and 0.08 ng/mL, respectively, while for rT3 – 5.4
pg/mL. The intra-assay coefficients of variation for T4,
T3, and rT3 were 5.8%, 6.2%, and 4.0%, respectively.
The hatchability and embryopathology were
statistically analysed by z test, while the results of TH by
two-way analysis of variance followed by Duncan’s
multiple range test. The statistical analyses were
performed using Sigma-Stat 2.03 (SPSS Science
Software Ltd., USA). The figures were prepared with
Grapher 4.0 (Golden Software Inc., USA). Because the
radioimmunoassay revealed that there were no
significant differences in plasma thyroid hormone levels
between male and female embryos during incubation
process, which is in agreement with previous findings
(31), the data from both sexes were combined. The
results were presented as means ± SEM. and considered
significant at P<0.05 and highly significant at P<0.01.
Results
The
results
of
the
hatching
and
embryopathological analyses are presented in Tables 1
and 2, respectively; and changes in TH levels are shown
in Fig. 1. A significantly higher mortality of embryos
immediately after P injection (i.e. on days 6-7 of
embryogenesis) occurred in paracetamol-treated group
(P<0.01; Table 1). There were no significant differences
in embryonic mortality between control and P-treated
groups from the 8th d till the day of hatch; however, the
total mortality of P-injected embryos was significantly
(P<0.01) higher (27.1% in P-treated group vs. 20.0%
and 21.2% in C1 and C0 groups, respectively; Table 1).
The hatchability of chicken embryos treated with P was
significantly (P<0.05) lower in comparison with the
values of both control groups. In P-injected embryos the
mean time of hatching was 507.2 ± 1.1 h and was
significantly higher than in C0 group (P<0.05; Table 1).
The pathological analysis of the dead embryos
of each group, revealed that P injection in ovo
significantly increased the liver damage in the form of
lobe hypoplasia (or atrophia), hypertrophia of lateral
lobes (lat. hypoplasia lobi hepatis et hypertrophia
consecutiva loborum lateralium.), and subcapsular
haemorrhages (lat. haermorrhagiae subcapsulares
hepatis), and the gizzard damage (megagizzard)
(P<0.05; Table 2).
In the control groups (C1 and C0) plasma T4
concentrations increased gradually from 0.53±0.15 and
0.33±0.08 ng/mL on day 11 to the maximal values of
16.4±2.02 and 29.4±3.28 ng/mL on day of hatch,
respectively (Fig. 1a). In embryos exposed to
paracetamol, a similar increase in T4 concentration
during embryogenesis was observed; however, on days
14, 16, and 18 the plasma levels of T4 for this group
were by 165%, 51%, and 37%, respectively, higher in
comparison with C1 (P<0.05). On day 20, a sharp
decrease in T4 concentration in the P-treated group was
found; the level of T4 on day 20 in P-injected embryos
was 1.71 and 2.68 times lower in comparison with C1
and C0 groups, respectively (P<0.01). On the day of
hatch, the level of T4 in the experimental chicks
increased; however, it was still by 33% lower than that
in non-injected chicks (C0 group, P<0.01, Fig. 1a).
The plasma T3 concentrations gradually
increased in both control groups (C1 and C0) from
0.32±0.07 and 0.14±0.08 ng/mL on day 11 to 3.68±0.60
and 3.14±0.17 ng/mL on day of hatching, respectively
(Fig. 1b). In the embryos treated with paracetamol, the
T3 concentrations on day 11 were 6.33 and 3.47 times
lower than in C1 and C0 groups, respectively (P<0.01).
539
maximal levels of 167.1±18.2 and 225.3±15.1 pg/mL at
the time of piping (day 20), respectively (Fig. 1c).
Subsequently, the levels of rT3 sharply decreased toward
hatching (54.7±7.2 and 32.2±6.1 pg/mL in C1 and C0
groups, respectively). In the P-treated group, the
statistically (P<0.01) higher concentrations of rT3 in
comparison with both control groups were found only
on day 14 (increase by 233% and 131%, respectively).
The significantly higher levels of rT3 in comparison with
C0 (by 58%) and C1 (by 31%) groups were also noticed
on days 18 and 20, respectively (P<0.05; Fig. 1c).
On day 14, the concentrations of T3 increased
and were by 88% and 79% higher than those in C1 and
C0 groups, respectively (P<0.01). On day 18, the T3
level in the experimental group was 1.82 times lower
than in the C0 group (P<0.05). On the other hand, on
day of hatching, T3 concentrations in the P-treated
chicks were by 37% and 62% higher in comparison with
the values of C1 and C0 group, respectively (P<0.01,
Fig.1b).
The concentrations of rT3 on day 11 were
below the sensitivity of the analytical method. In the
control groups, (C1 and C0) plasma rT3 levels increased
from 11.8±2.8, and 16.0±3.2 pg/mL on day 14, to the
Table 1
Effect of paracetamol on the mortality and hatchability of chicken embryos
Group
Item:
C1 (n*=80)
C0 (n*=85)
a
Mortality - days 6-7 (%)
Mortality – days 8-17 (%)
Mortality – days 18-21 (%)
Total mortality (%)
Hatchability (%)
Mean time of hatching ± SE (h)
P (n*=70)
b
4.7
4.7a
11.8a
21.2 a
78.8a
504.4±1.1a
15.7c
4.3a
7.1a
27.1b
72.9b
507.2±1.1b
6.3
3.8a
10.0a
20.0a
80.0a
505.1±1.0ab
P- eggs injected with 4 mg of paracetamol on the 6th d of embryogenesis; C1 – eggs injected with aqua pro injectione;
C0 – eggs non-treated; * - without eggs used for blood sampling
a, b, c - values marked with different letters differ significantly (P<0.05)
Table 2
Effect of paracetamol on frequency of malformations in chicken embryos mortalised
between days 18 and 21 of incubation
Item
Embryos living at 18th d
Type of detected malformations
Malpositions
Teratosis
Liver damage: lobe hypoplasia (or atrophia)
and hypertrophia of lateral lobes,
subcapsular haemorrhages
Megagizzard
Incomplete retraction of the yolk sack
C0
76
%
10.53a
3.95a
Group
C1
72
%
12.50a
2.78a
P
56
%
8.93a
1.79a
1.32ab
0.00b
5.36a
1.32ab
9.21
0.00b
11.11
3.57a
7.14
a, b – values marked with different letters differ significantly (P<0.05). For further explanations, see Table 1.
540
(a)
40
C0 (non-treated)
C1 (aqua pro injectione)
Paracetamol (4 mg/egg)
T4 [ng/ml]
30
b
20
ab
10
ab
ab
ab
0
E11
(b)
E14
E16
E18
E20
H
6
ab
T3 [ng/ml]
4
2
ab
b
ab
0
E11
(c)
E14
E16
E18
E20
H
300
a
rT3 [pg/ml]
200
b
100
ab
0
b.s.
E11
E14
E16
E18
E20
H
Fig. 1. Effect of paracetamol in ovo injected at the 6th d of incubation on plasma thyroxine (T4) (a),
triiodothyronine (T3) (b) and reverse-triiodothyronine (rT3) (c) levels in embryonic chicks. Each value represents the
mean ± SE from 5-7 determinations; a - P<0.05 in comparison with C1 (control injected with aqua pro injectione); b P<0.05 in comparison with C0 (control non-injected).
541
Discussion
The present study showed that paracetamol in
ovo injected at the first week of incubation affects the
embryo development, decreases chick hatchability and
delays the time of hatching. These unfavourable effects
are accompanied by significant alterations in
iodothyronines concentration in blood plasma of
embryonic chicks.
In the reported study, the embryotoxic effect of
paracetamol was observed. This implication is
confirmed by the significantly higher embryo mortality
in the paracetamol treated group in comparison with
both control groups at the first two days following
injection. It can also be supported by the higher
frequency of the embryo malformations caused
especially by the elevation of the liver and stomach
damage in the P-treated group. Although the death of the
embryo after in ovo injection (as a manipulation) can
often result from damage and disruption of the
embryonic homeostasis, the applied substance is of more
importance. Moreover, the embryos sensibility to the
manipulation seems to be associated with the stage of its
development (7). Many authors described the
teratogenic effect (i.e., underdevelopment, heart
damage, chorioallantoic haemorrhage, and malpositions)
of growth stimulators, antibiotics, and other xenobiotics
injected in ovo during the first week of chick
embryogenesis (3, 4, 24, 32). Lin et al. (18) showed that
injection of N-nitrozofenacitine (a derivative of
paracetamol) in ovo on the 6th d of chicken
embryogenesis evoked the feather and claw
malformations, and caused brain damage. The toxic
effect of paracetamol on the chicken hepatocytes in vitro
has also been reported (19) but the mechanisms and
circumstances, in which paracetamol damages
hepatocytes, have been not described. However, it has
been postulated that thyroid hormones can sensitize the
liver cells to hepatic toxins. This hypothesis can be
supported by the investigations of Hartleb et al. (13)
who reported two cases of human liver damage
mediated by paracetamol and accompanied by increased
plasma level of thyroid hormones.
In the performed experiment, a gradual increase
in circulating iodothyronine concentrations observed
during chicken embryogenesis under controlled
conditions was consistent with data of the earlier studies
(9, 22, 24, 26, 29, 30, 32). The lowest concentrations of
T4 and T3 were found on day 11 of the chick
embryogenesis, while the highest one was on the day of
hatch when the chicken embryo had already been
switched from chorioallantoic to lung respiration (9, 11).
The plasma T4 concentrations were low until day 16 of
embryogenesis, and then it rose exponentially toward
the day of hatch. The most outstanding finding of this
study was the effect of paracetamol on iodothyronine
concentration in blood plasma. Paracetamol increased T4
concentration in the middle phase of embryogenesis (i.e.
between the 14th and 18th d); however, during the
hatching period, T4 concentration was significantly
lower in paracetamol-treated chicken embryos. These
changes were accompanied by a significant delay of the
hatching process in paracetamol treated group. The
correlation between the thyroid hormones concentration
in blood plasma and a rate of hatching was also
observed in chicken embryos exposed to dioxin-like
polychlorinated biphenyls (24) and electromagnetic field
(26). It seems likely that the observed changes in T4
concentration in blood of chicken embryos following
paracetamol treatment are probably associated with a
negative effect of this preparation on the hypothalamopituitary-thyroid axis. It cannot be excluded that the
decrease in T4 during the last phase of embryogenesis, is
connected with a competitive effect of paracetamol
compound with T4 binding to blood transport proteins
involved in the distribution of thyroid hormones in the
organism (15, 23). In the case of T3, a sharp increase in
concentration of this hormone in paracetamol-injected
embryos was found at the day of hatch. It may resemble
both the function of the thyroid gland, which secretes T3
(2, 21) and the activity of the hepatic and renal
iodothyronine deiodinases: type I (D1), which catalyses
both outer (ORD) and inner (IRD) ring deiodination, and
type III (D3) that is responsible for the IRD of T4 and T3
into
3.3’,5’-triiodothyronine
(rT3)
and
3,3’diiodothyronine (3.3’-T2), respectively (8, 9, 22, 32).
The gradual increase in rT3 concentration before
hatching is probably a result of the high activity of
deiodinase D3 in the liver (8, 9). It cannot be excluded
that alterations in iodothyronines concentration in blood
plasma of paracetamol-treated embryos are also
associated with the hepatotoxic effect of this
preparation.
In conclusion, the results obtained indicate that
paracetamol exerts unfavourable effects on chick
embryogenesis: (i) increases embryo mortality; (ii)
decreases hatchability, in addition, (iii) delays the time
of hatching. This effect is probably associated with
thyroid hormone alterations following P-treatment in
late embryogenesis. Further investigations are needed to
elucidate the mechanism(s), by which the paracetamol
affects the function of the thyroid gland and the
peripheral metabolism of thyroid hormones in
embryonic chicken.
Acknowledgments: The paper was supported
by DS 3210/ZHZiŚH/2006 and BW 2223/KFZ/2006.
References
1.
2.
3.
4.
Abdel-Fattah K. I., Bobek S., Sechman A.: Serum pattern
of thyroxine (T4), 3,3',5-triiodothyronine (T3) and 3,3',5'triiodothyronine (rT3) in fed and fasted cocks following
TRH stimulation. J Vet Med A 1991, 38, 401-408.
Astier H.: Thyroid gland in birds: Structure and function.
In: Avian Endocrinology, edited by A. Epple, M.H.
Stetson, Academic Press, New York, 1980, pp. 167-189.
Borzemska W., Karpińska E., Szeleszczuk P., Binek M.,
Malicka E., Kosowska G., Malec H.: Evaluation of
toxicity of stimulators in a biological test on chick
embryos. Med Wet 1995, 51, 745-747.
Borzemska W., Kosowska G., Malec H., Piusiński W.,
Niezgoda J., Malec L.: Attempts to establish the toxicity
542
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
of Galium aparine seeds using a biological test, i.e. chick
embryos and chickens. Med Wet 1991, 47, 366-368.
Borzemska W.B.: Vade mecum of avian diseases. PWRiL
Warszawa, 1984, pp. 11-14.
Boyd E.M., Bereczky G.M.: Liver necrosis from
paracetamol. Br J Pharmacol 1966, 26, 606-614.
Bruggeman V., Swennen Q., De Ketelaere B., Onagbesan
O., Tona K., Decuypere E.: Embryonic exposure to
2,3,7,8-tetrachlorodibenzo-p-dioxin in chickens: effects of
dose and embryonic stage on hatchability and growth.
Comp Biochem Physiol C 2003, 136, 17-28.
Darras V.M., Van Der Geyten S., Kühn E.R.: Thyroid
hormone metabolism in poultry. Biotechnol Agron Soc
Environ 2000, 4, 13-20.
Darras V.M., Visser T.J., Berghman L.R., Kühn E.R.:
Ontogeny of type I and type III deiodinase activities in
embryonic and post hatched chicks: relationship with
changes in plasma triiodothyronine and growth hormone.
Comp Biochem Physiol 1992, 103A, 131-136.
Davidson D.G., Eastham W.N.: Acute liver necrosis
following overdose of paracetamol. Br Med J 1966, 27,
497-499.
Decuypere E., Kühn E.R.: Thyroid hormone physiology
in Galliformes: Age and strain related changes in
physiological control. Am Zool 1988, 28, 401-415.
Dikstein S., Zor U., Ruah D., Sulman F.G.: Stimulatory
effect of paracetamol on chicken growth. Poult Sci 1966,
45, 744-746.
Hartleb M.: Do thyroid hormones promote hepatotoxicity
to acetaminophen? Am J Gastroenterol 1994, 89, 12691270.
Iqbal A., Decuypere E., Kühn E.R., Abd El Azim A.:
Plasma iodohormone concentrations in early and late
hatched chicks incubated at different temperatures. Med
Sci Res 1989, 17, 169-170.
Ishihara A., Sawatsubashi S., Yamauchi K.: Endocrine
disrupting chemicals: interference of thyroid hormone
binding to transthyretins and to thyroid hormone
receptors. Mol Cell Endocrinol 2003, 31, 105-117.
Kołaciński Z., Ruciński P.: Paracetamol: therapeutic
action, pathogenesis and treatment of acute poisoning
complicated by severe liver damage. Przegl Lek 2003, 60,
218-222.
Laub D.N., Elmagbari N.O., Elmagbari N.M., Hausburg
M.A., Gardiner C.S.: Effects of acetaminophen on
preimplantation embryo glutathione concentration and
development in vivo and in vitro. Toxicol Sci 2000, 56,
150-155.
Lin J.K., Yen J.Y., Chang H.W., Lin-Shiau S.Y.: Nnitrosophenacetin: its synthesis, characterization,
mutagenicity, and teratogenicity. J Natl Cancer Inst 1984,
72, 863-869.
Lindenthal J., Sinclair J.F., Howell S., Cargill I., Sinclair
P.R., Taylor T.: Toxicity of paracetamol in cultured chick
hepatocytes treated with methotrexate. Eur J Pharmacol
1993, 228, 289-298.
20. Lum J.T., Wells P.G.: Pharmacological studies on the
potentiation
of
phenytoin
teratogenicity
by
acetaminophen. Teratology 1986, 33, 53-72.
21. McNabb F.M.A.: Thyroids. In: Sturkie’s Avian
Physiology, edited by G.C. Whittow, San Diego, London,
Boston, New York, Sydney, Tokyo, Toronto, Academic
Press, 2000, pp. 461-469.
22. Reyns G.E., Venken K., Morreale De Escobar G., Kühn
E.R., Darras V.M.: Dynamics and regulation of
intracellular thyroid hormone concentrations in
embryonic chicken liver, kidney, brain, and blood. Gen
Comp Endocrinol 2003, 134, 80-87.
23. Rodighiero V.: Drug pharmacokinetics in thyroid
dysfunction. Minerva Endocrinol 1985, 10, 97-113.
24. Roelens S.A., Beck V., Maervoet J., Aerts G., Reyns
G.E., Schepens P., Darras V.M.: The dioxin-like PCB 77
but not the ortho-substituted PCB 153 interferes with
chicken embryo thyroid hormone homeostasis and delays
hatching. Gen Comp Endocrinol 2005, 143, 1-9.
25. Scanes C.G., Hart L.E., Decuypere E., Kühn E.R.:
Endocrinology of the avian embryo: An overview. J Exp
Zool 1987, Suppl. 1, 253-264.
26. Sechman A., Niedziólka J., Lis M., Rząsa J.: Changes in
thyroid hormone levels in chicken embryos exposed to
extremely low frequency electromagnetic field. Arch
Geflügelk 2006, 70, 41-47.
27. Sechman A., Rząsa J., Paczoska-Eliasiewicz H.: Effect of
non-steroidal aromatase inhibitor on blood plasma
ovarian steroid and thyroid hormones in laying hen
(Gallus domesticus). J Vet Med A 2003, 50, 333-338.
28. Stark K.L., Lee Q.P., Namkung M.J., Harris C., Juchau
M.R.: Dysmorphogenesis elicited by microinjected
acetaminophen analogs and metabolites in rat embryos
cultured in vitro. J Pharmacol Exp Ther 1990, 255, 74-82.
29. Thomes R.C., Hylka V.W.: Plasma iodothyronines in the
embryonic and immediate post hatched chicks. Gen.
Comp Endocrinol 1977, 32, 417-422.
30. Thomes R.C.: Ontogenesis of thyroid function and
regulation in the developing chick embryo. J Exp Zool
1987, Suppl. 1, 273-279.
31. Thomson J.S., Prescott L.F.: Liver damage and impaired
glucose tolerance after paracetamol over dosage. Br Med
J 1966, 27, 506-507.
32. Van Der Geyten S., Van Der Rynde I., Segers I.B., Kuhn
E.R., Darras V.M.: Differential expression of
iodothyronine deiodinases in chicken tissues during the
last week of embryonic development. Gen Comp
Endocrinol 2002, 128, 65-73.
33. Vesely D., Vesela D.: Embryotoxic effects of a
combination of zearalenone and vomitoxin (4dioxynivalenole) on the chick embryo. Vet Med (Praha)
1995, 40, 279-281.
34. Weeks B.S., Gamache P., Klein N.W., Hinson J.A.,
Bruno M., Khairallah E.: Acetaminophen toxicity to
cultured rat embryos. Teratog Carcinog Mutagen 1990,
10, 361-371.