Effects of isochaborat testosterone on the kidney rats

Histopatholgical Effects of Testeosterone

Isocaprorate on Kidneys of the Male Albino Rats

"Thesis"

Submitted for partial Fulfilment of Ph.D. degree in Basic Medical Sciences

(Anatomy and Embryology)

By

Mohamed Radhy Ahmed Abd Elbaqy

Assistant Lecturer of Anatomy, Department of Anatomy and Embryology

Faculty of Medicine, AL-Azhar University, Damietta

Supervised by

Prof. Dr. Gamal Sayed Ahmed Desouki

Professor of Anatomy and Embryology

Faculty of Medicine- AL-Azhar University-Cairo

Dr. Ahmed El-Sayed Ahmed Amer

Lecturer of Anatomy and Embryology

Faculty of Medicine- AL-Azhar University- Damietta

Faculty of Medicine- AL-Azhar University

2022

Acknowledgements

Praise to ALLAH, the merciful and the compassionate for all the

countless gifts have been offered. Of these gifts, those persons who gave me the

precious hands, so I have been able to fulfill this review.

I am greatly honored to express my thanks and deepest gratitude to, Prof.

Dr. Gamal Sayed Ahmed Desouki, Professor of Anatomy and Embryology,

Faculty of Medicine- AL-Azhar University, who provided valuable advice and

critical discussions during the whole research period supervised this work. He

also encouraged me in during all preparation stages of this thesis. I am most

grateful to him for his help understanding, support and assistance. He was of

great help to me even before this thesis. His supervision will never be forgotten.

I am greatly honored to express my thanks and deepest gratitude to, Dr.

Ahmed El-Sayed Ahmed Amer, Lecaturer of Anatomy and Embryology,

Faculty of Medicine- AL-Azhar University, for giving me the honor of working

under his supervision, for his continuous encouragement with kind guidance

throughout the whole work, for his encourage creative, comprehensive advice

and support until this work came to existence.

Mohamed Radhy Ahmed Abd Elbaqy

I

2022

List of contents

Acknowledgements.................................... Error! Bookmark not defined.

List of contents ...................................................................................... II

List of Tables ....................................................................................... III

List of Figures...................................................................................... IV

List of Abbreviations............................................................................. V

Introduction............................................................................................ 1

Aim of the work ...................................................................................... 3

Review of Literature ......................................................................... 4-42

Materials and Methods ................................................................... 43-59

Results .............................................................................................. 60-82

Discussion ........................................................................................ 83-88

Summary and conclusion …………………………………………...76

Recommendations ................................................................................ 77

References ........................................................................................ 78-98

........................................................................................... 99 الملخص العربي

II

List of Tables

Table N. Suspect Page N.

Table (1) Data from Food and Drug Administration draft guidelines (FDA) 48

Table (2) Final body weight of the studied rats 52

Table (3) Morphometry among the studied groups 54

Table (4) Some biochemical parameters related to kidney function 55

Table (5) Final body weight distribution among the studied groups 59

III

List of Figures

Figure No. Title Page No.

Figure (1) Kidneys in a cadaver 6

Figure (2) Kidneys in situ 7

Figure (3) Internal anatomy of the kidney 9

Figure (4) Urogenital System of rat 12

Figure (5) Steroid pathways in Leydig cells 13

Figure (6) Regulation of Leydig cell steroidogenesis by luteinizing hormone 15

negative-feedback loop

Figure (7) Chemical structure of testosterone 16

Figure (8) Chemical structures of some synthetic (AAS) 17

Figure (9) Mechanism of action of exogenous anabolic steroids 18

Figure (10) Flowchart of positive and negative effects of (AAS) administration 20

Figure (11) LM of kidney of control male rat 200x 53

Figure (12) LM of kidney of control male rat 400x 54

Figure (13) EM of the DCT of kidney of control group rats 2000x 55

Figure (14) LM of kidney treated with (Sustanon ®250) 50 mg/kg B.W 200x 56

Figure (15) LM of kidney treated with (Sustanon ®250) 50 mg/kg B.W 400x 57

Figure (16) EM of of kidney treated with (Sustanon ®250) 50 mg /kg B.W 58

(2000X)

Figure (17) EM of of kidney treated with (Sustanon ®250) 50 mg /kg B.W 59

(4000X)

Figure (18) LM of kidney treated with (Sustanon ®250) 100 mg/kg 200x 60


Figure (20) EM of of kidney treated with (Sustanon ®250) 100 mg /kg (2000X) 62

Figure (21) EM of of kidney treated with (Sustanon ®250) 100 mg /kg (4000x) 63



Figure (24) EM of DCT of kidney treated with (Sustanon ®250) 150 mg /kg. 66

(2000X)

Figure (25) EM of DCT of kidney treated with (Sustanon ®250) 150 mg /kg.

(4000X)

67

IV

List of Abbreviations

AASs

AR

AREs

DHT

FAI

FSH

GD

GnRH

Hand E

HDL

HPG

IGF

LH

MMP

ND

PSA

RAAS

ROS

SHBG

T/E

T/LH

TM

TU

UGT

VEGF

WADA

Anabolic-Androgenic Steroids

Androgen Receptor

Androgen Response Elements

Dihydrotestosterone

Free Androgen Index

Follicle Stimulating Hormone

Gender Dysphoria

Gonadotropin-Releasing Hormone

Hematoxylin and Eosin

High-Density Lipoprotein

Hypothalamic-Pituitary-Gonadal

Insulin-Like Growth Factor

Luteinizing Hormone

Matrix metalloproteinases

Nandrolone Decanoate

Prostate-Specific Antigen

Renin-Angiotensin-Aldosterone System

Reactive Oxygen Species

Sex Hormone-Binding Globulin

Testosterone to Epitestosterone

Testosterone/Luteinizing Hormone

Transgender Males

Testosterone Undecanoate

Uridine Diphospho-Glucuronosyl Transferase

Vascular Endothelial Growth Factor

World Anti-Doping Agency

V

Introduction

Introduction

It has been reported that young people start using anabolic androgenic

drugs at the age of 16 and researches conducted on the issue show that the

reasons for using this drug are that they want to have a muscular body and

increase their sportive performance (Al-Aubody and AL-Diwan, 2018).

Testeosterone isocaprorate under trade name (Sustanon ®250) is one of

the most commonly used anabolic androgenic drugs to increase skeletal

muscle mass and strength. This drug is ablend of four testosterone derivatives.

Little is known about the effects of this drug on the kidneys at the cellular

level (Ahmed, 2019).

Abusing anabolic androgenic drugs by athletes is a serious negative

phenomenon which is documented by a number of investigators in many

countries around the world including Arab countries. The main factor

responsible for the continuation of this problem among the athletes and the

youth is the absence of the official restrictions on pharmacies selling androgenic

drugs such as (Sustanon ®250) (Abd Hamza and Rashid, 2017).

Unfortunately, many athletes especially in the power sports like

bodybuilding and weight lifting administrate illegally high doses of these drugs

to obtain huge increase in muscular mass and improve their performance during

the international sport competitions ( Rosca Stancu et al., 2020).

Other studies attempted to investigate the adverse effects induced by

Testeosterone Isocaprorate (Sustanon ®250) abuse which is a potent synthetic

testeosterone derivatives. In several countries it was administered

intramusculary and used to increase the growth rates of farm animals. The mode

of action of Anabolic Androgenic Steroids (AAS) in promoting growth is not

1

Introduction

fully understood, but may increas the necrosis, degeneration in epithelial lining

of renal tubules and dilated Bowmanʼs space in male rats with increased the

serum testosterone level and increased production of free radicals which almost

has a central role in the regulation of creatinine clearance in the kidney

(Majhool and Zenad, 2016).

(Sustanon ®250) is a trade name of Testeosterone Isocaprorate which

owned by Organon Pharmaceuticals for an oil-based injectable blend of four

testosterone compounds:

30 mg testosterone propionate

60 mg testosterone phenylpropionate

60 mg testosterone isocaproate

100 mg testosterone decanoate (De Souza et al., 2017).

2

Aim of the Work

Aim of the work

The aim of this study was to determine the effects of Testeosterone

Isocaprorate (Sustanon ®250) by observation changes of histological and

ultrastructurals of kidneys of males' albino rats.

3

Review of Literature


Anabolic Androgenic Steroids (AAS) commonly known as anabolic steroids

which are a large group of molecules including endogenously produced androgens

such as, testosterone as well as synthetically manufactured derivatives (Al-Aubody

and Al-Diwan, 2018).

Testosterone and Nandrolone Decanoate (ND) are the most commonly abused

androgens. AAS used were and still widespread due to their ability to improve muscle

growth for athletes' performance and minimizing androgenic effects. Indeed,

androgens are able to increase the size of muscle fibers as well as muscle strength and

while their used was initially restricted to professional bodybuilders, nowadays it has

become more popular among recreational athletes (Tirla et al., 2021).

AAS have anabolic properties which widely used for therapeutic purposes.

Indeed, AAS had a role in the treatment of chronic kidney disease and osteoporosis in

postmenopausal women, as well as terminal stage of breast cancer. However, used of

AAS for the forbidden by the World Anti-Doping Agency (WADA). However, AAS

used was still very popular and 1–3% of US inhabitants have been estimated to be

AAS users ( Althobiti et al., 2018).

However, AAS have side effects involving all organs. Therefore, their abuse

was considered a public health issue. In this regard, an increased awareness is needed

among the population and healthcare workers, both for diagnostic, therapeutic and

prevention purposes of AAS (Tirla et al., 2021).

4


1.Anatomy of human kidney

The kidneys are pelvi-abdominal organs placed retroperitoneally in the upper left

and right abdominal quadrants and part of urinary system. Their shape resembles a

bean, where we can describe the superior and inferior poles, as well as the major

convexity pointed lateraly, the minor concavity pointed medialy (Liao et al., 2020).

The main function of the kidney is to eliminate excess body waste products,

fluids and salts by filltring mechanism and products of many metabolisms which make

kidneys key in the regulation of acid-base balance, blood pressure and many other

homeostatic parameters (Curry et al., 2020).

The kidneys have their anterior and posterior surfaces. The anterior surface faces

towards the anterior abdominal wall, where as the posterior surface is facing the

posterior abdominal wall (Tsujimoto et al., 2020).

1.1.External anatomy

The kidneys are located between the transverse processes of (T12-L3) vertebrae,

with the left kidney typically positioned slightly higher than the right. This is because

the liver forcing the right kidney a bit down. The superior poles (extremities) (T12) of

both kidneys are more medial pointed towards the spine than the inferior poles

(extremities) (L3). The hilum of the kidney usually projects at the level of the (L2)

vertebrartery Thus, the ureter is seen paravertebrally starting from the (L2) and going

downwards (Ma et al., 2020).

The medial border of the kidney contains the hilum of the kidney, which is the

entry and exit point for the kidney vessels and ureter (Ong et al., 2020).

The most anterior vessel is the renal vein which exits the kidney, just behind it is

the renal artery that enters in, and The most posterior is the exiting ureter. It is important

to remember this order of vessels and ducts since this is the only thing that will make

5


you able to orient the kidney and differentiate the left one from the right when they are

outside of the cadaver (figure 1) (Barwinska et al., 2020).

Figure (1): Kidneys in a cadaver: From superior to inferior and from anterior to posterior, you will find the renal vein,

followed by the renal artery, and ending with the ureter (Barwinska et al., 2020).

The kidney is protected by layers that surround it which are:

The fibrous capsule (renal capsule).

The perinephric fat (perirenal fat capsule).

The renal fascia which besides the kidneys also encloses the suprarenal gland

and its surrounding fat (Chacon‐ Caldera et al., 2020).

Outside the fascia is the most superficial layer of fat tissue called the

perinephric fat. This layer sits posterolateral to each kidneys and separates it from the

muscles of the posterior abdominal wall (Kosovic et al., 2020).

6


Figure (2): Kidneys in situ (Minuth, 2020).

1.1.1.Relations

1.1.2.Anterior surface of right kidney

The highest portion of the superior pole is covered with the right suprarenal

gland. The superior one-half is in contact with the layer of peritoneum that separates it

from the liver. This potential space that separates the liver from the right kidney is

called the hepatorenal pouch of Morison (fig. 2) (Minuth, 2020).

The center is touched by the retroperitoneal posterior wall of the 2 nd part

of duodenum (Wang et al., 2020).

The lateral part of the inferior pole is directly contacted with the right colic

flexure (Kosovic et al., 2021).

The rest of the inferior pole is associated with the peritoneum of the small

intestine, more precisely the jejunum (Van Daal et al., 2020).

1.1.3.Anterior surface of left kidney

Since the abdominal organs are not paired, the left kidney is not related to the

same organs as the right kidney (Peired et al., 2020).

7


The anterior surface of the left kidney has the following relations: the highest

part of the superior pole is covered with the left suprarenal gland. The inferior portion

of the superior pole contacts with the peritoneum of the stomach medialy

and spleen lateraly (Brand et al., 2020).

Just inferior to the stomach and spleen impressions, where the left kidney

directly contacts with the pancreas. The lateral part of the inferior half is directly

associated with the left colic flexure and descending colon (Krebs et al., 2020).

The medial part of the inferior half and the inferior pole are contacted by the

peritoneum of the jejunum (Phillips et al., 2020).

1.1.4.Posterior surface relations

The posterior surfaces of both kidneys are related to certain neurovascular

structures and muscles which are subcostal artery, 11 th and 12 th ribs, subcostal,

iliohypogastric, ilioinguinal nerves, diaphragm, psoas major, quadratus lumborum and

transversus abdominis muscles (Ding et al., 2021).

The superior half of each kidney is covered by diaphragm, which is why the

kidneys move up and down during respiration (Li et al., 2020).

The muscular relations of the inferior half where the medial stripe represents the

impression of the psoas major muscle, the central represents the impression of the

quadratus lumborum muscle and the lateral represents the impression of the transversus

abdominis muscle (Shirk, 2020).

1.2.Internal anatomy

The parenchyma of the kidney consists of the outer renal cortex, and inner

renal medulla (Davies et al., 2020).

8


Figure (3): Internal anatomy of the kidney (Xie et al., 2020).

The main unit of the medulla is the renal pyramid. There are 8-18 renal pyramids

in each kidney that on the coronal section look like triangles lined next to each other

with their bases directed toward the cortex and apexes toward the hilum. The apex of

the pyramid projects medial toward the renal sinus. This apical projection is called

the renal papilla and it opens to the minor calyx (fig.3) (Xie et al., 2020).

The minor calyces unite to form a major calyx. Usually, there are two to three

major calyces which unite to form the renal pelvis. The pyramids are separated by

extensions of the cortex called the renal columns (Parvin et al., 2020).

The pyramids contain the functional units of the kidney, the nephrons, which

filter blood to produce urine which then is transported through a system of the

structures called calyces which then transport the urine to the ureter (Schroeder et al.,

2020).

9


1.2.1.Nephron

Ultrastructurally, the nephron is the functional representative of the kidney. Each

nephron contains a renal corpuscle and renal tubule (Wen et al., 2020).

The glomerulus is a web of arterioles and capillaries, with a special filter which

filters the blood that runs through the capillaries (Maly et al., 2020).

The glomerular membrane is designed in a way in which it is not permeable for

big molecules in blood, but it is permeable to the smaller substances (Ullah et al.,

2020).

So in the filtered fluid that goes to the renal tubule, we have both necessary and

unnecessary substances. Because of this, the tubules are designed in a way that

they reabsorb the necessary substances and carries them back to the blood whereas they

do not absorb but rather secrete unnecessary substances (Jang et al., 2020).

In this way, the consistency of blood is preserved and no important substances

are lost. On the other hand, the products of cellular metabolism and drug metabolites

are eliminated from the blood which prevents their depositing in the body and potential

toxicity (Liu et al, 2020).

1.3.Vasculature and lymphatic drainage

1.3.1.Arteries

Each kidney is supplied by a single renal artery, arises from the abdominal

aorta. Both renal arteries arise just below the superior mesenteric artery. In addition to

the renal artery, accessory renal arteries are present too (Hughson et al., 2020).

When the renal arteries enter the kidneys through the hilum, they split into

anterior and posterior branches. The posterior branch supplies the posterior part of

kidney, whereas the anterior branch devides into five segmental arteries, each supplying

a different renal segment. The segmental arteries then branches into the interlobar

arteries, which further branch into the arcuate arteries. Finally, the arcuate arteries

branch into the interlobular arteries which branch off even further by giving afferent

10


arterioles to run blood past to the glomerulus for blood filtration (Barwinska et al.,

2021).

Each kidney has a single renal vein which conducts the blood out of the kidney

and is positioned anterior to the artery. The renal veins drains to the Inferior Vena Cava

(IVC), so the right vein is shorter because the I.V.C. runs closer to the right kidney. The

left renal vein passes anteriorly to the aorta just below the trunk of the superior

mesenteric artery. Concerning lymphatic drainage, each kidney drains into the lateral

aortic (lumbar) lymph nodes, which are placed around the origin of the renal artery

(Kurts et al., 2020).

1.3.2.Innervation

The kidneys are innervated by the renal plexus. This plexus provides input from

the lower thoracic splanchnic nerves (sympathetic) and from the vagus nerve

parasympathetic. The sensory nerves from the kidney travel to the spinal cord at the

levels (T10-T11), which is why the pain in the flank region always rises suspicions that

something is wrong with the corresponding kidney (Tanabe et al., 2020).

2.Anatomy of rat kidney

2.1.Urogenital System

The excretory and reproductive systems of vertebrates are closely integrated

with the urogenital system. However, they do have different functions: the excretory

system removes wastes and the reproductive system produces gametes (sperm and

eggs) (Andersen et al., 2020).

2.2.Excretory Organs

The primary organs of the excretory system are the kidneys. Locate these large

bean shaped structures located toward the back of the abdominal cavity on either side

11


of the spine. Renal arteries and veins supply the kidneys with blood (Akison et al.,

2020).

Trace these vessels to where they connect to the aorta and IVC. Locate the

delicate ureters that attach to the kidney and lead to the bladder ( Velosa et al., 2021).

The cortex (the outer area) and the medulla (the inner area). The urethra carries

urine from the bladder to the urethral orifice (Tomar et al., 2020).

The small yellowish glands embedded in the fat atop the kidneys are the adrenal

glands (fig. 4) (Packialakshmi et al., 2020).

Figure (4): Urogenital System of rat (Packialakshmi et al., 2020).

12


3. Biochemistry

3.1.Steroidogenesis

Testosterone in males is derived from cholesterol and synthesized in the Leydig

cells located in the testicular interstitium (Fig. 5) (Arazi et al., 2017).

Figure (5): Steroid pathways in Leydig cells ( Arazi et al., 2017).

Approximately 95% of circulating testosterone in men was produced by the

Leydig cells and the remaining is derived from the adrenal gland. The testes in a

normal man secretes about 6-7 mg testosterone daily. In contrast, testosterone in

females is mainly produced in the ovaries and adrenal glands, but in about 10 times

lesser amount (Matsumoto et al., 2022).

After testicular secretion, testosterone is disposed along four major pathways,

one direct pathway where testosterone binds to the androgen receptor of skeletal

muscles and one pathway where a testosterone is converted to the more potent

13


androgen 5α dihydrotestosterone (DHT), characteristically expressed in the prostate

but also at lower levels in skin and the kidney (Jayasena et al., 2022).

Testosterone production follows a diurnal rhythm, with a peak concentration in

the morning between 7 and 10 am, with declining concentration during the day, and

rising again at night during sleep. It has been reported that food intake can lower

circulating testosterone levels up to 30% compared to fasting conditions (Hallak et

al., 2020).

Despite this evidence, most samples for testosterone analysis are today not

taken in a fasting state, as the reference ranges used in clinical practice are not based

on fasting values (Fortin and Moravek, 2020).

Epitestosterone is a naturally occurring 17-hydroxy epimer of testosterone.

Epitestosterone is produced by the testis, but has no biological activity (Saitoh et al.,

2017).

Epitestosterone is neither a metabolite nor a precursor of testosterone. The

nearly constant ratio of urinary testosterone to epitestosterone (T/E) of approximately

1 made it attractive as a reference substance in detection of exogenous administered

testosterone (Jia et al., 2022).

3.2. Regulation of testosterone synthesis

The circulating levels of testosterone in males are regulated by the

hypothalamic-pituitary-gonadal (HPG) axis, via a negative feedback loop (Zhang et

al., 2021).

14


Figure (6): Regulation of Leydig cell steroidogenesis by luteinizing hormone and rapid negativefeedback

loop. Gonadotropin releasing hormone (GnRH) stimulates synthesis and secretion of both

luteinizing hormone (LH) and folliclestimulating hormone (FSH) (Forsdahl et al., 2020).

Gonadotropin-releasing hormone (GnRH), released from the hypothalamus,

stimulates the synthesis and secretion of the gonadotropins luteinizing hormone (LH)

and follicle stimulating hormone (FSH) from the anterior pituitary gland (Fig. 6)

(Forsdahl et al., 2020).

LH stimulates the synthesis of testosterone in both sexes, and FSH along with

intra-testicular testosterone (Devi et al., 2018).

LH is mainly regulated by testosterone via negative feedback. The negative

feedback on FSH is affected by inhibin, a gonadal hormone produced by the Sertoli

cells in the testicles, which control the secretion of FSH (Solheim et al., 2021).

15


3.3.Synthetic derivatives of testosterone

Testosterone ingested orally in its unmodified form has no significant effect due

to the first-pass effect of the kidney. To circumvent this, synthetic AAS with

modifications of the testosterone molecule have been designed to reduce the rate of

metabolism, maximize the anabolic effect, and minimize the undesired androgenic

side effects (Rejtharová et al., 2017a).

Figure (7): Chemical structure of testosterone (4-androsten-17-ol-3-one) (De Moura Ribeiro et al.,

2018).

There are three main classifications of androgen analogs. Class A modifications

are esterification of the 17-hydroxyl group with any of the several carboxylic acid

groups (testosterone cypionate). The longer carbon chains increase the lipophilic

properties that makes the molecule more soluble in lipid vehicles. Intramuscular (I.M.)

injection of testosterone esters result in a gradual release, thereby slowing the

absorption of testosterone. Class B analogs have been alkylated at the 17-hydroxy

position. Class C are produced by modification of the A, B or C ring (Fig. 7) (De

Moura Ribeiro et al., 2018).

16


Figure (8): Chemical structures of some synthetic anabolic androgenic steroid (Tircova et al., 2019).

Alkylated analogs and those with modified ring structure are relatively resistant

to hepatic metabolism and are therefore available for oral use (Fig. 8) (Tircova et al.,

2019).

4. Physiology of AASs

The anabolic androgenic effects are related to the androgen receptor (AR)

signaling action. Androgen receptors are widespread in human tissues and organs (

Salonia et al., 2020).

In particular, free testosterone is transported into target tissue cell cytoplasm,

binding to the AR takes place either directly or after conversion to

5αdihydrotestosterone (DHT) (Mulrooney et al., 2019).

17


After dimerization the complex binds to specific promoter areas of target genes

called Androgen Response Elements (ARE), influencing the transcription process

(Rejtharová et al., 2017).

4.1.Pathophysiology of AASs

The most relevant mechanisms that lead to the increase of AAS in circulation

are administration of testosterone or its synthetic derivatives or administration of

drugs that raise endogenous testosterone production. The mechanism of action of AAS

in supraphysiological doses is characterized by the impairment of testosterone

biosynthesis in tissues ( Fig. 9) (Yoon et al., 2019).

Figure (9): Mechanism of action of exogenous anabolic steroids: an anabolic steroid is transported

into the target tissue cell cytoplasm where it can either bind the androgen receptor, or be reduced by

the cytoplasmic enzyme 5-alpha reductase. The N-receptor complex undergoes a structural change

that allows its translocation into the cell nucleus, where it directly binds to specific nucleotide

sequences of the chromosomal DNA The produced DNA interferes with the physiological

biosynthesis of testosterone (Yoon et al., 2019).

18


AAS exert their effects by activating androgen receptor (AR) signaling. Several

parts of the body are involved because of the presence of ARs in many tissues. Indeed,

high dose AAS displace glucocorticoids from their receptors, decrease proteins

breakdown in muscles, leading to an increase in muscle mass and muscle strength

(Rejtharová et al., 2018).

The inhibition of glucocorticoid action is also due to the stimulation of growth

hormone (GH) and insulin-like growth factor (IGF)-1 axis. High doses of AAS exert

an antiestrogenic effect due to a down-regulation of androgen receptors and a

competition with estrogens with their receptors (Cai et al., 2019).

In this regard, the beneficial effect of physical activity in diminishing oxidative

stress as a consequence of the up regulation of antioxidant enzymes. Such data have

also been confirmed that investigated the effects of a supraphysiological

administration of testosterone enanthate (500 mg). In this study an impairment of

endothelial function as a consequence of the dysfunction of antioxidative capacity

following testosterone administration was demonstrated (Christou et al., 2019).

Furthermore, oxidative stress plays a leading role in AAS mediated

neurotoxicity androgens may be neuroprotective in cases of low levels of oxidative

stress, however, they may increase brain damage in cases of elevated oxidative stress

(Christoffersen et al., 2019).

AAS related damage is also associated with apoptosis activation. Indeed, it was

demonstrated that supraphysiological concentrations of AAS may induce

neurotoxicity by involving the apoptotic process and neurodegeneration (Aljubori,

2019).

Moreover, environmental and inflammation stress can lead to proteotoxic

damage and dysregulate heat shock proteins. In this regard, an activation of the

extrinsic pathway of the apoptosis in the vascular smooth muscle cells in rats treated

with testosterone was observed (Marocolo et al., 2019).

19


Figure (10): Flowchart of positive and negative effects of anabolic-androgenic steroid (AAS)

administration. Prolonged and high doses of testosterone and his derivatives lead to serious

consequences in all body tissues and organs (Bazm et al., 2019).

The results of a study demonstrated that the presence of AAS metabolites in

urine may be a predictive factor of cardiac changes in AAS abusers. Oxidative stress,

apoptosis, inflammation are responsible for multi-organ damage in AAS abusers (Fig.

10) (Bazm et al., 2019).

20


Moreover, testosterone has anti-inflammatory effects and improves insulin

sensitivity because of its capacity to reduce the expression of pro inflammatory

cytokines, and reduce the circulation of inflammatory cells (Lazuras et al., 2019).

Recent clinical and experimental studies proved that the increased activity of

the renin-angiotensin-aldosterone system (RAAS) plays a pivotal role in the

pathogenesis of cardiological diseases (Elboga et al., 2019).

AAS are characterized by the activation of protein synthesis. A similar

mechanism was described in skeletal muscles: increased protein synthesis, a decrease

in protein breakdown, the elevated formation of new myotubes and myonuclei lead to

an increase in muscle mass and strength as well as an increased exercise capacity

(Ansah and Apaak, 2019).

5. Use and abuse of AAS

5.1.Androgenic disorders

The primary clinical use of testosterone is replacement therapy for androgen

deficiency. This includes male hypogonadism (HG), to a decrease in testosterone

synthesis. Testosterone deficiency can either results from primary testicular disorder

or occur secondary to hypothalamic-pituitary dysfunction, or as a combination of both

the defects (Da Justa Neves and Caldas, 2017).

The clinical diagnosis of hypogonadism is based on consistent symptoms and

signs of androgen deficiency in combination with a subnormal serum testosterone

concentration. A serum testosterone level < 8nmol/L suggests deficiency, while a

level above 12 nmol/L is considered normal (Alves et al., 2018).

Testosterone is available in three different formulations for clinical replacement

therapy. Long-acting I.M. injections of testosterone undecanoate (TU) (Nebido®)

1000 mg every 12th week is the most commonly used treatment regimen (Mullen et

al., 2020).

21


Transdermal preparations have the advantage to mimic the normal

physiological diurnal rhythm. This preparation is applied to the skin once a day in

doses of 50-100 mg (1%) testosterone. Oral TU is not normally used in clinical

therapy, due to the low bioavailability necessitating administration three times a day

(Albano et al., 2021).

Monitoring of long-term testosterone replacement therapy should according to

the Endocrine Society Guidelines include measurements of total serum testosterone,

haematocrit and prostate-specific antigen (PSA) at 3 to 6 months and at 12 months and

annually after initiating testosterone therapy (Liu and Wu, 2019).

There has been increase in testosterone in most countries over more than a

decade. Testosterone prescription in Egypt has increased three fold over 13 years

(2006-2019), rising from 6600 to 17,100 individual male patients 40 years or older

(Harvey et al., 2019).

Testosterone is also used off-label in cross-sex hormone treatment in females

with gender dysphoria (GD). GD is defined as the feeling of discomfort in individuals

whose gender identity differs from their sex assigned at birth (Anawalt, 2019).

The number of persons with a GD diagnosis has increased during the last five

years. The increase has been most pronounced among children and adolescents aged

13-17, especially among individuals assigned female at birth (Santos and Coomber,

2017).

The treatment for individuals diagnosed with GD includes psychotherapy,

cross-sex hormone treatment and sex reassignment surgery if the patient desires. The

physical changes induced by testosterone replacement therapy in females-to-males

(transgender males (TM)) (Zahnow et al., 2018).

The Clinical Practice Guidelines for treatment of gender dysphoric persons,

suggest that total serum testosterone should be monitored every third months during

the first year of hormone therapy and then once or twice yearly (Baggish et al., 2017).

22


5.2.Abuse of AAS

The main reason for using AAS is the desire to increase muscle mass and

strength to enhance athletic performance. Other motives reported by males in a study

at an out-patient clinic specialized in treatment of addiction in adults, were to become

more aggressive/braver, to alleviate insecurity or in preparation of committing a crime

(Christoffersen et al., 2019).

Cognitive function may also be impaired by AAS abuse. Weight lifters exposed

to AAS had lower cognitive functions (Kanayama et al., 2020).

AAS are used in complex programs of so called “cycling”, stacking and

pyramiding”. Cycles of 6-12 weeks are often used with complete abstinence inbetween

in the attempt to minimize side-effects. However, continuous use is also

frequent (Anawalt, 2018).

In pyramiding, a low initial dose of AAS is administered which is gradually

increased, often 5-100 times the therapeutic doses, and towards the end of the cycle

tapered off. The rationale for this abuse pattern is the expectations to avoid withdrawal

symptoms (Smit and Mortali, 2018).

5.3.Administration of AAS

AAS are available in a wide range of various preparations. The most commonly

used form of testosterone administration is I.M. injections of testosterone esters.

Testosterone esters administered as a depot injection diffuses slowly into the

bloodstream and even if the cleavage process by esterase enzymes starts immediately,

the testosterone ester is still detectable in blood (Kanayama et al., 2020).

The elimination of I.M. testosterone esters is suggested to be absorption ratelimited

depending on the length of the ester side chain, testosterone enanthate showed

a half-life of 4.5 days compared to 29.5 days for testosterone undecanoate (Bjrnebekk

et al., 2017).

23


5.4.Multisubstance use

Some athletes consume multiple drugs in addition to AAS which can interact

adversely with AAS. Polydrug assumption makes it hard to attribute the observed

effects to a single drug. AAS effects are also related to sex, dose and duration of

administration. In this regard, most of the effects are observed after long-term

administration (Bonnecaze et al., 2020).

Studies have reported that the abuse of AAS is often combined with the misuse

and abuse of other drugs. The reasons given by AAS users for combining AAS with

use of other drugs were to enhance the effects of AAS or to counteract the side-effects

of AAS was used to increase endurance and burn fat (Torrisi et al., 2020).

Furthermore, a mixed drug abuse was observed in autopsied AAS users, who

also were found in violent death than users of other drugs, suggesting a particular high

risk for AAS users to get involved in violence or to develop depressive symptoms

(Pope et al., 2017).

Several mechanisms are involved in AAS adverse effects. This is possibly due

to the widespread presence of androgen receptors AR in the body and to the

impairment of biosynthesis, transformation and degradation of endogenous steroids.

AAS bind to a specific type of androgen receptor and by the time the receptors are

saturated, AAS in supraphysiological doses may lead to secondary effects (Mulrooney

et al., 2019).

As we mentioned before the prolonged misuse and abuse of AAS can leads to

several adverse effects, some of which may be even fatal especially the ones regarding

the renal failure and sudden cardiac death (Solimini et al., 2017).

The toxicological investigations executed mostly on urine samples but also on

blood, by performing several screening tests, showed the presence of AAS and/or their

metabolites in urine specimens in 12 cases; in one case sustanon was detected in

blood, while in another case stanozolol was detected in a hair sample (Harvey et al.,

2020).

24


Autopsy plays a pivotal role in the study of AAS adverse effects and organs

damage related to their abuse. Moreover, autopsy studies may provide useful

information regarding the pathophysiology of the effects of AAS long-term

administration (Hauger et al., 2020).

C.N.S. and A.A.S.

The neurotoxic action of AAS is associated with both membrane AR and G-

protein coupled receptors. Furthermore, several studies highlighted the role of

apoptosis in determining brain damage. Indeed, it was demonstrated that high

concentrations of methandienone and 17-a-methyltestosterone provoke detrimental

effects on neuron cell cultures expressing AR, leading to cell death (Hearne et al.,

2021).

The presence of apoptosis in brain areas of rats treated with long-term

administration of nandrolone was suggested in a recent study. Furthermore, it was

found that daily injections of stanozol in male adult rats for 28 days led to

histopathologic changes in the hippocampus (Zahnow et al., 2020).

Recent evidence, by administrating neuropsychological tests to weightlifters

both AAS users and nonusers, demonstrated a cognitive disfunction due to long-term

high AAS exposure. In this regard, oxidative stress and apoptosis due to AAS abuse

may lead to neurodegeneration (Börjesson et al., 2020).

AAS in supraphysiological concentrations influence several central nervous

system functions, The underlying mechanisms involve neurotransmission by affecting

the synthesis and degradation of neurotransmitters, as well as neurotransmitter

metabolism (Ven et al., 2020).

In addition, an animal study suggested that long-term administration of ND

leads to anxiolytic behavior and memory impairment. Moreover, chronic AAS

administration changes neurotransmitter expression (Westlye et al., 2017).

25


Lastly, AAS may induce NMDA receptor phosphorylation in order to increase

excitatory neurotransmission, resulting in an increment of aggression. Long-term

research is needed to clarify the mechanisms and the organic and social processes

involved in neuropsychiatric effects of AAS abuse (Havnes et al., 2020).

Cardiovascular System

Not with standing the elevated morbidity and mortality, cardiac and metabolic

consequences of AAS abuse are still unclear. Cardiac injury is the most frequent

consequence of the administration of exogenous steroids, compared with the

remaining body tissues and organs (Bjrnebekk et al., 2019).

Moreover, it was demonstrated that after administration of AAS treated animals

lost the adaptive response of exercise-induced amelioration of antioxidant activity

(Bates et al., 2019).

When performing an autopsy in a sudden death case involving a young athlete,

attention to the physical phenotype is mandatory in order to suggest AAS abuse and

perform a detailed examination of the heart (Zahnow et al., 2017).

Other studies found increased low-density lipoprotein (LDL) cholesterol levels

after long-term AAS administration, underlining the promotion of athe rogenesis of

these substances. An increased sympathetic activity was observed after AAS

administration (Struik et al., 2018).

AAS users show multi organ dysfunction compared to non-users. Long-term

training associated with AAS administration reduces left ventricle relaxation

properties. In this regard, the use of AAS is associated with the loss of the effects on

left ventricle function (Smit et al., 2020).

Atrial fibrillation is the most frequent event but ventricular arrhythmias and

sudden cardiac death were described in literature (De Ronde and Ferretti, 2020).

In addition, 3% of AAS users had myocardial infarction as a consequence of

atherosclerotic disease. Experimental data showed that in animal treated with AAS

26


there were increased thrombotic stimuli. The hypothesized mechanisms are proarrhythmic

effects of AAS, induction of myocardial ischemia, structural changes

(Christiansen and Liokaftos, 2017).

New imaging tools, such as magnetic resonance, may give fundamental

information regarding myocardial tissue in these cases (Nicholls et al., 2017).

Clinicians must be aware of the mechanisms involved in cardiotoxicity, the

pathological and clinical consequences, as well as the diagnostic tools to highlight

cardiac damage in AAS abusers, in order to consider AAS abuse in differential

diagnosis and undertake primary and secondary prevention in their patients (Havnes

et al., 2021).

Liver

Hepatotoxicity is one of the most frequent side effects of AAS abuse. AAS

induced hepatotoxicity was hypothesized to be related to oxidative stress in hepatic

cells (Sessa et al., 2020).

Two of the most common kidney consequences following supraphysiological

doses of AAS are peliosis and cholestasis (Chang et al., 2018).

In addition, animal studies demonstrated that bile accumulation can be a

consequence of the reduction of his transportation ability. However, AAS associated

cholestasis is not characterized by the presence of necrosis and inflammation lead to a

regenerative signal in AAS induced Hepatotoxicity (Havnes et al., 2020).

A correlation between hepatocellular adenoma and androgen steroid therapy

was described in the literature and the risk of androgen-associated kidney tumor seems

to be related to the dose and the potency of AAS administration. (Bates et al., 2019).

Urinary System

Several studies highlighted that prolonged androgen exposure has a direct toxic

effect on kidneys, especially glomerular cells, causing accumulation of mesangial

27


matrix, podocyte depletion and structural adaptations. In this regard, kidney tissues are

characterized by the expression of ARs. AR activation leads to cell growth and

hypertrophy in the kidney (Frude et al., 2020).

Prolonged administration of ND in mice has been shown to cause dosedependent

oxidative kidney stress and damage. Mice kidneys treated with ND

exhibited increased lipid peroxidation and decrease antioxidant enzyme activity

(Vaskinn et al., 2020).

Morphological changes were observed in mice treated with ND. Three months

after I.M. injection of androgen, several histopathological alterations were detected:

glomerular atrophy and fragmentation, tubular wall rupture, vacuolar degeneration of

the epithelium lining of the proximal convoluted tubules and blood hemorrhage

between the tubules, basal lamina thickening in distal convoluted tubules and tubes

with only the basal lamina, many hyaline cylinders, some areas of necrosis,

eosinophilic cell cytoplasm, which is a sign of chronicity and vascular congestion,

were found in kidney samples (Ding et al., 2021).

As in other tissues and organs, oxidative stress, apoptosis and inflammation

play a pivotal role in urinary system damage. This information is fundamental for

therapeutic and prevention measures (Ribeiro et al., 2018).

Muscoloskeletal System

Muscle mass seems to be influenced by AAS administration. In fact,

testosterone, by binding to AR, produces an increased production of IGF-1, a

decreased expression of myostatin (Ganson and Cadet, 2019).

Moreover, the decreased expression of Vascular Endothelial Growth Factor

(VEGF) may play a role in skeletal damage due to AAS, as a consequence of poor

remodeling. Nevertheless, AAS could also be involved in tendon damage (Wang et

al., 2021).

28


It was demonstrated that ND increased tendon remodeling despite decreases in

Matrix metalloproteinases (MMP) activity in rat tendons. However, AAS related

MMP dysregulation still needs to be better clarified. Esthetic purposes, increase of

muscle mass and strength are one of the most frequent reasons why young people and

athletes are AAS abusers (Bjrnebekk et al., 2021).

Reproductive System

Androgens play a pivotal role in the development of male reproductive organs.

A recent study focused on Leydig cell cultures treated with ND, demonstrating an

impairment of testosterone production (Nagata et al., 2020).

Never theless, recent findings support the hypothesis that increased frequency

and duration of high-dose AAS lead to sexual dysfunctions following discontinuation

(Al-Harbi et al., 2020).

Animal histological studies of liver demonstrated hepatotoxicity was

hypothesized to be related to oxidative stress in hepatic cells due to AAS use (Sessa

et al., 2020).

Apoptosis has been reported to play an important role in the regulation of germ

cell populations in the adult testes. The correlation between apoptosis and high AAS

doses and exercises has recently been experimentally assessed in animal models

(Zelleroth et al. 2019).

According to the length of use of AAS and the period since the last drug

administration prior to the survey, changes in sperm parameters were observed in a

study among bodybuilders compared to healthy volunteers (Abrahin et al. 2017).

According to recent data, 20% of patients who were being treated for

symptomatic hypogonadism had previously used AAS. Management strategies for

male infertility secondary to AAS induced hypogonadism should focus on the

underlying hypogonadal state (Greenway and Price, 2018).

29


According to a recent study, chronic AAS abuse should be considered when a

muscular man presents with hypogonadism, onset of gynecomastia (Pope et al.,

2021).

Hematologic Consequences

Before the introduction of recombinant human erythropoietin, AAS were used

in the treatment of anemials; indeed, AAS are capable of increasing erythropoietin

secretion. AAS abuse has been recurrently associated with an increased risk of

thrombosis (Hernández-Guerra et al., 2019).

However, the association has primarily been based on case reports. Increased

LDL and decreased HDL are linked to an increased cardiovascular risk. Mild, but

significant, increases in mean RBC s , hematocrit, Hg , and WBC s concentrations in 33

men were described after IM testosterone enanthate, 200 mg every 3 or 4 weeks for 24

weeks (Hill and Waring, 2019).

The influence of AAS on plasma concentration and function of coagulation

factors depends on the substance and the dose of the AAS (Sandvik et al., 2018).

A recent report suggested a possible correlation between AAS abuse and

immunodeficiency that may be related to a mimicking action of corticosteroid activity

(Harvey et al., 2022).

AAS and Cancer

The biochemical mechanism of AAS is similar to that of testosterone. AAS

bind to DNA sequences. In a recent review regarding androgen effects on cellular

functions, it was stated that a combination among genetic and epigenetic factors is the

cause of toxicity (Vilar Neto et al., 2021).

However, AAS related genotoxicity still remains unclear. Epigenetic molecular

mechanisms, which lead to a genetic transcription control, are: DNA methylation,

histone and chromatin (Chegeni et al., 2021).

30


Furthermore, during their catabolism, AAS reveal their oxidative role, increase

reactive oxygen species (ROS) production, which are highly unstable, , form covalent

bonds with DNA bases, and may induce genetic damage (Zoob et al., 2021).

It has been suggested that the incidence of cancer in different tissues is strictly

positively correlated to the number of stem cell divisions in the lifetime occurring in

them (Salerno et al., 2018).

The side effects on the natural synthesis of AAS play a role in hormonal

changes and it could be suspected to be at the base of certain carcinogenic

mechanisms (Brennan et al., 2018).

Given that it was demonstrated a correlation between AAS abuse and cancer,

the prevention of its abuse and the information campaigns in gyms and among young

athletes are mandatory (Wood and Serpa, 2020).

Determination of AAS in biological matrices

Exogenous AAS

In forensic toxicology investigations and in anti doping testing, detection of

AAS is mainly performed in urine. Although urine in general is a very suitable matrix

for determination of several substances with long detection windows, there are some

issues particularly in testosterone testing (Huml et al., 2021).

The metabolic reactions include phase I and phase II metabolism aiming to

convert the compounds into less potent, more polar and water soluble metabolites

(Kildal et al., 2022).

Urinary testosterone

Detection of exogenous testosterone is based on the determination of

testosterone glucuronide/epitestosterone glucuronide ratio (T/E). A T/E ≥12 together

with a ratio of testosterone/luteinizing hormone (T/LH) >400 nmol/IU is considered as

31


a sign of illegal administration of testosterone in forensic investigations (Machek et

al., 2020).

This methodology is not directly applicable to females, because oral

contraceptive therapy suppresses LH secretion (Schneider et al., 2022).

This is however a complex and expensive technique and is not used in forensic

doping investigations (McBride et al., 2018).

Serum testosterone

The circulating testosterone is to approximately 97-98% bound to plasma

proteins. In male is 44% and in females 66% of testosterone bound with high affinity

to sex hormone-binding globulin (SHBG) and the remaining major part is with much

lower affinity bound to human serum albumin, leaving only 1-2% as free circulating

testosterone (Lykhonosov and Babenko, 2019).

The free hormone hypothesis, which has been questioned, states that only

unbound testosterone is biologically active in target tissues. It has also been suggested

that SHBG bound testosterone can act on prostate and testicles (Butzke et al., 2022).

Although various procedures have been described for the measurement of free

testosterone. These methods are too complex and time consuming for routine use in

clinical laboratories (Nelson et al., 2022).

The accuracy of the calculations largely depends on the methods used for

measurement, SHBG concentrations, choice of affinity constant and other factors

(Magnolini et al., 2022).

Recently it was shown that the binding of testosterone to SHBG is a more

complex process including multi-step interactions and that this new model better

correlate to equilibrium dialysis in both men and women (Zaami et al., 2021).

32


Salivary testosterone

Saliva is attractive as a diagnostic matrix because salivary steroid levels are

supposed to reflect the free circulating levels in plasma as only free neutral lipidsoluble

and unconjugated molecules are able to pass through the acinar cells of the

salivary glands into the saliva (Bertozzi et al., 2019).

The transfer of free biomolecules from the circulating blood through the cell

membrane to oral fluids occurs through different mechanisms depending upon the

physiochemical properties of the molecule (Ip et al., 2019).

However, there are several factors that can influence the process of sample

collection. The largest confounder of salivary concentrations of drugs is blood leakage

into the oral mucosa (Souza et al., 2017).

Increased testosterone levels were observed immediately after tooth brushing

and remained elevated for 30 minutes. In testosterone testing, samples should be

visually inspected for blood contamination (Benjamin et al., 2020).

In addition, quantitative testing results can be affected by food and drink intake.

It was however concluded that this is not an important source of error in testosterone

measurement, due to the rapid passage by passive diffusion (Havnes et al., 2021).

The most common sample collection methods used for measurement of steroid

hormones have over time been cotton or synthetic swabs and collection of whole

saliva by passive drool (Bejtkovský and Snopek, 2021).

It has been suggested that synthetic swabs absorb testosterone and that the

results with cotton swabs might be due to interfering substances present in the cotton

(Corona et al., 2022).

33

Materials and Methods


All experiments were approved by the ethical committee of Al Azhar

University, Egypt and were done in accordance to the NIH recommendation and

guidelines of Committee for the Update of the Guide for the Care and Use of

Laboratory Animals (2011).

1.Materials

1.1.Animals used and housing:

The study was conducted using 40 mature male albino rats.

All rats were healthy, weighing (250 – 350) gm. and 8-10 weeks old at

the time when the experiment started. The animals were kept in 12/12 hours

light/dark schedule during the experimental study. The rats were fed with

standard laboratory chow containing 0.5% NaCl, 22% protein and 4-6% dietary

fat and allowed to drink water.

Rats will be obtained from the animal house of Medical Nile Center for

Experimental Research in Mansoura

The animals were bred and housed in plastic cages (56 x 39 x 19 cm)

bedded with wooden chips in groups of ten rats per cage in a good ventilated

room under normal day and night cycles and a room with controlled

temperature of (24 ± 3o) C, in the animal house of Medical Nile Center for

Experimental Research in Mansoura. Rats will be housed in spacious wire mesh

cages appropriate temperature.

(Sustanon ®250) ampoules obtained from Medical Research Center in

Mansoura university (250 mg/ml) and we use sesame oil from Medical

Research Center in Mansoura university.

34


During the present study, three doses of (Sustanon ®250) have been

selected which were 50, 100 and 150 mg/kg of the animal body weight (B.Wt.).

Animals weight ranged between 250-350 gm.

Animals had free access to laboratory chows and tap water, maintained

on a12:12-hour light – dark cycle and housed in an animal room where the

temperature of 22-26°C was controlled.

1.3.Experimental design

The rats were acclimatized to the cage and handling for one week then

will be randomly distributed into four groups (first group served as control and

the other groups as the treated groups). Each group consisted of ten rats per

cage.

Group (I): Control group [was be injected with normal saline

intramuscular injection (which is used to dilute (Sustanon ®250) for

injection)].

Group (II): (was be treated with (Sustanon ®250) intramuscular

injection about 50 mg/kg of body weight for two months one dose

weekly) (Brunton et al., 2018).

Group (III): (was be treated with (Sustanon ®250) intramuscular

injection about 100 mg/kg of body weight for two months one dose

weekly) (Harvey, 2011).

Group (IV): (was treated with (Sustanon ®250) intramuscular injection

about 150 mg/kg of body weight for two months one dose weekly).

1.4.Duration of this experiment

All treatments were last for two months.

35


(Sustanon ®250) was being administered weekly for two months based

on the body weight.

1.5.Specimens collection

At the end date of experiment (the two months) of treatment, all rats were

scarified after giving deep anesthesia by with phenobarbital (50 mg/kg body

weight).

The rat abdomen was opened, the kidney was dissected out, cut into 2-3

mm segments.

The kidneys were excised and processed for light and electron

microscopic examination.

The changes in the kidney associated with the dose of sustanon of each

group of rats were studied and examined using light and electron microscopic

study.

Light microscopic studies to assesed the histological structure of the

cortical and medullary regions of the kidney.

Electron microscopic studies to assesed epithelial cells of renal tubeules

and interstitial changes.

Used (Sustanon ®250) (250mg/ml) and the volume used was 1ml in

different concentrations and sesame oil.

Slides were examined under the light and electron microscopes in low

and high magnifications.

36


1.6.Chemicals:

A-Drug used:

Testosterone isocaproate under treade name of (Sustanon ®250) which is

an oil-based injectable blend of four testosterone compounds provided by

Organ on Pharmaceuticals in Egypt.

Each ampoule contained 1ml of oily solution of (Sustanon ®250).

According to the manufacturer, 1mL of (Sustanon ®250) consisted of four

testosterone ester compounds which included:

30 mg testosterone propionate

60 mg testosterone phenylpropionate

60 mg testosterone isocaproate

100 mg testosterone decanoate (De Souza et al., 2017).

B-Drug doses:

Recommended theraputic Human Equivalent Dose (HED) of

(Sustanon ®250) is used according to (mg / kg / week).

1. Three doses of (Sustanon ®250) have been selected which were 50,100

and 150 mg/kg of the rats body weight Bwt. (Maravelias et al.,2020)

The doses were calculated by this equation which based on Body Surface

Area (BSA)

Animal dose (mg / kg) =

Human Equivalent Dose (HED) (mg / kg) / conversion factor

(Anroop and Shery 2016).

37


Species

Body

Weight (kg)

Body

Surface Area

(m2)

Conversion

Factor

Adult Human 60 1.6 1.00

Dog 10 0.5 1.85

Rabbit 1.8 0.15 3.08

Rat 0.15 0.025 6.17

(Table 1) Data obtained from Food and Drug Administration draft guidelines (FDA).

2. Methods:

Specimens from the kidnyes of all rats were excised and prepared for

histological examination by the light and electron microscope.

Histological examination of the kidneys tissues

1.1.light microscopic examination:

After sacrificing the rats, their kidneys were dissected and cut into small

pieces (0.5 cm).

Staining was used:

Hematoxylin and Eosin (Hand E) stain (Feldman and Wolfe 2014).

In the following sections, the basic steps in performing an Hand E stain

are outlined.

1.1.1.Remove the Wax

Following the preparation of a paraffin section, all the elements are

infiltrated with and surrounded by paraffin wax which is hydrophobic and

impervious to aqueous reagents. The majority of cell and tissue components has

38


no natural color and is not visible. The first step in performing an Hand E stain

is to dissolve all the wax away with xylene (a hydrocarbon solvent).

1.1.2.Hydrate the Section

After thorough de-waxing, the slide is passed through several changes of

alcohol to remove the xylene, and then thoroughly rinsed in water. The section

is now hydrated so that aqueous reagents will readily penetrate the cells and

tissue elements.

1.1.3.Apply the Hematoxylin Nuclear Stain

The slide is now stained with a nuclear stain such as Harris hematoxylin,

which consists of a dye (oxidized hematoxylin or hematein) and a mordant or

binding agent (an aluminum salt) in the solution. Initially this stains the nuclei

and some other elements a reddish-purple color.

1.1.4.Complete the Nuclear Stain by “Blueing”

After rinsing in tap water, the section is “blued” by treatment with a

weakly alkaline solution. This step converts the hematoxylin to a dark blue

color. The section can now be rinsed and checked to see if the nuclei are

properly stained, showing adequate contrast and to assess the level of

background stain.

1.1.5.Remove Excess Background Stain (Differentiate)

On most occasions when Harris hematoxylin is employed, a

differentiation (destaining) step is required to remove non-specific background

staining and to improve contrast. A weak acid alcohol is used. After this

treatment, blueing and thorough rinsing is again required. Staining methods that

include a destaining or differentiation step are referred to as “regressive” stains.

39


1.1.6.Apply the Eosin Counterstain

The section is now stained with an aqueous or alcoholic solution of eosin

(depending on personal preference). These colors many nonnuclear elements in

different shades of pink.

1.1.7.Rinse, Dehydrate, Clear and Mount (Apply Cover

Glass)

Following the eosin stain, the slide is passed through several changes of

alcohol to remove all traces of water, and then rinsed in several baths of xylene

which “clears” the tissue and renders it completely transparent. A thin layer of

polystyrene mountant is applied, followed by a glass coverslip. If the stain and

all the subsequent steps have been properly performed, the slide will reveal all

the important microscopic components and be stable for many years.

In short: Tissue Processing used for light microscopic examination was

(Paraffin Method). Specimens were prepared by gross fixed then perfused in

10% neutral buffered formaline for 48-72 hours, followed by a dehydration

using a series of graded ethanol in ascending concentrations (50%, 70%, 95%,

and 100%), immersed in xylene for clearing, infiltrated in paraffin wax, and

finally embedded in paraffin wax. 4-6 micrometer thick paraffin sections were

obtained by using Rotary Microtome to inspect for histoarchitectural changes at

(200x and 400x ) magnification (Bancroft et al., 2013).

2-Electron microscopic examination:

According to the method described by Layton and Suvarna for the

electron microscopic analysis, cortex and medulla of kidneys tissues was

processed for electron microscope (Bancroft et al., 2013).

In the following sections, the basic steps to prepared slides are outlined.

40


2.1. Fixation:

The rats were perfused through the left ventricle with iced salin

containing heparin (Cal-heparin 5000 IU, Amoun, Egypt) to wash out blood,

followed by mixture of 2.5% gluteraldehyde and 2% paraformaldehyde in 0.2

M sodium cacodylate buffer at ph 7.4 (Perfusion done at a rate of 180 mm HG

for 30 second then at rate of 100mm Hg for another 30 minutes).

2.2.Preparation of chemicals: 0.2 M cacodylate buffer (ph 7.4):

(Humason1979).

A stock solution was prepared dissolving of a weight of 4208 g of

sodium cacodylate (Sigma Aldrich, Cairo, Egypt) in 1000 ml distilled water. A

working solution was prepared by adding 2.7 ml of 0.2 M HCL to 50 ml of

stock solution and volume was made up to 200 ml with distilled water.

2.3.Fixative solution: (Humason1979)

A weigh of 9 g paraformaldehyde was dissolved in 450 ml 0.1 M

cacodylate biffer on magnetic stir aided by addition of 3~4 pellets of sodium

hydroxide till the solution cleared, then the ph was readjusted to 7.4 by adding

drops of concentrated HCL. The solution is then cooled down on ice. 50 ml

glutaradehyde was then added to the cold solution.

2.4.1% Osmium tetraoxide:

Commercially available 4% Osmium tetraoxide ampoules (Sigma

Aldrich, Cairo, Egypt) diluted in 0.1 M cacodylate buffer 1:4 (v:v) and were

used freshly prepared.

41


2.5.Method used in perfusion: (Humason1979)

The equipment used in perfusion comprises: two 2000 ml glass flasks

(for the saline and the fixative respectively), each equipped with an outlet at

bottom, elevated 1.2 meter above the animals; two sets of plastic tubing with a

flow regulator; a plastic” Y” connector tubing and the connection were kept free

of air bubbles.

After anaesthesia, the animals were attached to a perfusion tray by

means of masking tape. The thorax was opened to expose the heart. An incision

in the left ventricle was made and the cannula was inserted in the ascending

aorta through the left ventricle. The cannula was clamped inside the aorta with a

haemostatic clamp.The right atrium was opened and the saline was allowed to

flow at a maximum rate for 30 seconds to 2 minutes (180mm Hg)

The perfusion with the fixative was started maximum rate and the flow

of the saline was decreased to zero. After 1~2 minutes the perfusion rate was

slowed down and maintained at a pressure of 100 mm Hg for 30 minutes.

2.6. Post-fixation, dehydration and embedding:

The kidney was cut into 2-3 mm segments, immersed in fresh fixative at

4 ºC for further 24 hours. The segments were then washed in 0.1 M cacodylate

buffer two times 10 minutes each and then transferred into a secondary fixative

consisting of 1% osmium tetraoxide in 0.1 M cacodylate buffer for afuther 2 h

(on ice). After this, they were dehydrated through graded alcohol series, cleared

in propylene oxide, and finally embedded in Epon 812.

42


2.6.1.Preparation of epon 812: (Humason1979)

Stock solution:

For accuracy of measurement, the gravimetric rather than volumetric

measurements were used in preparation.

Mixture A

A weight of 50 g epon 812 was mixed with a weight of 90 g DDSA

(Dodecenyl succinic anhydride) in an 8-oz bottle, which was capped and shaken

vigorously until the two components were completely mixed.

Mixture B

A weight of 100 g epon 812 was mixed with a weight of 75 g MNA

(Nadic Methyl Anhydride) in an 8-oz bottle, which was capped and shaken

vigorously until the two components were completely mixed.

Working solution:

Mixtures A and B were mixed in ratio of 3:2 to obtain a block of

medium hardness. Then 0.14 g (ml) of DMP-30 (2,4, 6-tridimethylaminomethyl

phenol) was added per 10 g of this mixture.

2.7.Method used in dehydration and embedding

(Humason1979)

The kidney was divided into small blocks (~1mm) using clean razor

then dehydrated in ascending concentration of ethanol as follows:

• Ethanol 50% 10 minutes



43






The specimen then cleared in three changes of propylene oxide 15

minute each. The specimens then impregnated in two successive mixtures of'

propylene oxide and epon {(1:1) for one hour, then, (2:3) for six hours} and

finally in epon alone for 12 hours at room temperature.

Tissue specimens were embedded with epon in a flat silicone rubber

molds and then polymerized at 60 ºc for 48 hours after appropriate orientation

using fine wooden stick.

2.8. Sectioning:

Rectangular flat blocks containing the specimens, were removed from

the moulds and trimmed by clean razor.

Both semi-thin (1-µm) and ultra-thin (~80nm) sections from the kidney

were cut using ultra microtome (Reichert-Jung, Licea Germany) using glass

knifes for semi-thin sectioning and diamond knife (Reichert-Jung, Licea

Germany) for ultra-thin sectioning.

The glass knifes were prepared by cutting triangular pieces with sharp

apical edge from 5 square inch glass plates of 6 mm thickness using glass

cutting device (Reichert-Jung, Licea Germany).

2.9. Staining:

The semi-thin sections were mounted on glass slide and stained with

Toluidine blue. Thin sections were mounted on cupper microgrids of 200

meshes (Reichert-Jung, Licea Germany) and stained with 0.2% lead citrate and

aqueous solution of uranyl acetate.

44


2.9.1.Preparation of Stains and methods used in Staining:

Uranyl acetate: (Bancroaft and Stevens 1996).

A weight of 3.75 g uranyl acetate was dissolved in 50 ml distilled water

on magnetic stirrer for 10 minutes and kept covered by aluminum foil at 4 °C.

Lead citrate: (Reynolds 1963).

A weight of 1.33 g Lead nitrate was mixed with weight of 1.76 g

Sodium citrate and dissolved in 30 ml distilled water (Co 2 -free) (Co 2 was

removed from water by using vacuum desiccator).

The mixture was shaken vigorously at intervals for 30 minutes in a 50-

ml volumetric flask. The completion of conversion of lead nitrate to lead citrate

is indicated by the appearance of a uniform milky suspension. To this

suspension 8 ml of 4% sodium hydroxide was added. The mixture was then,

made up to 50 ml with distilled water and then mixed by inversion until lead

citrate dissolved and the solution cleared up completely (pH 12).

2.9.2.Technique of double staining: (Humason 1979).

A small quantity of fresh sodium hydroxide pellets was placed at one

side of an absolutely clean plastic Petri dish to produce a Co 2 -free atmosphere,

the dish was covered.

The grid with mounted sections was floated, section side down, on a

drop of 2% aqueous uranyl acetate solution for about 5 minutes in a second

dish. By the time the staining with uranyl acetate was completed, the

atmosphere in the first dish would be Co 2 free.

The grid was rinsed in distilled water. 2 drops, each slightly larger than

the grid, from lead citrate solution were placed in the first dish, The first drop

45


was not used. The grid, section side down, was placed on the top of the drop of

lead solution for 3-10 minutes.

After staining the grid is rinsed thoroughly and consecutively in Co 2

frees 0.02 N sodium hydroxide solution and Co 2 free distilled water.

2.9.3. Examination:

Thin sections mounted on cupper microgrids of 200 meshes (Reichert-

Jung, Licea Germany). Ultrathin sections were observed at 80 kV using a JEOL

JEM -2100 at EM Unit, Mansoura University, Egypt.

3.Preparation of Stains and methods used in Staining:

3.1.Toluidine blue: (Bancroaft and Stevens 1996)

Preparation:

A weight of 1g of Brax (sodium tetraborate) and 100 g of toludine blue

were dissolved in 100ml of distilled water. The solution was filtered after

complete dissolving of the dye using 0.35µm filter.

Technique:

Few drops of the staining solution were placed on the slides, which were

heated on a hot plate at 60°C. Then, excess solution was washed off in running

tap water.

Examination:

Semi-thin sections mounted on glass slides were photographed using

Olympus ® digital camera installed on Olympus ® microscope with 1/2 X photo

adaptor.

46


4. Digital morphometric study

The result images were analyzed using computer assisted digital

image analysis on Intel Core I5 ® based computer using VideoTest ®

Morphology ® software (Russia). 2 slides from each rat's were prepared, 5

random fields from each slide were analyzed.

4.1.The software routine of quantification includes:

Step 1: Image acquiring form the camera using a u-tech ® frame grabber.

Step 2: scaling the image using the length bar in electron micrographs.

Step 3: measuring the lengths in the electron micrographs using the

scaled measurement tool of the program.

Step 4: All results were exported as .XLS as length in um.

5. Statistical analysis

Data were tabulated, coded then analyzed using the computer program

Microsoft® Excel for windows. (Microsoft Inc., USA).

5.1.Types of statistics:

Analytical statistics:

The results were expressed as means ± standard error of mean (M ± SE) and

statistical analyses were carried out using statistically available software of

statistical package for social science (SPSS) version 11.5. Followed by

comparison tests for comparisons between the groups.

In the statistical comparison between the different groups, the

significance of difference was tested using analysis of variance (ANOVA) to

compare between more than two groups of numerical (parametric) data

P values; as regards the Probability

47


P value >0.05 was considered statistically non-significant

P value <0.05 was considered statistically significanterior

P value <0.000 l was considered highly significanterior

All graphic representations of the data were performed with Microsoft

Excel for windows. (Microsoft Inc., USA).

Descriptive statistics:

Descriptive statistics were calculated in the form of mean ± Standard deviation

(SD).

Data were expressed as mean and standard deviation (Mean ± S.D.) One-way

analysis of variance (ANOVA) was performed to test for significance followed

by post hoc tests for comparisons between the groups. P values ≤ 0.05 and 0.01

were considered statistically significanterior

Also, the blood samples were be taken from the animal before scarifying

and analyzed for creiatnen clearance to detect the level of kidneys damage

biochemically. The blood analysis was being performed in Mansoura

international hospital. The data of the blood analysis was be managed

statistically in all groups and discussed according to (Petrie and Sabin, 2019).

The results of this study were analyzed statistically using SPSS software

(version 24 for windows).

Biochemical analysis:

Blood samples were taken from eye vein, and biochemical analysis

performed in fresh heparin treated serum.

Serum was obtained by centrifugation at 3000 RPM for 15 minutes and

stored at -4°C for Serum creatinine, uric acid, urea and total protein levels were

48


achieved automatically were analyzed by using commercial kits from Medical

Nile Center for Experimental Research Company in Mansoura by using fully

automatic biochemistry analyzer (model bt35i), while serum albumin

concentration was determined by spectrophotometric method using albumin kit

(BIOLABO, France). The absorbance was measured at 630 nm.

49

Results

Results

Gross features:

Final body weight of the studied rats

It has been noticed that the maximum body weight found in the

control group 226.2 ± 14.6 g (206 – 249) gm than in other groups.

There was a highly significant difference among the studied groups

according to body weight as it showed highly significant decrease in the (Sustanon

®250) injected groups (p= 0.001) (Table 2).

Regarding kidney weight, there was statistical decrease in kidney

weight in (Sustanon ®250) injected groups in comparison with control group.

Table (1): Final body weight of the studied rats

Group

Number of

Final body weight F-Test p-value

rats

Mean ± SD (Range)

Group 1 Control 10 226.2 ± 14.6 (206 – 249) 18.7 0.001**

Group2 (50 mg/kg b.wt.) 10 215.5 ± 11.24 (203 – 227)

Group3(100 mg/kg b.wt.) 10 198 ± 10.11 (190 – 201)

Group 4(150 mg/kg b.wt.) 10 173.9 ± 11.2 (158 – 189)

* *Statistically highly significant difference (P ≤ 0.001).

(Table 1) showed the final body weight among studied groups.

50

Mean ± SD

Results

300

250

200

226.2

215.5

198

173.9

150

100

50

0

Group 1 Control

Group 2 (50 mg/kg

b.wt.)

Group 3 (100

mg/kg b.wt.)

Group 4 (150

mg/kg b.wt.)

Series1 226.2 215.5 198 173.9

Table 2: Final body weight distribution among the studied groups. illustrated the

morphometry among the studied groups.

Morphometry among the studied groups:

It has been found that there was a highly significant difference

according to capillary tuft area, bowman space, proximal convoluted tubules

diameter and distal convoluted tubules diameter among the studied groups.

Groups

* *Statistically highly significant difference (P ≤ 0.001).

Table (3): Morphometry among the studied groups (Oneway anova)

Capillary tuft area

(µm)2

Mean ± SD

Bowman space

(µm)

Mean ± SD

Prox. tubule

diameter

(µm)Mean ± SD

Dist. tubule

diameter (µm)

Mean ± SD

Group 1 Control 1108.8 ± 187.2A 9.263 ± 1.6 A 26.6 ± 3.5 A 46.72 ± 5.8 A

Group 2

(50 mg/kg b.wt.)

Group 3

(100 mg/kg b.wt.)

966.34 ± 147.36 7.117 ± 1.8 28.5 ± 3.4 49.64 ± 5.9

711.25±119.77A 6.446 ± 1.9 A 30.8 ± 3.2 A 63.19 ± 6.5 A

Group 4

632.1 ± 103.3 A 5.674 ± 2.3 A 35.7 ± 3.3 A 66.21 ± 7.6 A

(150 mg/kg b.wt.)

F 6.688 20.72 9.17 9.561

P 0.001*** <0.001 *** <0.001 *** <0.001

***

A: indicate significant difference between each group and control gp (p<0.05)

51

Results

Biochemical analysis

The present investigation has shown non-significant increase of serum

albumin, urea and uric acid in (Sustanon ®250) injected groups in comparison to

control group. With respect to serum total protein and creatinine, (Sustanon

®250) caused non-significant increase in the first dose and significant increase

in the last two doses compared to control group (Table 4).

All these changes were approximately increased dose dependently.

Kidney

parameters

Group 1

Control

Group 2 (50

mg/kg b.wt.)

Group 3 (100

mg/kg b.wt.)

Group 4 (150

mg/kg b.wt.)

Test of

Significance

Albumin(mg/dL) 3.27 ±

0.07 A

3.36 ± 0.10 A 3.4 ± 0.09 A 3.7 ± 0.12 A F=4176.9

P=<0.001

Total proteins

(mg/dl)

6.8 ±

0.11 a

7.2 ± 0.12 a 7.2 ± 0.13 a 7.7 ± 0.14 a F=86.24

P=<0.001

Urea (mg/dl) 5.4 ± 1.4 a 5.6 ± 0.05 5.7± 0.09 a 5.71 ± 0.12 a F=23.3

p<0.001

Uric acid (mg/dl) 1.8 ±

0.09 a

2.3 ± 0.2 a 3.4 ± 0.09 a 3.7 ± 0.12 a F=438.5

p<0.001

Creatinine(mg/dl) 0.5 ±

0.02 a

0.6 ± 0.06 a 2.2 ± 0.15 a 2.3 ± 0.2 a F=581.4

p<0.001

Data presented as mean ± S.E???, * =P<0.05.

A a: indicate significant difference between each group and control gp (p<0.05)

Table (4): Some biochemical parameters related to kidney functions

52

Histological structure of the kidney's tissues

Results

Group (I):

Control group [injected with normal saline intramuscular injection (which

is used to dilute (Sustanon ®250) for injection)] shows that:

By Light microscopic examination with Hematoxylin and Eosin stain

showed that normal histological structure of the cortical and medullary regions of

the kidney. Normal glomeruli and normal renal tubules with normal Bowman’s

capsule and bowman's space (Fig. 15) and (Fig. 16).

By Electron microscopic examination showed that normal cellular structure

of the epithelial cells of the kidney tubules, three lining cells with their spherical to

oval nuclei (N), basal cell membrane infoldings carrying elongated mitochondria

(M), some cytoplasmic vacuoles (V) and somewhat thin basal lamina (Fig. 17).

53

Results

(Fig. 11): Light micrograph of kidney of control male rats showed normal

histological structure with normal glomeruli (stars) and normal renal tubules (black

arrows). Hand E, 200x.

54

Results

(Fig. 12): Light micrograph of kidney of control male rats showed normal

histological structure with normal glomeruli (stars) and normal renal tubules

(black arrows). Hand E, 400x.

55

Results

(Fig. 13): Transmission electron micrograph of the distal convoluted tubule

of kidney of control group rats showing three lining cells with their spherical to

oval nuclei (N), basal cell membrane infoldings carrying elongated mitochondria

(m), some cytoplasmic vacuoles (v) and somewhat thin basal lamina (arrows).

(4000x).

56

Group (II):

Results

(Treated with IM injection of (Sustanon ®250) about 50 mg/kg/B.W. for

two months one dose weekly) showed a mild dilatation of the kidney tubules and

degeneration of kidney tubular epithelial lining cells (Fig. 18), (Fig. 19) and (Fig.

20). with the Interstitial fibrosis and neutrophil infiltrate. (Fig. 21) and (Fig. 22).

(Fig. 14): Light micrograph of kidney histological changes in male rats

treated with (Sustanon ®250) 50 mg/kg/B.W. shows the necrosis in epithelial

lining of renal tubules (head arrow). Hand E, 200x.

57

Results


treated with (Sustanon ®250) 50 mg/kg/B.W. shows the necrosis in epithelial

lining of renal tubules (head arrow) and dilatation of kidney tubules (arrowes).

Hand E, 400x.

58

Results

(Fig. 16): Transmission electron micrograph of kidney histological changes

in male rats treated with (Sustanon ®250) 50 mg/kg/B.W. shows the Interstitial

fibrosis and neutrophil infiltrate red arrow. (2000x)

59

Results


in male rats treated with (Sustanon ®250) 50 mg/kg/B.W. shows the necrosis in

epithelial lining of renal tubules (red arrow) and dilatation of kidney tubules (black

arrowes). (4000x).

60

Group III:

Results

(Treated with (Sustanon ®250) intramuscular injection

about 100 mg/kg B.W. for two months one dose weekly) shows necrosis of lining

of renal tubules and disappearance of Bowman’s space caused by proliferation of

mesengial cells (Fig. 22)and (Fig. 23) with Interstitial fibrosis and Collagen

bundles run parallel to each other (Fig. 24), (Fig. 25) and (Fig. 26).


treated with (Sustanon ®250) 100 mg/kg/B.W. shows necrosis of lining of renal

tubules and disappearance of Bowman’s space caused by proliferation of

mesengial cells (head arrow) Hand E, 200x.

61

Results


treated with (Sustanon ®250) 100 mg/kg/B.W.shows necrosis of lining of renal


mesengial cells (head arrow) Hand E, 400x.

62

Results


in male rats treated with (Sustanon ®250) 100 mg/kg/B.W. shows Interstitial

fibrosis and Collagen bundles run parallel to each other white arrow (2000x).

63

Results


in male rats treated with (Sustanon ®250) 100 mg/kg/B.W. shows Increased

mesangial matrix (M) white arrow (4000x).

64

Group IV:

Results

(Was treated with (Sustanon ®250) intramuscular injection

about 150 mg/kg/B.W. for two months one dose weekly). shows proliferation of

the cells of the lining layer of renal tubules and some fat vacules deposited

between cells. (Fig. 26) and (Fig. 27) with Increased mesangial matrix and cells

that (Fig. 28)and (Fig. 29).

(Fig. 22): Light micrograph of kidney histological changes in male rat

treated with (Sustanon ®250) 150 mg/kg B.W. shows proliferation of the cells of

the lining layer of renal tubules (black arrows) H and E, 200x.

65

Results

(Fig. 23): Light micrograph of kidney histological changes in male rat

treated with (Sustanon ®250) 150 mg/kg B.W. shows proliferation of the cells of

the lining layer of renal tubules. H & E, 400x.

66

Results

B

M

M

(Fig. 24): Transmission electronic micrograph of kidney histological

changes in male rat treated with (Sustanon ®250) 150 mg/kg B.W. shows

Increased mesangial matrix and cells. ( M:mesangial matrix, MC: Mesangial cells,

BM: Capillary basement membrane) (2000X).

67

Results


in male rats treated with (Sustanon ®250) 150 mg/kg/B.W. shows elongated

mitochondria, some cytoplasmic vacuoles (stars), necrosis of lining of renal


mesengial cells (red arrows) (4000x).

68

Discussion

Discussion

In recent years, the intentional abuse of anabolic androgenic drugs

especially the testosterone derivatives by athletes has increased rapidly in many

countries to become a serious negative phenomenon (Abd Hamza and Rashid,

2017).

Most famouse synsetic anabolic androgenic drugs is Testeosterone

Isocaprorate (Sustanon ®250) which can be given directly or in food.

The current study aimed to determine the effects of Testeosterone

Isocaprorate (Sustanon ®250) by observation changes of histological and

ultrastructurals of kidneys of males' albino rats for two months.

The data have shown that:

Regarding kidney weight, there was statistical decrease in

kidneys weight in (Sustanon ®250) injected groups in comparison with control

group.According to a recent study that performed a increase in kidney sizes of

AAS users, compared to non-using weightlifters was observed (Kanayama et

al., 2020). Which was supported by our study but using the experimental

animals such as the albino rats.

Regarding capillary tuft area, there was statistical decrease in capillary

tuft area in (Sustanon ®250) injected groups in comparison with control

group.A recent study conducted on Wistar rats treated with testosterone

cypionate for two months demonstrated a decrease in capillary tuft area in the

69

Discussion

animals’ tissues through the increase of fat vacules in kidney that may play a

role in acute renal failure (Rosca et al., 2019).

There was statistical decrease in Bowman space in (Sustanon ®250)

injected groups in comparison with control group. Another study confirmed a

decrease in Bowman space in kidney after two weeks only of high doses of

Testosterone cypionate intravenous administration (Arora et al., 2019). Our

study ranged doses from 50 to 150 mg/week and the duration was two months

and the role of adminstration was intramuscular injections .

There was dilatation in Prox. tubule diameter in (Sustanon ®250)

injected groups in comparison with control group. were significantly increased

in the last two doses of (Sustanon ®250) (100 and 150 mg/kg b.wt.) injected

rats when compared with control rats. The latter changes were approximately

occurred in dose dependent pattern.

The last dose (150 mg/kg b.wt.), which used in this research, caused a

significant increase in the level of serum albumin, while in the other two lower

doses, the level of serum albumin increased non-significantly in comparison to

the serum albumin of the control rats.

The significant elevation in means of these three compounds

indicated defects in the functions of the kidney as a result of injection of the rats

with doses of (Sustanon ®250) .

This result is consistent with the study of (Abd Hamza et al.,

2009), who reported a markedly significant increase of the serum level of the

blood compounds related to the renal functions (blood urea, uric acid,

70

Discussion

creatinine, albumin….etc) when (Sustanon ®250)

male guinea pigs (Abd Hamza et al., 2009).

injected intramuscular in

The kidney results of this study seem to support results obtained by

Habscheid (Habscheid et al., 1999) who recorded a kidney dysfunction in a

youth athlete, who had taken high doses of anabolic drugs for two months, and

may lead to renal failure.

The results of this study were similar to those obtained by Hoseini

(Hoseini et al., 2009) who noticed marked abnormalities in the kidney structure

caused by high doses of steroids and were different from those seen by Conway

(Conway et al., 2000) who suggested that (Sustanon ®250) had very rare if any

effects on kidneys.

In regard to the mechanism of action of these effects on the kidney as a

result of high doses of (Sustanon ®250) is still unknown. Nevertheless, a few

hypotheses are suggested. Welder (Welder et al., 1995) and Draisci (Draisci et

al., 2000) for example supposed that Testosterone toxic metabolites may cause

these effects.

The histological effects in rat kidney due to (Sustanon ®250)

administration, as revealed by the present work, was confirmed by (Vaskinn et

al.,2020). who found the biochemical results in which an elevation in creatinine

level was detected, Swelling of mitochondria in renal tubules cells, appearing of

condensed conformational shape of most of them and losing of mitochondrial

cristae may refer to an apoptotic cell death in response to (Sustanon ®250)

administration. (Vaskinn et al.,2020).

71

Discussion

Muraoka, (2001) reported that testosterone replacement in middle-aged

rats increased the rate of apoptosis of mesangial cells. In addition, renal tubules

cell apoptosis in male rats also increased with the level of testosterone

(Muraoka, 2001).

The increased production of free radicals as a result of Testosterone

administration (Hoffmann et al., 2005) may explain the cellular damage in

kidney and kidney cells as oxidative stress is the main cause of cell death

(Ramaekers et al., 1997).

In a previous paper, the authors detected a significant dose dependent

increase of malondialdehyde level (MDA) (a lipid peroxidation indicator) in

(Sustanon ®250) treated rat serum (Rasul and Aziz, 2012) after using similar

doses in rats. This may clarify one important mechanism of (Sustanon ®250)

action, which includes Testosterone derivatives, in inducing nephrotoxicity.

Moreover, a recent study found that in a population of AAS users the

weekly doses of AAS till 14 weeks ranged 750–1550 mg/week (da Cruz et al.,

2019).

Another study reviewed deaths of some albino rats after high mega doses

admistration of Testosterone Cypionate for one week only. The doses ranged

from 500 to 2000 mg/day except one male albino rate continues to ten dayes

regarding cause of death was venous thromboembolism after autopsy (Fink et

al., 2019). Our study ranged doses from 50 to 150 mg/week and the duration

was two months.

Another study showed that 35% of the users examined were found to be

positive for two or more AAS in connection with autopsy. Moreover, an

72

Discussion

association between the use of AAS and any steroids drugs was observed (Sessa

et al., 2018).

It was demonstrated that AAS increase the risk of premature death,

especially among subjects with other pathologies and/or psychiatric diseases. A

survey conducted in 21 gyms in Britain reported that 8% of respondents

declared having taken AAS in their life. Another study in the UK showed that

70% of the clientele in a health and fitness community were AAS users

(Piacentino et al., 2022).

Free and bio available testosterone can be calculated by mass action

binding algorithms, and the measured values of serum creatinine and serum

albumin. The Vermeulen equation has been the most widely applied. In a

previous study comparing five algorithms, large differences were found

between the results of the calculations. Furthermore, it was shown that

commonly used formulae over estimate serum creatinine in male relative to

equilibrium dialysis measurement (Teck and McCann, 2018).

The previous study was confirmed by the biochemical results in which an

elevation in creatinine level was detected in our results after administration of

testosterone cypionate by weekly doses pattern.

A recent study suggested a dose related oxidative stress in mouse kidneys

treated with prolonged doses of Testosterone Cypionate (Sustanon ®250). The

authors observed an increase in lipid peroxidation markers and an increases of

pro-inflammatory and pro-apoptotic markers associated with a decrease of

antioxidant enzymes, which could lead to secondary focal segmental

glomeruloscelerosis (Hauger et al., 2021).

73

Discussion

Another study showed that aggressive decrease of serum albumin in

albino arts after intramuscular injection of (Sustanon ®250) for two months

when combined it with another steroids which differ from the presesnt study

which obtained the non significant of serum albumin after intramuscular

injection of (Sustanon ®250) for two months (Kanayama et al., 2020).

A recent study conducted on 20 cases treated with testosterone cypionate

as testosterone replacement therapy for two months demonstrate that a decrease

in uric acid level in 70% of cases that may play a role in acute renal failure

(Alharbi et al., 2019).

A recent study suggested that all AAS do renal dysfucttion by

immunosupretion effects of any type of steroids. In this study three groups were

investigated: “corticosteroid” users, “glcosteroids” abusers and “Aging” people.

In this regard, miR-34 and miR-132 were considerably higher in the “AAS”

group ( Xie et al., 2020).

Prolonged administration of ND in mice has been shown to cause dosedependent

oxidative kidney stress and damage. Mice kidneys treated with ND

exhibited increased lipid peroxidation and decrease antioxidant enzyme activity

(Vaskinn etal.,2020).

74

Summary and conclusion


In summary, Abusing of anabolic androgenic steroid (AAS) athletes has

been increased. especially athletes in the power sports like bodybuilding and

weight lifting administrate illegally high doses of these drugs to have increase in

muscular mass and improve their performance during the international sport

competitions.

Testeosterone Isocaprorate (Sustanon ®250) is a potent synthetic

testeosterone derivatives. testosterone compounds: 30mg testosterone

propionate, 60mg testosterone phenylpropionate, 60mg testosterone isocaproate,

100 mg testosterone decanoate

In several countries it was administered intramusculary and used to

increase the growth rates of farm animals.

The present investigations were achieved to determine the effects of

Testeosterone Isocaprorate (Sustanon ®250) by observation changes of

histological and ultrastructurals of kidneys of male's albino rats. This

investigations included biochemical, histological and ultrastructural

observations.

Three doses of (Sustanon ®250) : 50, 100, and 150 mg/Kg. Bw. were

given to the rats and compared with the control. Dose dependent increase of

albumin, urea , uric acid, total proteins and creatinine were recorded after

(Sustanon ®250) administration weekly doses for two mounths.

The histological study was shown some nephrotoxic effects such as mild

dilatation of the kidney tubules and degeneration of kidney tubular epithelial

75


lining cells with the appearance of dead cells and inflammatory foci. necrosis of

lining of renal tubules and disappearance of Bowman’s space. Mainly in groups

III and IV.

The electron microscopic figures have confirmed the above histological

results and showed certain ultrastructural alterations such the Interstitial fibrosis

and neutrophil infiltrate with Increased mesangial matrix and cells. Mainly in

groups III and IV.

These histological and ultrastructure damages were in dose dependent

pattern which call the attention for the side effects due to abusing of

suprapharmacological doses by athletes.

76

Recommendations

Recommendations

1- Athelets should be Aware of side effects of AAS.

2- The diagnosis and management of chronic AAS use requires a good

rapport with the patient, clinical judgment, and shared decision-making.

3- Evidence is urgently required to support the development of effective

services for users and of evidence-based guidance and interventions to

respond to users in a range of healthcare settings.

4- there is a need to ensure that interventions are culturally appropriate to

the target groups.

5- Built on the long-standing dismissive approach towards the effectiveness

of AAS by elements of the health profession and an on-going ‘just say

no’ stance amongst some practitioners, it is evident that establishing trust

through listening to the AAS-using communities will be an essential

element of intervention and service development.

77

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الملخص العربي

الملخص العربي

تم زيادة تعاطي الرياضيين الستيرويد منشط الذكورة االبتنائية وخاصة الرياضيين في رياضات القوة مثل كمال

األجسام ورفع األثقال،‏ حيث يتعاطون جرعات عالية بشكل غير قانوني من هذه االدوية لزيادة الكتلة العضلية وتحسين

أدائهم خالل المسابقات الرياضية الدولية.‏

مركب التستوستيرون إيزوكابرورات ‏)سوستانون ®250( هو من مشتقات التستوستيرون االصطناعية القوية

ويتكون من 4 مركبات التستوستيرون وهم:‏

فينيلبروبيونات،‏

30

ملغ من بروبيونات التستوستيرون،‏

60

60

ملغ من التستوستيرون إيزوكابرويات،‏

100

ملغ من ديكانوات التستوستيرون.‏

ملغ من هرمون التستوستيرون

في العديد من البلدان يتم إعطاؤه عضلياً‏ واستخدامه لزيادة معدالت نمو حيوانات المزرعة.‏

تم إجراء الدراسة الحالية لتحديد تأثيرات هرمون التستوستيرون أيزوكابرورات ‏)سوستانون ®250( من خالل

مالحظة التغيرات النسيجية والخلوية للكلى في ذكور الجرذان البيضاء.‏ وشملت هذه الدراسة المالحظات البيوكيميائية

والنسيجية والبنية الخلوية.‏

تم اختيار عدد

فئران

40

من ذكور فئران االلبينو ونقسيمهم عشوائيا الى

تم اختيار ثالث جرعات من ‏)سوستانون ®250( :

4

مجموعات متساوية بحيث كل مجموعة

10

150 ،100 ،50

ملغم/كغم.‏ وزن الجسم.‏

وحقن الفئران الموجودة فى المجموعات الثانية والثالثة والرابعة عضليا بواقع مرة واحدة اسبوعيا لمدة شهرين

وتسجيل النتائج التى حدثت على الكلى ومقارنة النتائج مع المجموعة االولى.‏

تم تسجيل زيادة تعتمد على الجرعة في األلبومين واليوريا وحمض البوليك والبروتينات الكلية والكرياتينين بعد

تناول ‏)سوستانون 250( جرعات أسبوعية لمدة شهرين.‏

أظهرت الدراسة النسيجية بعض التأثيرات الكلوية مثل توسع خفيف في األنابيب الكلوية وتنكس الخاليا الظهارية

األنبوبية الكلوية مع ظهور الخاليا الميتة والبؤر االلتهابية.‏ نخر بطانة األنابيب الكلوية واختفاء حيز بومان.‏ بشكل رئيسي

في المجموعتين الثالثة والرابعة.‏

أكدت األشكال المجهرية اإللكترونية النتائج النسيجية المذكورة أعاله وأظهرت بعض التغيرات في البنية التحتية

مثل التليف الخاللي وارتشاح العدالت مع زيادة مصفوفة وخاليا مسراق الكبيبة.‏ بشكل رئيسي في المجموعتين الثالثة

والرابعة.‏

اظهرت النتائج زيادة نسبة الكرياتنين واليورك اسد فى الدم بشكل ملحوظ فى المجموعتين الثالثة والرابعة.‏

1

كانت هذه األضرار النسيجية والمجهرية االلكترونية في نمط يعتمد على الجرعة مما يسترعي االنتباه إلى اآلثار

الجانبية الناجمة عن إساءة استخدام الجرعات الدوائية للتستيستيرون ومشتقاته بجرعات ومدد غير محسوبة طبيا من قبل

الرياضيين.‏

التأثيرات النسيجية المرضية لهرمون التيستوستيرون

إيزوكابرورات على الكلى لدى ذكور الجرذان البيضاء

رسالة

مقدمة للحصول على درجة الدكتوراة

من الطبيب

محمد راضى احمد عبد الباقى

مدرس مساعد بكلية الطب جامعة األزهر - دمياط

فى مادة التشريح وعلم األجنة

تحت اشراف

أ.د.‏ جمال سيد احمد الدسوقى

أستاذ التشريح واألجنة

كلية الطب بنين

- جامعة األزهر بالقاهرة

د.‏ أحمد السيد أحمد عامر

مدرس التشريح واألجنة

كلية الطب - جامعة األزهر بدمياط

جامعة األزهر

2022

Effects of isochaborat testosterone on the kidney rats

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