Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
136
CONCOMITANT OCCURRENCE OF OXIDATIVE STRESS
WITH SUSTANON IN MALE RAT
Y.Z. Al-abdaly*, E .R. Al-Kennany ,E.K. Al-Hamdany**
Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine,
University of Mosul, Mosul, Iraq
Department of Pathology and Poultry Diseases , College of Veterinary Medicine, University
of Mosul, Mosul, Iraq
Keywords: heart, glutathione, malondialdehyde .
Corresponding Author:[email protected]
ABSTRACT
The present study was conducted to investigate the effect of androgenic–
anabolic steroids Sustanon 20 mg/ kg on oxidative stress. Twenty male rats were
divided into four groups treated for 4 weeks as once weekly treatment. The 1st group
considered as control group, the 2nd group injected with sustanon 20 mg/kg
intramuscularly, the 3rd group injected with sustanon intramuscularly 20 mg/kg and
then orally dosage of vitamin E 100 mg\kg, and 4th group got orally dosage of vitamin
E 100 mg/kg. The results revealed significant decrease in glutathione level in the
serum and tissue of brain and liver in sustanon group as compared with control, while
there is significant increase in malondialdehyde level in the serum of sustanon group
as compared with control The result showed no different between the groups treated
with sustanon alone and with sustanon and vitamin E.
All the pathological changes have been recorded for the sustanon group only
while there were no pathological changes for other groups. The gross pathological
changes showed congestion of heart and liver in sustanon group.. The microscopic
changes of heart showed vacuolar degeneration of cardiac cells and edema between it.
Liver showed necrosis of hepatic cells, infiltration of inflammatory cells. Lungs
revealed pneumonia with thickening of wall of alveoli and bronchioles by
inflammatory cells, and emphysema of alveoli.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
137
We conclude from this study that sustanon in high dose have an effect on
increase oxidative stress, macroscopic and microscopic pathological changes in heart,
liver and lung, and no effect of vitamin E in this study.
INTRODUCTION
Several clinical studies showed that administration of anabolic-androgenic
steroids (AAS) in the pharmacological doses has a beneficial effect in many cases and
is considers safe drug (1). On the other hand, the long-lasting administration of high
doses of steroids can seriously harm health (2,3). The effects of high doses of AAS
for a long time may cause various side effects (4). One of those harmful effects is
induced oxidative stress due to disturbance in the balance between oxidants and
antioxidants (5). Persistent oxidative stress may lead to pathological cases and
development sever pathologies (over 150 disorders) (6).
Overdoses of AAS cause cardiovascular disorders such as hypertrophy of the
left ventricle of the heart, arterial hypertension, cardiac arrhythmia, blood clotting,
coronary blood flow disability, myocardium inflammation, acute coronary
inefficiency, cardiac infractions, arterial sclerosis, circulatory failure and heart attacks
which may end of sudden death (2). It can also cause some mental instability in the
form of a maniac-depressive states expressed by uncontrolled nervousness or deep
depression associated with some suicidal inclinations (3). Oral synthetic steroids, such
as stanozolol, reduce high-density lipoprotein (good fat) and increase low-density
lipoprotein cholesterol (bad fat) more than injected testosterone at similar doses(7).
Sustanon is described as a long acting anabolic androgenic drug(8). Regardless of
these unique pharmacological structure and properties of Sustanon, only few studied
attempted to investigate the adverse effects induced by overdose of Sustanon abuse.
This is absolutely not correct as there are many studies which confirmed the
occurrence of irreversible adverse effects among the abusers such as cardiac disorders
and chronic hepatitis (9,10,and 11). Testosterone replacement therapy has also been
shown to reduce circulating levels of inflammatory mediators, such as TNF-α and IL-
1β as well as total cholesterol in patients with established coronary artery disease
(CAD) and testosterone deficiency (12,13).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
138
Primary aims of this study to assess the effects of toxic dose of sustanon and
concomitant with oxidative stress, the role of vitamin E, and histopathological
changes of heart, lung and liver.
MATERIALS AND METHODS
Experimental design and treatment:
Twenty male albino rats, weighing between 250-300 g were maintained in
clean plastic cages in the laboratory animal room (23 ± 2 °C). Daily dark/light cycle
(12/12 hrs) rats were randomly divided in to four groups treated for 4 week
administered as once weekly. The first group considered as control, the second group
deeply injected with 20 mg/kg sustanon (19) intramuscularly and third group injected
with 20 mg/kg sustanon intramuscularly then orally dosage with vitamin E 100 mg
animal , fourth group orally dosage with vitamin E 100 mg animal . (21).
Chemicals and dose preparations:
Sustanon 250% from Organon Oss Holand company, it was dissolved in
sesame oil for dose preparations; volume of dose is 2 ml/kg.
Oxidative stress parameter Malondialdehyde (MDA) content as indicator of
lipid peroxidation was determined in the serum, by a colorimetric method according
to (14).
The reduced form of glutathione (GSH) was determined in the tissue and
serum by colorimetric method according to (15).
Collection of blood samples and organs:
Measurements: At the end of the treatment period (4weeks), each animal of the four
groups was anesthetized by inhalation of drops of Diethyl Ether in a piece of cotton.
Blood samples were collected from eye choroid venous plexus according to (16). Five
ml of blood have been collected and immediately placed in clean tubes and then
centrifuged for serum collection, three serum samples were separated from each
animal sample and also animals was anatomical examine for study morphological
changes and then organs (heart, liver, brain and lungs) were collected for biochemical
and histopathological examination.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
139
Histopathological Examination
For light microscopic investigations, specimens from heart, liver, and lungs
were fixed in 10% phosphate buffer formalin, dehydrated in alcohols and embedded
in paraffin. Five micron tissue sections were stained with hematoxylin and eosin stain
for general histopathological examination. One slide was prepared for each rat.
Statistical Analysis
The results were expressed as means ± S.E.M. All data were done with the
Statistical Package for Social Sciences (SPSS 11.0 for windows). The results were
analyzed using one way analysis of variance (ANOVA) between different treatment
groups. Statistical significance was set at p < 0.05.
RESULT
There were significant decreased in the serum GSH observed among treated
Groups when compared with control group, were as serum MDA level was increased
when compared with control group (Table 1).
Table 1: Effect of Sustanon on glutathione and malondialdihyde in serum of rats
Groups GSH (Micromole/ml) MDA (Nanomal/ml)
control 2.55 ± 0.07 0.9 ± 0.13
Sustanon 20 mg kg-1 1.2 ± 0.08* 2.79 ± 0.09*
Sustanon 20 mg kg-1
+ vit E 100 mg animal-1
1.03 ± 0.12* 2.29 ± 0.13*
vit E 100 mg/ animal 2.8±0.04 1.0±0.1
Number of animals in each group was five (n = 5), data are expressed
as (MeanSE.); *: Significant difference (p < 0.05) comparing to control group.
Brain GSH were significant decreased in treated groups when compared with control
group, were as liver GSH level was decreased in sustanon group when compared
with control group (Table 2).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
140
Table 2 Effect of Sustanon on glutathione in tissue of Rats
Groups Brain Liver
control 2.8 ± 0.07 2.9 ± 0.15
Sustanon 20 mg kg-1 1.5 ± 0.08* 1.4 ± 0.09*
Sustanon 20 mg kg-1
+ vit E 100 mg animal-
1.4 ± 0.12* 2.6 ± 0.12
vit E 100 mg/ animal 2.4 ± 0.12 2.6 ± 0.12
Number of animals in each group was five (n = 5), data are expressed
as MeanSE. *: Significant difference (p < 0.05) comparing to control group.
Pathological Changes
The gross examination showed that the heart was congested and enlarged and
liver congestion (Fig. 1), whereas the lung showed inflammation, congestion and
purulence formation (Fig. 2).
The microscopic changes of heart showed vacuolar degeneration of cardiac
cells, hyalinization of cardiac muscles fibers and edema between it, infiltration of
inflammatory cells, and congestion (Fig. 3). Liver showed necrosis of hepatic cells,
infiltration of inflammatory cells, and congestion of central vein (Fig. 4). Lungs
showed pneumonia with thickening of wall of alveoli and bronchioles by
inflammatory cells, emphysema of alveoli, and congestion of pulmonary blood
vessels (Fig. 5).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Fig. 1: Gross appearance of rat (sustanon group) showed the heart congested and
enlarged (A) and liver congestion (B).
Fig. 2: Gross appearance of rat (sustanon group) showed the lung with inflammation,
congestion and purulence formation.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Fig. 3: Microscopic appearance of heart of rat (sustanon group) showed vacuolar
degeneration of cardiac cells ↑, hyalinization of cardiac muscles fibers↑↑ and edema
between it, infiltration of inflammatory cells (C), and congestion (D). H&E stain.
105X.
Fig. 4: Microscopic appearance of liver of rat (sustanon group) showed necrosis of
hepatic cells (A), infiltration of inflammatory cells (B), and congestion of central vein
(C). H&E stain. 195X.
D
↑
↑
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
143
Fig. 5: Microscopic appearance of lung of rat (sustanon group) showed pneumonia
with thickening of wall of alveoli and bronchioles by inflammatory cells (A),
emphysema of alveoli, and congestion of pulmonary blood vessels (B). H&E stain.
105X.
DISCUSSION
Based on the available literature, the current study is believed to be as one of
the prior studies which investigated the oxidative stress and its relationship with heart,
lung and liver histopathological changes induced by high dose of Sustanon. However,
there are many studies which confirmed the irreversible adverse effects such as
cardiac disorders and chronic hepatitis (1,2). The histopathological changes is
concomitant with increase malodialdihid and decrease glutathione in sustanon groups
that is agreement with (17) Animals body prevent oxidative damage, ranging from
antioxidant enzymes to low molecular weight antioxidants, and also specific cellular
components that repair molecules which is oxidative damaged. A defect in the
balance between pro-oxidants and antioxidants lead to oxidative damage (17).
Pathogenesis of overgrowth of skeletal muscles and heart muscle hypertrophy
in subjects taking large doses of AAS is a complex process. It consist of many factors
including the insulin-like growth factor 1 (ILGF1) and tissue growth factor 1 (TGFβ1)
A
B
B
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
144
(1) that may be the cause for heart hypertrophy in our study in group treated
with sustanon.
In high doses, androgens tend to raise LDL cholesterol levels and lower HDL
cholesterol levels. That’s one of the things that gave testosterone its bad reputation.
but in other circumstances, the situation is very different men with the lowest
testosterone levels tend to have the highest cholesterol levels. Anabolic androgenic
steroids suggest could be possible new risk factor for causing a disease termed
toxicant associated steatohepatitis (TASH) (18). The possible reason for ineffective
role for vitamin E may be law dose which use in experiment The severe lung injury
that report in our study may be because of heart injury or decrease the immunity of
lung because of sustanon treatment (20) and hepatopathologic changes agreement
with another study (19) that showed Sustanon for 15-30-60 day induce hepatotoxic
effect in male rat. These changes are irreversible and progressive for one month after
end of drug treatment (19).
CONCLUSION
The high dose of sustanon can cause severe defects representing by increase
oxidative stress and pathological damages of heart, lung and liver. Also there is no
effect of vitamin E in decrease of oxidative stress effect done by over dose of
sustanon.
ACKNOWLEDGEMENTS
The research was supported by collage of veterinary medicine – University of Mosul.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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ترابط حدوثیة الاجھاد التأکسدی مع السستانون فی ذکور الجرذان
یمامة زھیر صالح العبدلی * , انتصار رحیم الکنانی,** انتصار خزعل الحمدانی **
*فرع الفسلجة والکیمیاء الحیاتیة والادویة، کلیة الطب البیطری، جامعة الموصل، موصل، العراق.
** فرع الامراض وامراض الدواجن، کلیة الطب البیطری، جامعة الموصل، موصل، العراق.
الخلاصة
أجریت الدراسة الحالیة لمعرفة تأثیر السستانون ٢٠ ملغم/کغم على الإجھاد التأکسدی. تم تقسیم ٢٠ من
ذکور الجرذان إلى أربع مجامیع عولجت لمدة ٤ أسابیع مرة واحدة اسبوعیا. المجموعة الأولى اعتبرت مجموعة
سیطرة ، المجموعة الثانیة حقنت بالسستانون ٢٠ ملغم/کغم حقن اً عضلی اً المجموعة الثالثة حقنت عضلی اً
100 ملغم/کغم، اما المجموعة الرابعة E بالسستانون ٢٠ مجم / کغم ثم جرعت مباشرة عن طریق الفم بفیتامین
100 ملغم/کغم . أظھرت النتائج انخفاضا کبیرا فی مستوى الجلوتاثیون E فقد جرعت عن طریق الفم بفیتامین
فی مصل الدم والأنسجة فی الدماغ والکبد ، فی حین أن ھناک زیادة کبیرة فی مستوى بیروکسید الدھن فی مصل
المجموعة المعاملة بعقارالسستانون.
أظھرت النتائج عدم وجود اختلاف بین المجموعات التی عولجت بالسستانون وحده والمجموعة المعاملة
کما أظھرت التغیرات المرضیة العیانیة احتقان القلب والرئة اما التغییرات المجھریة E بالسستانون مع فیتامین
للقلب فقد اظھرت التنکس الفجوی فی الخلایا العضلیة القلبیة والوذمة البینیھ. کما حدث نخر فی الخلایا الکبدیة ،
وارتشاح الخلایا الالتھابیة، بینما أظھرت الرئتین وجود التھاب رئوی مع تثخن جدار الاسناخ والقصیبات
الھوائیة بالخلایا الالتھابیة والنفاخ الرئوی للاسناخ. .
نستنتج من ھذه الدراسة أن السستانون فی الجرع العالیة یکون لھ تأثیر فی زیادة الإجھاد التأکسدی مع حدوث
فی ھذه الدراسة. E تغیرات عیانیة ومیکروسکوبیة فی القلب والرئة، فی حین لم یحدث أی تأثیر لفیتامین
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