EFFECT OF CERTAIN ANTIOXIDANTS ON CEREBRAL ISCHEMIA INDUCED IN IRRADIATED ...

175 Pages·2008·6.91 MB·English

Introduction
EFFECT OF CERTAIN ANTIOXIDANTS ON CEREBRAL
ISCHEMIA INDUCED IN IRRADIATED RATS
A THESIS PRESENTED
BY
Engy Refaat Abd El-Aziz
(B. Pharm. Sciences - Ain Shams University)
Pharmacist in Drug Radiation Research Department
National Centre for Radiation Research and Technology
Atomic Energy Authority - Egypt
Submitted for The Degree of Master in Pharmaceutical Sciences
(Biochemistry)
Under The Supervision of
Prof. Dr. Amal A. Abd El-Fattah Prof. Dr. Mona A. El-Ghazaly
Prof. of Biochemistry Prof. of Pharmacology
Faculty of Pharmacy Deputy Head of the Biotechnology Division
Cairo University National Centre for Radiation Research and Technology
Atomic Energy Authority
Egypt
Ass. Prof. Dr. Maha M. El-Sawalhi
Ass. Prof. of Biochemistry
Faculty of Pharmacy
Cairo University
FACULTY OF PHARMACY
CAIRO UNIVERSITY
2008
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Introduction

ﻡﻴﺤﺭﻟﺍ ﻥﻤﺤﺭﻟﺍ ﷲﺍ ﻡﺴﺒ
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Introduction
CONTENTS
Subjects Page
1. Aim of the work…………………………………………………………............... 1
2. Introduction…………………………………………….……………………..…..
3
2.1.Radiation…………………………………………………………………..…...
3
2.1.1.Types of radiation……………………………………………………………..
4
2.1.2. Mechanism of radiation induced tissue damage………………………….....
5
5
2.1.3. Biological effects of ionizing radiation……………………………………....
6
7
2.1.4. Protection against radiation hazards…………………………….…………….. 8
2.2. Cerebral Ischemia/Reperfusion………………………………………….……. 11
2.2.1. Types of cerebral ischemia……………………...…………………….….. 11
2.2.2. Pathophysiology of Ischemia/Reperfusion injury……………………….…. 12
2.2.3. Factors involved in ischemia /reperfusion induced tissue damage….……… 16
2.2.3.1. Energy failure……………………………………………………….… 16
2.2.3.2. Disturbance of calcium homeostasis………………………….............
17
2.2.3.3. Mitochondrial dysfunction………………………………………………
18
2.2.3.4. Role of excitatory amino acids…………………………………….….
19
2.2.3.5. Role of cytokines………………………………………………………..
20
2.2.3.6. Role of free radicals………………………………………………….….
22
2.3. Naturally occurring antioxidants……………………………………………….
23
2.3.1. Glutathione and related enzymes………………………………..…...
24
2.3.2. Vitamin E……………………………………………….....................
26
2.3.3. Coenzyme Q …………………………………………………..……
10 28
2.3.4. Rutin……………………………………………………………….
31
33
3. Materials and
33
Methods………………………………………………………….…………………
33
3.1. Materials………………………………………………………….………..
33
3.2. Design of the experimental work………………………………..................
3433
3.2.1. Irradiation of animals……………………………………………..…….
33
3.2.2. Induction of cerebral ischemia/reperfusion………………………………….
35
3.3.3. Experimental groups……………………………………................................
35
3.3. Tissue sampling…………………………………………………..………........
35
3.3.1. Brain ……………………………………………………………....................
35
3.3.1.1. Prepration of cytosolic fraction for the determination of calcium concentration
and the activities of LDH, GPx and GR enzymes……………………………..........
35
3.3.1.2. Preparation of brain homogenate for the determination of MDA and GSH
levels......................................................................................................................... 36
3.3.2. Serum for determination of TNF-α and IL-1β levels………………..………. 37
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Introduction
3.4. Methods…………………………………………………………................… 37
3.4.1. Determination of lactate dehydrogenase activity in cytosol of brain………....
37
3.4.2. Determination of concentration of calcium in cytosol of brain…….…….….
39
3.4.3. Determination of malondialdehyde level in brain homogenate……………...
41
3.4.4. Determination of reduced glutathione level in brain homogenate…………...
43
3.4.5. Determination of glutathione peroxidase activity in cytosol of brain...…..…
45
3.4.6. Determination of glutathione reductase activity in cytosol of brain..……..…
47
3.4.7. Determination of serum level of tumor necrosis factor-α….…………..….....
49
3.4.8. Determination of serum level of interleukin-1β…………..………..………...
52
3.5. Statistical analysis…………………………………………………………….…
55
4. Results………………………………………………………………………. 56
5 . Discussion……………………………………………………..……………..
80
6. Summary and conclusion…………………………………….………………. 98
7. References……………………………………….…………………..………… 104
~ Appendix……….………………………………………………………………
~ Arabic summary………………………………………………………………
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Introduction
LIST OF FIGURES
Figure Title Page
No.
(1)Direct and indirect radiation effects on key biological molecules of
cells……………………………………………………………………………….. 5
(2)Ionizing radiation is highly damaging and can lead to a number of diseases and
even cellular death………………………………………………………………. 6
(3)Production of oxygen and nitrogen free radicals and other reactive species in
mammalian cells………………………………………………………………… 10
(4)Schematic illustration of cellular ionic changes occurring during the ischemia
induced periods of metabolic depression with bioenergetic stress (phase 1) and
complete energy failure with anoxic depolarization (phase 2)………………........ 13
(5)Schematic illustration of degradative changes occurring during ischemia induced
neuronal destruction (phase 3). …………….………….......................................... 14
(6) The sequence of events occurring during ischemia and reperfusion……….…..
15
(7) Schematic diagram representing events leading to ischemic brain injury.…..… 21
(8) Ischemia/reperfusion presents numerous opportunities for formation of reactive
oxygen/ nitrogen species and resultant tissue injury………………………........... 22
nd resultant tissue injury………………………....
(9) Glutathione and related enzymes………………………………………………. 25
(10)Co-operative interactions of vitamin E, vitamin C, glutathione and glutathione
peroxidase and their metabolic coupling to NADPH generating pathways………
27
(11)Coenzyme Q is an important cofactor in facilitating electron transport from
10
complexes I and II to complex III in the mitochondrial respiratory
chain..……………………………………………………………………………. 29
(12) Standard curve of calcium……………………………………………………. 40
(13) Standard curve of malondialdehyde………………………………….………. 42
(14) Standard curve of glutathione……………………………………………...… 44
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Introduction
(15) Standard curve of tumor necrosis factor-α…………………………….……. 51
(16) Standard curve of interleukin-1β…………………………………………….. 54
(17)Effffeecctt ooff cceerreebbrraall isicshchememiai/ar/erieopneartfeuds ioorn acseuptea riartely or in combination with
fracc tliaocntaattee dd eohry dsrionggelen aisreradiation on brain cytosolic lactate dehydrogenase
activity and calcium level in male rats………………………………………. 58
Effect of cerebral ischemia/reperfusion separately or in combination with
Effect of cerebral ischemia/reperfusion separately or in combination with
(18)
fractionated or single irradiation on brain levels of malondialdehyde and
glutathione in male rats……………………………………………..……….
in male rats…………………………………………………………….
61
(19) Effect of cerebral ischemia/reperfusion separately or in combination with
fracfrtaiocntiaotneadt eodr aocru stein igrrlea diirartaidoina toino nt hoen b rbariani nc yctyotsoosloicli ca caticvtiitviietsie osf ogfl ugtaluthtaitohnieone
peropxeridoaxside aasned a gnldu tgaltuhtiaotnheio rneed urecdtauscet aisne m ina lmearaletsr…ats…………………..……………………………………
64
(20) Effect of cerebral ischemia/reperfusion separately or in combination with
fractionated or single irradiation on serum levels of tumor necrosis factor-α
anrdle uinktienr-l1eβu kinin m-1aβl ei rna tms…ale… r…ats…………………………………………………………………..…………………. …... 67
ytosolic lactate dehydrogenase activity and calcium level in male rats sub
(21)Effect of vitamin E, coenzyme-Q or rutin on brain cytosolic lactate
10
dehydrogenase activity and calcium level in male rats subjected to
fractionated irradiation and cerebral ischemia/ reperfusion………………….. 70
(22) Effect of vitamin E, coenzyme-Q or rutin on brain levels of
10
malondialdehyde and glutathione in male rats subjected to fractionated
irradiation and cerebral ischemia/ reperfusion.…..……………………………
73
(23) Effect of vitamin E, coenzyme-Q or rutin on brain cytosolic glutathione
10
peroxidase and glutathione reductase activities in male rats subjected to
fractionated irradiation and cerebral ischemia/reperfusion………………….
eroxidase and glutathione reductase activities in male ra 76
(24) Effect of vitamin E, coenzyme-Q or rutin on serum levels of tumor necrosis
10
factor-α and interleukin-1β in male rats subjected to fractionated irradiation
mor necrosis factor-α and interleukin-1β in male rats subjected to fraction
and cerebral ischemia/reperfusion…………………………………………… 79
LIST OF TABLES
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Introduction
LIST OF TABLES
Table Title Page
No.
……………………………………………………………………….
(1)Effect of cerebral ischemia/reperfusion separately or in combination with
fractionated or single irradiation on brain cytosolic lactate dehydrogenase
activity and calcium level in male rats……………………………………… 57
(2)Effect of cerebral ischemia/reperfusion separately or in combination with
fractionated or single irradiation on brain levels of malondialdehyde and
glutathione in male rats……………………….………………………………
60
(3) Effect of cerebral ischemia/reperfusion separately or in combination with
fractionated or single irradiation on brain cytosolic activities of glutathione
peroxidase and glutathione reductase in male rats…………………………… 63
(4)Effect of cerebral ischemia/reperfusion separately or in combination with
fractionated or single irradiation on serum levels of tumor necrosis factor-α
and interleukin-1β in male rats.………………………………………….…… 66
(5)Effect of vitamin E, coenzyme-Q or rutin on brain cytosolic lactate
10
dehydrogenase activity and calcium level in male rats subjected to fractionated
irradiation and cerebral ischemia/reperfusion………………………………… 69
(6) Effect of vitamin E, coenzyme-Q or rutin on brain of malondialdehyde and
10
glutathione in male rats subjected to fractionated irradiation and cerebral
ischemia/reperfusion…..………………………………………………………. 72
(7) Effect of vitamin E, coenzyme-Q or rutin on brain cytosolic glutathione
10
peroxidase and glutathione reductase activities in male rats subjected to
fractionated irradiation and cerebral ischemia/reperfusion….…………..….
75
(8) Effect of vitamin E, coenzyme-Q or rutin on serum levels of tumor necrosis
10
factor-α and interleukin-1β in male rats subjected to fractionated irradiation
and cerebral ischemiaL/rIeSpeTrf uOsioFn .A.…B…B…E…R…V…IA…T…IO…N…S………………… 78
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Introduction
LIST OF ABBERVIATIONS
ΔA change in absorbance
AMPA α-2-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid
ANOVA analysis of variance
BBB blood brain barrier
[Ca2+] intracellular calcium ion concentration
i
CAT catalase
CBF cerebral blood flow
CNS central nervous system
CoQ coenzyme Q
10 10
CuZn-SOD copper-zinc superoxide dismutase
DAGs Diacyl glycerides
DiPGE Dimethyl prostaglandin E
2 2
DTNB 5,5' dithiobis-(2-nitrobenzoic acid)
EAA excitatory amino acid
EDTA ethylene diamine tetra-acetic acid
ELISA enzyme linked immunosorbant assay
ER endoplasmic reticulum
ET-1 endothelin-1
FFA free fatty acid
γ-GCS gamma-glutamyl cystienyl synthetase
G6PDH glucose-6-phosphate dehydrogenase
GPx glutathione peroxidase
GR glutathione reductase
GSH reduced glutathione
GSSG oxidized glutathione
GST glutathione-S-transferase
Gy Gray
H O hydrogen peroxide
2 2
IL-1β interleukin-1β
IMP inositol monophosphate
i.p. intraperitoneal
[K+] extracellular potassium ion concentration
e
LDH lactate dehydrogenase
LDL low density lipoprotein
LTC Leukotriene C
4 4
MCAO middle cerebral artery occlusion
MDA malondialdehyde
Mn-SOD manganese superoxide dismutase
MPT mitocondrial permeability transition
MTH mitocondrial transhydrogenase
n1 neutron
0
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Introduction
NaN sodium azide
3
NF-κB nuclear factor-κB
NMDA N-methyl-D-aspartate
NO nitric oxide
NOS nitric oxide synthase
O molecular oxygen
2
-
O (cid:127) superoxide anion
2
OH(cid:127) hydroxyl radical
ONOO- peroxynitrite
PAF platelet activating factor
PARP poly(ADP) ribose polymerase
pH potential of hydrogen
PHGPx phospholipid hydropeoxide glutathione peroxidase
PLA phospholipase-A
2 2
PMNs polymorphnuclear leukocytes
PPP pentose phosphate pathway
PUFA polyunsaturated fatty acid
RNS reactive nitrogen species
ROS reactive oxygen species
SOD superoxide dismutase
TBA thiobarbituric acid
TBARS thiobarbituric acid reactive substances
TNF-α tumor necrosis factor-α
XDH xanthine dehydrogenase
XO xanthine oxidase
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Introduction
ACKNOWLEDGEMENTS
No words can express my gratitude and appreciation to Prof. Dr. Mona Abd El-
Lateef El-Ghazaly; Prof. of Pharmacology and Deputy Head of Biotechnology
Division, National Centre for Radiation Research and Technology; for the
suggestion of the research point, practical and financial support, instructive
supervision, subjective criticism and the continuous guidance and efforts that were
kindly and generously offered by her; throughout each stage of this study.
I would like to express my deep and sincere thanks to Prof. Dr. Amal Ahmed
Abd El-Fattah; Prof. of Biochemistry, Faculty of Pharmacy, Cairo University, for
her kind guidance, great patience, subjective and precise revision, as well as her
continuous valuable encouragement and support allover the supervision of this
work.
I am also very grateful for Ass. Prof. Dr. Maha Mahmoud El-Sawalhi; Ass.
Prof. of Biochemistry, Faculty of Pharmacy, Cairo University; for her contribution
in the supervision of this work and her help in the experimental work and writing of
the thesis.
I would like, also; to express my thanks to Prof. Dr. Mona M. Seif-El-Naser;
Prof. of Pharmacology, Faculty of Pharmacy, Cairo University; and to Dr.Rania
Mohsen ; lecturer of pharmacology, Faculty of Pharmacy, Cairo University for
teaching me the model of ischemia and the handling of brain tissue.
My gratitude is also extended to all my colleagues and friends at Drug Radiation
Research Department, National Centre for Radiation Research and Technology,
for their continuous and sincere help and support.
Last but not least; I would like to thank my family for their support and
encouragement which enabled this work to be completed, may God reward them
all.
Engy Refaat
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