https://doi.org/10.52973/rcfcv-e33230
Received: 26/01/2023 Accepted: 02/03/2023 Published: 13/03/2023
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Revista Científica, FCV-LUZ / Vol. XXXIII, rcfcv-e33230, 1 – 4
ABSTRACT
Atipamezole is a specic α2-adrenergic receptor antagonist, and
there exists insucient information on its use during pregnancy.
The aim of this study was to determine the embryotoxic activity of
Atipamezole through an in ovo method. During the rst stage of the
study, 210 fertile eggs were divided into seven groups of 30 fertile
eggs and placed in an incubator. On the seventh day of the rst stage,
no application was made to the control group. The second group
was administered 50 microliters (µL) of saline solution, while the
other groups were given Atipamezole at doses of 250, 125, 62.5, 31.25
and 15.62 micrograms·egg
-1
(µg·egg
-1
) in 50 µL saline solution. In the
second stage, according to the embryotoxic dose range determined
from the rst stage, 150 fertile eggs were divided into ve groups
of 30 fertile eggs and placed in an incubator. On the seventh day of
the second stage, no application was made to the control group.
Fifty µL of saline solution was administered to the second group.
The other groups were given Atipamezole at doses of 220, 190 and
160 µg·egg
-1
in 50 µL saline solution. After the incubation period, the
eggs hatched, and the embryonic mortality rates were calculated.
The mortality rate was determined to be 39.3% at the highest dose
(250 µg·egg
-1
= 5 miligrams·kilograms
-1
–mg·kg
-1
–) (P<0.05), while the
mortality rate at other doses was determined to be the same as the
control group (P>0.05). In conclusion, it can be stated that the dose
determined for Atipamezole in this study was very high compared
to the recommended doses and it can be used in pregnancy as a
benet-loss calculation when necessary. However molecular or
histopathological studies regarding the development of organ drafts
are necessary to determine the safety of its use during pregnancy.
Key words: Atipamezole; embryotoxicity; in ovo
RESUMEN
El atipamezol es un antagonista específico de los receptores
adrenérgicos α2 y no existe suficiente información sobre su uso
durante el embarazo. El objetivo de este estudio fue determinar la
actividad embriotóxica de atipamezol mediante un método in ovo.
Durante la primera etapa del estudio, se dividieron 210 huevos
fértiles en siete grupos de 30 huevos fértiles y se colocaron en una
incubadora. Al séptimo día de la primera etapa no se realizó ninguna
aplicación al grupo de control. Al segundo grupo se le administró
50microlitros (µL) de solución salina, mientras que a los otros grupos
se les administró atipamezol en dosis de 250; 125; 62,5; 31,25 y 15,62
microgramos·huevo
-1
(µg·huevo
-1
) en 50 µL de solución salina. En la
segunda etapa, según el rango de dosis embriotóxica determinado a
partir de la primera etapa, se dividieron 150 huevos fértiles en cinco
grupos de 30 huevos fértiles y se colocaron en una incubadora. Al
séptimo día de la segunda etapa, no se realizó ninguna aplicación
al grupo de control. Se administraron 50 µL de solución salina al
segundo grupo. Los otros grupos recibieron atipamezol en dosis
de 220; 190 y 160 µg·huevo
-1
en 50 µL de solución salina. Después
del período de incubación, los huevos eclosionaron y se calcularon
las tasas de mortalidad embrionaria. Se determinó que la tasa de
mortalidad era del 39,3% con la dosis más alta (250 µg·huevo
-1
=
5miligramos·kilogramos
-1
–mg·kg
-1
–) (P<0,05), mientras que la tasa
de mortalidad con otras dosis se determinó que era la misma que
la del grupo de control (P>0,05). En conclusión, se puede armar
que la dosis determinada de atipamezol en este estudio fue muy
alta en comparación con las dosis recomendadas y se puede
utilizar en el embarazo como un cálculo de pérdidas y ganancias
cuando sea necesario. Sin embargo, los estudios moleculares o
histopatológicos relacionados con el desarrollo de borradores de
órganos son necesarios para determinar la seguridad de su uso
durante el embarazo.
Palabras clave: Atipamezol; embriotoxicidad; in ovo
Determination of Embryotoxic effects of Atipamezole using in ovo model
Determinación de los efectos embriotóxicos del uso de Atipamezol en modelo in ovo
Rahmi Canbar
1
* , Muhittin Uslu
2
, Mustafa Sedat Arslan
3
and Harun Kızılay
4
1
Necmettin Erbakan University, Faculty of Veterinary, Department of Pharmacology and Toxicology. Konya, Turkey.
2
Yozgat Bozok University, Department of Laboratory and Veterinary Health, Sefaatli Vocational College. Yozgat, Turkey.
3
Selcuk University, Faculty of Veterinary Medicine, Department of Anatomy. Konya, Turkey.
4
Selcuk University, Faculty of Pharmacy, Department of Pharmacology. Konya, Turkey.
*Corresponding author: rahmicanbar@erbakan.edu.tr
Embryotoxic effects of Atipamezole / Canbar et al. _________________________________________________________________________________
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INTRODUCTION
α2-adrenergic receptors are found in many tissues and organs such
as the central nervous, cardiovascular and digestive systems [4].
Norepinephrine has certain regulatory effects on the central nervous
system by binding to α2-adrenergic receptors [17, 19]. When agonists,
such as Xylazine, Detomidine, Medetomidine and Dexmedetomidine,
bind to the α2-adrenergic receptors in the central nervous system,
the release of Norepinephrine is prevented and sympathetic tone is
decreased but sedation and analgesia is increased [21, 25]. Clinically,
it has been reported that α2-adrenergic receptor agonists slow
the heart rate with long-term hypotension after hypertension and
causes side effects such as decreased cardiac output, kidney and
liver damage, shock and respiratory depression [9, 23]. Some studies
revealed that the effects of α2 receptor agonists are similar to each
other, but the duration of action varies depending on the dose [9].
Antagonists of this receptor, including Atipamezole, Yohimbine and
Tolazoline can be used to reduce the frequency of side effects caused
by α2-adrenergic receptor agonists in pets [23].
Atipamezole (4–(2–ethyl–2,3–dihydro–1H–inden–2–yl)–1H–imidazole)
is a specic α2–adrenergic receptor antagonist that rapidly reverses
the undesirable side effects caused by α2–adrenergic receptor
agonists during the sedation phase in the Veterinary eld [10, 17]. In
addition, one study reported that it has proved useful in the Veterinary
eld for Amitraz poisoning [18]; in a study conducted in dogs (Canis
lupus familiaris), Atipamezole was reported to be successful for
treating Amitraz poisoning when administered intramuscularly at
a dose of 50 micrograms·kilograms
-1
(µg·kg
-1
) [13]. In another study
conducted in alpine mountain goats (Rupicapra rupicapra), the optimal
anesthetic dose was 2.6–3.6 miligrams· kilograms
-1
(mg·kg
-1
) Xylazine,
and 0.26–0.36 mg·kg
-1
Atipamezole was used to reverse the ecacy
of Xylazine [8]. An investigation conducted in mice (Macaca mulatta)
stated that the effects of ethanol could be antagonized to a large
extent using Atipamezole [20]. In Atipamezole toxicity studies,
the letal dose 50 (LD
50
) was reported to be higher than 30 mg·kg
-1
in
genetically modied Naval Medical Research Institute (NMRI) mice
and Sprague-Dawley rats (Rattus) for intravenous, subcutaneous and
intraperitoneal exposures. While calculating the LD
50
it was reported
that heart and lung damage occurred in the dead animals. When a
100 mg dose is administered to humans, restlessness, shivering,
coldness and hypersalivation are observed, and the amount of plasma
Norepinephrine increases, causing an increase in systolic and diastolic
blood pressure [17]. There is no information regarding the safety of
Atipamezole in pregnancy in target species [25].
Poultry embryos are frequently preferred for the investigation
of embryotoxic and teratogenic effects of drugs and chemicals [5,
6, 14, 24]. This methodology has the advantages of knowing the
developmental stages of the chicken (Gallus gallus domesticus)
embryo, simplify of application and providing cheap and reproducible
results. With the use of high sample sizes of chicken embryos, it is
statistically superior to the studies on mammalian species. Using this
method can help guide future prenatal toxicity studies in mammals
and minimizes the number of test subjects as well as the pain suffered
by the subjects. As a result, ethical rules, legal restrictions and animal
rights are not contradicted [16]. Disadvantages of the poultry embryo
toxicity methodology as it relates to mammalian toxicity studies
include its lack of a maternal–fetal relationship that is observed in
mammals and the pharmacokinetic disparities observed with the
differences in chicken eggs compared to mammalian embryos [15].
However, its positive aspects include the need for little laboratory
equipment, simplify of application, short time of experimentation, low
cost and the understanding that morphogenetic events are similar
in all living things [12, 15].
One study indicated that medication should be administered to the
eggs in the early embryonic period to determine the toxicological dose
limits. However, if the teratological effects of the metabolites that
occur from the drug metabolism in the liver are being investigated,
exposure during later developmental periods, during which the liver
and kidneys complete their development, are preferred [16]. The liver
is formed by the fourth day in poultry embryos, and the induction of
enzymes increases after the seventh day [11].
Although Atipamezole has been reported to be safe in pregnant cattle
(Bos taurus) [2, 3] it has been determined by the manufacturing company
that the information regarding that statement is insucient [26].
In this study, it was hypothesized that the toxic effects of Atipamezole
on embryos in the in ovo model are dose dependent. The aim of the
study was to determine the possible embryotoxic effects of Aipamezole
using an in ovo model.
MATERIALS AND METHODS
Fertile chicken eggs were obtained from a commercial enterprise
(Anadolu Damizlik, Konya, Turkey). During the study, the incubation
periods were completed in an egg incubator (Imza Teknik, Konya,
Turkey). On the seventh day of incubation, fertility was checked under
light, and non-fertile eggs were removed from the groups. Fertile
eggs were added to replace the non-fertile eggs, and treatment
groups were comprised 30 eggs each. A commercial formulation of
Atipamezole (Antisedan™ inj, Zoetis, Istanbul, Turkey) was used in
the study. All doses were applied at a volume of 50 microliters (µL).
The study was planned and performed in two stages.
Experimental design and animal practices
Stage 1: Determination of embryotoxic dose limit
In this study, 210 fertile eggs were randomly divided into seven
groups of 30 fertile eggs and placed in an incubator. On the seventh
day of stage one, the rst group was treated as the control group, and
saline with no Atipamezole was applied to the second group, which
served as the vehicle control. Groups 3−7 received Atipamezole at
doses of 250, 125, 62.5, 31.25 and 15.62 µg·kg
-1
(5, 2.5, 1.25, 0.625,
0.3125 mg·kg
-1
) respectively. After the incubation period of 21 days,
the eggs hatched, and the numbers of live and dead embryos were
recorded (İmza Teknik, Konya, Turkey).
Stage 2: Determination of embryotoxicity
In the second stage, 150 fertile eggs were randomly divided into ve
groups of 30 fertile eggs and placed in an incubator. On the seventh
day of stage two, the rst group was treated as the control group, and
saline with no Atipamezole was applied to the second group, which
served as the vehicle control. Groups 3−5 received Atipamezole at
doses of 220, 190 and 160 µg·egg
-1
, respectively. These were within
the embryotoxic dose limits determined from the rst stage. After
the incubation period of 21 days, the eggs hatched, and the numbers
of live and dead embryos were recorded.
_______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIII, rcfcv-e33230, 1 – 4
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Statistics
Mortality rates were calculated using the Abbott method. Intergroup
embryonic mortality rates were evaluated for differences using the
Chi-square test (SPSS 22.2, IMD SPSS,Armonk, USA) [5].
RESULTS AND DISCUSSION
In this study, Atipamezole caused an observed death rate of 39.3%
at a dose of 5 mg·kg
-1
(TABLE I).
Atipamezole is a specic α2-adrenergic receptor antagonist that
rapidly reverses the undesirable side effects caused by α2-adrenergic
receptor agonists during the sedation phase in the veterinary eld
[10, 17]. There is insucient information regarding the use of the
drug during pregnancy [26].
In this study, it was determined that Atipamezole caused embryotoxic
activity at a high dose (5 mg·kg
-1
) at a rate of 39.3% (P<0.05), which
was significantly different from the other treatment groups. No
statistical differences were detected among the other treatment
groups (P>0.05, TABLE I). The recommended application doses of
Atipamezole are 0.03–0.04 mg·kg
-1
in cats (Felis catus) and dogs and
0.03−0.06 mg·kg
-1
in horses (Equus caballus) and cattle (Bos taurus)
[25]. In a study conducted in humans, Atipamezole was administered
at a dose of 0.05 mg·kg
-1
against Dexmedetomidine [1]. In a different
study, it was shown that Atipamezole did not exhibit negative effects
on offspring and pregnancy at the 0.162 mg·kg
-1
dose in two pregnant
cattle who had been administered Medetomidine [3]. It was reported
that the administration of Atipamezole at a 0.045 mg·kg
-1
dose in cattle
during the last two months of pregnancy did not have a negative effect
on calves and that premature birth or periparturient disorders were
not observed [2]. It was revealed that Atipamezole administered at
a dose of 0.75 mg·kg
-1
changed creatine kinase activity compared to
non–pregnant females in a study conducted on pregnant reindeer.
Furthermore, abortion was not observed within 10−18 hours after
application of Atipamezole at a 6.3 mg/goat dose in the transport
of hook horned mountain goats [7, 22]. From this study, it should be
considered that the Atipamezole was used at a dose of 5 mg·kg
-1
, which
is high compared to the recommended doses.
CONCLUSIONS
From this study, Atipamezole may be embryotoxic in high doses
(5mg·kg
-1
). However, the drug ecacy of embryos on organ primordia
has yet to be investigated. In future studies, it is suggested that
researchers conduct molecular and histopathological studies on
embryo development.
Ethical committee
The study was approved by the ethics committee of Selcuk University,
Faculty of Veterinary Medicine, Experimental Animals Production and
Research Center (SUVDAMEK) with the number 2021/35.
ACKNOWLEDGEMENTS
For this study, no nancial support was received from any institution
or company. The summary of the research was presented orally at the
II International Veterinary Pharmacology and Toxicology Congress,
7–10 September, 2022, Balikesir University, Turkey and the summary
was published in the booklet.
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TABLE I
Death rates from Atipamezole administration
Doses
(µg·egg
-1
)
NAE NDE N
Death rate
(%)
Alive rate
(%)
Actual
death rate
(Abbott
method)
Chest-1
Control 28 2 30 6.7
b
93.3 −
SF control 29 1 30 3.3
b
96.7 −
250 17 13 30 43.3
a
56.7 39.3
125 28 2 30 6.7
b
93.3 0
62.5 29 1 30 3.3
b
96.7 −3.6
31.25 28 2 30 6.7
b
93.3 0
15.62 28 2 30 6.7
b
93.3 0
Chest-2
Control 29 1 30 3.3
b
96.7 0
SF control 30 0 30 0
b
100 0
220 28 2 30 6.7
b
93.3 3.5
190 29 1 30 3.3
b
96.7 0
160 29 1 30 3.3
b
96.7 0
NAE: Number of alive embryos, NDE: Number of dead embryos,
a, b
: Different
letters in the same column represent statistical signicance (
P<0.05)
Embryotoxic effects of Atipamezole / Canbar et al. _________________________________________________________________________________
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