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Republic of Iraq
Ministry of Higher Education and Scientific Research
University of Basrah
College of Veterinary Medicine
Department of Physiology, Pharmacology and Chemistry

Effect of Lycopene In Vivo and In Vitro on Some

Physiological and Histological Changes Induced by
Monosodium Glutamate in Rats

A Thesis

Submitted to the Council of The College of Veterinary Medicine,

University of Basrah as Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy in (Veterinary Physiology)

By:
Manal Nasser Abd Al Hassen
B.S. (1998), MSC. (2004)

Supervised by:
Assist. Prof. Dr. Adel M. Al Zobidy

Prof. Dr. Eman Aboud Al Masoudi

2019 A.D 1441 A.H

1
 ‫صدق هللا العلي العظيم‬

‫سورة الكهف اآلية (‪)901‬‬

‫‪2‬‬
 Confirmation of Supervisors

We certify that this thesis was prepared under our supervision at the
Department of Physiology, College of Veterinary Medicine/ University of
Basrah as a partial fulfillment of the requirements for the degree of
Philosophy of Doctorate in Veterinary Medicine Science /Veterinary
Physiology.

Signature Signature
Assist. Prof. Prof.
Dr. Adel M. Al-Zobidy Dr. Eman Aboud Al-Masoudi

Date:
Date:

Recommendation of the Head of the department

In view of the available recommendation, I forward this thesis for debate
by the examining committee.

Signature
Prof. Dr. Muna H. AL-Saeed
Head of the Department of Physiology,
Pharmacology and Chemistry
College of Veterinary Medicine
University of Basrah
Date:

3
 Examining Committee Decision
We are the members of examining committee, after reading this thesis (Effect Of Lycopene
In Vivo And In Vitro On some Physiological And Histological Changes Induced By
Monosodium Glutamate in rats) and examining the student (Manal Nasser Abd Al Hassen ) in its
content found it adequate as a partial fulfillment of the requirements for the degree of Doctorate of
Philosophy in Science of Veterinary Medicine (Physiology).

Signature:
Chairman
Prof. Dr. Jassim M.A. Al-Kalby
College of Veterinary Medicine/
University of Basrah
Signature: Signature:
Member Member
Prof. Dr. Alaauldeen Subhi M. Al-Sallami Prof. Dr. Fawzi Sadam Mehson
Science faculty/ College of Veterinary Medicine/
University of Kufa University of Basrah

Signature: Signature:
Member Member
Asst. Prof. Dr. Ayyed Hameed Hassen Asst. Prof. Dr. Nawras Abdelah Alwan
College of Veterinary Medicine/ College of Veterinary Medicine/
University of Kerbala University of Basrah

Signature: Signature:
Member & Supervisor Member & Supervisor
Asst. Prof. Dr. Adel M. Al-Zobidy Prof. Dr. Eman Aboud Al-Masoudi
College of Veterinary Medicine/ College of Veterinary Medicine/
University of Basrah University of Basrah

Approval of the College committee on postgraduate studies

Signature:
Prof. Dr. Kamal M. AL-Saad
Dean of College of Veterinary Medicine/University of Basrah

4
 Dedication
This thesis is dedicated to:

My dear husband Emad, who leads me through

the valley of darkness with light of hope and
for being always my biggest support.
My parents, the reason of what I become
today.
My beloved and wonderful kids: Fatima, Abd
Al wahed and Ali, whom I can’t force myself
to stop loving and kept me motivated and
smiling during the difficulties.
My brothers and sisters, I am really grateful to
all of you.
All my family .The symbol of love and giving.
All the people in my life who touch my heart.

Manal

5
 Acknowledgments

Acknowledgments

First of all, I am deeply grateful to Allah. I would like to express my sincere gratitude
to my supervisors, Assist. Prof. Dr. Adel M. Al Zobidy and Prof. Dr. Eman A. Al
Masoudi for their support , helpful suggestions and writing for this thesis and motivation.

I Owe to the current Dean of Veterinary Medicine College, University of Basrah, Prof.
Dr. Kamal M. Al-Saad. Also , to Dr. Hazeem T. Thuni . I Owe to the Head of
Department of Physiology, Pharmacology and Chemistry in the College of Veterinary
Medicine, University of Basrah, Dr. Muna Al Saeed.

I like to thank the following scholars: Prof . Dr. Majdy F. Majeed,Prof. Dr. Muna Al.
Saeed and Assist Prof. Dr. Nawres Abd Al Ellah Alwan at the College of Veterinary
Medicine for their assistance in statistical analysis and analyzed of histopathological
part of study.

I greatly thank my sister Ethar N. Abd Al Hassen (pathologist technician) in of Basrah

general hospital , for her help in histological sections preparations. Also, I Owe to Prof.
Dr. Abdul Ellah Abd Al Hussen at College of Pharmacy, University of Basrah .

Also, I express my gratitude to the following people for their friendship and scientific
help during the project work: Dr. Ameer Abd Al Raheem , Dr. Nadeera F. Neama , Dr.
Rafid Dolab for their helpful in my study .

In addition, I would like to thank my best friend Dr. Rawaa H. Al Myahi in College of
Pharmacy, for her help in the experiment of isolated neurons cells.

My appreciation also goes to the staff of Department of Laboratory and Clinical

sciences, especially the secretary Ahmad and Mr. Abd Allah for their great effort.

Manal

6
7
 Summary

We used seventy adult male and twenty adult female rats . The present study was
carried out in the animal house in the Collage of Pharmacy, University of Basrah, Iraq.
The study divided into two experiments as following:

The First experiment: It aimed to investigate the effect of lycopene on some

physiological and biochemical parameters in male rats treated with monosodium
glutamate such as: body weight, antioxidant activity, blood parameters, biochemical
parameters and some hormones as well as the histological study of liver, kidney, and
the brain. We used Sixty adult male rats of 4th month age were divided randomly into six
groups (10 rats in each) as follow: The First group (Control): rats were given 0.25ml of
normal saline orally by oral gavage for 30 days. The Second group (G2): rats were given
0.25 ml of MSG (20 mg/Kg BW) by oral gavage for 30 days. The Third group (G3): rats
were given 0.25ml of MSG (20mg/kg BW) by gavage orally for 15 days and after that
the animals given 0.25ml of lycopene (200mg/kg BW) by oral gavage for other 15 days.
The Fourth group (G4): rats were given 0.25ml of lycopene (200mg/kg BW) by oral
gavage for 15 days followed by 0.25ml MSG (20 mg/kg BW) given orally for another
15 days. The Fifth group (G5): rats were given 0.25ml of lycopene (100mg/kg BW) by
oral gavage and after one hour the same animals had been given (0.25ml) of MSG (20
mg/kg BW) by oral gavage for 30 days. The sixth group (G6): rats were given
0.25ml of lycopene (200mg/kg) daily by oral gavage and after one hour the same
animals had been given 0.25ml of MSG (20mg/kg BW) by gavage for 30 days. At the
end of the first experiment, the animals, sacrificed and The blood sample collected for
physiological and biochemical analysis, in addition to histological (the liver, kidney and
brain) were examined histologically.

The results showed a significant decrease in body weight gain in groups treated
with lycopene as a compared with the control group and G2. Most blood parameters
shown a significant increase in G2, while the groups which treated with lycopene showed
a significant decrease as a compared with the control and G2 groups. Most treated groups
have a significant increase of liver enzymes and total protein also total bilirubin,

8
creatinine, urea and uric acid. Regarding the lipid profile ,all parameters (except high
density lipoprotein) showed a significant increase in most treated groups, especially G2.
Adrenocorticotropic hormones showed a significant increase in most treated groups. On
the contrary, in cortisol and Triiodothyronine. But Thyroxin showed a significant rise in
G2 only. Regarding the antioxidant activity, the Glutathione peroxidase and Superoxide
dismutase showed a significant decrease in the whole treated groups as compared with
control. Whereas in malondialdehyde, showed a significant increase in G2 as compared
with the control and other treated groups. Concerning the histopathologic study, sections
of most treated groups has been affected.

The second experiment: The purpose of this experiment to isolate primary

hippocampus and cortical neurons cells from prenatal pulps rat at age (E16-18) days of
pregnant and to investigate the effect of MSG and lycopene on the cell viability of
these cells, depending on dose and time of 7 th days of incubation. The results showed a
significant decrease cell viability in MSG treated neurons as compared with untreated
cells in according time depended or time. While the lycopene treatment cell showed no
significant differences as compared vehicle(dimethylallyl diphosphate) treated cells.
Furthermore, to prove that isolated cells are neurons, we did Immunocytochemistry
study and the results proved it.

9
 List of contents

No. Subject Page No.

Summary I-II

List of contents III-IX

List of figures X- XI

List of tables XII

List of abbreviation XIII-XV

Chapter One: Introduction

Introduction 1-3

Aims of Study 3

Chapter Two: Review of Literatures

2-1 Monosodium Glutamate 4

2 -1.1 Physical and Chemical Properties of Monosodium Glutamate 4-5

2-1.2 Sources of Monosodium Glutamate 6

2-1.3 Kinetics and Metabolism of Monosodium Glutamate 6-9

2-1.4 Side Effect of Monosodium Glutamate 9

2-1.5 Monosodium Glutamate as a Flavor Enhancer 10

2-1.6 Toxicity of Monosodium Glutamate 10-11

2-1.7 Neurological Effects of Monosodium Glutamate 11-12

2-1.8 Effect of Monosodium Glutamate on Body Weight 12-13

2-1.9 Effect of Monosodium Glutamate on Oxidative Stress 14-15

2-1.10 Effect of Monosodium Glutamate on Blood Parameters 15-16

2-1.11 Effect of Monosodium Glutamate on Serum Lipid Profile 16-17

2-1.12 Effect of Monosodium Glutamate on Liver Function Tests 17-18

2-1.13 Effect of Monosodium Glutamate on Serum Total Protein and Bilirubin 19

Concentration

2-1.14 Effect of Monosodium Glutamate on Kidney Function Tests 20-21

2-1.16 Effect of Monosodium Glutamate on Some of Hormones Levels 21-22

11
2-1.17 Histopathological effect of Monosodium Glutamate 22-24

2-2 Lycopene 24-25

2-2.1 Properties of Lycopene 25-26

2-2-2 Biosynthesis of Lycopene and its Role in Photosynthesis 26-27

2-2.3 Bioavailability and Pharmacokinetics of Lycopene 27-28

2-2.4 Sources of Lycopene in Diet 28

2-2.5 The Biological Action of Lycopene 29

2-2.6 Lycopene as Antioxidant 29-30

2-2.7 Effect of Lycopene on Body Weight 30-31

2-2.8 Effect of Lycopene on Blood Parameters 31

2-2.9 Effect of Lycopene on Biochemical Parameters 32-33

2-3 Tissue Culture 33-34

2-3.1 Cell Cultures Types 34

2-3.1.1 Primary Cell Culture 34

2-3.1.2 Continuous Cell Lines 35

2-3.1.3 Features of Cell Line 36

2-3.1.4 Maintaining Cells in Tissue Culture 36

2-3.2 Primary Neuronal Cell Cultures 36-37

2-3.3 Cell Viability 38

2-3.4 Effect of Monosodium Glutamate on Primary Neuronal Cell Culture 38-39

2-3.5 Effect of Lycopene on Primary Neuronal Cell Culture 39

2-6 Immunocytochemistry 39-40

Chapter Three: Material and Methods

3-1 Chemicals and Biological Materials 41-43

3-2 Instruments 43-44

3-3 Study Design 44-45

Scheme (1) Diagram Illustrating of Whole Study Design 44-45

3-4 Animal Housing and Management 46

3-5 Experiment Design 46

11
3-5.1 First Experiments 46

3-5.1.1 Body Weight Variation Measurements 47

3-5.1.2 Collection of Blood Sample 47-48

3-5.1.3 Hematological Study 48

3-5.1.4 Antioxidant Enzymes Measurement 49

3-5.1.4.1 Measurements of Superoxide Dismutase (SOD) 49

3-5.1.4.2 Estimation of Glutathione Peroxidase (GPX) 49-50

3-5.1.4.3 Serum Malondialdehyde Estimation (MDA) 50-51

3-5.1.5 Biochemical Study 51

3-5.1.5.1 Serum Alanine Aminotransferase (ALT)Estimation (U/l) 51-52

3-5.1.5.2 Serum Aspartate Aminotransferase (AST) Estimation (U/I) 52-53

3-5.1.5.3 Serum Alkaline Phosphatase (ALP) Determination (U/I) 53-55

3-5.1.5.4 Serum Total Protein (TP) Determination (g/dl) 55

3-5.1.5.5 Serum Total Bilirubin (TB) Determination (mg/dl) 56

3-5.1.5.6 Estimation 0f Uric Acid(mg/dl) 56-57

3-5.1.5.7 Serum Urea Estimation (mg/dl) 57-58

3-5.1.5.8 Estimation of Creatinine(mg/dl) 58-59

3-5.1.5.9 Serum Total Cholesterol Estimation 59-60

3-5.1.5.10 Estimation of Triglyceride 60-61

3-5.1.5.11 Estimation of Serum Lipoprotein Cholesterol 61

3-5.1.5.12 High-Density Lipoprotein Cholesterol 61-62

3-5.1.5.13 Low-Density Lipoprotein Cholesterol 62

3-5.1.5.14 Very Low-Density Lipoprotein 62

3-5.1.6 Hormones Assay 62

3-5.1.6.1 Adrenocorticotropic Hormone (ACTH ) 63-64

3-5.1.6.2 Cortisol Hormone 64-65

3-5.1.6.3 Thyroid Stimulating Hormone (TSH) 65-66

3-5.1.6.4 Estimation of Total Thyroxin (tT4) 66-67

3-5.1.6.5 Estimation of total Triiodothyronine (T3) 67

3-5.1.7 The Histological Study 68

12
3-5.2 Second Experiment 68

3-5.2.1 Primary Cortical and Hippocampus Neuronal Cultures 68

Schematic(2) Diagram Illustrating The Procedures for Neurons Culture 69

3-5.2.2 Preparation of Poly-D-Lysine (PDL) 70

3-5.2.3 Coating Plastic Cell Culture Dishes via Poly-D-Lysine (PDL) 70

3-5.2.4 Preparation and Coating Poly-D-Lysine (PDL) and Fibrinogen of Glass Dishes 71

3-5.2.5 Neuronal Dissection and Culture 71-74

3-5.2.6 Preparation Dose Testing 74

3-5.2.7 Time-Dependent Injury of Neurons by MSG and Lycopene Treatment 74

3-5.2.8 Viability Assay Using Acridine Orange 75

3-5.2.9 Immunocytochemistry 75-76

3-5.2.9.1 Fixation 75

3-5.2.9.2 Immunostaining of Cells Seeded as Monolayers 76

3-6 Statistical analysis 76

Chapter Four : Results

4-1 First Experiment 77

4-1.1 Effect of MSG alone or with Lycopene 77

4-1.1.1 Body weight 77

4-1.1.2 White Blood Parameters 78

4-1.1.3 Red Blood Cells Parameters 78-79

4-1.1.4 Oxidative Stress and Antioxidant Activity 80

4-1.5 Biochemical Parameters 80

4-1.5.1 Liver Enzyme 80-81

4-1.5.2 Kidney Function 82

4-1.5.3 Lipid Profile 82-83

4-1.6 Some of Hormones. 83-84

4-1.7 The Histopathological Investigation 85

4-1.7.1 Liver 85-88

4-1.7.2 Kidney 89-92

4-1.7.3 The Brain 93-96

13
4-2 Second Experiment 97

4-2.1 Cell Viability by Trypan Blue Exclusion 97

4-2.2 Dose-Dependent Injury of Mature Neurons 97-101

4-2.3 Time-Dependent Injury of Mature Neurons 101-106

4-2.4 Immunostaining of Cells 106-107

Chapter Five : Discussion

5-1 First Experiment 108

5-1.1 Effect of MSG Alone Or With Lycopene On: 108

5-1.1.1 Bodyweight Changes 108-110

5-1.1.2 Blood Parameters 110-113

5-1.1.3 Oxidative Stress and Antioxidant Activity 113-114

5-1.1.4 Biochemical Parameters 115

5-1.1.4.1 Liver‟s Enzymes 115-117

5-1.1.4.2 Kidney‟s Function Tests 117

5-1.1.4.3 Lipid Profile 118-119

5-1.1.4.4 Some of Hormones 119-121

5-1.1.5 The Histopathological Investigation 122

5-1.1.5.1 Liver 122-123

5-1.1.5.2 The kidney 123-124

5-1.1.5.3 The Brain 124-125

5-2 Second Experiment 125

5-2.1 Cell Viability by Trypan Blue Exclusion 125-126

5-2.2 Dose-Dependent Injury of Mature Neurons 126-128

5-2.3 Time-Dependent Injury of Mature Neurons 129-130

5-2.4 Immunostaining of Neurons Cells 130-131

Conclusions 132

Recommendations 133

References 134-165

Summary in Arabic ‫أ‬-‫ب‬

14
 List of figures

No. Title Page

2-1 Chemical Structure of MSG 5

2-2 Chemical Structure of Lycopene 26

4-1 Section of Control Liver Rats 86

4-2 Section of Liver Rats treated with MSG (20 mg/kg) 86

4-3 Section of Liver Rats treated with MSG (20 mg/kg) then Lycopene (200 mg/kg) 87

4-4 Section of Liver Rats treated with Lycopene (200 mg/kg) then MSG (20 mg/kg) 87

4-5 Section of Liver Rats treated with Lycopene (100 mg/kg) and MSG (20 mg/kg) 88

4-6 Section of Liver Rats treated with Lycopene (200 mg/kg) and MSG (20 mg/kg) 88

4-7 Section of Normal Kidney Rats 90

4-8 Section of Rats Kidney treated with MSG (20mg/kg) 90

4-9 Section of Rats Kidney treated with MSG (20mg/kg) then Lycopene(200mg/kg) 91

4-10 Section of Rats Kidney treated with Lycopene (200mg/kg) then MSG (20mg/kg) 91

4-11 Section of Rats Kidney treated with Lycopene (100mg/kg) and MSG (20mg/kg) 92

4-12 Section of Rats Kidney treated with Lycopene (200mg/kg) and MSG (20mg/kg) 92

4-13 Section of Normal Brain Rats 94

4-14 Section of Rats Brain treated with MSG (20mg/kg) 94

4-15 Section of Brain Rats treated with MSG (20mg/kg) then Lycopene (200mg/kg) 95

4-16 Section of Brain Rats treated with Lycopene (200mg/kg) then MSG (20mg/kg) 95

4-17 Section of Brain Rats treated with Lycopene (100mg/kg) and MSG (20mg/kg) 96

4-18 Section of Brain Rats treated with Lycopene (200mg/kg) and MSG (20mg/kg) 96

4-19 Trypan Blue Dye Exclusion Test 97

4-20 Neurons Cells (Control) Untreated 98

4-21 Neurons Cell Treated With (250 & 500)µm With MSG 99

4-22 Neurons Cells Which Treated With DMSO (Vehicle)And Lycopene(250 & 500)µm 99

4-23 Fluorescence micrograph showing dose-dependent injury of mature neurons 100

MSG(250-500)µm or lycopene(250-500)µm damage of neurons by acridine dye .

4-24 Time-Dependent Injury of Neuronal Cells by 250 µm MSG. 102

4-25 Time-Dependent Injury of Neuronal Cells by 250 µm Lycopene. 103

4-26 Fluorescence micrograph showing time-dependent injury of neuronal cells analyze 104-105

15
 damage of neurons by acridine dye

4-27 Immunocytochemistry/Immunofluorescence-Tuj 1 Staining of Neuron-Specific Class 107

III Beta Tubulin(primary antibody) in differentiated neural cells

List of table

No. Title Page

2-1 Different between primary cells and cell line 35

3-1 Chemicals and their suppliers 41-43

3-2 Instruments used in study and their suppliers 43-44

4-1 Effect of MSG alone or with lycopene on body weight of male rats 77

4-2 effect of MSG alone or with lycopene on white blood cells parameters of male 78
rats.

4-3 effect of MSG alone or with lycopene on red blood cells parameters of male rats. 79

4-4 effect of MSG alone or with lycopene on some antioxidant enzyme superoxide 80
dismutase (sod), and glutathione peroxidase (GPX) and of male rats

4-5 effect of MSG alone or with lycopene on liver function enzyme such AST, ALT, 81
ALP, total protein and bilirubin of male rats

4-6 effect of MSG alone or with lycopene on creatinine, urea and uric acid of male rats 82

4-7 effect of MSG alone or with lycopene on lipid profile of male rats 83

4-8 effect of MSG alone or with lycopene on some of hormones of male rats 84

4-9 dose-dependent injury of mature neurons by MSG (250-500 µm)compared control 101
and lycopene (250-500 µm)compared DMSO (vehicle).

4-10 time-dependent injury of mature neurons by MSG (250µm)compared control and 106
lycopene (250µm)compared DMSO (vehicle) after 30min,1h and 2h.

16
 List of Abbreviations

Symbol Description
ACTH Adrenocorticotropine Hormone
ALP Alkaline Phosphatases
ALT Alanine Aminotransferases
ANOVA Analysis of Variance
AN Arcuate Nucleus
AMPK Adenosine Monophosphate Activated Protein Kinase
AST Aspartate Aminotransferases
BBB Blood Brain Barrier
BMI Body Mass Index
BW Body Weight
CAT Catalase
CHD Coronary Heart Disease
CH2NO5P Carbamoyl Phosphate
CNS Central Nervous System
CRF Corticotrophin Releasing Factor
CRS Chinese Restaurant Syndrome
CVD Cardio Vascular Disease
Cyp2e1 Cytochrome P450 2e Polymorphism In Feline Liver
DAPI 4,6-Diamidino-2-Phenylindole
DMAPP Dimethylallyl Diphosphate
DMSO Dimethyl Sulfoxide
DNA Deoxyribonucleic Acid
EDTA Ethylenediaminetetraacetate
ELISA Enzyme Linked Immunosorbent Assay
E16-18 Embryo At Age 16-18 Days
FAD/WHO Expert Committee on Food Additive
FASEB Federation of American Societies for Experimental Biology
FDA Food and Drug Administration
FITC Fluorescein Isothiocyanate
FL Femtoliter
FR Free Radical
GFR Glomerular Filtration Rate
GH Growth Hormone
G6PD Glugose-6-Phosphate Dehydrogenase
GGT Gamma Glutamyl-Transferases
GMP Guanosine -5-Mono-Phosphate
GPO Glycerol -3-Phosphate Oxidase
GPX Glutathione Peroxidase
GSH Glutathione
GST Glutathione-S-Tranferase
HB Hemoglobin Concentration
HBSS Hanks‟ Balanced Salt Solution
HFD High-Fat Diet
HPA Hypothalamus-Pituitary-Adrenal Axis
HMG CO A 3-Hydroxyl-3-Thylglutaryl Coenzyme A
H2o2 Hydrogen Peroxide

17
ICC Immunocytochemistry
IFIC International Food Information Council
IGF1 Insulin-Like Growth Factor 1
IMP Inosine -5-Mono-Phosphate
IP Intraperitoneal
IPP Isopentenyl Pyrophosphate
Ld50 Median Lethal Dose
IUPAC International Union of Pure and Applied Chemistry
LDH Lactate Dehydrogenase
LH Luteinizing Hormone
LPO Lipid Peroxidation
MDA Malondialdehyde
MCV Mean Corpuscular Volume
MCH Mean Corpuscular Hemoglobin
MCHC Mean Corpuscular Hemoglobin Cocentration
MSG Monosodium Glutamate
MTT Dimethylthiazole
MVA Mevalonic Acid
NDS % Normal Donkey Serum
NOHA Nutrition For Optimal Health Association
PBS Phosphate Buffered Saline
PCV Packed Cell Volume
PFA Paraformaldehyde
PNS Peripheral Nervous System
POD Peroxidase
PVN Periventricular Nucleus
Q.A.S Quality Assurance Statement
RBC Red Blood Cell
ROS Reactive Oxygen Species
RT Room Temperature
SOD Superoxide Dismutase
SPSS Statistical Package for the Social Sciences
SE Standard Error
TCA Tricarboxylic Acid Cycle
TC Total Cholesterol
TG Triglyceride
TRH Thyroid Releasing Hormone
TSH Thyroid Stimulating Hormone
TUJ 1 Β-Tubulin Iii
WBC White Blood Cell
XTT Methoxy Nitro Sulfophenyle

18
19
 Introduction

Monosodium glutamate (MSG) is a vital flavor component

responsible for the real improving taste. It is the sodium salt of
glutamate and it is just glutamate, as well as sodium and water. The
glutamate is the greatest public amino acids present in nature,
regarded as a chief constituent of numerous proteins in addition
peptides, existing in most tissues. Glutamate is also formed in the
body showing a crucial role in human metabolism. Approximately
every food has glutamate that forming a main constituent of
furthermost natural protein foods such as milk, meat, fish, and some
vegetables (Fernstrom, 2000).
From seaweed and other plant sources MSG was extracted.
Currently, it is synthesized in many countries round the world over a
natural fermentation route by molasses from sugar beets or sugar cane,
as well as starch and corn sugar, MSG is used to improve the natural
tastes of seafood, meats, poultry, snacks, stews and soups. When
added to foods, it offers additive purpose like to obviously occurring
free glutamate (Yamaguchi and Ninomiya,1998).
However, large amounts of it may cause a sensation of facial
pressure, chest pain, headaches, excessive fluid retention, burning
sensation, and sweating . Study has exposed that the body use of
glutamate as a transmitter and there are glutamate responsive tissues
all over encephalopathy related with hepatic disease (Biodun and
Biodun,1993).

21
 Today, the safe concentration of MSG in foods and its toxicity in
the human is still an arguable matter (Beyreuther et al., 2007). Higher
doses of MSG have showed neurotoxic effect for example, destruction
neurons happening through hypothalamic nuclei through their changes
in the hypothalamus pituitary axis in animals (Seo et al., 2010).
Furthermore, too much MSG intake may perhaps producing injury of
the liver and the kidney (Ortiz et al., 2006).

Results indicate that free glutamate separated from MSG possibly

will action on convinced receptors in the central nervous system or
peripheral nervous system neurons, triggering histopathological
variations (Iamsaard et al., 2014).
Lycopene is a main constituent of red-colored fruits and
vegetables, comprising of 40 carbon atoms connected through
unsaturated and conjugated double bonds (Holzapfel et al., 2013).
Lycopene has been described to have anti-inflammatory, anticancer
effects. Lycopene consumption has been found to be related with the
decreases in the frequency of prostate cancer, breast cancer, and lung
cancer (Michaud et al., 2000; Koh et al., 2010). Lycopene is present
in tomatoes as an acyclic form of beta carotene, whose eating can
evolve high lycopene concentrations in blood. These concentrations
have contrariwise effects associated with the risk for several kinds of
cancers such as prostate cancer and pancreatic cancer, it has a
protective effect against myocardial infarction, and cancer of the
digestive system (Kohlmeier et al.,1997). The mechanism of lycopene
by which it can effect carcinogenesis in the prostate is still being
examined. In vitro and animal studies, they have recommended that
21
the mechanism implicated affect the evolution of the cell cycle (Karas
et al., 2000), and the altitudes of antioxidant enzymes (Breinholt et
al., 2000), cell proliferation (Levy et al., 1995) and communication
junction, in different cancer cell lines (Stahl et al., 2000) .
Lycopene has an antioxidant action and everyday eating can
support the antioxidant system and prevent lipid peroxidation in
humans (Visioli et al ., 2003). Lycopene is one of the greatest
powerful antioxidants and the most main carotenoid in human plasma
and it is supposed to be one of the active compounds accountable for
the health profits of tomato (Mein et al., 2008). While the
epidemiological sign shows a consistent relationship between tomato
products or lycopene and lower cardiovascular disease risk (Sesso et
al., 2003; Jacques et al., 2013). Lycopene is also found in other
sources such as: Gac fruit (Momordica cochinchinensis), tomato
products, including tomato juice, ketchup, pizza sauce, watermelon,
papaya, pink grapefruit, and pink guava (Ishida and Chapman, 2004).

Aims of the Study

In recent times, fear of MSG had augmented due to the opposite

reactions and virulence of MSG, with few and limited literature about
the interaction of MSG and lycopene, so this study aimed to
investigate the role of lycopene on some hematological, biochemical
and histological changes in liver, kidney, brain as well as
hippocampus and cortical neurons induced by MSG.

22
23
 Reviews of Literature

2-1 Monosodium Glutamate

Monosodium glutamate (MSG) is a commonly used flavor enhancer

derived from glutamic acid, a naturally occurring amino acid in a diversity of
food products (Stanska and Krzeski, 2016). MSG has a specific taste
umami, which was first deliberated a predominant taste in Asia and much later
in Western cultures. This molecule was identified about100 years ago as the
fifth basic taste, in addition to sweet, sour, salty, and bitter (Kurihara, 2015).
MSG is found in high-protein food products, such as meat or fish, and also
in certain types of Hydrolyzed protein foods such as sodium caseinate,
vegetable protein and autolysis yeast become common. Each hydrolyzed
protein contains free glutamic acid which had similar neurotoxic properties
and taste improving potential just like free glutamic acid found in MSG. In
the 1970s, the companies changed the MSG in the hydrolyzed vegetable
protein and autolysis yeast in baby food. In the latest 30 years, usage of MSG
has seriously increased. MSG found in different concentrations in foods such
as cow milk, apple, human milk, eggs, beef, chicken, almond, carrot and onion
(Singh, 2005).
2 -1.1 Physical and Chemical Properties of Monosodium
Glutamate

Monosodium glutamate is usually made as a crystalline white powder

through fermentation methods via syrup from sugar beet or else sugar cane,
from starch hydrolyses and in natural proteins such as defatted soybean flakes
and wheat gluten, that have a specific taste called savory sweetness which is
different from the taste of monosodium glutamate and it is well-known as a
salt added to food to improve the flavor (Zealand, 2003).
24
 The taste of this salt is composed of saltiness, sweetness, bitterness, and
sourness, (Umami Taste). It is frequently popular in Chinese, Korean, and
Japanese food. The best delectable concentration for it, is between 0.2 – 0.8%
with the biggest tasty dose for humans is about 60mg/kg body weight
(Walker and Lupien, 2000).

MSG is freely soluble in water on the other hand, it is meanly soluble in

ethanol, MSG is considered stable and not hygroscopic which doesn‟t alter in
form ,otherwise feature through sustained packing at room temperature. It
does not break down during usual food handling or cooking nonetheless in
acid state (pH 2.2-2.4) as well as at a in elevation temperatures ,MSG is
incompletely dry and altered towards 5-pyrrolidone-2- carboxylate
(Ninomiya, 1998). International Union Pure and Applied Chemistry (IUPAC)
name MSG, Sodium 2-amino pentane dioate. Its solubility in water 74 g/100
ml, melting point 2320C, molecular weight 187.13, molecular formula is
C5H8NO4Na (figure 2-1), appearance is white crystalline powder, physical
state is solid, and powder, it is practically odorless with pH 6.7 - 7.2 (5%
solution) and sparingly soluble in alcohol (Zealand, 2003).

Figure (2-1): Chemical Structure of MSG (Cited by Zealand, 2003)

25
2-1.2 Sources of Monosodium Glutamate

Most food in nature contain glutamate, such as: fish, meat, milk, poultry
in addition to vegetable. Commonly, foods enriched by protein like cheese
also meat have huge quantities of certain glutamate, but furthermost
vegetables comprise moderately a little amounts. Conversely, even with their
lesser protein substances, the vegetable has a tendency to have consistently
greater levels of free glutamate chiefly in tomatoes, potatoes and in addition
peas, numerous prepared and processed food for instance sauces as well as
seasonings, most of restaurant nutriments furthermore encompass an
important levels of free glutamate (Yoshida, 1998).

In Australian or New Zealand consumers nearby is no information

accessible for intake of MSG. Documents from the United Kingdom shows an
average consumption is 590 mg/day, extreme (97.5th percentile consumers)
consuming 2.33 gm/day in extremely tested restaurant meal, eating equally
great as 5000 mg or extra may possibly (Yang et al., 1997).

2-1.3 Kinetics and Metabolism of Monosodium Glutamate

Glutamate occupies a central position in human intermediary metabolism.

It serves as a substrate for protein synthesis and glutathione synthesis, MSG
is an amino group donor in the synthesis of other amino acids through
transamination and is involved in regulating the urea cycle. Glutamate is also
a precursor of glutamine. This reaction catalyzed by glutamine synthetase has
a central function in amino acid metabolism. The main pathway to transform
free ammonium into glutamine for transport in the blood stream. Glutamate
acts as an excitatory neurotransmitter in the central nervous system (CNS)
and is the precursor of the neurotransmitter γ-aminobutyric acid (GABA).

26
Glutamate plays a significant role in the development of the nervous system
Glutamate is an important source of energy for certain tissues,particularly the
intestines (mucosa) (Chairman, 2005). There are numbers of essential
metabolic parts of glutamate as: The substation used for protein synthesis : for
example one of the best copious amino acids existing in flora, containing
about 10 to 40% through bulk of utmost proteins, L- glutamic acid is a critical
substrate used for protein creation. Glutamic acid has physical as well as
chemical features make MSG a major supplier for the minor arrangement of
proteins, that is α-helices (Young and Ajami, 2000).

The transamination through α-ketoglutarate: L-glutamate synthesis

beginning with an ammonia also with α-ketoglutarate which are intermediary
of the TCA. This response is necessary in the biosynthesis of all amino acids.
Glutamate is also regarded as a subscriber in the biosynthesis of further
amino acids over and done with transamination replies (Zealand, 2003). A
pioneer of glutamine from glutamate. A glutamine is made via glutamine
synthetase, this is an imperative reaction in amino acid breakdown. A chief
way used for transforming NH3 to glutamine for transference through the
blood stream (Reeds et al., 2000).

The substrate that used for glutathione manufacture; In all animal cells
glutathione consists of glutamic acid, glycine and cysteine, serves as a
reluctant of tri peptide toxic peroxides through glutathione peroxidase,
glutathione is suggested to transport amino acids through cell membranes.
The precursor of N-acetyl glutamate: The CH2NO5P synthetase which is a
vital of controlling the enzyme through the urea cycle certifying the
percentage of urea production in consensus per rates of amino acid de
amination (Brosnan, 2000). There are two main sources of dietary glutamate,

27
breakdown of consumed protein or after eating of diets that have quantities of
unbounded glutamate also, logically existing, otherwise supplementary in
the usage of MSG. In the gut, glutamate is captivated through active transport
system, this process could be competitively reserved depending on Na
concentration ( Wijayasekara and Wansapala, 2017) .

Dietary protein contain glutamic acid which break down into free amino
acids, in addition to minor peptides, both of them are absorbed into mucosal
cells. Peptides are hydrolyzed to free amino acids and a number of the
glutamate is metabolized, while the extra glutamate performs in the portal
blood in order to metabolized by the liver (Stoll et al., 1998). Previous study
exhibited that 95% of nutritive glutamate existing through mucosa
metabolized in the main passage, 50% performed by way of portal CO2, with
smaller extents such as lactate also alanine, a glutamate is a distinct chief
supplier for gastric energy creation. Other documents have shown10% of
alimentary glutamate is combined with mucosal protein production. While, a
residue presence for the production of proline, arginine as well as glutathione.
Actually, wholly these materials are derived completely from dietary
glutamate. The level of plasma glutamate is related with of MSG ingestion,
the composition of the dosing vehicle and the circumstances of administration
of the dose can significantly influence the circulating glutamate in response to
oral ingestion (Ninomiya, 1998).

Generally, the suggestion of the amount of glutamate concentrations

increase in plasma is be affected by the size and dose in addition to the kind
of dose up vehicle, for example water reasons more than a varied lunchtime;
a time consumption of diet, abstained showed a more reaction through a
meal; also macronutrient structure of the simultaneous food. The

28
concentration of glutamate in breast milk were rather great, also effected
modestly via MSG breakdown (Battaglia, 2000). In the brain, the main
neurotransmitter is glutamate, the blood brain barrier well eliminates passive
influx of plasma glutamate, when the plasma altitudes existed more than
twenty times values. Subsequent an oral dose of MSG (2g /kg) BW, brain
glutamate significantly increased, the common of the glutamate which was
using via the brain, it is derivative after the confined production glutamine
also in Tri carboxylic acid cycle (TCA), as well as a substantial portion
resulting since the reutilizing protein in the brain (Smith, 2000).

2-1.4 Side Effect of Monosodium Glutamate

The side effect oral consumption of MSG in acute, self-limited, and

temporary such as headache, burning feelings in the hind of the neck,
forearms and chest, facial pressure, chest pain, nausea, numbness in back
of neck, drowsiness, palpitation, tightness, burning, weakness in face,
warmth upper back, neck in addition to arms and bronchospasm (Carmen,
2001).

Federation of American Societies for Experimental Biology (FASEB)

illustrated that this index of symptoms is established on recommendation
reports collected by the Food and Drug Administration (FDA) adversarial to
the reaction observing method as well as appraisal of the texts is constructed
on financial records that are objective and not demonstrable, intelligences of
other indicators, like ventricular tachycardia, arrhythmias and atrial
fibrillation were not assumed whichever acceptance through the FASEB,
there was only situation record that needed assenting indication involving
responses to MSG satisfied nutrients (FASEB, 1995).

29
2-1.5 Monosodium Glutamate as a Flavor Enhancer

Umami taste are provided from food additives which categorized as a

flavor enhancer. The salts of glutamate are : monosodium glutamate,
monopotasium glutamate, monoammonium glutamate, ribonucleotides,
complexes and also name Disodium Inosine -5-Mono-Phosphate (IMP) and
Disodium Guanosine -5-Mono-Phosphate (GMP) have flavor enhancing
properties merely the free form of glutamate, in its L-configuration exhibit.
And for this purpose, it is commonly used as a flavor enhancer in the food
handling industry (Populin et al., 2007).

Codex alimentary glutamate and its salts, monopotassium glutamate,

monosodium glutamate, monoammonium glutamate, calcium diglutamate and
magnesium diglutamate are characterized as a taste enhancer for every food.
However, some foods are not improved by adding glutamate, i.e. sweetened
foods in specific and mainly bitter food (Heyer et al., 2004). The perfect
concentration of umami taste usually as far as sweetness and saltiness. Many
studies are obvious that most people are sensitive to its flavor-enhance
properties (Yeomans et al., 2008). In European region the studies carried out,
propose that the best concentrations (0.6–1.2%) of MSG are possible to be
greater than those described by Asian consumers (Bellisle, 2008).

2-1.6 Toxicity of Monosodium Glutamate

In recent history there are two main estimates of the toxicity of MSG
which have been assumed in current history. Food and Drug Administration
(FDA) in 1958, confirmed that glutamate is generally accepted as a safe
component, with further commonly used food constituents such as salt,
baking powder and, vinegar. At this period, an adequate daily intake was

31
(0–120 )mg/kg BW which assigned, including the L-glutamic acid
counterparts of the salts; which also reflect supplementary to intake as of
all non-additive food sources. A more broad safety assessment was made in
198, showed that glutamate has a small acute toxicity in common
conditions demonstrated by the review and the Joint FAD/WHO Expert
Committee on Food Additive (JECFA). However the oral lethal dose (LD 50)
in rats and mice is 15.000–18.000 mg/kg BW. (Walker and Lupien, 2000).
The oral LD50 for construction hypothalamic lesions is about 500 mg/kg in the
neonatal mouse given by gavage, while the major pleasant dose for humans is
nearly 60mg/kg through greater doses producing nausea (Samuels, 1999).

MSG is a silent poison existing in our diet, chiefly our kids‟ nutrition, it
can cause drop in the cerebral functions then changing the serum and brain
serotonin levels. Moreover, it may cause progressive changes in the brain of
the male albino rats and at lesser doses MSG can produce a significant rise
in weight; failure in cerebral function, nevertheless alterations in serum or
brain serotonin levels otherwise pathological alterations in the brain (El-
Kholy et al., 2018).

2-1.7 Neurological Effects of Monosodium Glutamate

The glutamate in the brain serves as a neurotransmitters adding to its

common role in protein and energy metabolism. They are stored in nerve
endings and used via nerve cells to excite or inhibit other target cells or nerve
cells, such as endocrine or muscle cells (Mattson, 2008). The highly doses of
MSG have harmful effect on brain function, the quantity of MSG which are
injected differ from 0.5 g/kg to larger dosages of 4 g/kg of the neonate‟s body
weight, prompting brain lesions and a diversity of additional physiological

31
special effects in rodents (Mattson, 2008). The investigation of the latent
neurotoxicity is the chief constituent of an integrity estimate, using
information starting with 59 discrete investigations about mice, rat, rabbits,
hamsters, duck, guinea pigs as well as dogs, the results showed a great extent
of care that crucial necrosis in the hypothalamus was detected via rabbits
also rodents when subcutaneous or intravenous intake of glutamate otherwise
next actual great oral dosages through gavage (Lopez-Perez et al.,
2010).Currently several scientists identified that MSG kills brain cells and
causes neuroendocrine syndromes. In laboratory animals, while originals a
confrontational reactions in humans. The scientists found different disease
conditions such as Alzheimer's disease and seizures disease which associated
with the glutamate cascade (Eweka et al., 2011). Other study has been shown
that MSG has neurotoxic effects causing in brain injury (Eweka and Adjene,
2007). Therefore, it established that MSG acts as a powerful neurotoxin by
affecting the chemical conformation of hippocampus that activates
neurodegenerative pathways. Similar toxic effects of MSG were observed in
cerebellar cortex of male rats. It initiated that the treated rats with 3 g/kg/day
areas of degeneration progress in cortex which were enclosed by granule cells
and pyknotic Purkinje (Beas-Zarate et al., 2002).

2-1.8 Effect of Monosodium Glutamate on Body Weight

Previous study on mice had been showed that MSG performed effective
role in prompting obesity (Morris et al., 1998). In humans, a study was showed
(752 healthy Chinese) that relationship between MSG consumption and
obesity, which was found to be really associated with improved body mass
index (BMI). MSG consumers developed increased weight as contrast with
non-users, which was a conclusion autonomous of physical activity and whole

32
energy intake (He et al., 2008). However other study showed that MSG
ingestion was not linked with body weight increase for the period of five years,
without altering the parameters of food objects and rice eating, a 5% rise in
weight was establish. On other hand when these factors were adjusted, obesity
due to MSG consumption was reduced (He et al., 2010). A study accompanied
349 human focuses from Thai population designated that great doses of MSG
produced obesity and metabolic syndrome which was independent of
supplementary major factors corresponding whole energy intake in addition to
level of physical activity (Insawang et al., 2012). Further hypotheses have
suggested that the MSG mechanisms impact going on the metabolic rate, the
latent linkage MSG as well as obesity consist of MSG influence proceeding
energy balance through promoting delectableness of food and via disturbing
the signaling cascade of leptin action in the hypothalamic (Hermanussen and
Tresguerres, 2003; He et al., 2011).

Early postnatal of rats and mice which have been treated with MSG
indicated to the advance of obesity and insulin resistance in adult animals
(Macho et al., 2000; De Carvalho et al., 2002). Rats that treated with MSG
exhibit hypertrophic type of obesity, hyperleptinemia, hyperinsulinemia
reduced serum Insulin growth hormone I, raised serum triglycerides and
cholesterol, diminished insulin-stimulated glucose which transported into
adipocytes in soleus muscle in vitro (Pinterova et al., 2001; Zorad et al., 2003).
Adding to that, adipose tissue, plasma membranes: MSG have been promoted
obese rats which showed a defective angiotensin II type I and insulin
receptors (Mori et al., 2008).

33
2-1.9 Effect of Monosodium Glutamate as Oxidative Stress

Monosodium glutamate induces oxidative stress (Ahluwalia et al., 1996).

The rise in oxidative stress can lead to changes in cell membrane lipids and
proteins producing several diseases like coronary heart disease (CHD),
diabetes, cancer etc. (Dhalla et al., 2000). The chronic MSG consumption can
be a source of reactive oxygen species (ROS) which lead to the excessive renal
metabolism of glutamate, diminished levels of main anti- oxidant enzymes and
developed lipid peroxidation have been confirmed in the kidneys of chronic
MSG exposure rats (Paul et al., 2012). In renal culture cells, high doses of
glutamate induce toxicity in these cells (Thomas et al., 2009).

In the structure of renal lipids the large quantity of long-chain

polyunsaturated fatty acids produces kidney damage by ROS (Leung et al.,
2008). That makes kidney tissues susceptible to damage via diverse
mechanisms such as the elevation of lipid peroxidation, protein alteration, and
DNA injury, which produce cell death. The involvement of ROS has been
testified in tubulo-interstitial, tubular, and glomerular alterations derivatives.
Other studies have described glutamate-induced oxidative impairment in tissues
such as brain and neurons, anywhere glutamate receptors α- ketoglutarate
dehydrogenase,and cystine-glutamat antiporter are the vigorous performers
(Kubo et al., 1997).

Previous studies showed that administration of MSG at a dose of 4 and 8

mg/g BW produced a significant rise in the lipid peroxidation in hepatic tissue,
besides significant increase in blood glutamate and glutamine level . The liver
was participated in metabolism and detoxification, thus it may be directly
affected by toxic chemicals and their metabolites. When the MSG treated rats

34
with 0.6 mg/g of BW used for sequential 10 days they will progress
symptoms of liver damage lead to significant increase in lipid peroxidation
(LPO) and activities of liver enzymes such as catalase, glutathione- S-
transferase and superoxide oxidase. In addition, the level of glutathione which
is the substrate for GST was decreased in liver consequent to amplified activity
of GST. It approves the evolution of oxidative damage produced by the creation
of ROS correspondingly. MSG administration lead to elevation of the activities
of ALT, AST, and GGT enzyme in serum (Tawfik and Al-Badr, 2012).

2-1.10 Effect of Monosodium Glutamate on Blood Parameters

Literatures have been shown intake of medicine compound or any drugs

can change usual range of hematological parameters (Ajagbonna et al., 1999).
Though, some of studies are dealing with the effects of MSG on blood
parameters are infrequent. Ashaolu et al. (2011) tests the effect of MSG on
hematological parameters by administration of Westar rat two doses of MSG
(2.75-5.5) mg/kg BW for 14 days, the results showed a significant decrease in
lymphocyte and neutrophil percentage and significant elevation in red blood
cells count, hemoglobin concentration, packed cell volume, mean corpuscular
hemoglobin, mean corpuscular hemoglobin concentration, mean cell volume,
compared with control group which all indicated of an anemic condition in the
treated animals. Another study also used two groups, each consist of 8 animal,
control and experiment groups, the second one administrated orally MSG in
dose 200 mg /kg day for 30 days result showed significant decrease in, RBC
count, WBC count, lymphocyte%, granulocyte %, monocyte %, MCH, and
MCHC (Ghadhban., 2017). Previous study was instituted that MSG had a
progressive relationship with variation in hemoglobin levels in men,
nonetheless not in women, amongst contributors who were anemic at reference

35
line, present a significant risk of anemia at continuation, independent of food
form and supplementary routine influences. The rise in hemoglobin was usually
perceived merely in those who are anemic at reference line, suggestive of that
anemic members required an enhanced nutrition with other events to control
anemia. Meanwhile only one study informed a relation between MSG and
hemoglobin, it is essential to carry out additional studies before illustration the
decision that MSG progresses the hemoglobin level decrease (Shi et al., 2012).

2-1.11 Effect of Monosodium Glutamate on Serum Lipid Profile

Previous study investigated a potential effect of MSG on serum lipids,

cholesterol, lipoproteins. The experiment was used 40 adult male rats. which
divided into 4 groups. First group is control. The second, third, and fourth
groups were administrated 2, 4 and 8 mg/g of MSG respectively in intake
water. After 8 weeks, blood was set from each rat exclusively for biochemical
assay. The results were showed that MSG significantly increased the level of
serum lipid profile in MSG-treated animals, with significant reduction in HDL
level. MSG ingestion produced hyperlipidemia and hyperlipoproteinemia
(Alwaleedi, 2016).

Yoshida, (1998) established that the subcutaneously MSG treated mice

(2mg/g) BW developed obesity in 9 weeks after birth and raised blood levels of
cholesterol, total cholesterol, and cholinesterase besides with triglyceride
content of liver compared to control mice. They illustrated an obvious fatty
variation in liver of „MSG obese‟ mice. Diniz et al. (2004) showed that adult
male Wister rats which were nourished by MSG alongside with normal diet,
they detected a rise in voluntary diet intake and food conversion ratio, they
settled that MSG triggered overfeed that prompted metabolic disorders related

36
with oxidative stress in lack of obesity that might be reduced using supplement
of dietary fibers. Meanwhile the hazard of atherosclerosis is prominent by
hyperlipidemia, so, MSG probably act as a contributing agent in the origination
of atherosclerosis (Schummer et al., 2008). Previous studies conveyed
noticeable rises in the level of serum VLDL, LDL and cholesterol in adult rats
(Singh et al., 2011; Inyang et al., 2012). The increased levels of serum
cholesterol underneath the effect of MSG, perhaps designates an deficiency of
cholesterol metabolism and consequent risk of CHD in rats (Keith et al., 1999).

2-1.12 Effect of Monosodium Glutamate on The Liver Function Test

The major glandular organ is liver in the body weighing between (1.4-1.6)
kg in human. It lies beneath the diaphragm in the right thoracic area of the
abdomen. It has a major role in metabolism in addition to many functions in the
body; glycogen storage, production of bile; plasma protein synthesis,
production an alkaline compound which assistances in digestion, as well as
detoxification of furthermost, substances (Gartner and Hiatt, 2000).

Meanwhile, the liver is convoluted in the performance of these various

functions, it may be at risk of injury which subsequent from toxic substances.
Onyema et al. (2006) established that the hepatotoxicity in rats prompted by
gavage of MSG for ten days (0.6 mg/g ) BW. The cytotoxic effect of MSG
induced liver damage and each of its enzyme such as AL, AST and ALT
released increasing their serum levels (Wissing and Khun, 2000). Other study
has been showed that MSG compact liver function and elevated serum ALT
and AST levels in rats (Giffen et al., 2002). Earlier studies have established that
MSG is lethal to the liver cells by generation of extra ammonium ions and ROS
(Stadtman et al., 2000).

37
 Excess of ammonium ion is identified to cause the development of ROS,
which react with polyunsaturated fatty acids of cell membrane producing
damage of mitochondrial and plasma membranes which caused leak of liver
enzymes such as AST and ALT (Eweka et al., 2011). Study by Ortiz et al.
(2006) showed the administered of MSG, at dose of 4 mg/g BW by intra-
peritoneal injection of rats. They were sacrificed the animal after 15 min post
injection showed elevation levels of ALT and AST, which indicate that the
serum concentration of these enzymes varies with the hepatic impairment. The
cytotoxic effect of MSG stimulate tissue injury and enzyme discharge lead
increasing their serum levels.

Furthermore, previous studies have shown that MSG diminished liver

function and raised serum ALT and AST levels in rats (Tawfik and Al-
Badr,2012). Solomon et al. (2015) they used mice which treated orally with
MSG (0.1, 0.25 and 0.5 gm/kg)BW per day for 21 days. The results showed
the level of AST and ALT were raised up in the blood of group 3 mice treated
with MSG, suggesting liver injury. Another study used four groups, group one
served as the control whereas groups two, three and four were provided by
foods accompanied with MSG at doses of (0.5, 1 and 5 mg/ kg) BW of diet
respectively for 28 days. The results showed a significant increase in ALP and
ALT activities but there was no significant difference in the levels of both
serum ALP and ALT actions in the group which fed 0.5% MSG when
compared with the control group however there were a significant increase in
the other treatment groups ,which suggest that MSG at the levels of
supplementation in the diets of the rats had increased the liver function
enzymes in the serum as the level of MSG increased in the diet (Akanya et al.,
2015).

38
2-1.13 Effect of Monosodium Glutamate on Serum Total Protein and
Bilirubin Concentration

Liver is the chief site of the production of plasma proteins. A disruption of

protein synthesis as a result of diminished hepatic function will lead to a
reduction in their plasma concentration, drop of the protein concentration in the
MSG treated rats that specify a reduction of the synthetic function of the liver
and rise rate of protein dissolution, abnormalities of lipids and lipoprotein
showed a chief role in the pathogenesis and development of atherosclerosis and
cardiovascular diseases (Chrysohoou et al., 2004). One study demonstrated that
the daily consumption of MSG has showed an increase in direct and indirect
bilirubin ,serum total protein when compared to control rats (Eweka et al.,
2011).

Furthermore, another study described that the raised levels of bilirubin

(hyperbilirubenimia) are perceived when there was liver diseases, potential
erythrocyte hemolysis, reduced uptake by the liver as well as decreased
conjugation, oxidation yields might cause damage of the membrane structure
with consequential erythrocyte (Ibrahim and Dalia, 2012). Sandharbh et al.
(2015) used forty Wistar albino rats divided into four groups ; group I served as
a control and received distilled water but group II, III and IV administered
MSG at the dose level of 70 mg/100 gm of body weight daily for a period of 30
days, 45 days and 60 days, respectively, results of that study was showed
significantly increase in the group III and group IV level of total protein and
bilirubin .

39
2-1.14 Effect of Monosodium Glutamate on Kidney Function Tests

The plasma creatinine and plasma urea concentration are defective indices
of glomerular filtration rate (GFR). By comparison with urea, an increase in
plasma creatinine is virtually always a concern of condensed GFR and so has a
renal origin, though the reduced GFR (renal disease) is correspondingly linked
with increased plasma urea concentration, there are additional non-renal
circumstances that can provide increased plasma urea, moreover, a
studydemonstrated that the diurnal intake of MSG showed an increase in serum
uric acid, urea, creatinine, and urea/creatinine ratio concentrations compared
with control group (Piacenza et al., 2009; Vinodini et al., 2010).

Perceived that food additives produced changes in kidney cell lining of

convoluted tubules in addition that of Bowman‟s corpuscles. Further, study
observed that there is a promotion in kidney functions parameters next to
administration of MSG which produced modifications in kidney functions.
These damages could also be accredited to renal blood flow and glomerular
filtration rate (GFR) and the changes in the threshold of tubular reabsorption
(Khadiga et al., 2009).

Yousef and Bakoban, (2012) study metabolic defects and oxidative

impairment prompted many diseases beneath the effect of MSG risk, used
twogroups, each group contained 20 rats, for four weeks, The control group on
drinking water, the second group is injected (4mg/kg) BW intraperitoneal (I.P.)
of pure MSG from first week to third week and withdrawal in fourth week.
The results showed alteration in serum levels of creatinine, blood urea nitrogen,
uric acid, compared with control group from first to third week, but after
withdrawal of MSG, these tests have enhancement results.

41
 Finally, MSG may cause an adverse effect on kidney function tests which
might be due to oxidative stress. Abass and Abd El-Haleem, (2011) were
studied the toxic effects of MSG on rats cerebrum and kidneys. They took three
groups each containing 12, control group received 2 ml normal saline orally for
28 days and MSG group which received (830 mg/ kg) BW orally for the same
period. The results showed increase in the serum creatinine and blood urea
nitrogen. Other study was administrated MSG doses of (1.5 mg/kg) BW to rats
for four weeks. MSG had effect on kidney functions as serum creatinine, serum
urea and uric acid were significantly increased an adverse effect on renal
functions might be due to oxidative stress induced by MSG on renal tissue
(Elatrash and Abd El-Haleim, 2015) .

2-1.16 Effect of Monosodium Glutamate on Some of Hormones Levels

An usual reaction of the stress process is produced when stimulus of the

hypothalamo-pituitary-adrenal axis (HPA), various kinds of stress affect the
sensitivity of the HPA axis and excite the discharge of corticotrophin
releasing hormone (CRH) from the periventricular nucleus (PVN) in the
hypothalamus. The pituitary gland is create and release of adrenocorticotropic
hormone (ACTH), along with increases of CRH, in turn, ACTH also
stimulates the release of corticosterone in animals and glucocorticoids in
humans from the adrenal cortex (Marin et al., 2007).

Brain stimulants as well as excitatory amino acid neurotransmitters have

also been involved in the chronic activation of the HPA axis, while the
specific role of neurotransmitters in stress reply is uncertain, there is
increasing preclinical instruction of the HPA axis (Mathew et al., 2001).

41
 Glutamate is the furthermost copious excitatory amino acid
neurotransmitter in the brain has been identified to trigger the HPA axis and
prompt ACTH altitude (Zelena et al., 2005).

Certain authors -significant alterations in thyroid morphology when long

period of MSG usage in neonatal rats (Miskowiak and Partyka,
1999).Furthermore, an image of amplified thyroid activity was stated in adult
rats that received MSG (4 mg/gm) BW for one week and the effect was
detected one month after the last dose(Rani et al., 2013).Newly, a study
performed immunocytochemical effect on GH, LH, FSH, and Prolactin
immune reactive cells in the pituitaries of mice also have been given injection
in the perinatal period using MSG, documents on MSG action on pituitary-
thyroid axis are deficient (Iamsaard et al., 2014). Another study was
performed on male rats, subcutaneously injected on days 2, 4, 6, 8, and 10 of
postnatal life with MSG at a dose of (4 mg/g )BW When the animals reached
the ages of 6 or 12 months, weight of pituitary were determined, TSH
immune reactive cells were detected. The results showed a reduction in the
weight of the pituitary. The amount of TSH-immuno reactive cells in the
pituitary stayed unaffected, the number of TSH-positive cells remained
unchanged (Miskowiak and Partykal, 2000).

2-1.17 Histopathological Effect of Monosodium Glutamate

Monosodium glutamate have several harmful effect at higher dose on the

kidney of adult rats, when have been given 3 and 6 g of MSG for the period
of 14 days. The results were seen by kidney microanatomy which exhibited
variable marks of cytoarchitectural alteration and decrease in the of renal
corpuscles in the treated groups as compared with control group (Schiffman,

42
2000). Another study have showed that when a large doses of MSG were
administered to neonatal rats may cause steatohepatitis and sign of pre-
neoplastic modifications in the liver (Nakanishi et al., 2008). Alternative
study has been shown that exposure to MSG in large doses through the
neonatal period may result in steatohepatitis and evidence of pre neoplastic,
Forty adult male Swiss albino mice was allocated in four groups as ( A, B, C
and D) of 10 mice each group. Group A is control and give normal saline
.while, groups B, C and D administered MSG at these doses (0.5, 1.0 and 1.5)
mg/kg BW per day. respectively. dissolved in normal saline for 28 days. On
day 29 of the study animals were sacrificed, and the liver and kidneys were
removed, weighed and processed for histological examination. The results
showed a significant increase in the relative liver weight at 1.0 and1.5 mg
MSG /kg BW and a relative increase in kidney weight taking place at 1.5
mg/kg BW, this was supplemented by a dose- dependent increase in body
weight compared to control which failed to reach statistical significance
(Onaolapo et al., 2013).

The liver and kidney histology showed a loss of normal liver architecture
with changing degrees of disorder and apoptotic cell death compared to
controls. The kidneys of MSG-exposed mice showed contraction of the renal
glomerulus and thickening of the walls of the renal tubules. Another study
investigated the effect of MSG on the ovaries of adult Wistar rat. They were
used three groups A, B and C (eight for each group), A and B treated groups
were given 0.04mg/kg BW and 0.08 mg/kg of MSG mixed with the grower's
marsh, respectively. The control group (C); received equal amount of feeds
(Growers' mash) without MSG for more than fourteen days.

43
 The results were showed sign of cellular hypertrophy, progressive and
atrophic modifications with more stark changes in the group that received
0.08mg/kg of MSG (Eweka et al., 2011).

Ogbuagu et al. (2018) determined the LD50 and organ toxicity of MSG.
Thirty adult albino Wistar rats of both sexes were used in this study. The
experiment was achieved on 15 rats (divided into 5 groups) received 500
mg/kg, 750 mg/kg, 1000 mg/kg, and 1250 mg/kg of MSG mixed with
the feeds, respectively. With infinite supply of drinking water daily for 8
weeks. While the control group received an equal amount of feeds without
MSG. The result of histological examination were showed huge necrosis in
the liver and lungs, fatty change in the spleen and progressive modifications
of heart muscle cells. There were asymmetrical spaces of several shapes, sizes
and necrosis in the kidney.

2-2 Lycopene

Lycopene are red carotenoid dye in tomatoes and other tomato-based

products such as watermelon, pink grapefruit, guava and papaya, lycopene
were a cyclic arrangement of beta-carotene lacking pro-vitamin A action
(Bramle, 2000). It has involved considerable attention throughout current
times because it is useful in falling the oxidative stressing coronary heart
diseases and additional chronic diseases (Upritchard et al., 2000; Agarwal et
al., 2001;Rissanen et al., 2002).

The fresh tomato fruits contain lycopene which occurs fundamentally

in the all-trans structure. The chief reasons of tomato lycopene dissolution
through processing are isomerization and oxidation(Ferreira et al., 2005(.

44
 Current epidemiological studies have recommended that the intake of
tomatoes and tomato-based food produces diminution the risk of cancer
(pharynx, oral cavity, esophagus, rectum, stomach, urinary bladder, colon,
prostate and breast) in human)Vaishampayan et al., 2007; Tang et al., 2009).

Tomatoes institute an essential farmed harvest worldwide are vital part of

human food. They are grown-up for their eatable fruits which can be
consumed fresh in cooked or salad, peeled or made into pastes ketchup, soup
or powdered or juice in some canning trade. In West Africa, tomatoes are
used as condiments for stew, which are steady features in African meals
(Olajire et al., 2007).

2-2.1 Properties of Lycopene

Lycopene has been synthesized by microorganisms, plants nevertheless via

animals, however it a natural pigment, it is an acyclic isomer of beta carotene.
Lycopene is an extremely unsaturated hydrocarbon comprising two
unconjugated and eleven conjugated double bonds. it also suffers cis-trans
isomerization prompted by sunny, thermal energy, and chemical reactions
(Nguyen and Schwartz, 1999). The red color of lycopene is due to its
numerous conjugated carbon double bonds. All double bond diminish the
energy necessary for electrons to be converted into advanced energy
situations permitting the particles to absorb visible light of gradually
elongated wavelengths (Ganesh et al., 2016).

Lycopene has the molecular formula C 40H56 as figure (2-2) which first of
all specified by Willstatter and Escher (1910) who offered their study and
improve that lycopene is an isomer of the carotenes, lycopene has molecular
weight 536.85 Da, melting point 172-173ºC, it is dark-red sticky liquid, easily

45
soluble in n-hexane and ethyl acetate; moderately soluble in acetone and
ethanol; but insoluble in water, the chemical names is (psi)Ψ,Ψ-carotene or
all-trans-lycopene (Beer, 2006).

Figure ( 2-2) : Chemical Structure of Lycopene (Cited by Beer, 2006)

2-2-2 Biosynthesis of Lycopene and Its Role in Photosynthesis

Lycopene has been synthesized through prokaryotic cyanobacteria and in

eukaryotic plants . The synthesis originates by C6H12O4 (MVA), that is
converted into C5H12O7P2 (DMAPP), later compressing 3 particles of (IPP)
C5H12O7P2 to provide the 20 carbon geranylgeranyl pyrophosphate, two
particles of this product are formerly condensed in a tail-to-tail arrangement
to provide the 40 carbon phytoene (C40H64).The first step in carotenoid
biosynthesis (Alda et al., 2009). Through a number of desaturation steps,
phytoene is altered into lycopene. The two isoprene groups of lycopene
cyclized to yield beta carotene, then converted into an extensive diversity of
xanthophyll. Lycopene is vital pigments set up in photosynthetic pigment-
protein complexes in photosynthetic bacteria, fungi, algae and plant.

46
 They are accountable for the bright colors of vegetables and fruits, carry out
several functions in photosynthesis, and defend photosynthetic organisms
from extreme light injury. Lycopene is a main intermediate in the
biosynthesis of various chief carotenoids, such as beta carotene, and
xanthophyII (Sies, 1996). In plants, lycopene is biosynthesis generally (about
90%) for instance each E-isomer. The greatest accessible bases of lycopene
sustain an ordinary isomer spreading proportion. Lycopene was exposed
through diet handling lycopene suffers geometric isomerization,
accumulation the fraction of Z-isomers. Certain, lycopene can be isomerize to
Z-forms with an attendance of oil or heat, otherwise throughout drying up
(Xianquan et al., 2005).

2-2.3 Bioavailability and Pharmacokinetics of Lycopene

Lycopene absorption from food sources take place in the range of 10% to
30% in humans, and powerfully absorbed as soon as added with fat due to its
lipophilic features. After ingestion, lycopene is taken up by food lipid
micelles and combined into mucosa of the small intestine. The micelles are
crowded into chylomicrons, which are transported to the liver by the lymph
system (Ganesh et al., 2016). Furthermore, since lycopene is a fat-soluble
composite, the tissues picked up is enhanced once it is consumed per oil. Its
condensation in body tissues is greater than the whole carotenoids.
Lycopene is mostly spread to fatty tissues as well as organs such as the testes,
liver, and adrenal glands In distinction to the supplementary carotenoids, the
serum values are frequently compacted by alcohol consumption or smoking,
while its levels reduced with increasing age (Gerste, 1997).

47
 Lycopene distribution is reliant on chylomicron micelles intermediated
mechanism, and its movement starting from gastrointestinal tract towards
body tissues. Various routine hazardous, such as smoking, drinking of
alcohol, blood lipid stages, as well as the biological factors, like age or
hormonal station, have a stimulus to the absorption of lycopene (Holzapfel
et al., 2013). The lycopene isomeric form also affects the absorption, for
example all-trans lycopene formula is fewer absorbed as compared to cis-
isomeric arrangement, The existence of fat beside lycopene rises its
absorption (Naz and Butt, 2014).

2-2.4 Sources of Lycopene in Diet

The human also animals, do not synthesize lycopene, so they depend on

the nutritional sources. Carotenoids have been never synthesized inside them,
however they accomplish it absolutely from the food (Ganesh et al., 2016).
Nevertheless 85% of our nutritional lycopene derive from tomato and the
tomato-based products. The residue being found in pink grapefruit,
watermelon, papaya and guava. while, the tomato products such as ketchup,
soup, juice, pizza, spaghetti sauces are the major suppliers in the diet
(Goldfaden and Goldfaden, 2012). All-trans is the major isomeric formula of
lycopene in fresh tomatoes but trans to cis isomerization take place through
cooking/diet handling and storage (Tonucci et al.,1995).

2-2.5 The Biological Action of Lycopene

Even though the lycopene is not considered as a critical nutrient, an

investigation has shown that lycopene has different profits for health of
human. The oxidative lipids, DNA and proteins that already occurred are
impaired by lycopene. It is a powerful overcome of single oxygen (a reactive

48
formula of oxygen), which recommends that it may possibly have moderately
stronger antioxidant properties than further most important plasma
carotenoids (Madhava et al., 2011).Lycopene has been established to be a
strong and definite inhibitor to the proliferation of cancer cell (Nahum et al.,
2001).

2-2.6 Lycopene as Antioxidant

The oxidative stress is documented as one of the most important providers

to the improved danger of cardiovascular disease as well as cancer. Among
the public carotenoids, lycopene is identified as the furthermost strong
antioxidant, established through in vitro using other antioxidants,
concentration besides the partial pressure of oxygen (Frusciante et al., 2007;
Raiola et al., 2014).Lycopene has been established to be the best effective
antioxidant in vitro producing several investigators to accomplish that the
antioxidant properties of lycopene are in charge for disease inhibition. The
study of the antioxidant eminence of blood in rats publicized that certain
antioxidant enzymes such as superoxide dismutase (SOD), glutathione
peroxidase can be prompted by lycopene (Lauridsen et al., 2000).

The destruction of endothelial cells through oxidative stress is an

essential constituent to the etiology of atherosclerosis. Tang et al. (2009)
suggested that lycopene diminished the oxidative damage of endothelial cells
prompted by H2O2, reduced the expression of p53 (tumor antigen) and
caspase-3mRNA in damaged cells. In addition it diminished the apoptosis of
injured cells. These conclusions can describe why lycopene inhibits
atherosclerotic cardiovascular diseases. Moreover, the study of Lee et al.
(2000) confirmed that the intake of tomato foods using olive oil (without

49
sunflower oil) improved the antioxidant activity of human plasma,
endogenous anti oxidative enzymes like superoxide dismutase, glutathione
reductase and glutathione peroxidase can be promoted by lycopene (Subhash
et al., 2007). The antioxidant activity of lycopene has been briefly estimated
on its capacity to scavenge free radical, or to protect cell constituents against
oxidative impairment in cell culture, or in animal (Zhao et al., 2002).

The conjugated double-bond system of lycopene convenes strong

antioxidant activity together with the ability to reduce singlet oxygen and
peroxyl radicals. The singlet oxygen reducing activity of lycopene has been
shown to be more than that for other carotenoids, including alpha-carotene
(Clinton, 1998). In vivo, lycopene consumption has been related with
decreased levels of serum lipid peroxidation and low-density lipid (LDL)
peroxidation (Rao and Agarwal, 1998).

2-2.7 Effect of Lycopene on Body Weight

Lycopene is generally kept in the adipose tissues. The investigators have

suggested that, lycopene could diminish the construction of cytokins and
chimiokins (which perform a role in the common physiology of the body)
through the adipose tissue which would decrease the hazard of increasing
pathologies related with obesity (Gouranton et al., 2010). The considerable
weight loss detected in cancer might be due to that of cancer malabsorption,
cachexia, anorexia, in fact contributes to advanced wasting, particularly in
skeletal muscle as well as adipose tissue. Lycopene may have an excellent
antitumor and antioxidant profit, that might increase rats health digestive
function, the increase in food consumption might have assisted the increase in
body weight (DeBoer et al., 2007). Another study was showed that lycopene

51
administration had no significant effect on body weight loss (Aydin and
Çelik, 2012).

Bahcecioglu, et al. (2010) investigated the defensive effect of lycopene

used forty male rats, were divided into 4 groups, they were provided for
standard diet, high-fat diet (HFD), HFD plus lycopene at a dose of 2 mg/kg
BW in addition to the high-fat diet lycopene at a dose of 4mg/kg BW for a
period of 6 weeks. The results were showed there was no significant
difference in body weight between the groups that fed on the experimental
diets for 6 weeks.

2-2.8 Effect of Lycopene on Blood Parameters

Boeiraa et al. (2014) studied the effect of lycopene on signs of oxidative

stress on hematological and histopathological parameters used adult male
mice received lycopene (20 mg/kg BW) for ten days. The results were
showed that the treatment with lycopene for ten days did not alter the blood
parameters significantly (leukocytes, neutrophils, sticks, eosinophil‟s,
monocytes, lymphocytes and platelets number). Another study evaluate the
role of lycopene on hematological parameters in cadmium exposed mice with
administration of lycopene in a dose (20mg/kg) BW for 15 days against
cadmium treated group which received (0.32mg/kg BW). The results showed
a significant increase in all hematological parameters as compared to
cadmium treated group (Sharma and Vijaya, 2015).

2-2.9 Effect of Lycopene on Biochemical Parameters

The best biomarkers usually have been used for detecting the risk of
cardiac heart disease (CHD) are serum cholesterol level, low density
lipoprotein (LDL). The transported cholesterol via blood stream, is assumed to

51
perform a main role in the pathogenesis of arteriosclerosis that is the
fundamental disorder producing heart attack and ischemic strokes (Heller et
al., 1998). Because of its lipophilic nature, lycopene is concentrated in LDL
as well as VLDL fractions but not in HDL fractions (Stahl and Sies, 1996). It
has been shown that it diminishes the levels of LDL plus lipid peroxidation in
subjects that consumed tomato juice, tomato sauce and lycopene oleoresin
capsules (Agarwal and Rao, 1998).

Studies in vitro and in vivo have showed that lycopene can prevent
cholesterol synthesis by obstructing 3-hydroxy-3-methyl glutaryl Co- enzyme
A (Blum et al., 2005). Moreover, administration of lycopene to rats reduced
the serum urea and creatinine level (Ayhan et al., 2014). Furthermore,
Aidoud et al. (2016) they have showed a significant drop of serum ALT,
AST and ALP through a study on Wistar rats that administrated rich food of
lycopene mixture with olive oil for 4 weeks, the results revealed that the
lycopene added diet lead to the improvement of liver function by reducing the
hepatic enzyme levels and improving the antioxidant level of liver tissue.
Lycopene is considerably reduced the amount of the changed hepatic foci
prompt of glutathione S-transferase in the livers of rats, with decrease
proliferating cell nuclear antigen positive hepatocytes as well as reduced the
activity of extracellular signal. Both lycopene and tomato extract complement
prompted lipid peroxidation through the liver. Investigators perceived a
significant decline in the (CYP2E1), inflammatory emphases as well as
mRNA expression of pro inflammatory cytokines (Ben-Dor et al., 2001). The
clinical and in vitro, studies were showed that lycopene reduce liver damage
and certainly inhibited the progress of hepatocellular carcinoma (Seren et al.,
2008).

52
2-3 Tissue culture

Tissue Culture is a common expression used for the elimination of cells,

tissues, or organs from an animal then set into an artificial milieu to perform
growth. Tissue culture is accomplished as pure growth of tissue or cell
isolated from the organism. It is correspondingly well-known as procedures of
keeping tissues alive and developing in an suitable culture medium. The
growing tissues of alive organism that extracted outside the body is put in a
suitable culture medium, comprising combination of nutrient either in solid or
fluid form (Surachi, 1999).

The different types of cell can currently be grown in culture containing

connective tissues such as neural cells, epithelial tissues, fibroblasts, smooth,
cardiac and skeletal muscle, endocrine cells as well as various different kinds
of tumor cells (Merten, 2006). The best appreciated method to study the
functions, in addition to mechanism of actions of numerous cells was vitro
culture. A specific group of cells can be cultured in enormous amounts to
study their cellular actions, proliferations and differentiations. The cell culture
is extremely important to biotechnologist. The main zones that use animal cell
culture are; vaccine manufacturing, cancer investigation, drug selection and
enhancement, recombinant protein construction, stem cell biology, gene
treatment and in vitro fertilization technology. Many of nutritional factors can
be added to the medium to support growth such as serum, ca2+ ions, hormones
etc (Gabriella, 2011). Cells or tissues in most cases must be grown in culture
varying from days to weeks in order to achieve appropriate numbers of cells
used for analysis. In long-term culture maintenance of cells needs severe
adherence to sterile technique to circumvent contamination and possible
damage of valuable cell lines (Oyeleye et al., 2016).

53
2-3.1 Cell Cultures Types

Primary cell cultures can be obtained from adult tissue or embryonic,

usually have a limited lifespan in culture. The purpose of primary cultures is
to examine the cells have not been "changed". In any way ,but the obstructive
of primary cultures are the mixed nature of both preparation.
Continuous cell lines are irregular and are frequently transmuted cell lines
(Gabriella, 2011).

2-3.1.1 Primary Cell Culture

The primary cultures are freshly isolating cultures up to they are passage
or subculture. They are commonly heterogeneous, have a little growth
fraction, however they are further demonstrated the cell kinds when they are
derivative and the appearance of tissue certain properties. The principal step
in the primary culture is isolation of tissues from organ followed by
compilation of cells from the tissues. This is through addition of little trypsin
to the tissue for suitable breakdown and separation of cells. When trypsin is
added to any tissues in order to cut down the extracellular glycosidase and
proteases (Huang et al., 2010).

The external exposure proteins are break down by the action of trypsin
for separation of cells of the tissues so that produce individual cells. The cells
culture achieved when the digestive trypsin are incubated in existence or lack
of serum with culture medium (Oyeleye et al., 2016).

2-3.1.2 Continuous Cell Lines

A group of morphologically identical cells is a continuous cell line which

can be proliferated in vitro and are capable to reach to an indefinite number

54
of population through replications for an unlimited time. Continuous cell
lines are frequently originated from tumor tissue otherwise have been
purposely celebrated or transmuted (Quality Assurance Statement, 2012). A
cell line get up from a primary culture at the period of the first efficacious
subculture. The term cell line indicates that cultures from it contain lines of
cells initially current in the primary culture (Freshney, 2005). The cell lines
are depending on the life span of culture and are classified into two types
(Nema and Khare, 2012): Finite Cell Lines: Cell lines which include a
limited life span and withdrawal from the start to the finale, a circumscribed
number of cell categories (often 20-80 population doublings) be fit identified
as finite cell lines. Continuous Cell Lines: Cell lines altered beneath
laboratory environments or in vitro culture position give rise in the way of
continuous cell lines. The growth rate is fast as well as doubling-up time is 12
- 24 hours. Primary cells differ from cell lines according to (Freshney,
2000)as the following:

Table (2-1): Different between primary cells and cell line (Freshney, 2000)

Primary Cells Cell lines

1-Freshly isolated cells (from tissues) Originally primary, but then
transformed so keep growing
2-Hard to culture Easy to culture
3-Heterogeneous (mixed+ fibroblasts) cell Homogeneous (cloned) cell
population population
4-Finite number of passages(gradual loss of Infinite number of passages
function) (cancer-like)
5- More “physiological” Less “physiological”

2-3.1.3 Features of Cell Line

In order to use any cell line for the invention of biological product, one
must have facts of the following equipment associated to cell lines: Sex,

55
age and species of the donor tissue When cell lines for human, medical history
of the donor must be available. Culture history including methods which used
for the isolation of the tissues in cell line from which the line was derivative,
media used, passage history (Freshney, 2005).

2-3.1.4 Maintaining Cells in Tissue Culture

An appropriate temperature as well as gas combination (usually, 37°C, 5%

Co2)in the incubator. Culture environments differ extensively for every cell
type, and difference of circumstances for a specific cell type can result in
diverse phenotypes actually expressed .In addition to temperature and gas
mixture, the utmost usually varied factor through culture systems is the
growth medium. Formulas for growth media can be differ in pH, growth
factors, glucose concentration, and the existence of supplementary nutrient
constituents. The growth factors which used for supplement media are
frequently derivative from animal blood (Freshney, 2005).

2-3.2 Primary Neuronal Cell Cultures

The cortex and hippocampus have been fascinated neuroscientists for

many years because of their specific organizations ,functional roles in
knowledge, ignorance and remembrance (Costa et al., 2000). For instance the
most constructions in the brain, the complication of these structures produce it
to investigate and handle simply in vivo. The cell lines are derived from the
central nervous system precursors have been limited for the reason that the
neurons resulting from these lines are unsuccessful to summarize features of
central neurons, having the ability to form precise axons, dendrites in
addition to synapses. As a replacement for, the primary cell culture techniques
have been successfully established to study these neurons in vitro (Gerard et

56
al., 2012).Most well-known and extensively utilized techniques for the study
of cortical and hippocampal pyramidal neurons have been dissociated the
primary culture system founded by Kaech and Banker. (2006) used for the
neurons culture of embryonic rat. This culture system permits neurons to be
cultured in vitro with an extreme fewer complex milieu than utilized in vivo,
constructing them extremely, available to be used in explanations. The
incapacity to isolate the culture neurons from the brain of adult mammalian
and the common unsuccessfulness of neurons to regenerate in the lesion. The
brain has been funded on the conception that adult neurons do not regenerate
(Brewer, 1997).

To prove that isolated adult neurons are accomplished of regeneration,

four main technical complications must be dominate: Isolation of cells must
be from a convoluted network of thousands of adhesive connections, lacking
of effect of permanent destruction to cut off axons and dendrites. This may
possibly the primary advantage of beginning through embryonic tissue by
smaller amount connections. Disconnect away potential growth the inhibitory
factors required in the adult brain to keep homeostasis (Faissner and Steindler,
1995). Splitting the diverse cell types, exclusively neurons from glia, to be
capable to give experimental effects to precise cell types. The supplying of a
suitable substrate for supplement and satisfactory nutrients for continuous
viability in culture. To resolve the isolation difficulty by associating few
proteases to produce the isolated cells resulting trituration in an isolation
medium which are perfect for osmolality. This medium furthermore has anti-
oxidants to diminish reactive oxygen impairing the membranes, proteins and
DNA (Al-Zabin and Rohleffson, 2017).

57
2-3.3 Cell Viability

The establishment of assessing cells response to external factors is cell

viability and proliferation, the valuation methods are crucial for cells biology
and drug detection. The cell viability assessment is indispensible in order to
recognize cell reaction to the external factors (Hynes et al., 2003).
Therefore; several methods have been undertaken to estimate cell
viability, or in supplementary words cytotoxicity such as colorimetric
method like trypan blue or acridine orange and LDH release, MT, XTT
(Shokrzadeh and Modanloo, 2017). These methods can be constructed on
morphology alterations or variations in the permeability of cell membranes
interference of cell activities (Mickuviene et al., 2004).

2-3.4 Effect of MSG on Primary Neuronal Cell Culture

Xiong et al. (2009) they were studied the effect of MSG via neuronal
culture technique and cell injury assay by using mouse cortical neurons,
ordinarily used in vitro research for cell damage studies. They have
confirmed that incubation using MSG, at clinical appropriate concentrations,
prompted swelling and damage of mature neurons. This result may
moderately explain the headache made by MSG consumption. Glutamate has
the effectiveness of producing neuronal destruction through the retinal
ganglion cells as well as glutamate has created serious neurodegeneration as
described in MTT assay when incubation of retina using glutamate for 24 h
merely 34% of cells are viable subsequent to glutamate exposure for 24 h
(Namindla et al., 2016). However, in past texts exposes that the glutamate
yield stark neurodegeneration in 2 h time (Yukitoshi et al., 1995).

58
 Another study was showed that MSG solutions are used in 10, 30, 60, 90
µmol/dl concentrations the toxic dose in mouse caused neuron degeneration
(Walker, 1999). MSG solutions used in cell culture to discover the role of
MSG in mouse “in vitro”, less than 70 % of cells were established viable in
culture due to the results of cell viability assay (Sinem et al., 2017).

2-3.5 Effect of Lycopene on Primary Neuronal Cell Culture

Because the lycopene is lipophilic, its intake with fat rises relatively
without oil preparations in the food (Gartner et al., 1997). Moreover,
lycopene can pass via the blood brain barrier, because of its lipophilic nature,
proposing that it will inhibit oxidative stress which induced neurologic
lesions (Khachik et al., 2002; Wu et al., 2015). Hsiao et al. (2004) showed
that lycopene had defensive effect on main cerebral ischemia in rat models.
Another study was showed that lycopene decreased amyloid-β-induced
neurotoxicity and mitochondrial dysfunction in primary culture of cortical
neurons in rat (Qu et al., 2011).

Moreover, lycopene has inhibited oxidative stress, then diminishing the

mitochondrial enzyme activities, lycopene inverted neurochemical alterations
besides physiological defects in experimental animals in addition it has been
hypothesized that lycopene can suppress neuronal apoptosis through its
antioxidant activity (Sinwoo et al., 2017).

2-6 Immunocytochemistry

Immunocytochemistry (ICC) defined as the identification of a cell- or

tissue-bound antigen in situ, by means of a specific antibody–antigen
reaction, tagged microscopically by a visible label (Brook and Schumacher,
2001).

59
 ICC attribute to immunostaining of cultured cell lines or primary cells
comprising smears, swabs, and aspirates. By an Immunocytochemistry
detection of surface antigens (markers) on isolated cells (for example ,
membrane proteins on blood cells). The recognition is based on specific
antigen-antibody binding (immunoreactions). A specific antibody that was
produced by single B cell clone recognizes an epitope with (8-15) length
amino acid sequence in a protein. Successful immunocytochemistry needs (1)
preservation of the antigen in a form that is identifiable by the antibody, (2)
an appropriate antibody, and (3) an suitable label (Polak and Van Noorden,
1997).

61
61
 Materials and Methods
3-1 Chemicals and Biological Materials

The chemicals and biological materials which were used throughout the
study are listed in table (3-1) with their suppliers.
Table (3-1) Chemicals and Their Suppliers.
NO Chemicals Suppliers
1 Acridine orange Beijing Solaria science .China
2 ACTH kit ALPCO. America
3 Alkaline phosphate kit JOURILABS . Ethiopia
4 ALT kit JOURILABS . Ethiopia
5 Amphotericin B Beijing Solaria science .China
6 AST kit JOURILABS . Ethiopia
7 Beta tubulin Sigma-Aldrich .USA
8 Bilirubin kit JOURILABS . Ethiopia
9 Chloroform Noorbrok / England
10 Cholesterol kit . JOURILABS . Ethiopia
11 Creatinine kit JOURILABS . Ethiopia
12 Cortisol kit ALPCO. America
13 DAPI solution Beijing Solaria science .China
14 Double deionized distal water (ddH2O) Beijing Solaria science .China
15 DMSO Fishe chemical, USA
16 Donkey anti rabbit FITC Sigma-Aldrich .USA
17 Ethyl-alcohol Milpharm/London/UK
18 Fibronecin Sigma-Aldrich .USA
19 Formaldehyde TEDIA Company INC, USA
20 Gentamycin Beijing Solaria science .China
21 Glutathione peroxidase kit Elabascience Biotechnology, Inc. China
22 HCl Merck chemical, Germany

62
23 HDL Cholesterol kit JOURILABS . Ethiopia
24 Hibernate HE Brain Bit .UK
25 Hibernate HE-Ca Brain Bit .UK
26 Lycopene(pure) Shaanxi pioneer Biotech. China
27 Monosodium glutamate Sigma-Aldrich . Germany
28 NaOH Beijing Solaria science .China
29 NB Active 1 Brain Bit .UK
30 Normal donkey serum Sigma-Aldrich .USA
31 Normal saline Pioneer company, Iraq
32 Paraformaldehyde Torrance, USA
33 papain Brain Bit .UK
34 Phosphate buffer saline Beijing Solaria science .China
35 Poly-D-lysine Sigma-Aldrich .USA
36 Poly-L-ornithine Sigma-Aldrich .USA
37 Thermo clean Germany
38 Thiobarbituric acid Sigma-Aldrich .USA
39 Triglyceride kit JOURILABS. Ethiopia
40 Trypan blue Beijing Solaria science .China
41 Total protein kit JOURILABS. Ethiopia
42 TSH kit Sigma-Aldrich .USA
43 T3 kit Sigma-Aldrich .USA
44 T4 kit Sigma-Aldrich .USA
45 Superoxide dismutase kit Elabascience Biotechnology Inc China
46 Urea kit JOURILABS. Ethiopia
47 Uric acid kit JOURILABS. Ethiopia

63
 3-2 Instruments

The instruments and equipment which were used throughout the study
are listed in table (3-2).
Table (3-2) Shows the Instruments Used in Study and Their Suppliers:

No Instrument supplier
1 Auto cleave Yamato scientific, China
2 Cell dissociation sieve Sigma-Aldrich .USA
3 Centrifuge Genex ,Florida ,USA
4 Co2 incubator BINDER, Germany
5 Count 60 Genex ,Florida ,USA
6 DIA Reader ELX800 Spain
7 Digital Camera Olympus, USA
8 Dissection microscope Wiltronics, USA
9 Electronic Balance Electronic scale , China
10 Electronic sensitive balance Genex ,Florida ,USA
11 Fluorescence microscope Bio base, China
12 Inverted microscope Genex ,Florida ,USA
13 Laminar Flow Hood Genex ,Florida ,USA
14 LCD microscope Genex ,Florida ,USA
15 Micropipette Minipcr, UK
16 Petri dish Beijing Solaria science .China
17 Polish pasture pipette Brain bit, UK
18 Poly –D-lysine cover slips Brain bit, UK
19 Round cover slips Beijing Solaria science .China
20 Spectrophotometer Chrom Tech ,USA
21 TBS 4060R-CPPKG Canada
22 24 well plate Beijing Solaria science .China
23 water bath Memmert , Germany

64
3-3 Study Design

G1:Control (10
G3:10 male rats received G5:10 male rats
male rats
MSG(20mg/kg)BW for received
received 0.25
15 days then lycopene(100mg/kg)BW
ml normal
lycopene(200mg/kg)BW after 1 hr. received
saline for 30
for 15 days MSG(20mg/kg)BW for
days 30 days

G4:10 male rats received G6:10 male rats received

G2:10 male rats received lycopene (200mg/kg)BW lycopene(200mg/kg)BW.
MSG(20mg/kg)BW for 30 for 15 days then after 1hr. received
days MSG(20mg/kg)BW for MSG(20mg/kg)BW for
15 days 30 days

Sacrificed all animal for all groups and blood collection for biochemical(antioxidant activity
lipid profile ,liver function test, kidney function and some of hormones )organs(brain, liver
and kidney)were removed for histological examination

65
 II

Scheme (1) Diagram Illustrating of Whole Study Design

66
3-4 Animal Housing and Management:

Seventy adult male and twenty adult female rats were used of an average
body weight between (350±25) g. The animals were housed in individual
cages measuring (50X50 cm), at the animals house of Pharmacy Collage,
University of Basrah, Iraq. All animals were exposed to the same
environment including climate management, feeding and acclimatization on
place for two weeks before treatment. The animals were kept beneath optimal
condition (25±5ºC) and (12/12 hours light/dark cycle) during the study. The
rats feed standard pellets and distal water, and examined for any infection by
giving coarse of systemic antibiotics to make sure that they are healthy before
the beginning of the study.

3-5 Experiment Design

The study including two experiment as follow:

3-5.1 First Experiments

Sixty adult male rats with four month age were divided randomly into six
groups (10 rats for each group) as follow:

Frist group (control): Male rats were given 0.25ml normal saline orally by
gavage for 30 days.

Second group (G2) : Male rats were given 0.25 ml of MSG (20 mg/kg BW)
orally by gavage for 30 days.

Third group (G3): Male rats were given 0.25ml of MSG (20mg/kg BW) by
gavage orally for 15 days and after that the animals given 0.25ml of lycopene
(200mg/kg BW) by oral gavage for other 15 days.

67
Fourth group (G4): Male rats were given orally 0.25ml with lycopene
(200mg/kg BW) by gavage for 15 days and after that the animals were given
orally 0.25ml by MSG (20 mg/kg BW) for another 15 days.

Fifth group (G5): Male rats were given orally 0.25 ml of lycopene (100
mg/kg BW) by gavage then after one hour the same animals had been given
orally(0.25ml) of MSG (20 mg/kg BW) by gavage for 30 days.

Sixth group (G6): Male rats were given orally 0.25ml of lycopene (200
mg/kg) by gavage daily and after one hour the same animals had been
given 0.25ml of MSG (20mg/kg BW) by gavage for 30 days.

The experiment continues for one month after that we measure the
following parameters:

3-5.1.1 Body Weight Variation Measurements

The current experiment persistent for 30 days, the animals were weigh up
on zero days (pretreatment) then at the final of the experiment. Body weight
variation after 30 days of treatment was evaluated according to the following
equation:

Body weight change (g) = Final body weight (g) – Initial body weight (g)

3-5.1.2 Collection of Blood Sample

At the end of the first experiment, the animals anaesthazide by chloroform

and then sacrificed, the blood sample(10ml) were collected between 10:00 to
12:00 AM due to reduce the circadian deviation of hormones level, by heart
puncture (Parasurman et al., 2010). Blood samples were stored in plastic
sample test tube containing EDTA anticoagulant for hematological

68
parameters which done directly after collection, while another portion was
deposited in to tube without anticoagulant and allowed to clot at room
temperature ,then the blood samples were centrifuged at (3000 rpm) for 15
minutes and serum sample were separated in eppendrof tube and stored at (-

20٥ C) until used for biochemical analysis (Cray et al., 2009).

3-5.1.3 Hematological Study

The hematological tests were done in the Laboratory of Pharmacy Collage

in University of Basrah by using (Count 60 model Genex). The instrument
can measure and calculate 20 different parameters. The hematology and
analyzer contain solution (HC5-BAsolysc) contain cyanide free lyse reagent,
(HC- lyse CF) contain cyanide free lyse reagent (HC5-Eolse) contain cyanide
free lyse reagent and (HC-cleaner) cleaning solution used to clean fluidics
system and the instrument have a printer machine inside with thermal paper.
The hematological parameters used in the study were (RBCs, WBCs, Hb,
MCV, PCV, MCH, MCHC, Lym %, Mon % and Gran%).

Procedure:

Blood sample collected in tube contain an anticoagulant to prevent

forming clots that will clog the cell count, then position the sample tube in the
sample rotor and press start key, the sample rotor turn in moving the sample
inside the instrument and the probe draw (1ml) of blood sample from the tube.
The aspirating needle retracted after few second the rotor turns again
returning the blood sample tube after that we can remove the tube from
adapter of the rotor, the end of analysis was displayed in screen, including

69
automatically in the memory without any operator confirmation after that we
can printing the result by pressing printer key (Hoff and Rlagt, 2000)

3-5.1.4 Antioxidant Enzymes Measurement

3-5.1.4.1 Measurements of Superoxide Dismutase (SOD)

For determined The serum SOD had been used by ELISA kit (Elabascience
Biotechnology Inc. China ).

Procedure :

The procedure was done as the following steps

1. 50 μl of standard or sample was add to all well.

2. 50 μl of biotinated detection Ab was add to all well directly.

3. Incubated for 45 minutes at 370C.

4. Aspirated then wash 3 times.

5. 100 μl of HRP conjugated was add to all well then incubated for 30

minutes at 37٥C.

6. Aspirated and wash 5 times.

7. 90 μl of substrate reagent was add then incubated for 15 minutes at 37 0C.

8. 50 μl of stop solution was add to all well.

9. The absorbance was read on ELISA Reader at 450 nm directly after added
the stopping solution.
3-5.1.4.2 Estimation of Glutathione Peroxidase (GPX)

The serum GPX also is determined by ELISA kit (Elabascience

Biotechnology Inc. China), according to Flohé and Günzler, (1984) method.

71
Procedure:

The procedure was done as the following steps:

1.100 μl of sample or standard was add to every well then incubated for 90

min at 370C.

2. Eliminated the liquid then add 100 μl of biotinated detection Ab to both

well .

3. Incubated for 1 hour at 37 0 C.

4. Aspirated then wash 3 times.

5. 100 μl of HRP conjugated was add to all well then incubated for 30

minutes at 37 0 C.

6. Aspirated then washed 5 times.

7. 90 μl of substrate reagent was add then incubated for 15 minutes at 37 0 C.

8. 50 μl of stop solution was add to each well.

9. The absorbance was read on ELISA Reader at 450 nm proximately after

adding the stopping solution.

3-5.1.4.3 Serum Malondialdehyde Estimation(MDA)

The chief finale product of lipid peroxidation is MDA, will be done in

serum according to Yagi method (Yagi, 1998).

Principle of Measurement:

71
 The base of this on the spectrophotometer measurement. When
Thiobarbituric acid (TBA) reacts with MDA to produce thiobarbituric acid
reactive substance. Preparation of reagents:

20% trichloroacetic acid (TCA) 75ml

37% concentrated HCl 2 ml
Thiobarbituric acid 0.375 gm

Mixed and final volume was completed to 100 ml of DW

Procedure:

1. 1ml of serum is added to 2ml of reagent (TCA-TBA-HCl solution) then

mixed well.
2. Next boil the solution set for 15 min. in water bath at 100°C.
3. Centrifugation each of test tubes at 3000rpm. for 10-15 min. then removed
the precipitate.
4. Read the absorbance of the solution at 535nm. wave length against blank
(blank contain all reagents except serum).
The calculation:

A1-A0

MDA (µmol/L) = X 10

1.56

3-5.1.5 Biochemical Study

Both biochemical measurements were prepared on the serum when the

separation via using different enzymatic kits as follow:

3-5.1.5.1 Serum Alanine Aminotransferase (ALT) Estimation (U/l)

72
The Principle:

Determination of ALT founded on the next reaction (Schumann and

Klauke, 2003):

ALT

Oxoglutarate + L-Alanine L-Glutamate + Pyruvate

Pyruvate reacts with DNPH to produce a dyed hydrazine which can be

measure at 546 nm (530-550).

Procedure:

Reagent Blank Sample

Sample ---------- 0.1 ml
Solution 1 0.5 ml 0.5 ml
Distilled water 0.1 ml -----------

Mix and let stand exactly 30 min at 370C.

Solution 2 0.5ml 0.5ml

Mix and let stand exactly 20 min at 20-250 C.

NaOH 0.4N 5ml 5ml

Mix by gently inversion. read absorbance against blank tube after 5 min.

The Calculation:

Achieving the action of ALT through the serum as of the table which
mentioned in sheets of its kit.

3-5.1.5.2 Serum Aspartate Aminotransferase (AST) Estimation (U/I)

The Principle:

73
 Aspartate aminotransferase is measured by monitoring the concentration of
oxaloacetate hydrazone formed with 2,4-dinitrophenyl-hydrazine (Schumann
and Klauke, 2003).

AST
Oxoglutarate + L-Aspartate L-glutarate + Oxaloacetate

MD
NADH +H+ + Oxaloacetate NAD+ + L-Malate

The proportion of NADH intake is straight comparative to the AST action

via the sample.

Procedure:

Sample 0.1 ml
Working reagent 0.1 ml

Mix then subsequently 1 min incubation. Measure the alteration of optical

density for each min. (∆ O.D/ min) throughout 3 min.

The Calculation:

U/L= ∆ optical density per min x1746

3-5.1.5.3 Serum Alkaline Phosphatase (ALP) Determination (U/I)

Principle:

Colorimetric determination of the ALP activity according to the following

reaction (Tietiz, 1999):

74
 ALP
Phenyl phosphate Phenol + phosphate

The phenol liberated is measured in the presence of amin4antipyrine and

potassium ferricyanide. The presence of sodium arsenate in the reagent stops
the enzymatic reaction .

Procedure:

Serum Serum Blank Standard Reagent

Sample Blank
Reagent 1 2 ml 2 ml 2 ml 2 ml
Incubation for 5 min. at 370 C.

Serum 50µl ---- ----- -----

Reagent 2 ----- ---- 50µl -----
Incubation for exactly 15 min. at 370 C.

Reagent 3 0.5 ml 0.5 ml 0.5 ml 0.5 ml

Mix well

Reagent 4 0.5 ml 0.5 ml 0.5 ml 0.5 ml

Serum ------ 50µl ------ -------
D-water ------ ------ ------ 50µl
Mix. Let stand for 10 min. in the dark. Measure the dye concentration
is stable 45 min.
Calculation:

serum sample-serum blank

ALP (U/I) = X 142

Standard

75
3-5.1.5.4 Serum Total Protein (TP) Determination (g/dl)

Principle:

Serum proteins form a violet colored complex in the presence of copper

salt in alkaline solution (Biuret reaction),the intensity of the color is
proportional to the proteins concentration( Tietz, 2006).

Procedure:

Blank standard Sample

Sample ------ ------- 10 µl
Standard ------- 10 µl ------
Reagent 1 ml 1 ml 1 ml

Mix then after 10 minute. Incubation read the O.D in contrast to the blank,
the ending color is steady for at minimum 1 hour.

Calculation:
OD. of sample

TP (g/dl) = x ST. conc. (6gm/dl)

OD. of Standard

3-5.1.5.5 Serum Total Bilirubin (TB) Determination (mg/dl)

Principle:

In the existence of caffeine, TB reacts using Diazotized sulfanilic acid. In

the lack of caffeine, merely the Direct Bilirubin reacts.

Procedure:

76
 Adult pediatric

Blank Sample Blank Sample

R1 0.1 ml 0.1 ml 0.1 ml 0.1 ml

R2 ------- 25 µl ------- 25 µl
Mix well before the addition of R3

R3 0.5 ml 0.5 ml 0.5 ml 0.5 ml

S 100 µl 100 µl 20 µl 20 µl
Mix and incubate 5 minutes at 15-250 C

R4 0.5 ml 0.5 ml
Mix and read the optical density (O.D) after 5 minutes incubation against the Blank
sample. The final color is stable for least 30 min.

The Calculation:
TB (mg/dl)=OD x 10.8
3-5.1.5.6 Estimation of Uric Acid (mg/dl)

The Principle:

Uric acid is oxidized through uricase to allatoine then hydrogen peroxide

conferring to the subsequent reactions:

Uricase
Uric acid + O2 +2H2o2 Allantoine + Co2 +H2o2

peroxidase
2H2o2 + DHBS + 4-aminoantipyrine Red quinone +H 2O+HCl

Procedure:

Blank Sample Standard

Standard - ------ 20 µl

77
 Sample - 20 µl ------

Working 1 ml 1 ml 1 ml
Reagent
Mix very well then incubate 15 min. at room temperature otherwise 5 min.
at 370C at that moment read the (O.D) against the Blank, the pigment is
constant for 30 min.
Calculation:

O.D Sample
U.A. conc. = x Standard concentration (6mg/dl)
O.D Standard

3-5.1.5.7 Serum Urea Estimation (mg/dl)

The Principle:

Urea is hydrolyze through water besides Urease into NH3 and carbon
dioxide. The ammonia that produced is added to acted with hypochlorite in
addition salicylate to procedure a green compound

Urease
Urea + H2O 2NH3 +Co2

Procedure:

Blank Standard Sample

Standard ----- 10 µl ------
Sample ----- ------- 10 µl
Working reagent 1 ml 1 ml 1 ml
Mix well ,incubate for 5 min. at 37 0C.

R3 1ml 1 ml 1 ml

78
Mix, incubate for 5 min. at 370 C or else 10 min. at RT. Read the OD. of the
Standard as well as Sample against the Blank, the dye is unchanging for 1
hour.

Calculation:

O.D. Sample
Urea (mg/dl)= x St. conc.(50mg/dl)
O.D .Standard

3-5.1.5.8 Estimation of Creatinine (mg/dl)

Principle:

Creatinine in alkaline picrate solution, forms a color complex.

Procedure:

Blank Standard Sample

Distilled water 0.5 ml ------ -----
TCA Solution 0.5 ml 0.5 ml ------
Standard ------- 0.5 ml -------
Supernatant ------- ----- 1 ml
Reagent Mixture 1 ml 1 ml 1 ml
Mix well, let stand for 20 min. at room temperature, read the optical
density (O.D) of sample and standard against blank.

Calculation:

O.D Sample

Creatinine concentration (mg/dl)= X St. conc.(2mg/dl)

O.D Standard

3-5.1.5.9 Serum Total Cholesterol Estimation

79
 Measurement of serum total cholesterol through enzymatic and
colorimetric method using chemical kit Jourilabs (Tietz, 1996 and 1999).

Principle:

Cholesterol is measured by using of the subsequent enzymatic reaction:

Cholesterol esterase

H2O +Cholesterol ester Fatty acid + Cholesterol

Cholesterol Oxidase

O2 + Cholesterol H2 + 4 Cholesterol-3-one

peroxidase

2 H2O2 +4 amino antypirine Red quinone +4 H2O

Procedure:

Blank Standard Sample

Standard ---- 10µl ----
Sample ------ ---- 10µl
Working solution 1 ml 1 ml 1 ml
Mix ,incubate for 5 min. at 370C.Measure the optical density of standard
and sample against blank reagent, the color intensity is for stable 30 min.

Calculation:

O.D Sample
Cholesterol(mg/dl)= x St. conc.(200mg/dl)
O.D standard

3-5.1.5.10 Estimation of Triglyceride (mg/dl)

81
Principle:

The concentration of serum triglyceride was measured by using a special

chemical kit (JOURILABS. Ethiopia) based on (Stein, 1987):

Lipoprotein lipase
Triglycerides + H2O Glycerol +Fatty acid

Glycerol kinase
Glycerol +ATP Glycerol-3-phosphate
GPO
Glycerol-3-phosphate + O2 Dehydroxyacetone phosphate + H2O2
POD
2H2O2 + 4 aminoantipyrine + 4 CP Red quinone + 4 H2O

Procedure:

Blank Standard Sample

Standard -- -- 10 µl
Sample -- 10 µl --
Working reagent 1 ml 1 ml 1 ml
Mix, then incubate for 5 min at 370C.Read the OD .The paint is stable for
1hour at RT.

Calculation:

O.D. Sample
Triglyceride mg/dl= x Standard conc.(200mg/dl)
O.D. Standard

3-5.1.5.11 Estimation of Serum Lipoprotein Cholesterol

There are three varieties of lipoprotein cholesterol determination by the

following:

81
3-5.1.5.12 High-Density Lipoprotein Cholesterol

LDL, VLDL and chylomicrons are definitely triggered by

phosphotungestic acid also Magnesium ions and can be detached via
centrifugation. HDL persist in the supernatant which will be determined via
enzymatic technique (Tietz, 1999).

Procedure:

Sample 500µl
Reagent (R1) 1 ml
Mix then let to stand for 10 min at RT and centrifuge at 5000 rpm for 10 min.
Accumulate the supernatant then record it as a sample in the TC determination by
JOURILABS cholesterol kit.

Blank Standard Sample

Standard 150 mg/dl --- 50µl ----
Supernatant ---- ---- 50µl
Cholesterol R. 1 ml 1ml 1 ml
Mix, incubate at 25c for 20 min otherwise at 30 0 C used for 12 min, or at 37٥C for 10 min.
at that time read the OD. The color is steady for 30 min.

The Calculation:

O.D sample

HDL (mg/dl) = x 150

O.D standard

3-5.1.5.13 Low-Density Lipoprotein Cholesterol

LDL conc. can be determination via the following equation (Ram, 1996).

LDL= TC- (HDL+TG/5)

3-5.1.5.14 Very Low-Density Lipoprotein

82
 VLDL conc. was estimated by the following equation (Friedewald et al.,
1972).

VLDL=TG/5

3-5.1.6 Hormones Assay

The basic principle of an enzyme linked immunosorbent is to use an

enzyme to detect the binding of antigen (Ag) antibody (Ab). The enzyme
converts a colorless substrate to a colored product, indicating the presence of
Ag: Ab binding (Ma et al., 2006).

3-5.1.6.1 Adrenocorticotropic Hormone (ACTH )

Principle :

The ACTH Immunoassay is Enzyme-Linked ImmunoSorbent Assay

(ELISA) for the Estimation of the biologically active 39 amino acid chain of
ACTH. kit was used (ALPCO. America).

Procedure:

1.Adequate Streptavidin Coated Strips was sit in a container (accurate

concentration is illustrate on the vial label), At a minimum, put two wells to
work for as “blanks”. Refer to Step 9 for final plate reading.

2. 200 μL of calibrators, controls, as well as samples was put into the specify
well. Freeze (-20oC) the residual calibrators and controls immediately when
use.

3. 25 μL of Reagent 1 (Biotinylated Antibody) was add in all wells, which

comprise the calibrators, controls, and samples.

83
4. 25 μL of Reagent 2 (Enzyme Labeled Antibody)was add into each one of
the same wells. Shield the micro plate(s) by aluminum foil to avoid exposure
to light, then place it on rotator set at 170 + 10 rpm for 4 hours +30 min. at
room temperature (22-28٥C).

5.In the beginning a aspirate whole fluid then wash each well five times
through the Working Wash Solution,. The wash solution volume ought to be
set to dish out 0.35 mL into each well.

6. 150 μL of the ELISA Reagent B was add into both of the wells.

7. Suitable cover was use to bypass light exposure, put the micro plate(s) on a
rotator set at 170 + 10 rpm for 30 +5 min at room temperature (22-28٥C).

8. 100 μL of the Stop Solution was add through each of the wells. Mix
quietly.

9. Read the absorbance of the solution in the wells in 10 min by a micro plate
reader at 450 nm. Previous to reading, make sure both “blank wells” as stated
in Step 1 are occupied with 250 μL of distilled or deionized water. Read the
plate again using the reader at 405 nm counter to distilled or deionized water.

10. Via using the last absorbance values achieved in the prior step, create a
calibration curve via cubic spline four parameter logistics, or point-to-point
interpolation to enumerate the concentration of the ACTH.

3-5.1.6.2 Cortisol Hormone

Principle:

The subsequent enzyme immunoassay test follows the classic competitive

binding script. Competition take place between an unlabeled antigen and an

84
enzyme labeled antigen in place of a inadequate number of antibody binding
sites on the mico well plate . kit which also used (ALPCO. America).

Procedure:

1-Provid working solution of the cortisol-HRP conjugate then wash buffer.

2-Take off the required number of micro well strips. reseal the bag and return
every unused strips to the refrigerator.

3-Pipette the 20µl of each calibrator, control also specimen sample into
respectively labeled wells in identical.

4-Pipette 100 µl of the conjugate working solution into each well.

5-Incubate on a plate shaker (approximately 200 rpm) for 45 min. at room

temperature.

6-Wash the wells three times by 300 µl of diluted wash buffer per well then
tap the plate definitely against absorbent paper to ensure that it is dry.

7-Pipette 150 µl of TMB substrate into each well at timed intervals.

8-Incubate on a plate shaker for 15-20 min. at room temperature.

9-Pipette 50 µl of stop solution into each well at the same timed intervals as
in step 7.

10- Read the plate on a micro well plate reader at 450 nm within 20 min.
after addition of the stop solution.

3-5.1.6.3 Thyroid Stimulating Hormone (TSH)

Principle:

85
 Estimation of serum thyrotropin (TSH) concentration is commonly held as
a appreciated instrument in the identification of thyroid dysfunction; kit
which also was used (Sigma –Aldrich,USA).

Procedure:

1.Put the preferred number of coated strips into the holder.

2.Pipette 50 ml of TSH standards, control, then specimens into chosen wells.
3.Add 100 ml of prepared to use conjugate reagent to all wells. Shake for 10–
30 seconds.
4.Cover the plate and incubate for 60 minutes at room temperature (18–26
°C).
5.Eliminated liquid from all wells. Wash wells three times with 300 ml of 1x
wash buffer.
6.100 ml of TMB substrate was adding to all wells.
7.Incubation for 15 minutes at room temperature .
8.50 mL of Stop Solution was adding to all wells. Shaking the plate
temperately to mixture the solution.
9.Reading an absorbance on ELISA Reader at 450 nm in 15 minutes next
adding the stop solution.

3-5.1.6.4 Estimation of Total Thyroxin (tT4)

Principle:

Thyroxine (T4) is a beneficial marker for the diagnosis of hypothyroidism

as well as hyperthyroidism. The level of T4 is reduced in hypothyroid patients
and is amplified in hyperthyroid patients. The level of T4 is normal in
euthyroid individuals. kit which used(Sigma –Aldrich ,USA)

86
Procedure:

1. Setup the micro plate wells for all serum reference, control, and specimen
to be examined in duplicate. Change any unused micro well strips back into
the aluminum bag, seal, and store at 2–8 °C.

2. Pipette 10 mL of the standards, control, or specimen into the allocated well.

3. 100 mL of T4-Enzyme Conjugate solution was add to wholly wells .

4. Incubating for 60 minutes at room temperature with shaking.

5. Removed liquid from all wells. Wash wells three times with 300 mL of 1x
wash buffer

6. 100 mL of TMB substrate solution was adding to the whole wells.

7. Incubating, at room temperature, for 15 minutes.

8. Adding 50 mL of Stop Solution to all wells and gently mix for 15–20
seconds.

9. Reading the absorbance on ELISA Reader for each well at 450 nm within
15 minutes after adding the stop solution.

3-5.1.6.5 Estimation of Total Triiodothyronine (T3)

Principle:

Triiodothyronine (T3) is a suitable marker for the identification of

hypothyroidism and hyperthyroidism. The level of T3 is reduced in
hypothyroid patients and is increased in hyperthyroid patients, Graves‟
disease and pregnancy. Kit also used (Sigma –Aldrich ,USA)

87
Procedure:

1. Setup the micro plate wells for all serum reference, control, and specimen
to be examined in duplicate. Exchange every unused micro well strips back
into the aluminum bag, seal, and store at 2–8 °C.

2. 25 ml of the appropriate serum reference, control, or specimen was pipette

into the allocated well.

3. 100 ml of working T3-Enzyme Conjugate solution was add to all wells .

4. Cover the plate and incubate for 60 minutes at room temperature with
shaking.

5. Removed liquid from all wells. Wash wells three times with 300 of 1x
Wash buffer

6. 100 ml of TMB substrate solution was add to all wells.

7. Cover the plate and Incubate at room temperature for 15 minutes.

8. 50 ml of Stop Solution was add to each well and gently mix for 15–20
seconds.

9. The absorbance was read on ELISA Reader of each well at 450 nm within
15 min after adding the stop solution.

3-5.1.7 The Histological Study

Animals were sacrificed at the end of the experiment and the organs
(brain, liver, kidney and lung) were wisely removed and washed by normal
saline as well as fixed in 10 % buffered formalin for 24 hrs. Dry out the
specimens using categorized sequence of ethanol and cleared in two

88
variations of xylene then set in in paraffin wax. The width of piece 5µm were
scratch by using a rotary microtome in addition fixed on clean slides for
histological examination subsequently stained the sections via Haematoxylin
and eosin (H&E) and estimate the tissue structure under a LCD light
microscope (Mescher, 2010).

3-5.2 Second Experiment

Through this experiment 10 adult male and 20 adult female were used,
each of one male and two female were put in separate cage then after ensure
the mating occur and the female become pregnant, then the pregnant female
was separated, anaesthetized and sterilized the abdominal by ethanol alcohol
spray , then scratch medially over the skin in addition to muscles by a couple
of scissors showing an uterus besides embryos and take away wholly fetuses,
then follow the steps of protocol of isolation of cortical and hippocampus
neurons from fetus and we examine the effect of MSG (500 and 250µM),
lycopene (500 and 250 µM) compared with control (untreated) and DMSO
(vehicle).

3-5.2.1 Primary Cortical and Hippocampus Neuronal Cultures

The method for culturing rat cortical & hippocampus neurons was
principally the same as the technique described by Pacifici and Peruzzi.
(2012).

89
 1-Stage of anesthesia 2-Anatomay stage 3-Expulsion of rat pups

4- Dissection of Brain Pups

5-Dissociation of Cortex& 6-Produced Neurospheres 7-Viability Assay

Hippocampi

8- Incubation for 7days then Treatment neurons via MSG and

lycopene then record results

Scheme (2) Diagram Illustrating the Procedure for Neurons Culture

91
3-5.2.2 Preparation of Poly-D-Lysine

1. Five ml of sterile ddH2O was adding to 5 mg of PDL to acquire a stock

solution of 1 mg/ml.

2. Mixture stock solution using pipet for numerous times.

3. Immediately using or store PDL Solution at 2-8 °C.

3-5.2.3 Coating Plastic Cell Culture Dishes via Poly-D-Lysine (PDL)

1. Dilute the PDL stock solution by sterile ddH2O to the final concentration of
10 μg/ml.

2. Pipette adequate solution into a 60 mm dish to cover culture superficial

area (3 ml for a 60 mm dish).

3. Shake slightly to confirm even coating of the culture surface.

4. Incubate coated plates at room temperature (RT) overnight.

5. On the following day, generally the day of dissection, remove the Poly-D-
Lysine Solution via aspiration and wash permanently with 3 ml of sterile
ddH2O. Replication this step. After the second wash, remove water finally
through aspiration.

6. Plates can be stored at 4 °C for up to three weeks.

91
3-5.2.4 Preparation and Coating Poly-D-Lysine (PDL) and fibrinogen of
Glass dishes

1. Mixture PDL (1 mg/ml) and fibrinogen (1 mg/ml) stock solution in

sterilized ddH2O to the ultimate concentration of 10 and 5 μg/ml,
correspondingly.

2. Pipette sufficient solution into glass dishes to shelter the culture surface
area (3 ml for each one.

3. Shake gently to make sure coating of the culture surface.

4. Incubating coated dishes at room temperature overnight.

5. On the following day, eliminate the Poly-D-Lysine-fibrinogen coating

solution by aspiration then wash briefly twice with 1 ml of sterile ddH 2O.
Next the second wash, take away whole water by aspiration.

6. Glass dishes can be stored at 4 °C for three weeks.

3-5.2.5 Neuronal Dissection and Culture

1. Heated Neurobasal/B27 complete medium in a 37 °C in water bath.

2. Three milliliter of icy Hibernate E solution was adding to four culture
dishes (60 mm ) as well as thirteen milliliter to a fifteen milliliter BD Falcon
in height clearness polypropylene conical tube.

3. From (25-30) ml of cold dissection medium (hank balance solution HBS)

was adding to each of three 100 mm culture dishes. These plates, comprising
a large volume of medium, it will be used to wash the embryos directly after
their elimination from the amniotic sacs

4. Euthanizing an E16-18 timed pregnant rat .

92
5. Spray inferior abdomen by 70% ethanol then scratch medially concluded
the skin also muscles by a pair of scissors showing the uterus and embryos.

6. Taking away the whole fetuses and put them in a sterile 100 mm dish
covering an excess of cold dissection medium (25-30 ml).

7. Scratching embryos by a minor pair of scissors starting amniotic sac

formerly put them through the another 100-mm dish comprising icy dissection
medium.

8. Wash-down the embryos at RT through moderately orientated the (100mm

)dish for 5-10 sec. At that time, removal the washed embryos to the third (100
mm) dish comprising dissecting medium. Double washes in additional
medium is commonly adequate in order to eliminate entirely drops of blood.
Conversely, wash one other stage using a new 100 mm dish comprising about
25-30 ml of icy dissection medium.

9. With a stereomicroscope then hooked forceps, remove both embryo's brain

through removal the skin then cranium. Put whole brain via any of the
(60mm) dishes (usually, no other than 5 brains for every one dish) through
cold Hibernate E. Valid these dishes on ice.

10. Take individual dish at a stage then, beneath a dissecting microscope,

isolated hemispheres and separate a cerebral cortices eliminating a midbrain
also meninges.

11. Scratch brains laterally, extract hippocampi, also following the procedure
lower to separate hippocampal neurons.

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12. Accumulate wholly the separated cortices in a 15ml conical tube
comprising (13 ml ) of icy Hibernate E. Dispensation of a cerebral cortices
on ice up to wholly the separations are done. As of their slight size, separated
hippocampi can be placid in a 1.5 ml Eppendorf tube in place of a 15ml
tube. Next to this stage

cortices or hippocampi can be placed in the cryo tube vial comprising (1 ml )

of Hibernate E and 2% B27 and Gentamicin (50μg/ml) plus Fungizone (250
ng/ml) in an amount of 2-4 cortices or 2-4 hippocampi for each vial. Brain
tissue can be kept at 4 °C in the dark used for ready for one. When desired,
usage fine forceps to removal the brain tissue to a tube containing Hibernate E
then follow the procedure to separate neurons.

13. Allocate the tube in a tissue culture hood. Let the cortices and
hippocampus to calm down to the bottom of each tube, at that time prudently
eliminate a supernatant.

14. Thirteen ml from new Hibernate E was adding to the (15) ml conical tube,
permit cortices and hippocampus settle at the lowest of the tube and wisely
eliminate the supernatant. Reprise this step 2 extra times then, subsequently
the last wash-down, carefully eliminate wholly medium.

15. Moderately trituration (4-5 times) the cortices and hippocampus in 2 ml

Neurobasal/B27 complete medium using a fire-polished glass Pasteur
(approximately 1 mm in diameter), be careful to avoid bubbles.

16. Repeat another 4-5 times with a sterile glass Pasteur pipette smaller in
diameter (i.e. a pipette about 1/2-3/4 mm in diameter). Do not use a Pasteur
pipette smaller than this or it will destroy the cells.

94
17. Permit remaining pieces of tissue (in general very few, if any) to settle.

18. Transfer the upper single-cell suspension to a new 15-ml tube, leaving
behind settled pieces of tissue. Additional dilute the cell suspension up to 10-
12 ml with Neurobasal/B27 compl ete medium.

19. Mix fine and dilute cells for counting by adding 10 μl of cell suspension
to 40 μl of 50x Counting Solution (trypan blue) in a 1.5 ml Eppendorf tube.

20. Plate cells on PDL-coated plates at the density of 5.0 x 104/cm2.

21. Usually, we cut up 9-10 fetuses for each experiment, for instance nearly
13 x 106 neurons are derivative from each E16-18 fetus. If more embryos are
required, take care that the complete procedure does not latest more than 2
hours.

23. Neurons can be used for experiments after 5-7 days in vitro, even though
exact time depends on anticipated differentiation stage.

24. For lengthy culturing, exchange culturing medium every week with newly
prepared Neurobasal/B27 complete medium.

3-5.2.6 Preparation Dose Testing

Two dose were used in cortical and hippocampus primary culture

neurons. Stock solution of MSG were prepared at 250 and 500 µM in 100%
sterile distal water while, the stock solutions of Lycopene were prepared also
at 250 and 500 µM in Dimethyl sulfoxide (DMSO), compared with two
control, one of them treatment by sterile distal water, other was vehicle of
DMSO. Then each stock solutions of MSG and lycopene were diluted in
appropriate culture medium to the indicated concentration.

95
3-5.2.7 Time-Dependent Injury of Neurons by MSG and Lycopene
Treatment

Mature neurons were treated with a 500 and 250 µM dose of MSG.As well
as 500 and 259 µM dose of lycopene. The alteration in cell morphology was
checked at diverse time points, the incubation with MSG or lycopene at zero,
30 min. and one hr. the neurons was observed.

3-5.2.8 Viability Assay Using Acridine Orange

Reagents:

1 μl Acridine Orange Stock (5 mg/ml)

1 ml PBS or 1 ml HBSS

Method:

1) Adding Acridine Orange and PBS or HBSS then mix well.

2) Storing at room temperature for up to 2 weeks.

Procedure:

1) Diluting cells with equal volume of Acridine Orange and PBS or HBSS

2) Immediately look at cells under fluorescence microscope.

Red Cells = Dead

Green Cells = Live

Calculation:

Survival (%) = green cells/(green + red cells)

96
3-5.2 .9 Immunocytochemistry

3-5.2.9.1 Fixation

Cells were washed with PBS and fixed in 4% paraformaldehyde (PFA) for
15-20 min. at room temperature. After fixation, cells were washed three times
with PBS for 5 min. to eliminate fixative, and stored at 4°C prior to
immunolabelling (Al-Mayyahi, 2017).

3-5.2.9.2 Immunostaining of Cells Seeded as Monolayers

After fixation, specific staining was blocked (blocking solution: 5% normal

donkey serum ( NDS), 0.3% Triton-X-100 in PBS) for 30 minutes at RT.
Primary antibodies were diluted as follows in blocking solution: β-tubulin
III/TUJ-1 1:1000 added to the cells and incubated overnight at 4°C. Stained
cells were washed three times in PBS, blocked for 30 min at RT and
incubated with the appropriate FITC conjugated secondary antibody in
blocking solution (1:200 dilution, RT; 2 h).Cells were then washed three
times with PBS at RT, and mounted with DAPI (Al-Mayyahi, 2017).

3-6 The Statistical Analysis

The results were analyzed usage one- way (ANOVA) test in an entire
this study. Least significant different test (LSD) was calculated to test the
differences between groups for (ANOVA). The whole statistical calculations
were achieved by the assistance of the statistical SPSS (SPSS Inc.) version (
21.0). The data were expressed as means ± standard error (Al-Rawi, 2000).

97
98
 Results
4-1 First Experiment
4-1.1 Effect of MSG alone or with Lycopene on:
4-1.1.1Bodyweight
According to the results shown in table (4-1), revealed no significant
difference in final body weight in G2,G3 and G6 as compared with control
group ,while a significant decreased (p< 0.05) in final body weight in G4 and
G5 compared with control and other treated groups. On other hand the
significant (p< 0.05) decreased in final body weight was recorded in G5
compared with control and all treated groups. Whereas no a significant
difference were observed in body weight gain between G2 and G3 as
compared with control group. While a significant decreased(p< 0.05) in body
weight gain was recorded in G4 and G5 as compared with control and treated
groups. The significant decrease (p< 0.05) in body weight gain was recorded
in G5 as compared with control and all other treated groups .
Table (4-1): Effect of MSG alone or with Lycopene on Body Weight of Male Rats.
(Mean ± SE),( No.=6).
parameters Body weight (gm)/month

Groups Initial body Final body Body weight

weight weight change
G1 Control (normal saline) 327.00±22.95 402.50±14.40 75.50±12.82
a ab
G2 MSG (20mg/kg)for 30 days 320.67±21.41 401.83±16.15 81.17±11.69
a a
G3 MSG(20mg/kg) for 15 days then 323.33±5.54 401.00±4.95 77.67±5.99
lycopene(200/kg)for 15 days a ab
G4 lycopene(200mg/kg)for 15 days 335.50±10.98 386.83±15.86 51.33±8.21
then MSG(20mg/kg)for 15 days b c
G5 lycopene (100mg/kg) and after 1hr. 328.83±10.44 355.00±8.82 26.17±3.79
MSG (20mg/kg) for 30 days c d
G6 lycopene (200mg/kg) and after 1hr. 337.17±22.98 400.00±25.32 62.83±6.77
MSG (20mg/kg) for 30 days a bc
LSD NS 13.56 15.67
The different small letters refer to significant differences at (p< 0.05).

99
 4-1.1.2 White Blood Cells Parameters

The results in table(4-2) showed a significant increase (p< 0.05) in

WBC count in G2 and G4 as compared with the control and the other treated
groups. It is also showed a significant increase (p< 0.05) of lymphocytes
percentage in G2,G4, G5 and G6 as compared with the control. While G3
showed no significant differences as compared with control. Furthermore, it
is showed a significant increase (p< 0.05)in monocytes percentage in G3 as
compared with control and all other treated groups. Finally, the results of
granulocytes percentage showed a significant decrease (p< 0.05)in G2,G4,G5
and G6 as compared with control. But, there were no significant differences
between G3 and control.

Table (4-2): Effect of MSG alone or with Lycopene on white blood cells parameters
of male rats. (Mean ±SE), (No.=6).

parameters white blood cells parameters

WBCs(×103) Lym.% Mon.% Gran.%
Groups
G1 Control (normal saline) 5.14±1.21 58.14±3.80 6.40±0.71 36.13±3.71
b b bc a
G2 MSG (20mg/kg)for 30 days 7.96±1.38 73.14±4.31 6.54±0.81 21.11±4.43
a a bc c
G3 MSG(20mg/kg) for 15 days then 3.70± 1.29 55.69±4.03 10.06±0.76 35.17±4.14
lycopene(200/kg)for 15 days b b a a
G4 lycopene(200mg/kg)for 15 days then 8.99±1.49 75.22± 4.66 4.50±0.88 20.08±4.79
MSG(20mg/kg)for 15 days a a c cd
G5 lycopene (100mg/kg) and after 1hr. 4.10±1.49 65.69±4.66 4.94±0.86 28.89±4.79
MSG (20mg/kg) for 30 days b ab c b
G6 lycopene (200mg/kg) and after 1hr. 5.38±1.21 76.09±3.80 7.22±0.71 16.70±3.91
MSG (20mg/kg) for 30 days b a b d
LSD 2.49 10.21 2.09 3.50
The different small letters refer to significant differences at (p< 0.05).

111
 4-1.1.3 Red Blood Cells Parameters

The results in table (4-3), showed a significant increase (p< 0.05) of RBCs
count in G3 and G6 as compared with control and other treated groups. It
also in Hb conc. revealed a significant increase (p< 0.05) in G3 as compared
with control and all other groups, but there is a significant decrease (p< 0.05)
in G5. The same table showed that PCV percentage is a significant increase
(p< 0.05) in G3 as compared with all treated group. Regarding the (MCV), it
showed is a significant decrease (p< 0.05) in G2 as compared with G3,G5
and G6 and control. Furthermore, the results demonstrated a significant
decrease (p< 0.05)of (MCH) in G2 and G4 as compared with control and
rest groups. Finally, that the results of MCHC is also showed that there is a
significant decrease (p< 0.05)in all treated groups compared with control .

Table (4-3): Effect of MSG alone or with Lycopene on Red Blood Cells Parameters of
male rats. (Mean ±SE), (No.=6).

parameters
Red blood cells parameters
Groups RBCs Hb PCV MCV MCH MCHC
(×106) g/dl % fL pg g/dl

G1 Control (normal saline) 5.47±0.47 11.10±1.01 35.57±2.93 57.66±1.15 20.16±0.48 35.40±0.75

b bc b ab a a
G2 MSG (20mg/kg)for 30 4.71±0.53 10.53±1.15 32.96±3.32 52.66±1.15 17.06±0.54 32.91±0.85
days b bc bc c c b
G3 MSG(20mg/kg) for 15 7.12±0.50 13.19±1.07 41.16±3.11 58.35±1.08 20.95±0.51 33.40±0.79
days then lycopene a a a a a ab
(200/kg)for 15 days
G4 lycopene(200mg/kg) for 5.45±0.58 11.55±1.24 35.42±3.59 55.07±1.24 17.95±0.59 32.62±0.92
15 days then MSG b b bc bc bc b
(20mg/kg)for 15 days
G5 lycopene (100mg/kg) 5.34±0.58 10.27±1.24 31.41±3.59 57.74±1.24 18.79±0.59 33.13±0.92
and after 1hr. MSG b c c ab b b
(20mg/kg) for 30 days
G6 lycopene (200mg/kg) 5.67±0.47 11.09±1.01 34.04±2.93 59.48±1.02 18.60±0.48 32.43±0.75
and after 1hr. MSG ab bc bc a b b
(20mg/kg) for 30 days
LSD 1.58 1.16 4.14 3.00 1.21 2.14
The different small letters refer to significant differences at (p< 0.05).
111
4-1.1.4 Oxidative Stress and Antioxidant Activity

According to the results in table (4-4), which showed a significant

decrease (p< 0.05) of antioxidant enzyme SOD in all treated groups as
compared with control group, it is also seen, that there is a significant
decrease (p< 0.05) of GPX in G2 as compared with the control and other
treated groups. The results are is also revealed that there is a significant
increase (p< 0.05) of MDA in G2 as compared with control and other
treated groups.

Table (4-4): Effect of MSG alone or with Lycopene on some antioxidant enzyme
superoxide dismutase (SOD), and Glutathione peroxidase (GPx) and MDA of male
rats.(Mean ± SE), (No.=6).
Parameters SOD GPX MDA
mEq/L u/mg nmol/g
Groups
G1 Control (normal saline) 55.46±1.98 3576.3±230.9 0.66±0.27
a a b
G2 MSG (20mg/kg)for 30 days 45.60±1.11 2287.0±258.2 1.59±0.36
b c a
G3 MSG(20mg/kg) for 15 days then 46.52±0.38 3272.1±230.9 1.01±0.25
lycopene (200/kg)for 15 days b b b
G4 lycopene(200mg/kg) for 15 days 48.47±0.77 3154.3±231.4 0.89±0.36
then MSG (20mg/kg)for 15 days b b b
G5 lycopene (100mg/kg) and after 47.44±1.10 3284.0±275.3 1.10±0.39
1hr. MSG (20mg/kg) for 30 days b b b
G6 lycopene (200mg/kg) and after 46.60±0.85 3287.4±220.9 0.55±0.13
1hr. MSG (20mg/kg) for 30 days b b b
LSD 5.00 247.78 0.48
The different small letters refer to significant differences at (p< 0.05).

4-1.5 Biochemical Parameters

4-1.5.1 Liver Enzymes
In the table (4-5), AST enzyme revealed a significant increase (p< 0.05)
in all treated groups compared with control ,and the more significant change
was observed in G3.Furthermore (ALT) enzyme is also showed a significant

112
 increase (p< 0.05) in all treated groups, and the more significant change was
observed in G4 as compared with control. The same table indicated
that, there was a significant increase (p< 0.05) of alkaline phosphatase
(ALP) in all treated groups as compared with the control group, and that
more changes observed in G3. The total protein revealed a significant
increase (p< 0.05) in G2 compared with control and other groups. The results
are also demonstrated that ,there is a significant increase (p< 0.05) of total
bilirubin in all treated groups as compared with the control, and the more
significant change was observed in G6.

Table (4-5) Effect of MSG alone or with Lycopene on liver function enzyme such
AST, ALT, ALP, Total protein and bilirubin of male rats.(mean± SE),(No.=6)
Parameters AST ALT ALP Total Total
IU/L IU/L IU/L protein Bilirubin
Groups g/dl Mg/dl

G1 Control (normal saline) 30.38±14.93 11.17±1.87 16.10±1.10 4.81±0.58 1.46±0.13

d c e b c

G2 MSG (20mg/kg)for 30 175.83±40.90 78.17±1.54 23.06±2.63 18.21±1.18 3.18±0.20

days b a c a ab
G3 MSG(20mg/kg) for 15 293.83±39.23 67.00±2.24 33.41±2.67 4.84±0.35 3.09±0.28
days then lycopene a b a b ab
(200/kg)for 15 days
G4 lycopene(200mg/kg) for 117.00±35.06 79.00±1.26 20.66±3.14 5.11±0.80 2.81±0.44
15 days then MSG c a d b b
(20mg/kg)for 15 days
G5 lycopene (100mg/kg) and 162.33±33.92 72.00±1.83 28.81±5.66 5.37±0.29 3.21±0.34
after 1hr. MSG (20mg/kg) b ab b b ab
for 30 days
G6 lycopene (200mg/kg) and 111.50±37.31 74.50±1.12 20.82±3.22 4.34±0.45 3.53±0.34
after 1hr. MSG (20mg/kg) c ab d b a
for 30 days
LSD 32.73 10.44 4.51 2.40 0.51
The different small letters refer to significant differences at (p< 0.05).

113
4-1.5.2 Kidney Function Tests

Depending on the results in table (4-6) the creatinine, is significantly

increased (p<0.05) in all treated groups as compared with the control, and
the more significant change was observed in G2. The results
demonstrated, a significant increase (p<0.05) of urea level in G2 and G4 as
compared with the control, while G3 showed a significant decrease (p<0.05)
of urea as compared with the control and other treated groups. Regarding the
uric acid, there was a significant increase (p<0.05) in G2 as compared with
the control and other treated groups.

Table (4-6) Effect of MSG alone or with Lycopene on creatinine, urea and uric acid of
male rats.(mean± SE)(No.=6).

Parameters Creatinine Urea Uric acid

Mg/dl mmol/dl Mg/dl
Groups
G1 Control (normal saline) 2.21±0.04 12.55±1.31 3.06±0.43
c b d
G2 MSG (20mg/kg)for 30 days 2.95±0.27 14.13±1.50 4.73±0.16
a a a
G3 MSG(20mg/kg) for 15 days then 2.80±0.12 9.77±0.86 3.23±0.11
lycopene (200/kg)for 15 days ab c cd
G4 lycopene(200mg/kg) for 15 days then 2.55±0.03 14.58±1.21 3.66±0.40
MSG (20mg/kg)for 15 days b a bcd
G5 lycopene (100mg/kg) and after 1hr. MSG 2.71±0.07 12.67±1.19 3.56±0.13
(20mg/kg) for 30 days ab b bcd
G6 lycopene (200mg/kg) and after 1hr. MSG 2.81±0.05 13.42±1.70 3.96±0.19
(20mg/kg) for 30 days ab ab b
LSD 0.33 1.48 0.64
The different small letters refer to significant differences at (p< 0.05).

4-1.5.3 Lipid profile

The results in the present study demonstrated that, there is a significant

increase (p<0.05) of total cholesterol level in all treated groups compared
with the control groups as seen in table (4-7).

114
 The same table showed a significant increase (p<0.05) of triglyceride
level in G2 and G4 as compared with the control, while G3,G5 and G6
appeared near to value of control group. Regarding the HDL concentration
the results revealed that, there is a significant decrease (p<0.05) in G2
compared with the control and other treated groups. It is also seen that, there
is a significant increase (p<0.05) of LDL level in all treated groups as
compared with the control. Finally, the results showed a significant increase
(p<0.05) of VLDL level in all treated groups as compared with the control
and its more significantly increased in G2.

Table (4-7) Effect of MSG alone or with Lycopene on Lipid profile of male
rats.(mean± SE(No.=6).
Parameters TC TG HDL LDL VLDL
Mg/dl Mg/dl Mg/dl Mg/dl Mg/dl
Groups
G1 Control (normal saline) 105.18±10.16 155.44±7.11 38.72±3.83 33.18±6.20 31.88±3.55
b c a c c
G2 MSG (20mg/kg)for 30 122.42±6.58 224.34±4.55 27.57±2.85 42.57±8.92 45.08±1.32
days a a c ab a
G3 MSG(20mg/kg) for 15 114.13±12.42 172.16±2.98 35.98±1.36 43.17±19.17 34.83±2.48
days then lycopene a bc ab ab bc
(200/kg)for 15 days
G4 lycopene(200mg/kg) for 110.68±5.48 176.99±6.00 35.97±1.52 39.33±9.05 35.99±2.79
15 days then MSG ab b ab b b
(20mg/kg)for 15 days
G5 lycopene (100mg/kg) and 116.05±12.67 159.37±3.11 36.13±2.80 43.17±13.60 37.20±2.53
after 1hr. MSG (20mg/kg) a bc ab b b
for 30 days
G6 lycopene (200mg/kg) and 118.58±31.04 169.68±5.43 35.70±2.52 44.83±11.43 36.73±2.54
after 1hr. MSG (20mg/kg) a bc b a b
for 30 days
LSD 13.23 19.23 2.78 4.63 4.01
The different small letters refer to significant differences at (p< 0.05).

4-1.6 Some of Hormones

115
 The results in table (4-8) showed that , there is a significant increase
(p<0.05) of adrenocorticotropic hormone level (ACTH) in all treated groups
compared with the control. It is also showed that, there is a significant
decrease (p<0.05) of cortisol hormone level in all treated groups as compared
with the control. In addition to that, the result demonstrated no significant
differences of thyroxin stimulating hormone (TSH) level between all groups.
While it indicated that there is a significant decrease(p<0.05) of
triiodothyronine (T3) in all treated groups as compared with the control.
Finally, it is also showed that, there is a significant increase (p<0.05) of
tetraiodothyronine (T4) in G2,G4 and G5 as compared with the control and
other treated groups.

Table (4-8) Effect of MSG alone or with Lycopene on Some of Hormones of male
rats.(mean± SE)(No.=6)
Parameters ACTH Cortisol TSH T3 T4
Pg/ml Ug/dl mL/L Ng/dl ug/dl
Groups
G1 Control (normal saline) 4.26±0.80 85.99±9.99 0.02±0.009 3.15±0.55 44.41±5.49
c a a b
G2 MSG (20mg/kg)for 30 8.95±3.88 47.36±10.84 0.02±0.008 1.35±0.12 56.37±4.95
days ab bc b a
G3 MSG(20mg/kg) for 15 7.03±2.08 37.01±4.50 0.01±0.009 1.42±0.12 42.33±1.52
days then lycopene b c b b
(200/kg)for 15 days
G4 lycopene(200mg/kg) for 15 7.34±2.27 51.42±8.98 0.01±0.002 1.67±0.08 46.77±3.13
days then MSG (20mg/kg)for b b b a
15 days
G5 lycopene (100mg/kg) and 6.89±2.42 38.08±5.25 0.01±0.007 1.51±0.08 45.82±2.44
after 1hr. MSG (20mg/kg) for b c b a
30 days
G6 lycopene (200mg/kg) and 10.88±4.02 47.16±10.37 0.01±0.001 1.29±0.06 44.82±3.31
after 1hr. MSG (20mg/kg) for a bc b b
30 days
LSD 2.58 12.50 NS 0.50 10.74
The different small letters refer to significant differences at (p< 0.05).

116
4-1.7 The Histopathological Investigation
4-1.7.1 Liver
The liver section showed normal structure and distribution of hepatocytes
and central vein as in figure (4-1). The cross sections of liver treated by MSG
(20mg/kg) for thirty days showed some histological changes that were at
variance with those obtained in the control, such as bleeding, loss of normal
structure of hepatocytes, loss of nuclei and dilation of central vein see figure
(4-2).The examination of liver tissue which treated with MSG (20mg/kg) for
fifteen days then lycopene (200mg/kg) for other fifteen days show vein
congestive, dilation of central vein, vaculation of hepatocytes and necrosis of
nuclei, as in the figure (4-3). While the section of liver rats treated with
lycopene (200 mg/kg) for fifteen days then MSG (20 mg/kg) for fifteen days
shows vaculation of cytoplasm, degeneration, Edema, necrosis of nuclei and
pyknotic, as in figure (4-4). But when investigation of liver rats treated with
lycopene (100 mg/kg) and MSG (20 mg/kg) for thirty days shows normal
central vein, normal hepatocytes and some of vaculation of cytoplasm as in
figure (4-5).Finally, the section of liver rats treated with lycopene (200
mg/kg) plus MSG (20 mg/kg) for thirty days shows absent the normal
structure, Fibrosis, some of vaculation of cytoplasm, aggregation of lipid
drops and bleeding figure (4-6).

117
 Figure (4-1) Section of control liver rats showed central vein (CV),
hepatocytes( H), sinusoids( S). H&E Stain. (100x).

Figure (4-2)Section of liver rats treated with MSG (20mg/kg) shows bleeding
(B), loss of normal structure of hepatocytes (LH),loss of nuclei (LN), dilation of
central vein (D).H&E stain, (400x).

118
 Figure (4-3) Section of liver rats treated with MSG(20 mg/kg) then lycopene (200
mg/kg) shows vein congestive (VC), dilation of central vein(D), vaculation of
hepatocytes, (V), necrosis of nuclei (N).H&E stain, (400x).

Figure (4-4) Section of liver rats treated with lycopene (200 mg/kg) then MSG (20
mg/kg) shows vaculation of cytoplasm (V), degeneration (D), edema (E), necrosis of
nuclei (N), pyknotic (P).H&E stain, (400x).

119
Figure (4-5) Section of liver rats treated with lycopene (100 mg/kg) and MSG (20
mg/kg) shows normal central vein (CV), normal hepatocytes (H), vaculation of
cytoplasm (VC) , H&E stain, (400x).

Figure (4-6) Section of liver rats treated with lycopene (200 mg/kg) and MSG (20
mg/kg) shows absent the normal structure (AB), fibrosis (F) , vaculation of
cytoplasm (VC), aggregation of lipid drops (A), bleeding(B). H&E stain, (400x).

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4-1.7.2 Kidney

The histopathological examination of sections kidney of control group

showing normal renal glomeruli, normal glomerular capsule and normal renal
convoluted tubules distal and proximal as in the figure ( 4-7).

Figure (4-8) appeared kidney section treated with MSG (20mg/kg)

showing bleeding, loss of normal structure, pyknotic, absent of convoluted
tubules, necrosis and degeneration. While in figure (4-9) perform section of
kidney rats treated with MSG (20mg/kg) then lycopene (200mg/kg) shows
necrosis of glomerulus, necrosis of tubules and hypertrophy in some tubules.
In section of kidney rats treated with lycopene (200mg/kg) then MSG
(20mg/kg) shows bleeding, loss of normal structure of tubules, hypertrophy in
some tubules, degeneration and necrosis as in the figure (4-10).While in
figure (4-11) seemed section of kidney rats treated with lycopene
(100mg/kg) and MSG (20mg/kg)shows shrinkage of glomerulus, loss of
normal structure of tubules, degeneration and necrosis. Finally in the section
of kidney rats treated with lycopene (200mg/kg) and MSG (20mg/kg) shows
shrinkage of glomerulus and necrosis, hyperplasia of tubules, degeneration
and necrosis, figure (4-12) .

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Figure (4-7) Section of normal kidney rats shows capsule (C), glomerulus (G),
distal convoluted tubules (D), proximal convoluted tubules (P). H&E stain,
(100x).

Figure (4-8) Section of rats kidney treated with MSG (20mg/kg) shows bleeding
(B), loss of normal structure (L), pyknotic (P), absent of convoluted tubules (A),
necrosis and degeneration (N). H&E stain, (400x).

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 Figure (4-9) Section of rats kidney treated with MSG (20mg/kg) then lycopene
(200mg/kg) shows necrosis of glomerulus (NG), necrosis of tubules (N),
hypertrophy in some tubules (H). H&E stain, (400x).

Figure (4-10) Section of rats kidney treated with lycopene (200mg/kg) then msg
(20mg/kg) shows bleeding(B), loss of normal structure of tubules (L), hypertrophy in
some tubules (H), degeneration and necrosis (D). H&E stain, (400x).

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 Figure (4-11) Section of rats kidney treated with lycopene (100mg/kg) and MSG
(20mg/kg) shows shrinkage of glomerulus (S), loss of normal structure of tubules (L),
degeneration and necrosis (D). H&E stain, (400x).

Figure (4-12) Section of rats kidney treated with lycopene (200mg/kg) and MSG
(20mg/kg) shows shrinkage of glomerulus and necrosis (S), hyperplasia of tubules
(H), degeneration and necrosis (D). H&E stain, (400x).

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4-1.7.3 The Brain

The section of control rat brain showed some of the typical layered figure
(4-13) appearance of the cerebral cortex such as follows: molecular layer;
external granular layer; pyramidal cell layer; internal granular layer;
ganglionic layer and multiform layer as well as gray matter, white matter,
pyramidal cell, capillary, axon and nerve cell. Figure (4-14) appeared section
of brain rats treated with MSG (20mg/kg) shows necrotic of some cells
bodies, necrosis of tissue structure, degeneration of some nerve cell and
degeneration of axon. while figure (4-15) seemed section of brain rats treated
with MSG (20mg/kg) then lycopene (200mg/kg) shows loss of gray matter,
necrosis of tissue structure and degeneration of some nerve cell and terminal
axon. Another section of brain rats treated with lycopene (200mg/kg) then
MSG (20mg/kg) shows hyperpigmentation of axon, aggregation lipid drops
and hypertrophy of microglia cells as in figure (4-16). Finally in both figures
(4-17) and (4-18) appeared section of brain rats treated with lycopene
(100mg/kg), (200mg/kg) and MSG (20mg/kg) shows normal structure
exception necrosis and degeneration of axon and spongy vaculation.

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 Figure (4-13) Section of control brain rats shows the typical layered appearance of
the cerebral cortex labeled 1-6 as follows: 1-molecular layer; 2-external granular
layer; 3-pyramidal cell layer;4-internal granular layer; 5-internal pyramidal layer; 6-
multiform layer. gray matter (G), white matter (W), pyramidal cell (P), capillary (C),
axon (A), nerve cell (N). H&E Stain, (100x).

Figure (4-14) Section of rats brain treated with MSG (20mg/kg)shows necrotic of
some cells bodies (N), necrosis of tissue structure (NS), degeneration of some nerve
cell (D), degeneration of axon (DA).H&E Stain, (400x).

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Figure (4-15) Section of brain rats treated with MSG (20mg/kg) then lycopene
(200mg/kg)shows loss of gray matter (LG), necrosis of tissue structure (N),
degeneration of some nerve cell and terminal axon (D). H&E Stain, (400x).

Figure (4-16) Section of brain rats treated with lycopene (200mg/kg) then MSG
(20mg/kg) shows hyperpigmentation of axon (H), aggregation lipid drops (A),
hypertrophy of microglia cells (M). H&E Stain, (400x).

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Figure (4-17) Section of brain rats treated with lycopene (100mg/kg) and MSG
(20mg/kg) shows normal structure exception the following necrosis of axon (N),
spongy vaculation (S). H&E. Stain, (400x).

Figure (4-18) Section of brain rats treated with lycopene (200mg/kg) and MSG
(20mg/kg) shows normal structure exception the following degeneration of axon
(D) and spongy vaculation (S). H&E Stain, (400x).

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4-2 Second Experiment

4-2.1 Cell Viability By Trypan Blue Exclusion

Measurement of viable cells and cell number during suspension cell

culture were used trypan blue exclusion showing very good (83%)agreement
in total viable cell number .

Figure (4-19) Trypan blue dye exclusion test show viable cells (black arrow) and non-
viable cells (orange arrow), (400x).

4-2.2 Dose-Dependent Injury of Mature Neurons

After treatment of mature neurons with MSG can cause cell swelling or
injury at 7th day after incubation, rats cortical and hippocampi neurons
cultured in well plates were incubated with different concentration of MSG
(250-500 µM) compared to control (untreated) shown in figures (4-20) were
revealed swelling , detachment also lysed cells at high concentration as
figures (4-21). But when treatment by different concentration of lycopene
(250-500 µM) compared as DMSO (vehicle) neuron cells had not showed a
significant (p<0.05) differences compared vehicle as figure (4-22), in this
experiment using inverted microscope for examination . Cell injury was
investigated by viability assay using acridine orange in 7 th day after the
incubation using fluorescence microscope. Incubation
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of neurons with MSG caused a dose-dependent increase the concentration as
shown in figures (4-23), indicating neuronal injury.

Figure(4-20) A , B and C Pictures represent neurons cells (control) untreated were

showing the cortical and hippocampi neurons cells after 7th days incubation ,using
inverted microscope, (100x-400x).

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 Figure (4-21) D and E Pictures represent neurons cell treated with MSG (250 and
500)µm respectively, caused swelling and detachment, using inverted microscope,
(100x-400x).

Figure(4-22) A , B and C Pictures represent cortical and hippocampi neurons cells

which treated with DMSO (vehicle)and lycopene (250 and 500)µm respectively, after
7th days incubation, using inverted microscope, (100x).

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 Control MSG(250)µM MSG(500)µM

DMSO (Vehicle) Lycopene (250)µM Lycopene (500)µM

Figure (4-23) Fluorescence micrograph showing dose-dependent injury of mature

neurons MSG (250-500)µm or lycopene (250-500)µm damage of neurons by acridine
dye. Cultured rat cortical and hippocampi neurons were treated with different
concentrations of MSG for 1h. alive cells were appeared green. Dead cell were
appeared red and orange. Increasing MSG doses decreased total number of live
neurons (green) but increased total number of dead neurons (red) or (orange).As the
same in lycopene, same experiments were repeated 3 times, using fluorescence
microscope, (400x).

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 As a result in table (4-9) which it had been shown there a significant
decrease (p<0.05) in cell viability of all treated concentration as a compared
with the control (untreated). But, the lowest significant value was reduced in
high dose of MSG. While there was no significant differences between two
concentration high dose of lycopene and low dose of MSG.

Table (4-9) Dose-dependent injury of mature neurons by MSG (250-500 µm)

compared to control and lycopene (250-500 µm) compared DMSO (vehicle). (Mean±
SE) n=3.

Groups Viability(%)

Control(untreated) 92.20±1.02
a
MSG(250)µm 63.33±3.67
b
MSG(500)µm 33.00±8.00
c
DMSO 74.67±0.88
b
Lycopene(250)µm 70.67±2.96
b
Lycopene(500)µm 60.33±3.53
b
LSD 16.97
The different small letters refer to significant differences at (p< 0.05).

4-2.3 Time-Dependent Injury of Mature Neurons

After determined whether MSG and lycopene induced cell injury is time-
dependent. Mature neurons were treated with MSG (250)µM, and lycopene
(250) µM using inverted microscope. The change in cell morphology was
monitored at different time (zero, 30 min,1h and 2h) points following the
incubation with MSG or lycopene. As shown in figure (4-24), after the MSG
treatment, a clear swelling of neurons was observed. After 1h of MSG
treatment, severe swelling of neurons was seen and detachment of cells. 2h
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after MSG treatment, neurons were completely lysed. Thus, neuronal damage
by MSG is time dependent. in lycopene the damage much less and didn‟t
lysed. while in lycopene 250µM,neuron cells had not effected only after 2h
of treatment but not lysed as figure (4-25).

MSG (250) at zero time MSG (250) after 30 min

MSG (250) after 1 h MSG (250) after 2h

Figure (4-24) Time-Dependent injury of neuronal cells by 250 µm msg. cultured rat
cortical and hippocampi neurons in 35mm dish were continuously monitored after
treatment with 250 µm msg. neurons started to swell even after 30 minutes of MSG
treatment. the swelling got worse at 1h. 2h after MSG treatment, neurons were lysed,
using inverted microscope, (100x).

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 Lycopene 250µM at zero time Lycopene 250µM after 30min

Lycopene 250µM after 1 h Lycopene 250µM after 2 h

Figure (4-25) Time-Dependent injury of neuronal cells by 250 µm lycopene. cultured
rat cortical and hippocampi neurons in 35mm dish were continuously monitored
after treatment with 250 µm lycopene. neurons show little swelling only after 2h of
treatment but not lysed, using inverted microscope, (100x ).

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 Control after 30 min Control after 1 hr. Control after 2 hr.

MSG (250)µm after 30min MSG (250)µm after 1 hr. MSG (250)µm after 2hr.

Figure (4-26) Fluorescence micrograph showing time-dependent injury of neuronal

cells analyze damage of neurons by acridine dye, the cells treatment with MSG
(250)µm compared control, alive cells were appeared green. dead cell were appeared
red and orange. Increasing the period of treatment led to decreased total number of
live neurons (green) and increased total number of dead neurons (red) or (orange),
same experiments were repeated 3 times, using fluorescence microscope, (400x).

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 DMSO (Vehicle) after 30min DMSO (Vehicle) after 1hr. DMSO (Vehicle) after 2hr.

Lyco.(250)µm after 30min Lyco.(250)µm after 1hr. Lyco.(250)µm after 2hr.

Figure (4-27) Fluorescence micrograph showing time-dependent injury of neuronal

cells analyze damage of neurons by acridine dye, the cells treatment with lycopene
(250)µm compared DMSO (vehicle), alive cells were appeared green. dead cell were
appeared red and orange. increasing the period of treatment led to decreased total
number of live neurons (green) and increased total number of dead neurons (red) or
(orange), same experiments were repeated 3 times, using fluorescence microscope,
(400x).

According to the results as shown in table (4-10). A significant decrease

(p<0.05)in the cell viability of neuron cells were recorded in MSG treated
group after 30 min, 1hr. and 2hr. compared with control group. However a
significant increase (p<0.05) in neurons cells viability were observed in

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DMSO and lycopene groups after 30min,1hr. and 2hr. compared control
group. Finally no significant differences in cells viability were absorbed in
lycopene and DMSO groups after 1hr. and 2hr.

Table(4 -10)Time-Dependent injury of mature neurons by MSG (250µm)compared

control and lycopene (250µm) compared DMSO (vehicle) after 30min,1h and 2h.
(Mean± SE)n=3.

Groups Viability after Viability after Viability after 2h

30min(%) 1h(%) (%)
Control(untreated) 92.20±1.02 89.80±1.01 85.80±0.95
a a a
MSG(250)µm 69.00±4.93 56.00±3.06 45.00±7.51
c c c
DMSO(vehicle) 79.17±0.83 78.67±0.88 69.83±1.50
b b b
lycopene(250)µm 86.00±1.00 70.33±3.28 64.00±3.79
ab b b
LSD 10.01 14.04 13.63
The different small letters refer to significant differences at (p< 0.05).

4-2.4 Immunostaining of Cells

Fluorescence micrograph (4-28) showing dissociated cell culture of
prenatal rat cortical and hippocampi neurons staining with beta- tubulin III
(green staining) which was detected in cell bodies and axons of these cells.

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Figure (4-28) Immunocytochemistry/Immunofluorescence-Tuj 1 staining of neuron-
specific class III beta tubulin (primary antibody) in differentiated neural cells. cells
were staining using conjugated secondary antibody anti rabbit FITC. (axon of
neuron) A, (body of neuron) D, using fluorescence microscope, (400x).

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131
 Discussion
5-1 First Experiment
5-1.1 Effect of MSG alone or with Lycopene on:
5-1.1.1Body Weight Changes
The results of the present study showed that the final body weight and
weight change of adult male rats which are treated with MSG (20mg/kg) for
thirty days, do not reach to a significant differences as compared with
control group, these results are in agreement with Shi et al.(2010); Thu
Hien et al .(2012), they suggested that MSG does not have effect on the
body weight. While in other study which disagreed with our results such as
a study from Thailand established that the ordinary consumption of MSG
was associated with an increase in prevalence of overweight (Insawang et al.,
2012).

The results of Pinterova et al. (2001) studying in which they found that
potency of MSG as an obesity prompting agent is advanced in hypertensive
rats as compared with normotensive Wistar Koyoto rats. Another study
done by He et al. (2008) who discovered that there is connection between
MSG consumption and obesity in 752 healthy Chinese humans. It was
established to be linked with increased body mass index (BMI). MSG
consumers had reportedly increased weight as compared with non-users, that
was a result liberated of physical activity and total energy intake. The
potential relationship between MSG and obesity comprises the MSG effect
on energy balance by increasing sweetness of food and by disturbing the
hypothalamic signaling cascade of leptin action (Hermanussen and
Tresguerres, 2003; He et al., 2011). In further study, mice are inject with
MSG to become obese. Researchers thought MSG led to injuries in the brain
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as well as obstructs giving out leptin. Leptin is a hormone that act as
pointers to the brain that you must had sufficient to eat. It shuts off your
hunger and rises your calorie-burning. The complications with leptin
signaling, named leptin resistance which are influenced in fatness (Nicholas,
2010). In our study, MSG does not have effect on body weight, this may be
due to the quality of food, the dosage used, are the period of treatment not
enough so there is change. While the results of the final body weight and
weight gain in adult male rats that co-treated with MSG (20mg/kg) and
lycopene (100mg/kg) for thirty days, and in adult male rats that treated with
lycopene (200mg/kg) for fifteen days followed by treatment with MSG
(20mg/kg) for other fifteen days showed a significant decrease of body
weight and weight gain as compared with control. This may be because the
effectiveness of lycopene in reducing inflammatory factors in obese and
overweight people (Luvizotto et al., 2013).

Lycopene is hydrophobic and there are great lipoprotein receptors in fat

tissues for it, the storage position for lycopene and other carotenoids is
adipose tissues (Hojjati and Razavi, 2011). Our results are agreed with the
results of Markovits et al., (2009); Gouranton et al., (2011) they revealed
that lycopene prevents an inflammatory processes in obesity and assist
weight loss through the decrease of cytokines expression and chemokines in
adipocytes, these results are agreed with Guerendiain et al. (2015) their
study presented that there is a direct association between increased lycopene
plasma levels with reduced adiposity and weight loss in adolescents with
overweight or obese . Jahromi et al. (2016) showed that lycopene is
significantly condense serum levels of ghrelin and reduced body weight in
female rats. Ghrelin as an effective peptide increasing hunger, is mostly

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produced in the stomach and lower amounts in the brain, the hypothalamus,
pituitary, adrenal cortex, pancreatic islet cells and other numerous tissues
(Van der Lely et al., 2004).

While in adult male rats treated with high dose of lycopene

(200mg/kg)either (co-treated)or followed treatment by MSG (G3 or G6) they
have no significant differences as compared with control, may be due to
high dose of lycopene. The explanation of this is the gap junction
communication permits only small molecular signaling between cell through
channels that are formed by gap junction proteins such as connexin 43.
Merely one in vivo study set up a positive connotation between gap junction
communication or connexin 43 expression with lycopene intake
(Krutovskikh et al., 1997). Kucuk et al. (2001) investigated the effect of
gavage lycopene on gap junction communication in rat liver. Remarkably,
the lower dose (5mg/kg/d) enhanced communication whereas the higher dose
(50mg/kg/d) inhibited communication. A randomized clinical pilot of
lycopene supplementation described a significant increase in connexin 43
protein levels. Generally, there is moderately slight in vivo confirmation to
support a rise in gap junction communication with lycopene consumption
(Erdman et al., 2009). However, lycopene (low dose) reduced body weight
may be due to decreases serum ghrelin levels or may be promoting leptin
level.

5-1.1.2 Blood Parameters

The results of total WBCs count and differential count as represented in

table (4-2) showed that adult male rats treated with lycopene (200mg/kg) for
fifteen days followed treatment with MSG (20mg/kg ) for another fifteen

133
days, and the adult male rats treated with MSG (20mg/kg ) for thirty days,
both revealed a significant increase in WBCs as compared with control and
other treated groups . These results agree with the results of Elatrash and Abd
El-Haleim, (2015); Ghadhban, (2017) they were reported that MSG could be
toxic and cause damaging changes in hematological and biochemical
parameters (Ashaolu et al., 2011; Meraiyebu et al., 2012). The increase in
WBC count could be triggering of the immune system in the existence of a
contaminant, which may in turn be an adaptive reaction of the organism,
resulting in an increased WBCs count, while the results of Calis et al.
(2016); Zafar and Shrivastava, (2017) disagree with our result. The difference
with other literatures as cited above may be due to some factors such as the
amount of administered or route of administration, and period of
experimental subjects. The same table showed that all other MSG- lycopene
treated groups revealed a significant increase in WBCs count as compared
control and MSG group, only in G3 showed a significant decrease as
compared with control and other treated groups, which may be due to
protective effect of lycopene. This result agreed with results of Boeiraa et
al., (2014) they suggested that protective effect of lycopene which did not
change significantly from normal values of blood parameters (leukocytes,
segmented neutrophils, eosinophil‟s, monocytes and lymphocytes).
Regarding lymphocytes percentage, each groups were treated with MSG
revealed a significant increase as compared with control and other treated
groups, but within normal range (Mary et al., 2008).

The increase in lymphocyte percentage in treated animals may be due to

that MSG is considered as a noxious agent or might be a concern of the
relations between MSG and gastrointestinal macrophages. Macrophages

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attend as antigen presenting cell, and the antigenic products (polypeptides) to
the helper T cells and B lymphocytes bringing about their activation
(Sembuligham, 2005) .Macrophages moreover discharge substances called
interleukin-1 cytokines, which brings almost the activation, proliferation and
promotion of the lymphocyte count (Sembulingham, 2005; Barrett et al.,
2010).

The findings of our study suggest that lycopene was helpful in reducing
the harmful effect of MSG. Our result agreed with the results of Ashaolu et
al. (2011) ; Ghadhban, (2017).While the results of Elatrash and Abd El-
Haleim, (2015) are disagreed with our results. The monocytes percentage in
groups of animals which were treated with MSG (20mg/kg) for fifteen days
followed by treatment with lycopene (200mg/kg) for another 15 days,
showed a significant increase as compared with control and all treated
groups. This increasing may be due to the effect of lycopene which
induced phagocytic cells in host defense. These results agreed with
Ogundeji et al., (2013) . While the Granulocytes percentage, in rats treated
by MSG (20mg/kg) for fifteen days followed by treated with lycopene
(200mg/kg) for another fifteen days showed approximated values to control
may be that due to the effect of lycopene. The other treated groups showed a
significant decrease as compared with control. These results are agreed with
result of Ghadhban, (2017) and disagreed with Elatrash and Abd El-
Haleim,(2015) .

That may be due to effect of lycopene which is useful in decreasing the

risky effect of MSG by maintaining optimum hematological values. The
(RBCs count, Hb concentration and PCV percentage) parameters, in
animals that treated by MSG (20mg/kg) for fifteen days followed by treated

135
 with lycopene (200mg/kg)for another fifteen days ,showed a significant
increase as compared with control and all the other groups but, within
normal range (Mary et al.,2008). This result may be due to an improving
effect of lycopene that cause the former effect. This result agreed with
Ibrahim and Banaee, (2014). Moreover , the other treated groups showed no
significant differences but if the difference exists it is very little. This result
may be due to the co treatment with lycopene (because lycopene inhibition
action on MSG) which agreed with Ashaolu et al., (2011) ; Kolawole,
(2013); Al-Harbi et al., (2014) . But disagreed with results of Ghadhban,
(2017) .

Furthermore, the other red blood parameters (RBCs indices) which

include MCV, MCH and MCHC, showed significant decreased in the group
of animal which treated only by MSG as compared with control. This result
agreed with Ibrahim and Banaee, (2014) ; Ghadhban, (2017). And disagreed
with Al-Harbi et al., (2014). The other groups which are co- treated with
lycopene, showed values near to control. MSG could be toxic to erythrocytes
and also cause deleterious changes in hematological parameters but the
existence of lycopene improved the results .

5-1.1.3 Oxidative Stress and Antioxidant Activity

The current study revealed that MSG caused a significant effect on

antioxidant enzyme (SOD, GPX) and the malondialdehyde activity
(MDA).The results revealed a significant decrease in SOD levels in all
treatment groups as compared with control. Our results are agreed with the
results of Singh and Ahluwalia et al. (1996). And disagreed with results of
Calis et al., (2016). According to a previous study that investigated the

136
group which was given MSG (2 g/kg), showed not different in the SOD
activities in the brain, liver, and kidney homogenates. In spite of the
antioxidant properties of lycopene that have been demonstrated both in vivo
and in vitro, it has not improved the harmful impact of MSG, induced
oxidative stress, lipid peroxidation and decrease the GPX and SOD level
(Wertz et al., 2004).

New studies have investigated other metabolic and noxious effects of

MSG, with an amount of reports performance the stimulation of oxidative
stress in altered tissues of investigational animals after administration of
prolonged doses of MSG ( Diniz et al., 2004 ; Farombi and Onyema., 2006 ;
Onyema et al., 2006).

Glutamic acid has submitted as one and only of the amino acids used by
the kidney during gluconeogenesis meanwhile the net uptake of main
gluconeogenic precursors such as lactate, glycerol, glutamate, glutamine and
other amino acids by the kidney accounts for the turnover of glucose by the
kidney (Singh et al., 2003). The amplified influx of materials into the kidney
has been related with numerous variations and oxidative stress. This has been
documented in more current reports in which hyperglycemia caused
oxidative stress in the kidney by the development of free radicals and
changed the antioxidant reactions catalyzed by ROS scavenging enzymes
(Koya et al., 2003).Oral administration of MSG (20 mg/kg),body weight for
thirty days cause a significant increase of the level of malondialdehyde
(representing lipid peroxidation) compared with control group as in table (4-
4).While the other groups treated with lycopene showed no significant
differences as compared with control group. Our results agreed with Singh
and Ahluwalia et al. (1996) ; Kushwaha and Bharti, (2015) ; Wei et al.,

137
(2016) , and disagreed with Al-Harbi et al., (2014). An increase in lipid
peroxidation subsequent MSG to administration in rat could be due to an
increase in the level of Glutamine. Glutamine is identified to recruits
alteration in redox would-be to cell and favor lipogenesis (Malik and
Ahluwalia, 1994). MSG is also conveyed to prompt hyperglycemia, as a
consequence to peroxidation of membrane lipids via glucose oxidation,
(Chaudhary et al., 1996). Lycopene supplementation inhibit the effects of
oxidative stress of MSG in Wistar rats, actually through reducing oxidative
damage to erythrocytes. deduction, lycopene supplementation is helpful in
reducing the effects of oxidative stress in animals (result from MSG in our
study). The increase in lipid peroxidation perceived in this study may be
official to a direct effect of amplified generation of ROS subsequent from
MSG treatment.

5-1.1.4 Biochemical Parameters

5-1.1.4.1 Liver Enzymes

An aminotransferase is plentiful in the liver and released into the blood

stream after hepatocellular damage, creating them as sensitive indicator of
liver damage (Al-Mamary, 2002). Plasma levels of aminotransferase was
used as a sign of impairment of the liver structural integrity because these
enzymes located in cytoplasm and they are released into the circulating
blood only when there is structural damage (Janbaz and Gilani, 2000 ;
Hagar, 2004). The results of AST, ALT and ALP are represented in table
with control group, in spite of the treated groups with lycopene either
together or followed . The presence of lycopene did not improved the
massive effect of MSG. Our result agreed with that of Ayhan et al. (2011) ;
Okediran et al. (2014) ; Akanya et al. (2015). It is also, disagreed with a

138
study done by Wei et al., (2016), who recommended that oral administration
of lycopene enhanced lipid profiles and ordinarily diminished the level of
serum AST, ALT.

It is also disagreed with Ochiogul et al. (2014) who suggested that

administration of MSG to West African Dwarf goats at the doses and routes
used in theirs study led to significant reductions in serum ALT and AST. The
breakdown of most amino acids and their derivatives take place to extent in
the liver and essentially deamination to yield ammonium ion that could be
toxic unless made fewer toxin through the reactions of the urea cycle. The
sodium moiety in MSG might be easily separated to produce free glutamate.
Therefore, the ammonium ion excess that may occur with glutamate or
monosodium glutamate consumption could impair the liver, and liberating
the transaminases; later it observed a rise in the plasma. The oxidative stress
that induces alteration in the membrane integrity, and changing in membrane
permeability resulting in leakage of intracellular enzymes (Mayes and
Bender, 2003).

In our study regarding the previous parameters we found that the co-
treatment has more effect on liver than single treatment that may be due to
the increased interference interaction between substances inside the liver.
Furthermore ,according to the same table total protein level showed a
significant increase in MSG group only as compared with control and other
treated groups. The results mean that co-treatment with lycopene improved
the harmful effect of MSG. This result is disagreed with Eweka et al. (2011);
Okediran et al. (2014). But it is agreed with Ochiogul et al. (2014) ; Oladipo
et al. (2015) .

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 The elevation of the total protein in MSG treated group may indicate
inflammation or liver and kidney disease. MSG may have supplied the
needed substrates (glutamate as an amino acid) , which the liver used in
synthesizing part of the extra serum proteins. Moreover, and depending on
table (4-5), bilirubin level, showed a significant increase in all treated groups
as compared with control group. Our results are disagreed with Tawfik and
Al-Badr, (2012) ; Oladipo et al. (2015), and it is agreed with Kolawole,
(2013) ; Zafar and Shrivastava, (2017).A significant decrement in
hematological parameters beside altitude in serum bilirubin indicate the
use of MSG consumption is harmful to hematopoietic system (Farombi and
Onyema, 2006 ; Ashaolu et al., 2011; Maluly et al., 2013 ; Hassan et al.,
2014).

MSG intake is accountable to prompt destruction of red blood

corpuscles in premature or in young phase. This reduced life span of RBCs
results in breakdown, which is set by less number of RBCs in blood. Later
the destruction of RBCs liberated bilirubin is released in the serum, which is
transformed in its conjugated form by liver (Guyton and Hall, 1996). The
significant elevation of bilirubin level in serum of the MSG treated groups is
an indication to that MSG intake contributes to the higher rate of
degradation of RBCs and low abilities of liver to purify the relevant amount
of bilirubin. In spite of lycopene is potential agent of hepatoprotection may
be by its well-known antioxidant defense however, this did not succeed in
improving the harm of MSG.

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5-1.1.4.2 Kidney Function Tests

The results represented in table (4-6) showed a significant increase of

creatinine in all the treated groups as compared with control group. While
, the co-treated groups with lycopene showed less changes than MSG group.
These results agreed with Inuwa et al. (2011) ; Elatrash and Abd El-
Haleim, (2015) ; Ateya et al. (2016) ; Al-Mousawi, (2017). But it is
disagreed with Kolawole, (2013) who noted that there is no significant
changes were observed in all the studied biochemical parameters, including
indices of hepatic and kidney functions when treated with MSG in different
concentration (5,10 and 15 mg/kg) for 28 days orally. Creatinine
concentration in serum increases when the capacity of the kidney to filter
fluid in the body drops. In general a significant elevation of creatinine is an
indication to unwell performance of kidneys. There is a possible connection
between MSG and renal failure, or may be caused by alterations in the
threshold of tubular reabsorption, renal blood flow and glomerular filtration
(El-Sheikh and Khalil, 2011).

While the urea and uric acid level are seen in the same table showed that
the group which treated with monosodium glutamate revealed a significant
increase as compared with control. While the other co-treated groups with
lycopene gave result near to control . This result agreed with Ateya et al.
(2016) and disagreed with Kolawole, (2013). The promotion of these
parameters in existing study means inability of the kidney to eliminate waste
product of MSG, but here, the lycopene has an ability to improve the effect
of MSG through its ability to decreased reactive oxygen species.

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5-1.1.4.3 Lipid Profile

The current study showed that daily oral administration of MSG

produced a significant increase in some physiological parameters such as
lipid profile, like TC and LDL as represented in the table (4-7), all the
treated groups showed a significant increase as compared with control
group. Our result agreed with Ahmed, (2016) ; Hareeri et al. (2017) ; El-
Ezaby et al. (2018).The study of Purwantoyo et al. (2018) showed that
consumption of tomato extract can decrease the levels of TG, TC, and LDL
this result is opposite to the results of our study, the pure lycopene that used
as co-treatment with MSG didn‟t decline the levels of TC and LDL. The
explanation for that, it may be due to the variation in concentration ,purity,
route of administration or duration of treatment .Furthermore, the MSG
treated group and some of co-treated groups with lycopene showed
significant increase of TG as compared with control group. While the other
co-treated groups with lycopene gave result near to control.

All the treated groups (except the group which are treated with MSG
followed by lycopene ) showed a significant increase of VLDL as compared
with control group. That‟s mean the co-treated groups with lycopene
enhanced the destructive effect of MSG .It is also seen in table (4-7) that
there is a significant decrease of HDL in MSG group as compared with
control and other co-treated groups with lycopene. This result is agreed with
Ahmed, (2016) ; Purwantoyo et al. (2018). The cholesterol pool in the
intestine originates from alimentary cholesterol and from biliary excretion.
About 50% of the intestinal cholesterol pool is reabsorbed by the intestine
and recirculate through the body (via the entero-hepatic circulation), and the
residue excreted in feces . Abnormal value of cholesterol, in the blood is

142
considered as symptoms of liver disease (Grigore et al., 2007).The recent
study recorded that the higher plasma TG could be linked to the increased
number of medium VLDL particles. It has been shown that medium VLDL
particles are related with gut fat area in obese individuals (Okasi et al.,
2005). MSG rises the synthesis of fatty acids and triglycerides from acetate.
This might be due to the carriage of acetate into the liver cell, leading to
increase substrate (acetate) availability.MSG consumption increases the
synthesis of cholesterol. Additional reason, that monosodium glutamate was
able to increase the activities of 3-hydroxyl-3-thylglutaryl coenzyme A
(HMG Co A) reductase (Okediran et al., 2015).

Moreover, the study of Thomas et al. (2009) described that there is

hyperlipidemia with a significant elevation in levels of serum TG and TC
in MSG treated rats and suggested that an alteration in glucose metabolism to
lipogenesis might be a reason for the hyperlipidemia. The rate limiting
enzyme in cholesterol biosynthesis resulting in increased synthesis of
cholesterol in the MSG treated rats. While the beneficial effect of lycopene
can be due to the antioxidant effect or by inhibiting lipid peroxidation and
decrease the production of cholesterol, LDL and triglycerides, lycopene is
also inhibit the molecular pathways in cholesterol metabolism. The
committed step in the biosynthesis of cholesterol and isoprenoids is catalyzed
by HMG-CoA reductase.

5-1.1.4.4 Some of Hormones

The HPA axis is stimulated by reaction to stress, numerous types of

stress disturb the sensitivity of the HPA axis as well as arouse the discharge
of CRF from the PVN in the hypothalamus. The creation then release of

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ACTH as of the pituitary gland concurrently increases with the promotion
of the discharge CRH. ACTH, consecutively, promotes the discharge of
glucocorticoids in humans or corticosterone in the animals from the adrenal
cortex (Marin et al., 2007).The table (4-8) showed that there is a significant
increase of ACTH in all treated groups as compared with control group.
Our result agreed with Quines et al. (2014). The co-treatment with lycopene
doesn‟t change the result. The result is disagreed with Haddad et al. (2013).
They report that lycopene and beta-carotene, inhibit the cell proliferation and
strongly suppress ACTH secretion. Excitatory amino acid neurotransmitters
have been concerned in the chronic prompt of the HPA axis . The accurate
role of these neurotransmitters in stress response is uncertain. There is
increasing preclinical indication that glutamate, which is an excitatory amino
acid, plays an essential role in the ruling of the HPA axis (Mathew et al.,
2001) Glutamate is a greatest plentiful excitatory amino acid
neurotransmitter via the brain and has identified to trigger the HPA axis
then prompt ACTH altitude (Zelena et al., 2005).Too much accrual of
glutamate in the extracellular spaces could lead to extreme stimulation of
glutamate receptors through the hypothalamic nuclei, the glutamate altitudes
in the plasma were amplified when consumption of MSG. Monno et al.
(1995) conveyed that four g/kg of MSG administer to adult rats through
gavage prompted a ( 4.2- to 8.9 ) fold rise extracellular glutamate levels
inside the hippocampus and hypothalamus, correspondingly. Because of that
certain brain areas (comprising the hypothalamus or circumventricular
organs) absence a blood brain barrier (BBB) glutamate levels might increase
in quantity leading to the promotion of the plasma glutamate conc., that
could ultimately trigger the HPA axis. In same table (4-8) showed a
significant decrease of cortisol in all the treated groups as compared with
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control group . Our result agreed with Eliane et al. (1997). But disagreed
with Quines et al. (2014).

The level of plasma cortisol in rats exposed to physical or emotional

stress have been shown caused increase or decrease or not change stress is a
frequent precipitating factor in major depression. Or may be due to MSG
consumption lead to degenerative in zona fascinate in adrenal gland (Abdo
et al., 2018) . ACTH proliferations the creation then release of totally
adrenal steroids, aldosterone, cortisol in addition to adrenal androgens. It is
the main modulator of cortisol. As the cortisol level in blood increases,
release of ACTH is inhibited directly at the pituitary level. Through this
mechanism, decreasing cortisol levels lead to elevated ACTH levels (Guyton
and Hall, 1996).There are numerous opinions to describe the quite low level
of cortisol containing the generation of negative feedback inhibition or
down-regulation of the CRH receptor through the hypothalamus due to the in
elevation CRH location (Raison and Miller, 2003).As glutamate causes
stress, and the altitudes of plasma cortisol in humans and corticosteron in
rats that were frequently exposed to physical or emotional stress will be
increased or decreased.

Furthermore, the same table (4-8) showed that there is no significant

differences of TSH in all the treated groups as compared with control. This
result agreed with Miskowiak and Partyka.(1993). Since TRH is formed
principally in the periventricular nuclei (De Greef et al., 1992), our results
acquired submit that this hypothalamic nuclei is not damaged by MSG. The
cells in certain regions of CNS are further resistant to neurotoxic effect of
glutamate than cells of arcuate nucleus (AN). This may perhaps be due to
different varieties of receptors, alterations in second messenger systems or

145
diverse levels in ion channel triggering (Van del Pol, 1991). The T3
hormone level represented in table (4-8) showed a significant decrease in all
the treated groups as compared with control group while T 4 hormone
revealed a significant increase in the MSG treated group only as compared
with control group. The other co- treated groups with lycopene give result
near the control. Our result disagreed with Miskowiak and Partyka. (1993) ;
Ateya et al. (2016). These changes in thyroid hormones (T3 and T4) might be
resulted from alteration in the pituitary – thyroid axis or may be due to the
effect of MSG on enzymes that are responsible for conversion T 4 to T3
especially the deiodenase enzyme.

5-1.1.5 The Histopathological Investigation

5-1.1.5.1 Liver

The histological study of liver in most treated animals showed

alterations in histological pattern marked by interruption of hepatic cords,
loss of nuclei, dilation of central vein, vaculation of hepatocytes, necrosis of
nuclei, presence of inflammatory cells within and around the central. Except
the group of animals treated with (20mg/kg) of MSG and (100mg/kg) of
lycopene showed normal central vein, normal hepatocytes , some of
vaculation of cytoplasm. Our result agreed with Onaolapo et al. (2013) ;
Mustafa, et al. (2016). They noted that MSG may be toxic to the
hepatocytes, so it effect the cellular integrity and producing defect in
membrane permeability as well as hepatocytes architecture. The degenerative
deviations observed in the rats‟ liver sections in this experiment may have
been caused by the cytotoxic effect of MSG on this organ.

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 Furthermore, lycopene in our experiment has protective effects in liver
tissue especially in low doses. This result agreed with Joram et al. (2001) ;
Abdel-Rahman et al. (2018). They suggested that lycopene is associated
with hepatic cells regeneration, consequently, it supports the cellular
membrane but increasing the enzyme leakage and preserves its function in
protein biosynthesis. The hepatic associating effect of oxygen and removal of
peroxyl radicals that was confirmed by the elevated antioxidant activities of
SOD, GPx, along with reduced MDA in MSG and lycopene co-treated rats.
Lycopene suppresses cell proliferation (Abdel-Rahman et al., 2018). Both
carotenoids are capable of prompting intercellular communication via gap
junctions, which has been connected with inhibition of transformed cell
proliferation (Levy et al., 1995).

Another potential description of the satisfactory effect of b-carotene

and lycopene in weakening hepatic fibrosis is their strong interaction with
ROS . The free oxygen radicals can lead to tissue injury by reacting with
polyunsaturated fatty acids in cell membrane, nucleotides and critical
sulfhydryl bonds in proteins. The amount of tissue damage depends on
accessible antioxidants such as tocopherol, ascorbic acid, beta-carotene,
glutathione, selenium and superoxide dismutase (Stahl and Sies, 1996).

5-1.1.5.2 The Kidney

In our study the kidney microanatomy in all treated groups as

compared to controls showed dilatation of Bowman‟s space, reduction of
glomerulus and necrosis, hyperplasia of tubules, degeneration and necrosis,
loss of normal structure of tubules, hypertrophy in some tubules, pyknotic,
absent of convoluted tubules and bleeding. All of these cells changes are

147
caused by ROS that resulting from MSG treatment. The previous studies
have shown that MSG causes oxidative stress and impairment of the kidneys
(Paul et al., 2012). These results agreed with studies carried by Ortiz et al.
(2006) ;Eweka , (2007) ; Onaolapo et al. (2013). The lycopene in the co-
treated groups didn‟t changes or enhance of the harmful effect of MSG. This
result disagreed with Daniel et al. (2015), they suggests the ability of
lycopene to protect against kidney injury.

The effects which observed in both the liver and kidneys could have
occurred because these organs are complicated in the metabolism of
glutamate or may be due to exacerbation of trans fat in the liver prompted
fatty liver disease through mice via a mechanism comprises an amplifying
the central adiposity in addition to changes in individually hepatic also white
adipose tissue gene expression (Collison et al., 2009).When the L-glutamate
reaches a great concentrations ,through the renal artery, the kidney attempts
to excrete it. The renal corpuscle takes the L-glutamate by the afferent
arteriole, it is absorbed, filtrated, and crosses the membrane injuring the cell.
The convoluted proximal tubules were further vulnerable to be impaired in
contrast to the distal convoluted tubules (Ortiz et al., 2006). The formation of
ROS in the kidney resulted by MSG treatment was seen as a main provider
to their nephrotoxic effects leading to cellular and functional damage.

5-1.1.5.3 The Brain

The histological examination of brain in most treated groups except

those co-treated with lycopene in a dose of (100 and 200mg/kg) with
(20mg/kg) of MSG for thirty days have shown an increase of neuronal
injury appeared as necrosis of some cells bodies, necrosis of tissue structure,

148
numerous degenerating pyramidal and granule neurons (with
vacuolations),with loss of distinct pyramidal and granule cell morphology as
compared with control and other treated groups. Our results are agreed with
Olakunle et al. (2016). They revealed that these changes are evidenced by
pyknosis, karyorhexis and karyolysis are classic histological deviations
related with cell death sequence in the brain. Glutamate prompted neuronal
death has been described to be mediated by both apoptotic and necrotic
mechanisms, with excitotoxic and oxidative damage as two separated
pathways by which neuronal death is induced. Excitotoxicity involves the
more activation of glutamate receptors by increasing doses of MSG, leading
to a combination of fast and late cytotoxic events (Farombi et al., 2006).
Glutamate causes over activation of N-methyl-d-aspartate and α amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid receptors leading to discharge
of Ca2+from intracellular stores, which in turn leads to stimulation of
mitochondria and the release of enzymes like phospholipase and protein
kinases causing the degradation of proteins and membranes and leading to
cell death ; accumulative doses of MSG improves the comfort with which
these events are produced (Matute et al., 2006).

While lycopene in co- treated groups (at same time) enhanced the harmful
effect of MSG on brain tissue probably because lycopene is a potent
antioxidant. This result agreed with Țigu1 et al. (2016). Continuing
treatment with lycopene significantly progresses the cerebral functions and
obstruct apoptosis, through preventing mitochondrial oxidative impairment,
then reduction in inflammatory signs and protective properties against
amyloid influenced neurotoxicity in rat cortical neurons (Prakash and Kumar,
2014).The increased consumption of lycopene with carotenoids(lutein+

149
zeaxanthin) rich diet might be linked to the falling of the oxidative stress
which are resulted from MSG.

5-2 Second Experiment

5-2.1 Cell Viability by Trypan Blue Exclusion
The Trypan Blue dye exclusion assay, the major method projected in
the literature, is deliberated the ordinary cell viability measurement method
(Leo et al.,2015). It is still the most commonly used style (Pamphilon et al.,
2013). Moreover, Trypan Blue combined with a haemocytometer lattice is
observed as the standard method for estimating the cell population mass
(Belini et al., 2013) for example the total number of living cells in the culture
(Muench et al., 2002), Trypan Blue is a cell membrane-impermeable particle
and so only pass in cells having compromised membrane. Furthermore, with
Trypan Blue the cell viability is determined indirectly by identifying cell
membrane integrity (Strobe, 2001). when trypan blue enter into the cell, it
connects to intracellular proteins and in bright field the dead cells seem blue
(apoptotic and necrotic cells) are not notable (Stoddart, 2011). But the color
of living cells residues unaffected. In our study we isolated neuron cells
from immature embryonic rat that‟s due to the failure of isolate and culture
neurons from the adult mammalian brain and the common failure of neurons
to regenerate the cut brain have contributed to the conception that adult
neurons do not regenerate.

Through neuronal culture procedure in addition to cell damage test, we

studied the effect of MSG on rat cortical neuron, a usually usage in vitro
preparation designed for cell harm study. We established that incubation
using MSG, on clinical significant concentration, prompted enlargement
also injury of mature neurons ,deduction may moderately convinced by MSG

151
consumption. Differentiated cells are hypothesized to have set genetic
switches which prevent redifferentiation.

However, adult neurons preserve massive ability for altering their

general due to the moderately simple architecture of the nerve cell
population in the hippocampus (Dent, 2007 ; Hu et al., 2008). Hippocampal
cultures are classically made from late-stage embryonic tissue. This tissue is
easier to dissociate and comprises less glial cells than does mature brain
tissue (Mao and Wang, 2001; Sanes et al., 2006). Isolation of hippocampal
neurons from embryonic tissue as well reductions damage to axons and
dendrites due to fewer adhesion links (Brewer , 1995). Although
hippocampal cultures are best generated from rats that due to the moderately
easier isolation of the hippocampus, mice can moreover be used with the
identical protocols if suitable maintenance is taken throughout tissue
isolation. When neurons are cultured, the capability to use progressive
molecular techniques to analyze subcellular localization and operating can be
working (Dadsetan et al., 2009).This can be principally beneficial when
investigating embryonic lethal transgenic mice as it delivers the ability to
study protein relations that would be consequence in the death of the
embryo(Kunze et al., 2011).

5-2.2 Dose-Dependent Injury of Mature Neurons

In our study ,we were used two different concentration for our each
treatment with MSG and lycopene (250 and 500)µM. as in many previous
sources studies such as (Xiong et al., 2009) they used different
concentration

151
(3,30, and 300)µM of MSG. But (Kim et al., 2002 ; Teodoro et al., 2013)
they used different concentration of lycopene. As in the MSG treatment
(figures4-20 and 21) , neuron cells were showing swelling , detachment
lysed cells at high concentration. This result is agreed with (Choi, 1985) .
Earlier studies have shown that incubation of neurons thru high
concentrations of glutamate (500 µM) were quickly prompts neurons cell
death. The result is agreed with Xiong et al. (2009) who showed that the
incubation using (300) µM MSG prompted a slight rise of LDH release and
damage. While it is disagreed with Sinem et al. (2017) who showed that the
non-toxic doses which are mentioned in the literature and the cytotoxic
effect of MSG was examined in mouse MSCs (Mesenchyme Stem Cells) so,
MSG appears as safe in usage of certain concentrations on stem cell
toxicity. In our study ,the result shown in table (4-9) revealed that there is a
significant decrease of cell viability (after 7th days incubation) in all treated
concentration (250-500)µM as compared with the control (untreated). But ,
the more change(the least percentage) was in high dose of MSG which was
equal 33% ,this result is agreed with Namindla et al. (2016) ,but disagreed
with Sinem et al. (2017) who established their result of cell viability on
mouse MSCs , they were found alive (at least 70% of cells) in (10, 30, 60,
90) µmol/dl concentrations .

In MTT analyses, no cytotoxicity was found Glutamate mediated

excitotoxicity is the main hazard factor complicated in neurodegenerative
diseases such as Parkinson‟s disease, dementia, amyotrophic lateral
sclerosis, glaucoma and diabetic retinopathy. The mechanism of the
excitotoxicity is imbalanced calcium homeostasis due to interruption in
neuronal calcium amplification; mitochondrial membrane depolarization in

152
reverberation to glutamate is a progressive phenomenon in cell death
(Vaarmann et al., 2003).

An endogenous neurotransmitter is glutamate required for a variation of

physiological functions of neurons. Amplified discharge of endogenous
glutamate has recommended in order to play a vital role in neuronal damage
related thru a quantity of neurological conditions Preceding study has
shown that incubation of neurons through in elevation concentrations of
exogenously everyday glutamate (500 µM) quickly prompts death of
neuronal cell (Xiong et al., 2009).

MSG administrated to experimental animals lead to selective

neurodegenerative impairment in certain areas of the brain. This
phenomenon can be compacted by the increase in calcium binding proteins
levels, including calretinin(involved in calcium signaling) which delivers
calcium ions buffering properties in neurons. The blood-brain barrier (BBB)
is not completely developing in young persons, therefore they may be
exposed to MSG toxic effects .Furthermore, when it is treated by different
concentration of lycopene (250-500 µM) the same table showed no
significant differences as compared vehicle (DMSO) .This result is agreed
with Sinwoo et al. (2017).Lycopene showed protective effects against
myeloid β-induced neurotoxicity in cultured rat cortical neurons (Qu et
al.,2011). Amyloid-β prompts its neurotoxicity in a different ways,
comprising accelerating mitochondrial dysfunction, ROS production, and
neuronal death (Nunomura et al., 2006).

In several studies, lycopene is observed as a potent antioxidant that

permits through the BBB by its lipophilic nature. Numerous studies have

153
shown that lycopene protects from specific neurodegenerative diseases
associated with oxidative damage (Khachik et al., 2002 ; Palozza et al.,
2010).Therefore, lycopene generates protection against environmental
neurotoxins and against extreme levels of certain components, through its
really potent antioxidant properties.

5-2.3 Time-Dependent Injury of Mature Neurons

In our study, we used MSG (250) µM, and lycopene (250 µM) and
examine the results under inverted microscope. The alteration in cells
morphology were checked at different time (zero,30min,1h and 2h) points
following the incubation with MSG or lycopene. Subsequently the MSG
treatment, a strong swelling of neurons saw, next 30 min of MSG treatment,
swelling of neurons was seen and detachment of cells next to one hour.
Whereas after 2h MSG treatment, neurons were completely lysed as shown
in figure (4-24). Therefore, neuronal injury by MSG is time dependent. The
result is agreed with Xiong et al. (2009). While, in lycopene the injury was
greatly fewer and didn‟t lysed, the neuron cells had not affected only after
2h of treatment but not lysed as showing in figure (4-25).

The result appeared in table (4-10) showed that there is a significant

decrease in the cell viability after 30 min in MSG and vehicle as compared
control (untreated). As for the viability after one hour , there is a significant
decrease in MSG only as compared control, while the viability after two
hours, it is shown that there is a significant decrease in all treated as
compared control , and the more change was already in MSG treatment.
That mean, the neurons cell viability become less, depended on time of
incubation, especially with MSG which is the most influential. Meanwhile

154
 the report of “CRS” in 1968, numerous investigations have established that
a connection concerning MSG consumption in addition to its side effects
like nuisances (Yang et al., 1997). Those studies, an oral dose of MSG at
(3g/kg) or greater produced restaurant syndrome next 30 min. Through
venous dose at (50 mg /kg) correspondingly created the same signs. While
the monosodium glutamate concentration in the blood stream is hard near
expect per oral doses of MSG, a straight intravenous injection of (50 mg/kg)
monosodium glutamate is predictable in order to produce the blood
concentration at (53) µM . Although the BBB has small permeability to
MSG, the existence of an elevation affinity glutamate carriers found on the
BBB capillary membrane might simplify an usage of MSG addicted to the
brain (Xiong et al., 2009). Our result that neurons swelling takings place
next to 30 min after MSG treatment is dependable with a time progression
of MSG produced headache via clinical study. The use of Dimethyl
sulfoxide (DMSO) as a vehicle in our experiment, and the DMSO is the
most commonly utilized as a solvent in vitro and in vivo administration of
lipophilic compounds (like lycopene) (Santos et al., 2003; Balakin et al.,
2006).Good experimental design commands that the drug-treated group be
compared to a group treated with vehicle only. However, depending on
concentration, DMSO alone can cause different and frequently antagonistic
effects, including anti-inflammatory (Capriotti and Capriotti, 2012), anti-
oxidant (Phillis et al., 1998a; Túnez et al., 2005), neuroprotective (Shimizu
et al., 1997; Di Giorgio et al., 2008).

In neuroscience investigation, DMSO is frequently used as a solvent

but the concentration must be limited to prevent vehicle-mediated
neurotoxicity (Julien et al., 2012). In summary, doses of DMSO less than 1%

155
are commonly neurotoxic to neurons and astrocytes in vitro. In contrast,
concentrations ≤0.25 have slight influence on neural cell morphology or
survival, at minimum within 48 h. For instance, the antioxidant properties of
DMSO may suppress the toxicity of anexperimental compound. Because
lycopene possession the potentials of antioxidant properties have less
influence on the neurons cells.

5-2.4 Immunostaining of Neurons Cells

Immunocytochemistry (ICC) refers to immunostaining of the cultured

cell lines or primary cells . ICC offers a semi-quantitative means of
investigating the comparative copiousness, conformation, and subcellular
localization of target antigens. Traditional ICC methods usage chromogenic
detection in which enzyme conjugated antibodies alter chromogenic
substrates to colored rapidly at the reaction position. In
immunocytochemistry/immunofluorescence (ICC/IF) assays, the cellular
antigens are imagined using either fluorochrome-conjugated primary
antibodies (direct detection) or a two-step method (indirect detection)
including an unlabeled primary antibody followed by a fluorochrome-
conjugated secondary antibody. By joining different fluorochrome-labeled
antibodies, multiplex ICC/IF can detect numerous antigens in the same
sample (Polak and Noorden, 1997).

Differentiation and maturing of neuronal cell kinds through normal and

neoplastic progress is accompanied by specific expression of microtubule-
associated proteins and tubulins .Because neuronal cell types were the only
cells identified with TuJ1 in previous studies. The neuronal cells perhaps
have significantly more class III ẞ tubulin than other cell types, its

156
accumulation of class III ẞ tubulin in neurons (David et al., 1999). Many
studies showed that class III ẞ tubulin, broadly used neuron-specific marker
(Stone et al., 1996a).Therefore, we used it in our study to detect these cells.

157
158
 Conclusions and Recommendations

Conclusions

Certain conclusions can be summarized from our present study as:

1. Monosodium glutamate caused harmful effect in all parameters.

2. Co-treatment with lycopene caused improvement in some
biochemical, and hormonal parameters more than the group treated
with the MSG alone and MSG followed by lycopene.
3. Co- treatment with lycopene caused improvement of the adverse
effect of MSG in liver, kidney and brain histopathological changes in
these organs.
4. Neuron cells have been isolated from cortical and hippocampus of
(E16-18) from rat have a high success rate .
5. Monosodium glutamate has so vigorous effect on viability of neurons
cells either in time dependent experiment or in dose dependent
experiment.

159
 Recommendations

Constructed on the results of the present study, as followed:

1. Studying the effect of MSG in female during gestational and

lactating periods and its effect on reproductive system.
2. Studying the effects of MSG on cell apoptosis, and on cancer cells.
3. Further studies must be done to extract lycopene from plants and
make comparison between them and standard lycopene in their
effects.
4. Studying the effect of MSG on other isolated cells.
5. Studying the isolated neurons cells from postnatal pups from other
animals and testing the effect of MSG on it.

161
161
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 ‫الخالصة‬

‫استخدم فً الدراسة الحالٌة سبعٌن ذكر بالػ وعشرٌن انثى بالؽة من الجرذان والتً وضعت فً البٌت الحٌوانً لكلٌة‬
‫الصٌدلة‪-‬جامعة البصرة‪-‬العراق الدراسة قسمت الى تجربتٌن وهً كالتالً‪:‬‬

‫التجربة االولى‬
‫تهدؾ دراسة تاثٌر الالٌكوبٌن على بعض المعاٌٌر الفسٌولوجٌة والبٌوكٌماوٌة لذكور الجرذان المعاملة بملح كلوتامٌت‬
‫احادي الصودٌوم وتشمل ‪ :‬وزن الجسم ‪ ,‬الفعالٌة المضادة لألكسدة‪ ,‬معاٌٌر الدم والمعاٌٌر الباٌوكٌمٌائٌة ‪ ,‬وبعض‬
‫الهرمونات فضال عن الدراسة النسٌجٌة لكل من الكبد والكلٌة والدماغ وقد استخدمت فً التجربة االولى ستٌن ذكر جرذ‬
‫بالػ بعمر اربعة اشهر وزعت عشوائٌا الى ستة مجامٌع هً‪ :‬المجموعة االولى(السٌطرة)‪ :‬وفٌها جرعت الحٌوانات‬
‫بمحلول ملحً(‪ ) ۰٥٢٬‬مل لمدة ثالثٌن ٌوم‪ .‬المجموعة الثانٌة‪ :‬عوملت الحٌوانات ب (‪ ) ۰٥٢٬‬مل من الملح كلوتامٌت‬
‫احادي الصودٌوم بجرعة (‪ ٢۰‬ملؽم‪/‬كؽم من وزن الجسم) بواسطة التجرٌع الفموي ‪ .‬المجموعة الثالثة‪ :‬عوملت الحٌوانات‬
‫ب(‪ ) ۰٥٢٬‬مل من الملح كلوتامٌت احادي الصودٌوم بجرعة(‪ ٢۰‬ملؽم‪/‬كؽم من وزن الجسم) بواسطة التجرٌع الفموي‬
‫لمدة خمسة عشرا ٌوما وبعد ذلك عوملت ب (‪ ) ۰٥٢٬‬مل من الالٌكوبٌن بجرعة (‪٢۰۰‬ملؽم‪/‬كؽم من وزن الجسم‬
‫بواسطة التجرٌع الفموي لمدة خمسة عشرا ٌوما اخر المجموعة الرابعة‪ :‬عوملت الحٌوانات ب (‪ ) ۰٥٢٬‬مل من‬
‫الالٌكوبٌن بجرعة(‪ ٢۰۰‬ملؽم‪/‬كؽم من وزن الجسم) بواسطة التجرٌع الفموي لمدة خمسة عشرا ٌوما وبعد ذلك عوملت ب‬
‫(‪ ) ۰٥٢٬‬مل من الملح كلوتامٌت احادي الصودٌوم بجرعة(‪ ٢۰‬ملؽم‪/‬كؽم من وزن الجسم) بواسطة التجرٌع الفموي لمدة‬
‫خمسة عشرا ٌوما اخرى‪ .‬المجموعة الخامسة ‪ :‬عوملت الحٌوانات ب (‪ ) ۰٥٢٬‬مل من الالٌكوبٌن بجرعة(‪٠۰۰‬‬
‫ملؽم‪/‬كؽم من وزن الجسم) بواسطة التجرٌع الفموي وبعد ساعة واحدة نفس الحٌوانات عوملت ب (‪ ) ۰٥٢٬‬مل من الملح‬
‫كلوتامٌت احادي الصودٌوم بجرعة(‪ ٢۰‬ملؽم‪/‬كؽم من وزن الجسم) بواسطة التجرٌع الفموي لمدة ثالثٌٌن ٌوم‪ .‬المجموعة‬
‫السادسة ‪:‬عوملت الحٌوانات ب (‪ ) ۰٥٢٬‬مل من الالٌكوبٌن بجرعة(‪ ٢۰۰‬ملؽم‪/‬كؽم من وزن الجسم) بواسطة التجرٌع‬
‫الصودٌوم‬ ‫من الملح كلوتامٌت احادي‬ ‫الفموي وبعد ساعة واحدة نفس الحٌوانات عوملت ب (‪ ) ۰٥٢٬‬مل‬
‫بجرعة(‪ ٢۰‬ملؽم‪/‬كؽم من وزن الجسم) بواسطة التجرٌع الفموي لمدة ثالثٌٌن ٌوما‪ .‬وفً نهاٌة التجربة خدرت ثم شرحت‪.‬‬
‫وجمعت عٌنات الدم الستخدامها فً التحالٌل الفسٌولوجٌة و الباٌوكٌمٌائٌة‪ .‬فضال عن الفحص النسٌجً للكبد و الكلٌة‬
‫والدماغ‪ .‬النتائج بٌنت بان كل من الالٌكوبٌن وملح كلوتامٌت احادي الصودٌوم سبب كل مما ٌلً‪:‬‬

‫اظهرت نتائج الدراسة انخفاض معنوي واضح فً معدل الزٌادة الوزنٌة للحٌوانات المعاملة (مع او‬
‫متبوعة)بالالٌكوبٌن مقارنة مع مجموعة السٌطرة والمجموعة المعاملة فقط بالملح كلوتامٌت احادي الصودٌوم‪ .‬اؼلب‬
‫نتائج معاٌٌر الدم اظهرت زٌادة معنوٌة فً المجموعة المعاملة بالملح كلوتامٌت احادي الصودٌوم ‪ ,‬بٌنما المجامٌع‬
‫المعاملة بالالٌكوبٌن اظهرت انخفاض معنوي مقارنة بمجموعة السٌطرة والمجموعة المعاملة فقط بالملح كلوتامٌت‬
‫احادي الصودٌوم‪ .‬اؼلب المجامٌع المعاملة اظهرت زٌادة معنوٌة فً انزٌمات الكبد‪ ,‬فضال عن الكرٌاتٌنٌن والٌورٌا‬

‫‪211‬‬
 ‫وحامض الٌورٌك اٌضا اظهر زٌادة معنوٌة فً المجموعة المعاملة بالملح كلوتامٌت احادي الصودٌوم فقط‪ .‬اما فٌما‬
‫ٌخص مستوى الدهون‪ ,‬كل المعاٌٌر ماعدا(مستوى الدهون العالً الكثافة) اظهرت النتائج ارتفاع معنوي فً اؼلب‬
‫المجامٌع المعاملة‪ .‬اظهرت نتائج هرمون المنشط لقشرة الكظرٌة ارتفاع معنوي فً اؼلب المجامٌع المعاملة‪ ,‬على‬
‫العكس تماما فً هرمون الثاٌروكسٌن والذي اظهر ارتفاع معنوي فً المجموعة المعاملة بالملح كلوتامٌت احادي‬
‫الصودٌوم فقط ‪ .‬باألخٌر فً الفعالٌة المضادة لالكسدة‪ ,‬كل من انزٌمً الكلوتوثاٌون والسوبر اوكسٌد دسمٌوتٌز‬
‫اظهرت النتائج انخفاض معنوي فً كل المجامٌع المعاملة مقارنة مع مجموعة السٌطرة بٌنما فً المالونالدٌهاٌد ‪,‬‬
‫المجموعة المعاملة بالملح كلوتامٌت احادي الصودٌوم اظهرت زٌادة معنوٌة مقارنة مع مجموعة السٌطرة والمجامٌع‬
‫االخرة‪ .‬تبعا للدراسة النسٌجٌة‪ ,‬اؼلب المقاطع فً المجامٌع المعاملة قد تأثرت بشكل واضح‪.‬‬

‫التجربة الثانية‬
‫الؽرض من هذه التج ربة هو عزل الخالٌا العصبٌة من منطقة القشرة و قرن امون من اجنة الجرذان بعمر ‪ٌ61-61‬وم‬
‫من الحمل‪ ,‬ودراسة تأثٌر الملح كلوتامٌت احادي الصودٌوم والالٌكوبٌن على حٌوٌة هذه الخالٌا المعزولة اعتمادا على‬
‫الجرعة المستخدمة ووقت المعاملة بعد سبعة اٌام من الحضانة‪ .‬والنتائج اظهرت انخفاض معنوي فً حٌوٌة الخالٌا‬
‫والوقت‪ .‬بٌنما المعاملة بملح كلوتامٌت احادي الصودٌوم بالجرعة مع الخالٌا ؼٌر المعاملة(السٌطرة) تبعا للجرعة و‬
‫الخالٌا المعاملة بالالٌكوبٌن لم تظهر اي اختالؾ معنوي مقارنة مع الخالٌا المعاملة بالمذٌب المستخدم(الحمال)‬
‫باإلض افة الى ذلك ‪,‬ألثبات ان الخالٌا المعزولة هً خالٌا عصبٌة ‪,‬قمنا بأجراء دراسة الكٌمٌاء النسٌجٌة المناعٌة لتلك‬
‫ذلك‪.‬‬ ‫اثبتت‬ ‫والنتائج‬ ‫العصبٌة‪,‬‬ ‫بالخالٌا‬ ‫خاصة‬ ‫مضادة‬ ‫اجسام‬ ‫باستخدام‬ ‫المعزولة‬ ‫الخالٌا‬

‫‪212‬‬

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