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THERAPEUTIC EFFICACY OF ATROPINE AGAINST
CHOLINESTERASE INHIBITORS TOXICITY IN CHICKS
STRESSED WITH HYDROGEN PEROXIDE
Yaareb J. Mousa*, Muna H. I. Al-Zubaidy*, Sawsen M. Ameen* and Fouad K.
Mohammad**
* Department of Physiology, Biochemistry and Pharmacology, College of Veterinary
Medicine, University of Mosul, Mosul, Iraq.
** Ministry of Higher Education and Scientific Research, Baghdad, Iraq.
(Received 15 May 2016 ,Accepted 26 may 2016)
Keywords: Atropine, Oxidative Stress, H2O2, Chicks.
ABSTRACT
The aim of this study was to evaluate therapeutic efficacy of atropine in case of
cholinesterase (ChE) inhibitors toxicity in chicks subjected to oxidative stress (OS) with
hydrogen peroxide (H2O2). H2O2 at 0.5 % in drinking water induced OS at age of 7-14
days in the chicks when given each day for 14 consecutive days. There was no change in
the acute median lethal dose (LD50) for diazinon (7.9 mg/kg, orally) for both H2O
(control) and H2O2 groups. The therapeutic efficacy of atropine was decreased (265 %)
when measuring the median effective dose (ED50) for treating the diazinon toxicity in
H2O group (2.6 mg/kg, i.m.) and in the H2O2 group (9.5 mg/kg, i.m.). The signs of acute
diazinon toxicity were found to increase in H2O2 groups when compared to H2O group
and there is slight decrease in plasma and whole brain ChE activities in H2O2 group when
when compared to H2O group. The data revealed a decline in atropine efficacy for
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treatment diazinon toxicity in chicks that suffered from OS status and it is recommended
here to increase the dose of atropine in this case.
INTRODUCTION
Atropine is the drug of choice which is used as an antidote for the treatment of
cholinesterase (ChE) inhibitors toxicity such as organophosphates and carbamates in
human and animals by blocking the muscarinic receptors, thereby diminishing the excess
acetylcholine from binding to it (1-3). Diazinon is an organophosphate insecticides that
induces toxicity through inhibiting acetylcholinesterase in living tissues (2,3). Studies
have recommended using the powerful oxidant agent hydrogen peroxide (H2O2) for
induction of OS in laboratory animals such rats and chicks (4-7). It was found that OS
modulates the pharmacological response to drugs in chicks and if H2O2 was given at 0.5
% in drinking water, it induces OS in 7-14 day-old chicks when given fresh each day (5-
7). H2O2 induces OS through its capacity to increase the reactive oxygen species (ROS)
and free radicals formation in the tissues (8,9). The aim of the present study was to
evaluate the therapeutic efficacy of atropine in case of ChE inhibitors (diazinon as a
model) toxicity in chicks suffered from OS induced by H2O2 since no previous studies
dealt with such a life threatening situation.
MATERIALS AND METHODS
Experimental animals and induction of OS
Day-old broiler chicks of both sexes (body weight 45-125 g) were used. They were kept
in animal house at a temperature of 32–35 °C with constant lighting, and wood shavings
as floor litter. The chicks had free access to drinking water and feed along the
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experiment. The Scientific Committee of the College of Veterinary Medicine at the
University of Mosul reviewed and approved the protocol of this study. To induce OS,
day-old chicks were either provided with tap water (H2O-control group) or with fresh
H2O2 (Thomas Baker Chemical Ltd., U.K.) in tap water as 0.5% v/v each day for two
weeks in order to produce an OS as reported in previous studies (4,5,7).
Determination of acute median lethal dose (LD50) of diazinon in H2O (control) and
H2O2 (stressed) groups
The acute LD50 of diazinon (60%, VAPCO, Jordan) was measured for both the
control and stressed groups according to the up-and-down method described before (10).
The initial dose of diazinon was 7 mg/kg, orally for both groups. The onset and signs of
acute toxicity of diazinon in the chicks during 24 hours were also recorded in this
experiment.
Measuring the median effective dose (ED50) of atropine in the control and stressed
groups for alteration of acute diazinon toxicity
After determination of the acute LD50 value of diazinon (7.9 mg/kg, orally), the
dose of diazinon (10 mg/kg, orally) was chosen that causes acute toxicity leading to
lethality in the chicks. The ED50 of atropine (1%, Alsharq for Veterinary Drugs, Syria)
was determined for both the control and stressed groups according to up-and-down
method (10). The onset and signs of acute toxicity of diazinon during 24 hours were
measured to evaluate the differences between these two groups.
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Therapeutic efficacy of atropine against acute diazinon toxicity in control and
stressed chicks with H2O2
Atropine was injected at 6 mg/kg, i.m. (this dose was chosen according to ED50 value of
atropine) immediately after diazinon dosing at 10 mg/kg, orally for both the control and
stressed chicks (6 chicks/group). The onset of acute diazinon toxicity, toxic signs and
toxicity scores were evaluated for both groups.
In vivo determination of ChE activity in plasma and whole brain of the control and
stressed chicks
Atropine was injected at 6 mg/kg, i.m. immediately after diazinon dosing at 10 mg/kg,
orally for both the control and stressed chicks (6 chicks/group). The chicks were
euthanized and blood samples and whole brain were collected after 2 hours of atropine
and diazinon administration. The ChE activity was determined according to a modified
electrometric method described earlier (11-13) for investigating the alteration on the level
of ChE. Whole brain was homogenized on an ice bath by using a homogenizer in a pH
8.1 barbital-phosphate buffer solution at 3ml/100 mg wet weight of whole blood (11,12).
To measure ChE activity, the reaction mixture composed of 3 ml distilled water, 0.2ml
plasma or whole brain homogenate and 3ml of pH 8.1 buffer. The initial pH of the
mixture (pH1) was measured by using a pH meter (Hanna, Romania), and then the
substrate 7.5 % acetylthiocholine iodide was added to the mixture and incubated at 370C
for 30 min. Then the pH of the reaction mixture (pH2) should be measured. The ChE
activity (ΔpH/30 min.) was calculated as follows:
ChE activity (ΔpH/30 min.)＝(pH1−pH2)−ΔpH of blank (The blank was without the
plasma or brain homogenate sample).
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Statistics
To compare the means of two parametric groups, the unpaired Student's t-test was
applied for statistical analysis (14,15). On the other hand, the nonparametric data of the
two groups were statistically analyzed by using Fisher exact probability test and Mann-
Whitney U-test (15,16). The level of significance was at p < 0.05.
RESULTS
Acute LD50 value of diazinon in H2O and H2O2 groups of chicks
The acute LD50 value of diazinon was 7.9 mg/kg, orally for both the control and
stressed chicks (Table 1). The onset of acute toxicity was decreased and the signs of
toxicity (which were salivation, dyspnea, depression, tremor, recumbency, paralysis,
convulsion and death) were exacerbated in the stressed group when compared to the
control group as demonstrated in Table 1.
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The ED50 value of atropine in the control and stressed groups of chicks
Table 2 shows that the ED50 value of atropine was effective in treating the
diazinon toxicity increased by 265 %, which demonstrates decreased effectivness in the
H2O2 (stressed) group (9.5 mg/kg, i.m.) when compared with the H2O (control) group
(2.6 mg/kg, i.m.).
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Table 2: ED50 of atropine for preventing the acute toxicity of diazinon in control and
stressed chicks with H2O2
Parameters Results
Stressed group
(0.5 % H2O2 in water)
Control group
(Tap water)
ED50 value 2.6 mg/kg, i.m. 9.5 mg/kg, i.m.
The range of the doses 2-4 6-10
Initial dose 4 mg/kg 6 mg/kg
Last dose 3 mg/kg 8 mg/kg
Increase or decrease in the dose 1 mg 2 mg
Number of chicks 6 (XXOOXX) 6 (OOXOXO)
% effect of OS on ED50 value of Atropine= 265 %
X= survival; O= death
Atropine was injected (i.m.) immediately after the diazinon administration at 10
mg/5ml/kg, orally
Therapeutic efficacy of atropine against acute diazinon toxicity in control and
stressed chicks with H2O2
The signs of acute diazinon toxicity were found to increase in the stressed groups when
compared to control groups of chicks by measuring the onset of acute signs, signs of
toxicity and toxicity scores which revealed also a decrease in atropine therapeutic
efficacy in the stressed chicks when compared to the control tap water group of chicks
(Table 3).
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Table 3: Therapeutic efficacy of atropine against acute diazinon toxicity in control and
stressed chicks with H2O2
Toxicity
scores
Onset of % Toxicity signs
toxicity
(min.)+
Groups
Paralysis
Recumbency
Dyspnea
Tremor
Defecation
Salivation
49.67 ± 100 50 16.67 16.67 100 33.33 14
2.88
Control
group (tap
water)
15.67 ± 100 50 66.67* 83.33* 66.67 33.33 18
2.04*
Stressed
group (0.5
% H2O2 in
water)
+ Data represented Mean ± S.E. for 6 chicks/group
* significantly different from the respective control (H2O group) at p < 0.05
Atropine was injected at 6 mg/5ml/kg, i.m. immediately after diazinon dosing at 10
mg/5ml/kg, orally
ChE activity in the plasma and whole brain of the control and stressed chicks
Table 4 describes a slight change in the plasma and whole brain ChE activity with more
decreased in enzyme activity for the stressed chicks when compared to the control chicks.
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Table 4: ChE activity in the plasma and whole brain in the control and stressed chicks with
H2O2
Groups Plasma Whole brain
ChE activity % inhibition
(Delta pH/30
min.)
ChE activity % inhibition
(Delta pH/30
min.)
Control group (tap 0.048 ± 0.005 --------------- 0.030 ± 0.005 ----------------
water)
Stressed group (0.5 % 0.035 ± 0.008 37 0.028 ± 0.009 7
H2O2 in water)
Data represented Mean ± S.E. for 6 chicks/group
Atropine was injected at 6 mg/5ml/kg, i.m. immediately after diazinon dosing at 10
mg/5ml/kg, orally
Blood samples and whole brain were collected after 2 hours of atropine and diazinon
administration
DISCUSSION
Many cases of poisoning and diseases in animals may be accompanied by
stressful conditions that may affect the pharmacological responses and therapeutic
efficacy of certain drugs. The goal of this study was to evaluate the therapeutic efficacy
of atropine in case of ChE inhibitors (diazinon as a model here) toxicity in chicks
suffered from the OS (as a model of stress) induced with a powerful oxidant H2O2 since
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no previous literature dealt with that a life threatening situation. Fresh H2O2 given each
day at 0.5 % in drinking water was found to induce OS in 7-14 day-old chicks old (4,5)
which were found by reduced glutathione and elevation of malondialdehyde
concentrations in the body tissues of chicks. Atropine is the drug of choice used as an
antidote for the treatment of ChE inhibitors (organophosphates and carbamates) toxicity
and preventing death in human and animals through blocking the muscarinic receptors (1-
3). Diazinon induces toxicity through inhibiting acetylcholinesterase in living tissues
(2,3). There was no change in the acute LD50 value for diazinon in both the control and
stressed groups of chicks and it may be atributed to acute organophosphate toxicity
during 2 hours after dosing and death in vulnerable chicks (17). The therapeutic efficacy
of atropine at all was decreased when measuring the ED50 of atropine for treating the
diazinon toxicity and death in the control and stressed chicks and this revealed that
atropine was not able to treat and counteract diazinon toxicity at the same time with the
presence of a stressful condition (OS induced with H2O2 as a model) because stressful
conditions have synergistic action with toxicity status that exacerbate this condition (18).
At the same time, the OS induced with H2O2 modulates the pharmacological response and
efficacy of drugs in animals (4-7). In the same manner, the signs of acute diazinon
toxicity were found to increase in stressed chicks with H2O2 when compared to groups
that received tap water. A similar effect was described in previous study (18). There was
a slight change in the plasma and whole brain ChE activity in stressed chicks when
compared to the control chicks which means that the OS status caused by H2O2 affect the
enzyme level and potentiates the toxicity status (18). The data revealed a decline and
modulation in atropine efficacy for treatment of ChE inhibitors (diazinon) toxicity in the
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chicks that suffered from OS status induced with H2O2. We recommend here an increase
the dose of atropine in order to improve the treatment in this case.
الفعالیة العلاجیة للأتروبین ضد التسمم بمثبطات خمیرة الکولین أستراز فی الأفراخ المجھدة
ببیروکسید الھیدروجین
یعرب جعفر موسى*؛ منى حازم ابراھیم الزبیدی*؛ سوسن محمد أمین* و فؤاد قاسم محمد**
* فرع الفسلجة والکیمیاء الحیاتیة والأدویة، کلیة الطب البیطری، جامعة الموصل، الموصل، العراق.
** وزارة التعلیم العالی والبحث العلمی، بغداد، العراق.
الخلاصة
تھدف الدراسة الى تقییم الفعالیة العلاجیة للأتروبین فی حالة التسمم الحاد بمثبطات خمیرة الکولین أستراز
فی نموذج أفراخ الدجاج والتی تعانی من الإجھاد التأکسدی المحدث باستخدام بیروکسید الھیدروجین حیث أنھ لا
توجد دراسات سابقة أخذت بنظر الاعتبار ھذه الحالة. وقد وجد أن إعطاء بیروکسید الھیدروجین بترکیز 0.5 ٪ فی
14 یوم عندما یعطى بصورة طازجة کل - ماء الشرب یعمل على إحداث الاجھاد التأکسدی فی أفراخ الدجاج بعمر 7
یوم. لم یکن ھناک تغییر فی الجرعة الممیتة الوسطیة الحادة للدیازینون ( 7,9 ملغم / کغم، عن طریق الفم) لکل من
المجموعة التی أعطیت ماء الشرب وتلک التی اعطیت بیروکسید الھیدروجین مع ماء الشرب. وعند قیاس الجرعة
الفعالة الوسطیة للأتروبین لکل من المجموعة التی أعطیت ماء الشرب ( 2,6 ملغم / کغم، فی العضل) وتلک التی
اعطیت بیروکسید الھیدروجین مع ماء الشرب ( 9,5 ملغم / کغم، فی العضل) فقد تبین أن ھاک انخفاض فی الفعالیة
العلاجیة ( 265 ٪) لعلاج التسمم الحاد بالدیازینون فی أفراخ الدجاج. وعند العلاج بالأتروبین فقد لوحظ زیادة
العلامات السمیة الحادة للدیازینون فی المجموعة المعاملة ببیروکسید الھیدروجین بالمقارنة مع المجموعة المعطاة
ماء الشرب وفی الوقت نفسھ ھناک تغییرات طفیفة فی نشاط خمیرة الکولین أستراز فی البلازما والدماغ الکلی
الدماغ لکلا المجموعتین. کشفت بیانات البحث عن انخفاض فی فعالیة الأتروبین العلاجیة من التسمم المحدث
بمثبطات خمیرة الکولین أستراز (الدیازینون) فی أفراخ الدجاج التی تعانی من الاجھاد التأکسدی المحدث ببیروکسید
الھیدروجین وینصح ھنا بزیادة جرعة الأتروبین لتعزیز الکفاءة العلاجیة من التسمم فی ھذه الحالة.
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