
Embryotoxic effects of Atipamezole / Canbar et al. _________________________________________________________________________________
2 of 4
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.