Damaging Effect of Cigarette Smoke in Gamma- Irradiated Rats: Possible Protection by Certain Natural Products

THESIS PRESENTED BY

Aliaa Hossam El-Deen Ashoub (B. Pharm. Sciences - Ain Shams University 2000) 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 (Pharmacology and Toxicology)

Under Supervision of

Dr. Sanaa A. Kenawy Dr. Mona A. El-Ghazaly Prof. of Pharmacology Prof. of Pharmacology Faculty of Pharmacy Chairman of National Centre for Radiation Cairo University Research and Technology Atomic Energy Authority-Egypt

Department of Pharmacology and Toxicology Faculty of Pharmacy Cairo University 2013

(

)

)النمل-٩١(

Acknowledgements

I would like to thank Dr. Sanaa A. Kenawy, Professor of pharmacology, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, for her diligent support and her keen supervision for this thesis. It wouldn’t have been possible to achieve this piece of work without her kind guidance, ongoing assistance and great encouragement.

I would like to extend my deepest gratitude towards Dr. Mona A. El Ghazaly, Professor of pharmacology and Chairman of the National Centre for Radiation Research and Technology (NCRRT), Atomic Energy Authority, whom I consider as my primary intellectual supervisor for suggesting the topic of this thesis and for helping me in formulating it. Dr. El Ghazaly has greatly influenced my way of thinking and has helped me grow as a researcher by offering her precious knowledge and experience. Her teaching will guide me to accomplish more in my future research projects. In addition to her practical help in ensuring that all the needed facilities and equipment were easily accessible to me during the practical part of this research.

My sincere thanks goes to Dr. Nour El-Din Amin Mohamed, Professor of biological chemistry at the National Centre for Radiation Research and Technology (NCRRT), Atomic Energy Authority, for his additional support and guidance during this research. It was my greatest pleasure to work with Dr. Mohamed and benefit from his supervision and valuable input.

I would like to thank all my colleagues at the department of drug radiation research at the national centre for radiation research and technology, Atomic Energy Authority, for their continuous help, support and stimulating energy.

Finally, I would like to thank my family for their endless love, support and endurance during my studies. Without them I could not have come this far. I thank them and Thank God for all of his mercy and love.

Aliaa H. Ashoub

Contents

Contents Page

List of tables ……………………………………………………………………………. III

List of figures ……………………………………………...…………………………... V List of abbreviations.………………………………………………………………….. VIII 1. INTRODUCTION 1 ……………………………………………..……………………………………….. Ionizing Radiation ……………………………………………………………… 1 - Mechanisms of Radiation Effects …………………………………………….. 1 - Factors affecting Radiation Hazards ……………...…………………………... 3 1. Related to the Ionizing Radiation …………………………………... 3 2. Related to the Biological Target ……………………………………. 3 - Various Effects of Radiation ………..………….…………………………….. 4 1. The Early Effects …………………………………………………… 5 2. The Delayed Effects ………………………………………………… 8 Cigarette Smoke ………………………………………………………………... 10 Effects of tobacco smoke on biological systems ……………………………….. 11 -Tobacco smoking and oxidative stress …………..……………………………. 19 Flavonoids………………………………………………………………………. 21 - Biological Effects of Flavonoids……………………………...………………. 21 Catechin ………………………………………………………………………… 24 Rutin…………………………………………………………………………….. 27 2. AIM OF THE WORK ………………..…………………………..………... 30

3. MATERIAL & METHODS ……………………………………………….. 32

Material ……………………………………………………...………………… 32 - Animals ……………………………..………………………………………… 32 - Drugs and Doses ...…………………………..………….……………………. 32 - Radiation Facilities …………………………………………………………… 32 - Chemicals and Reagents ……………………………………………………… 32 - Experimental Design …………………………………………………….…… 34 - Sampling ……………………………………………………………………… 35 1. Blood Samples ………………………………………….……………..… 35 2. Tissue Samples ………………………………………………………….. 35 . Preparation of Gastric Mucosa ……………………………………... 35

I Contents (Cont’d)

Methods ………………………………………………………….…………….. 36 - Preparation of Cigarette Smoke Extracts ……………………………………... 36 - Identification of Compounds from Cigarette Smoke Extracts …………...…… 37 . Thin Layer Chromatography Method ………………..…………….. 37 - The Measured Parameters …………………….…………………………….… 37 1. Determination of Serum Alanine Transaminase ………………….... 37 2. Determination of Serum Aspartate Transaminase ……………….… 39 3. Determination of Serum Alkaline Phosphatase ………………….… 41 4. Determination of Blood Glutathione (GSH) ……………………..… 42 5. Determination of Serum Malondialdehyde ……………………….... 44 6. Determination of Serum Nitric Oxide …………………………….... 45 7. Determination of Serum Lactate Dehydrogenase (LDH) ………..… 46 8. Determination of Myeloperoxidase (MPO) activity in Gastric Mucosa ………………………………………………………..……. 47 9. Determination of Serum Tumor Necrosis Factor-α (TNF-α) ………. 49 Statistical Analysis ……………………………………………………….....… 52

4. RESULTS …………………………………………………………………… 53

5. DISCUSSION ………………………………………………………………. 90

6. SUMMARY & CONCLUSIONS …………………………………… 104

7. REFERENCES ……………………………………………………………... 107

١ ………………………………………………………… ARABIC SUMMARY

II

List of Tables

Table Title Page Effect of Catechin (50mg/kg) or rutin (40mg/kg) administration on serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gastric 1 54 myeloperoxidase (MPO) activities and levels of blood glutathione (GSH), serum malondialdehyde (MDA), nitric oxide (NO) and tumor necrosis factor – alpha (TNF-α) in normal rats Effect of CSE (5mg/kg) administration on whole body gamma-irradiated rats at dose level of 0.5 Gy, 1 Gy and 2 2a Gy on alanine transaminase (ALT), aspartate transaminase 57 (AST) and alkaline phosphatase (ALP) serum activities in rats Effect of CSE (5mg/kg) administration on whole body gamma-irradiated rats at dose level of 2 Gy in presence of 2b catechin (50mg/kg) or rutin (40mg/kg) on alanine 58 transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) serum activities Effect of CSE (5mg/kg) administration on whole body gamma-irradiated rats at dose levels of 0.5 Gy, 1 Gy and 2 3a 67 Gy on malondialdehyde (MDA), nitric oxide (NO) serum levels and glutathione (GSH) blood level Effect of CSE (5mg/kg) administration on whole body gamma-irradiated rats at dose level of 2 Gy in presence of 3b catechin (50mg/kg) or rutin (40mg/kg) on 68 malondialdehyde (MDA), nitric oxide (NO) serum levels and glutathione (GSH) blood level Effect of CSE (5mg/kg) administration on whole body 4a gamma-irradiated rats at dose levels of 0.5 Gy, 1 Gy and 2 76 Gy on lactate dehydrogenase (LDH) serum activity Effect of CSE (5mg/kg) administration on whole body gamma-irradiated rats at dose level of 2 Gy in presence of 4b catechin (50mg/kg) or rutin (40mg/kg) on lactate 77 dehydrogenase (LDH) serum activity

III List of Tables

(Cont’d)

Effect of CSE (5mg/kg) administration on whole body gamma-irradiated rats at dose levels of 0.5 Gy, 1 Gy and 2 5a 81 Gy on the activity of myeloperoxidase (MPO) in gastric mucosa Effect of CSE (5mg/kg) administration on whole body gamma-irradiated rats at dose level of 2 Gy in presence of 5b 82 catechin (50mg/kg) or rutin (40mg/kg) on the activity of myeloperoxidase (MPO) in gastric mucosa Effect of CSE (5mg/kg) administration on whole body 6a gamma-irradiated rats at dose levels of 0.5 Gy, 1 Gy and 2 86 Gy on tumor necrosis factor-alpha (TNF-α) serum level Effect of CSE (5mg/kg) administration on whole body gamma-irradiated rats at dose level of 2 Gy in presence of 6b 87 catechin (50mg/kg) or rutin (40mg/kg) on tumor necrosis factor-alpha (TNF-α) serum level

IV

List of Figures

Figure Title Page The mechanisms of ionizing radiation effects on i 2 biological ii Mechanism of antioxidant effects of flavonoids 23 iii Molecular structure of catechin 24 iv Molecular structure of rutin 27 v Apparatus used for the preparation of CSE 36 vi Standard curve of ALT 39 vii Standard curve of AST 41 viii Standard curve of GSH 43 ix Standard curve of MDA 45 x Standard curve of MPO 49 xi Standard curve of TNF-α 51 Effect of CSE (5mg/kg) administration, gamma 1a irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination 59 on serum activity of alanine transaminase (ALT) in rats Effect of gamma irradiation (2 Gy), CSE (5mg/kg) and their combination alone or in presence of catechin 1b 60 (50mg/kg) or rutin (40mg/kg) on serum activity of alanine transaminase (ALT) in rats Effect of CSE (5mg/kg) administration, gamma irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination 2a 61 on serum activity of aspartate transaminase (AST) in rats Effect of gamma irradiation (2 Gy), CSE (5mg/kg) and their combination alone or in presence of catechin 2b 62 (50mg/kg) or rutin (40mg/kg) on serum activity of aspartate transaminase (AST) in rats Effect of CSE (5mg/kg) administration, gamma 3a irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination 63 on serum activity of alkaline phosphatase (ALP) in rats Effect of gamma irradiation (2 Gy), CSE (5mg/kg) and their combination alone or in presence of catechin 3b 64 (50mg/kg) or rutin (40mg/kg) on serum activity of alkaline phosphatase (ALP) in rats Effect of CSE (5mg/kg) administration, gamma 4a irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination 69 on serum level of malondialdehyde (MDA) in rats

V List of Figures

(Cont’d)

Effect of gamma irradiation (2 Gy), CSE (5mg/kg) and

their combination alone or in presence of catechin 4b 70 (50mg/kg) or rutin (40mg/kg) on serum level of malondialdehyde (MDA) in rats Effect of CSE (5mg/kg) administration, gamma 5a irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination 71 on blood level of glutathione (GSH) in rats Effect of gamma irradiation (2 Gy), CSE (5mg/kg) and their combination alone or in presence of catechin 5b 72 (50mg/kg) or rutin (40mg/kg) on blood level of glutathione (GSH) in rats Effect of CSE (5mg/kg) administration, gamma 6a irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination 73 on serum level of nitric oxide (NO) in rats Effect of gamma irradiation (2 Gy), CSE (5mg/kg) and their combination alone or in presence of catechin 6b 74 (50mg/kg) or rutin (40mg/kg) on serum level of nitric oxide (NO) in rats Effect of CSE (5mg/kg) administration, gamma irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination 7a 78 on serum activity of lactate dehydrogenase (LDH) in rats Effect of gamma irradiation (2 Gy), CSE (5mg/kg) and their combination alone or in presence of catechin 7b 79 (50mg/kg) or rutin (40mg/kg) on serum activity of lactate dehydrogenase (LDH) in rats Effect of CSE (5mg/kg) administration, gamma 8a irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination 83 on serum activity of myeloperoxidase (MPO) in rats Effect of gamma irradiation (2 Gy), CSE (5mg/kg) and their combination alone or in presence of catechin 8b 84 (50mg/kg) or rutin (40mg/kg) on serum activity of myeloperoxidase (MPO) in rats

VI List of Figures

(Cont’d)

Effect of CSE (5mg/kg) administration, gamma irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination 9a 88 on serum level of tumor necrosis factor-alpha (TNF-α) in rats Effect of gamma irradiation (2 Gy), CSE (5mg/kg) and their combination alone or in presence of catechin 9b 89 (50mg/kg) or rutin (40mg/kg) on serum level of tumor necrosis factor-alpha (TNF-α) in rats

VII List of Abbreviations

Abbreviation Full Name ACP Acid phosphatase ALP Alkaline phosphatase ALT Alanine transaminase AMs Alveolar macrophages ANOVA Analysis of variance ARE Antioxidant response element AST Aspartate transaminase BBB Blood brain barrier Ca2+ Calcium ion CAT Catalase CCE Chloroform cigarette extract CCl4 Carbon tetrachloride COPD Chronic obstructive pulmonary disease COX-2 Cyclooxygenase-2 CRP C-reactive protein CS Cigarette smoke CSE Cigarette smoke extract DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid ECE Ethanol cigarette extract EDTA Ethylene diamine tetra acetic acid EGCG Epigallocatechin gallate ECG Epicatechin gallate ELISA Enzyme-linked immunosorbant assay eNOS Endothelial nitric oxide synthase ETS Environmental tobacco smoke Gamma-GTP Gamma glutamyl transpeptidase GI Gastrointestinal GIT Gastrointestinal tract GSH Reduced glutathione GSH-Px Glutathione peroxidase GSSG Oxidized glutathione GST Glutathione-S-transferase GTC Green tea catechins Gy Gray HBeAG Hepatitis B e antigen HDL High density lipoprotein

VIII List of Abbreviations

(Cont’d)

Abbreviation Full Name HMG-CoA 3- Hydroxyl - 3- methyl glutaryl coenzyme a HPLC High performance liquid chromatography HTAB Hexadecyl tetramethyl ammonium bromide I/R Ischemia/Reperfusion IFN-γ Interferon-gamma IL-1 Interleukin-1 IL-1β Interleukin-1 beta IL-6 Interleukin-6 IL-8 Interleukin-8 IMT Intima-media thickness iNOS Inducible nitric oxide synthase LDH Lactate dehydrogenase LDL Low density lipoprotein LET Linear energy transfer LP Lipid peroxidation LPS Lipopolysaccharide MDA Malondialdehyde mGy Milligray MPO Myeloperoxidase mRNA Messenger ribonucleic acid MS Mainstream NAD Nicotinamide adenine dinucleotide NADP Nicotinamide adenine dinucleotide phosphate NCRRT National centre for radiation research and technology NF-κB Nuclear factor kappa-b nNOS Neoronal nitric oxide synthase NO Nitric oxide NO2 Nitrogen dioxide OH• Hydroxyl radicals PAH Polycyclic aromatic hydrocarbons PD Parkinson’s disease PMNs Polymorphonuclear leukocytes PO Per os PUFAs Polyunsaturated fatty acids

IX List of Abbreviations

(Cont’d)

Abbreviation Full Name RNA Ribonucleic acid ROS Reactive oxygen species rpm Revolution per minute SE Standard error SIDS Sudden infant death syndrome SOD Superoxide dismutase SS Side stream ST Smokeless tobacco STZ Streptozotocin TBA Thiobarbituric acid TBARS Thiobarbituric acid reactive substances TGs Triglycerides TMB Tetramethyl benzidine TNF-α Tumor necrosis factor alpha UC Ulcerative colitis UV Ultraviolet VIS Visible WHO World Health Organization XO Xanthine oxidase γ-radiation Gamma radiation μGy Microgray

X

INTRODUCTION

Introduction

Ionizing Radiation

Mechanisms, Factors Affecting Hazards and Effects

Ionizing radiation can be defined as any type of electromagnetic (such as X- or gamma) rays or particle radiation (such as neutron or alpha particles) with sufficient energy to ionize atoms or molecules; that is, to eject electrons from their outer orbitals (Eggermont et al., 2006).

Radiations may be directly or indirectly ionizing. All of the charged particles previously discussed including particles and heavy ions are directly ionizing; that is, provided the individual particles that have sufficient kinetic energy. They can directly disrupt the atomic structure of the absorber through which they pass, and produce chemical and biological changes. Electromagnetic radiations (X- and gamma rays) are indirectly ionizing. They do not produce chemical or biological damage themselves, but when they are absorbed in the material through which they pass they give up their energy to produce fast-moving electrons that stimulate the living cells to produce reactive oxygen species (ROS); which in turn can induce oxidative damage to vital cellular molecules including DNA, proteins and lipids (Spitz et al., 2004).

The amount or quantity of ionizing radiation, or as it is usually called, the dose, is measured in terms of the energy absorbed in the tissues. The unit of absorbed dose is the Gray (Gy) defined to be an energy absorption of 1 joule per kilogram. In some instances, the dose is very much less than one Gray and then the units milligray (mGy) or microgray (μGy) are used. There are 1000 mGy or one million μGy in 1 Gy. A total body dose of 5 Gy to a human being would most probably be fatal; the average natural background radiation to which one is exposed is about 3 mGy per year. Equal doses of different types of radiation do not necessarily produce equal biological effects. For example, 0.5 Gy of neutrons is more effective than 0.5 Gy of X-rays. In general, X-rays, gamma rays, and electrons are least effective for a given dose, while heavy ions are the most damaging. Neutrons fall somewhere in between (Hall and Kereiakes, 2001).

Mechanisms of Radiation Effects

Ionizing radiation exerts its effects on biological targets through two major mechanisms direct and indirect (Giambarresi and Walker, 1989).

1 Introduction

Fig. (i): The mechanisms of ionizing radiation effects on biological tissues (c.f. Giambarresi and Walker, 1989).

The direct effect theory or target theory proposes that ionizing radiation acts by direct hits on target atoms. All atoms or molecules within the cells, such as enzymatic and structural proteins and RNA, are vulnerable to radiation injury. However, DNA, is the principal target, in which ionizing radiation produces single- or double-stranded chromosomal breaks (Michaels and Hunt, 1978). The direct mechanism theory was found to be inadequate for explaining cellular radiation injuries.

The indirect theory proposes that ionizing radiation exerts its effect via radiolysis of cellular water, forming free radicals. These free radicals interact with atoms and molecules within the cells, particularly DNA, to produce chemical modifications and consequently harmful effects (Hollahan, 1987).

The cells in the vicinity of irradiated cells show effects that cannot be attributed to targeting by ionizing radiation tracks. Additionally, when cells are irradiated and later transferred to another medium, the cells in proximity in the new medium exhibit DNA damage, mutation, teratogenesis and carcinogenesis. Through cell-to-cell interaction, the directly irradiated cells communicate with adjacent cells and spread the effect of radiation to a larger number of cells. The mechanism is not clearly understood; however, gap junctional intercellular communication (Azzam et al., 2001) or release of soluble factors (such as cytokines) (Ramesh et al., 1996) from irradiated cells has been proposed. The bystander effect has been mainly described for densely ionizing radiation (such as alpha particles) (Iyer et al., 2000), but also is seen in low linear energy transfer (LET) radiation (such as gamma or X-rays).

2 Introduction

Factors Affecting Radiation Hazards Radiation injury can be modified by factors related to the ionizing radiation and the target tissues.

1- Related to the Ionizing Radiation

Certain factors related to radiation itself determine the various effects for the same radiation dose to biological organs. Various types of radiation differ in penetrability based on linear energy transfer (LET), which expresses energy loss per unit distance travelled (kiloelectron volts per micrometer). This value is high for alpha particles, lower for beta particles, and even less for gamma rays and X-rays. Thus alpha particles penetrate a short distance but induce heavy damage, whereas beta particles travel a longer distance but much shorter than gamma rays (Giambarresi and Jacobs, 1987( .

The radiation dose is obviously an important factor. In addition, a single dose of radiation causes more damage than the same dose being divided (fractionated). Collectively these two factors are expressed as dose per fraction (Beir, 1990).

Dose rate expresses the time for which dose is administered. The longer the duration for the same total dose, the better the chance of cellular repair and the smaller the damage (Beir, 1990). 2- Related to the Biological Target Certain properties of tissues and cells can significantly modify the biological effects of ionizing radiation.

Although all cells can be affected by ionizing radiation, normal cells and their tumors vary in their sensitivity to radiation. Slowly and rapidly growing cells have different radiosensitivity in relation to their movement through the cell cycle. Radiosensitivity varies with the rate of mitosis and cellular maturity.Blood-forming cells are very sensitive to radiation, while neurons, muscle and parathyroid cells are highly radioresistant. Within a given cell, the nucleus in general is relatively more radiosensitive than the cytoplasm (Kendall, 2000).

Some cells are known to have a higher capacity than others to repair the damage caused by ionizing radiation; consequently, the biological effects of the same radiation dose are different. Significant repair is known to occur quickly, within 3 h (El Gazzar and Kazem, 2006).

3 Introduction

The life cycle of the cell includes several phases: the pre-DNA synthetic phase (G1), the DNA synthetic phase (S), the post-DNA synthetic phase (G2), mitosis (M), and the identified phase of no growth (0), which represents the time after mitosis to the start of the G1 phase. All phases of the cell cycle can be affected by ionizing radiation. The radiosensitivity of a given cell varies from one cell-cycle phase to another. Overall, sensitivity appears to be greatest in G2 phase; irradiation during this phase retards the onset of cell division. Irradiation during mitosis induces chromosomal aberrations, i.e., breaks, deletions, translocations, and others. The sensitivity of a given cell-cycle phase also differs from one cell type to another and by alteration of radiation injury (Prasad, 1995). For example, the reproductive cells are most sensitive during the M phase, while damage to DNA synthesis and chromosomes occurs mostly when the cell is in the G2 phase. Recovery from sublethal damage occurs in all phases of the cell cycle. However, this is most pronounced in the S phase, which is also the most radioresistant phase (Ward, 1988). Molecular oxygen is known to have the ability to potentiate the response to radiation; this is known as the oxygen effect. The amount of molecular oxygen rather than the rate of oxygen utilization by the cells is the most important factor for increasing the sensitivity of cells to radiation. The probable mechanism is the allowance of additional free radicals, which enhance the damage of cells (Ward, 1988; McGee et al., 2010).

Various Effects of Radiation

Humans are constantly exposed to ionizing radiations both from natural source like cosmic rays (during space travel), radioisotopes found in the earth crust and also from a wide variety of artificial sources. Currently, ionizing radiation finds extensive application like power generation, developing new varieties of high-yielding crops, sterilization and enhancing the storage period of food materials (food irradiation) etc. With such a wide application of ionizing radiation, human exposure to these radiations has become inevitable. Further, ionizing radiations are also widely used in treating cancers of various tissues where it is used to kill or arrest the growth of tumor cells. Presently, more than one-half of all cancer patients are treated with radiation therapy. Despite a good therapeutic index, radiotherapy can cause tissue injury to normal tissues in long-term cancer survivors (Meister, 2005).

The biological effect of low-level radiation is extremely difficult to study in a controlled environment. The effects of high radiation exposure

4 Introduction to populations during accidents or nuclear war have been the main source of information. At low doses, radiation can trigger only partially understood effects that can lead to cancer or genetic damage. These effects take years or generations to appear. At high doses, the effect may become evident within minutes, hours, or days. It is important for physicians to be familiar with the early effects of high radiation doses (1 Gy or more to the whole body), since the possibility that people may be exposed to such doses is increasing (Eggermont et al., 2006).

1- The Early Effects

Following exposure to a large, single, short-term whole-body dose of ionizing radiation, the resulting injury is expressed as a series of clinical symptoms. The sequence of events can be generally divided into four clinical periods: 1. The prodromal period, up to 48 h, when the symptoms include anorexia, nausea, vomiting, and diarrhea 2. The latent period, from 48 h to 2–3 weeks after exposure, when the patient becomes asymptomatic 3. The manifest phase, from week 6 to week 8 after exposure, when variable symptoms appear based on the radiation dose 4. The recovery period: if the patient survives, recovery occurs from 6 weeks to several months after exposure. The presentation of these periods and their duration depend on the amount of radiation exposure (Prasad, 1995)

The symptoms of radiation sickness can be mild, such as loss of appetite and mild fatigue, or evident only on laboratory tests with mild lymphopenia (subclinical), or may be severe, appearing as early as 5 min after exposure to very high doses of 10 Gy or more and also include fatigue, sweating, fever, apathy, and low blood pressure. Lower doses delay the onset of symptoms and produce less severe symptoms or a subclinical syndrome that can occur with doses of less than 2 Gy to the whole body, and recovery is complete with 100% survival (Waselenko et al., 2004).

Because the haematopoietic system has a high level of cell turnover, it is among the most radiosensitive tissues in the body. The cells are affected and suppressed at relatively low doses of acute irradiation. Exposure to ionizing radiation induces a dose dependent decline in circulating haematopoietic cells, not only through reducing bone marrow cell production, but also by redistribution and apoptosis of mature cells (Whitnall et al., 2000; Zhou and Mi, 2005 ; Hosseinimehr et al., 2006).

5 Introduction

In general, the decline in lymphocytes and granulocytes occurs over a period of hours or days after irradiation. Platelets also decline over a period of days, consistent with their half-life (Dainiak, 2002). The primary cause of mortality during the early phases of radiation-induced haematopoietic syndrome is sepsis, resulting from opportunistic infection, due to low numbers of neutrophils and increased translocation of bacteria across the gastrointestinal mucosa. This is complicated by thrombocytopenia and concomitant haemorrhage and defects in the adaptive immune system resulting from apoptosis of lymphocytes and deficient lymphopoiesis (Whitnall et al., 2000). Mitotically active hematopoietic progenitors have a limited capacity to divide after exposure to a whole-body radiation dose greater than 2 or 3 Gy (Hall and Giaccia, 2006). After exposure, a hematologic crisis might lead to predisposition to infection, bleeding, and poor wound healing, among others. A predictable decline in lymphocytes occurs after irradiation, and in fact, there is a 50% decline in absolute lymphocyte count within the first 24 hours after exposure, followed by a further, more severe decline within 48 hours (Waselenko et al., 2004). Gastrointestinal syndrome occurs with still higher doses of 6–10 Gy which cause manifestations related to the gastrointestinal tract in addition to those of the bone marrow syndrome. Initially, loss of appetite, apathy, nausea, and vomiting occur for 2–8 h. These effects may subside rapidly. Several days later, malaise, anorexia, nausea, vomiting, high fever, persistent diarrhea, abdominal distention, and infections appear (Vitolo et al., 2004). During the second week of irradiation, severe dehydration, hemoconcentration, and circulatory collapse may be seen, eventually leading to death.

The gastrointestinal (GI) tract is among the most radiosensitive organ systems in the body. In addition to the intestinal epithelium crypt, radiation exposure damages supporting structures such as endocrine glands of the GI tract (Hauer-Jensen et al., 2007).

Radiation-induced enterocolitis is a form of acute radiation disease developing after the absorption of dose of 8 Gy and higher. It concerns an absolutely lethal clinical-pathological unit, necro-haemorrhagic enteritis and colitis, for which no causal therapy is known (Nguyen et al., 2002). The large morphological changes in small intestine mucosa develop rapidly and progressively during the first 4 days after irradiation with supralethal doses. Molecular biology explains the effect of irradiation on cells via the alteration of signaling pathways (Suzuki et al., 2001; Torii et al., 2004).

6 Introduction

Small intestine is one of the most radiosensitive organs (Smith and De Cosse 1986). Intestinal toxicity is a major type of complication. Whole abdominal irradiation causes inflammation in small intestine with submucosal edema, hyperemia and infiltration of lamina propria with activated inflammatory cells, such as macrophages and neutrophils (Guzman-Stein et al., 1989; Klimberg et al., 1990).

Radiation-induced damage to the salivary glands results in xerostomia (dry mouth). The affected patients have both compromised quantity and quality of saliva that can affect the emotional and systemic well-being of these individuals (Wright, 1987). Such patients suffer from increased oral infections, difficulty in speaking, difficulty in swallowing food, and problems with digestion. In the human, the parotid gland is more radiosensitive when compared to the submandibular gland (Baum et al., 1985). Various studies noted over a 50% reduction in parotid gland function within few days after exposure of the head and neck region to low doses of radiation as those reviewed in the study of Nagler, (2002). The high sensitivity of the parotid gland has been suggested to be due to the predominance of serous cells in this gland as these epithelial cells appear to be the most radiosensitive (Baum et al., 1985).

Central nervous system is generally resistant to radiation effects. A dose higher than 10 Gy is required to cause substantial effects on the brain and the nervous system. Symptoms include intractable nausea and vomiting, confusion, convulsions, coma, and absent lymphocytes. The prognosis is poor, with death in a few days (El Gazzar and Kazem, 2006).

When enough radiation is delivered locally to a certain part of the body, as in the case of radiation therapy, which focuses on a certain field, acute effects can appear in the exposed area. Examples include skin erythema (Archambeau, 1987) and inflammation in small intestine with submucosal edema, hyperemia and infiltration of lamina propria with activated inflammatory cells, such as macrophages and neutrophils (Guzman-Stein et al., 1989; Klimberg et al., 1990) after whole abdominal irradiation.

Radiation is one of the hazardous matters and pathophysiological changes in the skin caused by ionizing radiation include erythema, desquamation for early changes occurring within hours and weeks as well as dermal atrophy and telangiectasia for later effects (Chi et al., 2005). The effects of ionizing radiation on the skin have been the subject of extensive research (Lyng et al., 2004; Donetti et al., 2005; Fangman and Cook, 2005; Franchi et al., 2006). However, there is limited study

7 Introduction investigating the acute effects of gamma radiation on the skin (Hoashi et al., 2008). In addition, very few studies have addressed the effect of ionizing radiation on skin biomechanics and structure (Ranu et al., 1975; Ranu, 1991).

2- The Delayed Effects

Exposure to X and gamma radiations is most strongly associated with leukemia and cancer of the thyroid gland, breast, and lung. Associations have been reported at absorbed doses of less than 0.2 Gy. The risk of developing these cancers, however, depends to some extent on age at exposure. Childhood exposure is mainly responsible for increased leukemia and thyroid-cancer risks, and reproductive-age exposure for increased breast-cancer risk. In addition, some evidence suggests that lung-cancer risk may be most strongly related to exposure later in life. Associations between radiation exposure and cancer of the salivary glands, stomach, colon, urinary bladder, ovary, central nervous system, and skin also have been reported, usually at higher doses of radiation (1 Gy) (Ron, 1998; Ron et al., 1999; Brenner et al., 2000; Lichter et al., 2000; Yeh et al., 2001; Bhatia et al., 2002).

Genetic effects may include changes in the number and structure of chromosomes and gene mutations, dominant or recessive (El Gazzar and Kazem, 2006).

Experimental results using viruses, bacteria, yeast and mammalian cells have demonstrated a correlation between the radiosensitivity and the DNA content, at least for groups of biologically similar objects: the higher the amount of DNA, the more sensitive the object (Altman et al., 1970). These results already suggested the DNA to play a key role in the response to radiation. This hypothesis was proven also more directly for mammalian cells (Munro, 1970).

Radiation can cause various lesions by direct interaction with DNA or indirectly through damage induced by free radicals mainly resulting from the radiolysis of water molecules, the most abundant molecule in the cell (Valko et al., 2007). The hydroxyl free radical is known to react with all components of the DNA molecule, damaging both the purine and pyrimidine bases and also the deoxyribose backbone (Halliwell, 1996; Valko et al., 2007). Finally, irradiation induces several types of damage to DNA, including double and single-strand breaks, base and sugar damage as well as DNA–DNA and DNA–protein cross-links (Barker et al., 2005).

8 Introduction

The fetus is exposed to unavoidable (background) radiation from cosmic rays, terrestrial radiation from ground and building and naturally occurring radioisotopes that are inhaled or ingested. The total fetal dose from background radiation sources is 1 mGy or less during the entire pregnancy (Bentur, 2007). The embryonic stage is one of the most radiosensitive stages in the life of any organism. The classical triad of effects of radiation on the embryo is growth retardation, embryonic, fetal, or neonatal death, and congenital malformation. The probability of finding one or more of these effects is dependent upon radiation dose, the dose rate, and the stage of gestation at exposure (El Gazzar and Kazem, 2006). Stage of development is particularly important, since the organ which is differentiated at that time will be most vulnerable; this determines the type of abnormality or malformation that will be observed. During the first 2 weeks of conception the effect of radiation is an all or none effect, where the embryo is aborted. Following this period and up to 8 weeks the embryo is very vulnerable to congenital malformations (Bentur et al., 1991, El Gazzar and Kazem, 2006).

The stimulative biological effects produced by low-dose irradiation of organisms have been studied extensively because low-dose irradiation brings about effects that differ from those produced by high-dose irradiation (Lucky, 1982; Macklis and Beresford, 1991). The immune function is one of the radiosensitive systems and a high-dose irradiation is known to induce immunosuppressive effects. In contrast, several reports have suggested that low-dose irradiation enhances the immune function: continuous low-dose γ-ray irradiation induced enhanced proliferation of splenocytes (James and Makinodan, 1988) and higher cell populations in the spleen and thymus (Planel et al., 1992). The single low-doses of X-rays stimulated the plaque-forming cell reaction of the spleen (Liu et al., 1987). Mitogen-induced proliferation of rat splenocytes also is reported to be stimulated by temporary low-dose irradiation (Ishii et al., 1990). Fibrosis is one of the major late effects of both radiotherapy and accidental radiation exposure. Its major characteristic is the massive over production and deposition of extracellular matrix components. Fibrosis after exposure to radiation has been described in numerous tissues including skin (Bentzen et al., 1989), lung (McDonald et al., 1995), heart (Fajardo and Stewart, 1997), liver (Geraci and Mariano, 1993) and kidney (Cohen et al., 1996).

9 Introduction

Some studies report that inflammatory cytokines, such as IL-1β, TNF- α, and IL-6, are all induced by ionizing radiation. They significantly contribute to the disorders associated with radiotherapy in the blood (Holler et al., 1990), peripheral lymphoid tissues, and lungs (Rubin et al., 1995; Hong et al., 1999). In animal models, increased cytokine expression after irradiation has been reported in hematopoietic, lung, spleen, and other tissues (Johnston et al., 1996; Chiang et al., 1997; Zhou et al., 2001). Tumor necrosis factor-α(TNF-α), a typical cytokine, serves as an intermediator of the immune system, protecting the host from the attacks of various infections and cancer cells, and induces the death of cancer cells by causing apoptosis (Robaye et al., 1991). Recent studies have reported that radiation increases the expression of specific proteins including TNF- α (Hallahan et al., 1989; Chiang and McBride, 1991).

Cigarette Smoke

Definitions and Effects on Biological Systems

Cigarette smoke is a complex medium containing approximately 4000 different constituents (Hoffman and Wynder, 1986) separated into gaseous and particulate phases. The components of the gaseous phase include carbon monoxide, carbon dioxide, ammonia, hydrogen dioxide, hydrogen cyanide, volatile sulphur-containing compounds, nitrogen oxides (including nitric oxide, NO) and other nitrogen-containing compounds. The particulate phase contains nicotine, water and tar (Guerin, 1980).

Cigarette tar, which contains at least 1017 free radicals per gram (Pryor and Stone, 1993), is a stable substance that can accumulate iron that promotes the formation of highly reactive hydroxyl radicals (OH•) (Cosgrove et al., 1985; Pryor and Stone, 1993). The gas phase of cigarette smoke also contains short-lived ROS that are potent oxidants (Kamp et al., 1992).

Use of smokeless tobacco (ST) is quite popular in countries of the Far East, Middle East and Europe (Bates et al., 2003) and it is increasing in the USA (Changrani and Gany, 2005). The main types of ST in Western and Asian countries are chewing tobacco and oral snuff. In developing countries like India, the chewing tobacco is mixed with betel

10 Introduction leaves, areca nut, lime, and catechu and is sold as legally commercial products termed ‘‘gutkha.’’ The product is basically a flavored and sweetened dry mixture of areca nut, catechu, and slaked lime with tobacco (Richter and Spierto, 2003). The mixture is chewed slowly, and in this process the aqueous extract due to saliva is not only absorbed locally but also ingested to enter into the systemic circulation. The use of gutkha has been classified as carcinogenic to humans and may be associated with oral disease (Merchant et al., 2000; Stepanov et al. 2005). Smokeless tobacco use can be addictive, leading to oral leukoplakias (oral mucosal lesions) and gingival recession, and it may play a contributory role in the development of cardiovascular disease, peripheral vascular disease, hypertension, peptic ulcers and fetal morbidity and mortality (Critchley and Unal, 2003).

Environmental tobacco smoke (ETS) is the smoke inhaled by bystanders (also called passive smoking) and is a combination of smoke emitted from the smoldering cigarette (sidestream smoke) and smoke exhaled by the smoker (mainstream smoke) (Teague et al., 1994; Chan- Yeung and Dimich-Ward, 2003). ETS is qualitatively similar in composition to mainstream smoke and contains the same carcinogens, toxic compounds and oxidizers, albeit at lower concentrations than those inhaled by smokers (Chan-Yeung and Dimich-Ward, 2003). Exposure to ETS has been causally linked to lung cancer (Adlkofer, 2001; Johnson et al., 2001; Chan-Yeung and Dimich-Ward, 2003; Janson, 2004) and has some association with asthma in never-smoking adults (Chan-Yeung and Dimich-Ward, 2003; Janson, 2004). In children, ETS exposure has been linked to lower respiratory tract infections, asthma induction and exacerbation, as well as chronic respiratory symptoms such as wheezing and coughing (Gilliland et al., 2001; Larsson. et al., 2001; Chan- Yeung and Dimich-Ward, 2003; Janson, 2004).

It should be noted that the deleterious health effects that result from tobacco use are almost independent of the form of tobacco. Thus, cigarette smoking, cigar smoking, pipe smoking, chewing tobacco, and snuff inhalation produce many of the same adverse health outcomes in people.

Effects of tobacco smoking on biological systems:

The extensive use of tobacco and its associated problematic health issues have been a concern to mankind. The World Health Organization (WHO) estimates that approximately one-third of the global population

11 Introduction aged 15 years or older are smokers and each smoker consumes an average of 15 cigarettes daily. Cigarette smoking, the most common form of tobacco use, has been found to account for hundreds of thousands of premature deaths and chronic diseases annually (WHO, 1997). It is well established that cigarette smoking can increase the risk of chronic obstructive pulmonary diseases, cardiovascular diseases and several forms of cancer, in particular, cancers of lung, oropharynx, larynx and esophagus (Shopland, 1995; Mannino, 2003; Tsiara et al., 2003).

Epidemiological evidence suggests that cigarette smoking is also deleterious to other parts of the gastrointestinal tract (GIT). In this respect, cigarette smoking increases both the incidence and relapse rate of peptic ulcer diseases and delays ulcer healing (Ma et al., 1999). Smoking is also positively associated with cancers of the stomach, liver and colon (Michael et al., 2002). Despite its detrimental effects on the GIT, cigarette smoking is currently the most consistent epidemiological finding associated with lowered incidence of ulcerative colitis (UC), a kind of inflammatory bowel disease (Calkins, 1989).

In pregnant women and in pregnant laboratory animals, smoke impairs pregnancy outcome, fetus growth and development, and has adverse health effects in later life (Haustein, 1999; Florek et al., 2007).

A consistent association between smoking during pregnancy and reduced average birth weight, with the risk of having a low-birth-weight baby (under 2500 g) increased by 53% in light smokers (< 1 pack/day) and 130% in heavy smokers (≥1 pack/day) compared with nonsmokers, (Meyer et al., 1976). Follow-up studies on children born to mothers who smoked during pregnancy have indicated growth retardation at 5 years (Wingred and Schoen, 1974) and 7 years (Goldstein, 1972) of age. The relationship between cognitive and mental development in children born to mothers who smoked during pregnancy is less clear. The 1979 Surgeon General’s report indicated that the data suggest unfavorable effects of smoking during pregnancy on the child’s long-term growth, intellectual development, and behavioral characteristics (DHHS, 1979).

It has been concluded that cigarette smoking is a risk factor for human reproductive effects in both parents and infants (DHHS, 1988, 1990). Specifically, the Surgeon General report indicated that smoking during pregnancy may lead to an increased risk of ectopic pregnancy, spontaneous abortion, abruptio placentae, placenta previa, and preterm delivery (DHHS, 2001). Smoking among males has been suggested as a risk factor for erectile dysfunction (Forsberg et al., 1979), decreased

12 Introduction libido (Ochsner, 1971), and decreased sperm count (Raboch and Mellan, 1975). Furthermore, the Surgeon General report has reported that women who smoke during pregnancy have a decreased risk for preclampsia, but the risks for perinatal mortality (both stillbirth and neonatal deaths) and sudden infant death syndrome are increased among the offspring of women who smoke during pregnancy (DHHS, 2001).

Numerous clinical and animal studies have demonstrated adverse outcomes of active maternal smoking during pregnancy on fetal development. Teratogenic effects of active maternal smoking include fetal growth retardation, increased rates of spontaneous abortion, reduced postnatal growth, increased risk of sudden infant death syndrome (SIDS) and behavioral and cognitive abnormalities (Werler et al., 1985; Behnke and Eyler, 1993; Weitzman et al., 2002; Mitchell and Milerad, 2006; Rogers, 2008).

Numerous epidemiological studies have linked cigarette smoking with reproductive problems in women. For example, an association has been observed between women who smoke and increased incidences of ectopic pregnancy, tubal infertility, and spontaneous abortion (Mueller and Ciervo, 1998; Saraiya et al., 1998; Castles et al., 1999). These problems could be due to an effect on the oviduct, and indeed, recent in vivo and in vitro studies have shown an effect on oviductal functioning by cigarette smoke (Knoll et al., 1995; Magers et al., 1995; Knoll and Talbot, 1998; DiCarlantonio and Talbot, 1999; Riveles et al., 2003). Factors such as cigarette smoke that impair oviductal functioning can diminish or prevent fertility (Florek and Marszalek, 1999; Shiverick and Salafia, 1999). Inhalation of mainstream (MS) or sidestream (SS) smoke by hamsters, at serum cotinine levels that were within the ranges found in active and passive human smokers, adversely affected the ultrastructure of the oviductal epithelium (Magers et al., 1995).

The children of mothers who smoke gain weight at a slower velocity than the children of non-smokers, suggesting that smoking may affect milk production (Cabello et al., 1991). Studies of daily milk production between 1 and 3 months into lactation have shown that daily milk production is significantly lower in smoking mothers. When weight gain was measured over a 14-day period, it was observed that the children of smokers exhibited 40% lower mean weight gain than the children of non- smokers (Vio et al., 1991). In addition, active maternal smoking habits have been linked with breastfeeding failure, reducing milk production and breast feeding duration (Horta et al., 2001; Letson et al., 2002),both exclusive and mixed (Cabello et al., 1991), and the reduction in

13 Introduction breastfeeding duration is proportional to the intensity of maternal smoking (Horta et al., 1997; Nafstad et al., 1997; Haug et al., 1998). Cigarette smoking has been associated with increased incidence of stroke in both men and women (Bonita et al., 1999). Smoking cigarettes have been shown to be a risk factor for stroke (Bonita et al., 1999; Ueshima et al., 2004). One quarter of strokes are associated with smoking (Hankey, 1999). The risk of stroke is dose dependent and increases by increasing number of smoked cigarettes per day (Bonita et al., 1999). In addition to enhanced risk factors for brain ischemia, there is a growing body of evidence that nicotine alters blood brain barier (BBB) permeability characteristics that have a direct influence on stroke outcome and the pathophysiology of brain ischemia (Abbruscato et al., 2002; Hawkins et al., 2002; Abbruscato et al., 2004; Hawkins et al., 2004). More importantly, stroke outcome has been shown to be worsened by increased edema observed in a two week exposure to nicotine after focal ischemia in rats (Wang et al., 1997).

Cigarette smoking decreases fasting insulin levels and leads to a transient increase in blood glucose levels after an oral glucose challenge (Janzon et al., 1983; Grunberg et al., 1988). Moreover, several experimental studies have shown that exposure to cigarette smoke has negative effects on glucose and lipid metabolism in diabetic and non- diabetic subjects (Eliasson, 2003).

Cigarette smoke decreases serum HDL and increases cholesterol, triglycerides (TGs) and low denisty lipoprotein LDL (Neufeld et al., 1997; Freeman et al., 1998; Vanisree and Sudha, 2006). The elevation in cholesterol level induced by CS might be accounted for by the increase in HMG-CoA reductase activity (Latha et al., 1993). Furthermore, the CS-evoked increase in TG has been attributed to the reduction in lipoprotein lipase activity, the enzyme responsible for TG hydrolysis (Freeman et al., 1998). CS also increases lipid peroxidation and promotes the development of reactive oxygen species within the vasculature, which enhance oxidative modification of LDL (Yamaguchi et al., 2005; 2007). Oxidized LDL plays a key role in the formation of atherosclerotic lesions (Jessup et al., 2004).

Cigarette smoking is one of the 10 greatest contributors to global death and disease (WHO, 2002). The increased risk of lung injury due to smoking (e.g., chronic obstructive pulmonary disease (COPD) and cancer) frequently does not diminish after smoking cessation and persists in exsmokers (Kabat, 1996; Rutgers et al., 2000).

14 Introduction

Pulmonary effects of cigarette smoke include COPD, increased airway reactivity, exacerbations of asthma, and an increased frequency of pulmonary infections. These effects are considered to be due to the direct actions of cigarette-derived toxins and ciliotoxins causing connective tissue destruction, hypersecretion, pooling of mucus and blebbing of membranes of endothelial cells. Cigarette smoke also reduces levels of exhaled nitric oxide in active and passive smokers, suggesting that it inhibits NO production (Schilling et al., 1994; Kharitonov et al., 1995; Yates et al., 2001). In the study of Su et al., (1998) exposure to cigarette smoke extract was reported to inhibit the activity, protein and messenger RNA of NO (nitric oxide) synthase (eNOS) in pulmonary artery endothelial cells irreversibly.

Cigarette smoking has been associated with gastric and duodenal ulcers in both humans (Friedman et al., 1974; Harrison et al., 1978; Kato et al., 1992; Endoh and Leung, 1994), and animals (Toon et al., 1951; Robert, 1972). Smokers are more susceptible to gastric ulcer than non- smokers, and also healing rate of the ulcers may be delayed (Doll et al., 1958; Korman et al., 1981). In fact, hundreds of compounds have been identified in cigarette smoke, it is possible that more than one of these substances may exert adverse actions on the ulcers of smokers.

Epidemiological studies correlated the adverse effects of cigarette smoking to peptic ulcer diseases (Piper et al., 1982). Smoking is associated with an increased risk and a reduction in healing rate of gastric (Iwata and Leung, 1995) and duodenal ulcers (Friedman et al., 1974). Nevertheless, the exact mechanisms linking cigarette smoking with gastric ulceration have not been defined yet. Tobacco cigarette smoke is an important exogenous source of free radicals and a number of studies have shown that smokers are exposed to higher levels of oxidant stress with increased levels of plasma free radicals activity and reduced plasma levels of antioxidants (Clausen, 1992).

Kalra et al., (1991) have found a significant increase in free radical production in smokers compared with non-smokers. Smokers were also found to have reduced mucosal blood flow (Guslandi et al., 1995). The reduction of blood flow was associated with vascular constriction in the gastric mucosa leading to a reduction in mucosal oxygen and hemoglobin levels (Cho et al., 1994). In turn, this reduction of oxygen in the tissues may produce a number of oxygen free radicals which could induce cellular damage and even cell death.

15 Introduction

In fact, Chow et al., (1997) had also shown that cigarette smoke exposure increased MPO activity in gastric mucosa; this increased activity has been reported to play a significant role in gastric mucosal damage (Tepperman et al., 1991). It is postulated that xanthine oxidase (XO)-derived oxidants initiate the production and release of pro inflammatory mediators from endothelium which attract and activate neutrophils in reperfusion model. These activated neutrophils can secrete MPO which catalyzes the formation of hydroxyl radicals and induces lipid peroxidation (Kurose and Granger, 1994).

Cigarette smoke has been firmly established as an independent risk factor for lung cancer which is considered as one of the most malignant tumors with the highest incidence and mortality among others. As a grand disease that is threatening the whole human society, its mortality has reached to as high as 13% per year, and is still increasing consistently in some developing countries (Hecht, 1999, 2002; Alberg et al., 2005). Compared with nonsmokers, the risk of lung cancer incidence in smokers is 22 and 12 folds, in male and female; respectively (Malyankar and MacDougall, 2004).

Lung cancer induced by cigarette smoke is a complex process involving multiple events and steps. Some molecular pathogenetic studies on adverse effects of smoking have been undertaken successfully at the gene (DNA) and transcription (mRNA) levels (Osada and Takahashi, 2002), but its mechanism still remains unclear.

Cigarette smoking is responsible for ~30% of all cancer deaths in developed countries. In addition to lung cancer, cigarette smoking is an important cause of oral, oropharyngeal, hypopharyngeal, laryngeal and esophageal cancers as well as pancreatic cancer, bladder cancer and cancer of the renal pelvis (Baron and Roham, 1996). Other cancers related to cigarette smoking are cancers of the nose, stomach, renal body, liver, colon, cervix and myeloid leukemia (Baron and Roham, 1996; Chao et al., 2000). Smoking is the cause of at least 30% of all cancers and 87% of lung cancers (CDCP, 2002; ACS, 2003).

Cigarette smoke carcinogens are the likely cause of cancer in smokers (Hecht, 1999). Some compounds such as N-nitrosamines and polycyclic aromatic hydrocarbons (PAH) require metabolic activation while others such as acetaldehyde and ethylene oxide do not. In either case, smokers are exposed to a constant flux of electrophiles which react with DNA to produce adducts.

16 Introduction

Cigarette smoking has been recognized as a strong risk factor for the development of vascular disease such as hypertension, stroke and coronary heart disease (Kiyohara et al., 1990; Hawkins et al., 2002). Tobacco smoking is associated with increased intima-media thickness (IMT) of carotid and femoral arteries, elevated levels of circulating C- reactive protein (CRP), and correlates with atherosclerotic vascular disease. Although the inhaled smoke from the combustion of tobacco is known to cause the development of atherosclerotic vascular disease (Wallenfeldt et al. 2001), however the mechanisms behind this are still elusive.

A common feature of CS-associated cardiovascular pathology, including atherogenesis (Ross, 1986), myocardial infarction (Go et al., 1988) and pulmonary emphysema (Blue and Janoff, 1978) is the activation and adhesion of circulating leukocytes to the micro- and macrovascular endothelium. The mediators involved in these activation and adhesion processes include activated complement fragments (Hoover et al., 1980), oxygen free radicals (Del Maestro et al., 1982), lipid peroxidation products (Smiley et al., 1991), and arachidonic acid metabolites (Dahlén et al., 1981). All these mediators have been shown to be generated in the organism in response to CS exposure (Gillespie et al., 1987; Kobayashi et al., 1988; Harats et al., 1990).

Smoking is an important risk factor for chronic kidney disease. The urine albumin excretion rate correlates with the number of cigarettes smoked (Pinto-Sietsma et al., 2000). Epidemiological studies established a positive correlation between smoking and nephrosclerosis and glomerulonephritis (Ejerblad et al., 2004). Smoking also promotes the progression of hypertensive (Warmoth et al., 2005) and diabetic nephropathies (Orth, 2002).

Chronic cigarette smoking has important adverse effects on renal function in patients with primary hypertension, primary glomerular diseases and diabetic nephropathy, as well as in those undergoing chronic hemodialysis. It is an independent predictor of albuminuria in patients with primary hypertension and has been related to the onset of microalbuminuria or proteinuria, as well as to chronic renal failure in patients with diabetes; smoking also plays a pathogenetic role in the development of idiopathic nodular glomerulosclerosis (Orth et al., 1997; Orth, 2000; Markowitz et al., 2002).On the same line, Kasiske and Klinger (2000) reported that smoking more than 25 pack/years was associated with a higher rate of graft loss in renal graft recipients.

17 Introduction

There is considerable evidence that smoking tobacco products induces analgesia that is thought to be mediated by nicotine, a constituent of tobacco. Several studies have reported reduced pain and anxiety in smokers (Nesbitt, 1973; Fertig et al., 1986; Rau et al., 1993). Nicotine delivered by transdermal patch elevated pain thresholds in males but not females (Jamner et al., 1998). During withdrawal, chronic smokers commonly suffer from dysphoria, discomfort, irritability and other symptoms sometimes including pain (Shiffman and Jarvik, 1984; Hughes et al., 1992; Allen et al., 2000), with some studies reporting increased pain sensitivity during withdrawal (Nesbitt, 1973; Seltzer et al., 1974; Silverstein, 1982; Pomerleau et al., 1984), a factor that may contribute to the very high relapse rate (Stitzer and Gross, 1988).

While many studies have demonstrated increased thresholds for nociceptive responses in animals following acute administration of nicotine (Aceto et al., 1983; Christensen and Smith, 1990; Craft and Milholland, 1998; Carstens et al., 2001), few have examined the effects of chronic exposure to nicotine or cigarette smoke. Rats receiving chronic infusion of nicotine (6 mg/kg/day) over a 28-day period exhibited analgesia with development of tolerance, in some pain tests (Yang et al., 1992;Carstens et al., 2001) and spontaneous or precipitated withdrawal from chronic nicotine administration (24 mg/kg/day for 14 days) induced hyperalgesia in mice (Damaj et al., 2003).In addition, Daily exposure of rats to smoke from one cigarette (containing 2.65 mg nicotine) induced analgesia after the first exposure day, with the development of tolerance on subsequent days (Mousa et al., 1988). The baseline nocifensive threshold (tail flick test) was elevated 1 day after withdrawal from chronic exposure to smoke from one cigarette per day over a 43-week period, even though the animals remained tolerant to the analgesic effect of acute smoke exposure (Mousa et al., 1988).

Chronic tobacco smoke exposure inhibits a wide range of immunological functions of alveolar macrophages (AMs), including their capacity to phagocytose inert particles and to become activated for tumor cell killing (Harris et al., 1970; Thomassen et al., 1988). Furthermore, cigarette smoking has been shown to suppress the production of proinflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL- 6) and tumor necrosis factor-alpha (TNF-α) (Brown et al., 1989; McCrea et al., 1994). These cytokines play an important role in the immune response against pathogens, explaining, in part, the increased susceptibility of cigarette smokers to pulmonary infections (Brown et al., 1987).

18 Introduction

Indeed, smoking has already been identified as a potential risk factor for autoimmune diseases—for example, as a risk factor for Graves’ disease and autoimmune hypothyroidism (Prummel and Wiersinga, 1993; Vestergaard et al., 2002), rheumatoid arthritis(Papadopoulos et al., 2005), primary biliary cirrhosis (Gershwin et al., 2005), and accelerated atherosclerosis in patients with autoimmune rheumatoid arthritis (Shoenfeld et al., 2005).

An association between smoking and lower prevalence of Parkinson's disease (PD) was first noted by Dorn (1959) and confirmed by Kahn (1966) and Hammond (1966). Subsequent studies (Kessler and Diamond, 1971; Baumann et al., 1980; Godwin-Austen et al., 1982) supported the conclusion that smoking was associated with decreased prevalence of PD, although several investigators (Marttila and Rinne, 1980; Haack et al., 1981; Golbe et al., 1986; Riggs, 1992) still claimed that the association was spurious and a few groups failed to demonstrate a significant association (Sasco and Paffenbarger, 1990; Kuopio et al., 1999). Overall, however, several studies (Grandinetti et al., 1994; Gorell et al., 1999; Tan et al., 2003) tend to support that smoking is inversely related to PD.

Tobacco smoking and oxidative stress

Cigarette smoking is a preventable risk factor and introduces many chronic diseases which increase the risk of morbidity and mortality. However, it may be possible to mitigate the smoke-induced oxidative damage by ameliorating the defense mechanism. The toxicity of cigarette smoke is due to nicotine, cadmium, benzopyrene, oxidants and inducers of reactive oxygen species (ROS) like nitric oxide (NO), nitrogen dioxide (NO2), peroxynitrite and nitrosamines that initiate, promote, or amplify oxidative damage (Church and Pryor, 1985). Free radicals or ROS induced by cigarette smoke are thought to be responsible for the induction of many diseases, including toxicity in the lung, liver and kidney. The radical concentrations in cigarette smoke condensate are known to be maintained for a considerable time by redox reactions despite their short life time (Nakayama et al., 1989). Evidence suggests that the free radicals in cigarette smoke contribute to the adverse effects of smoking cigarettes (Leanderson and Tagesson, 1992; Cross et al., 1993; Pryor, 1997).Cigarette smoke causes lipid peroxidation, oxidation of protein thiols and alterations in protein carbonyls in plasma (Frei et al., 1991; Reznick et al., 1992). The plasma of cigarette smokers is known to be low in ascorbic acid (Chow et al., 1986; Schectman et al., 1989) and the

19 Introduction epithelial lining fluid of smokers showed a decreased level of vitamin E (Pacht et al., 1986), which is suggested to be due to the increased consumption of these antioxidants by cigarette smoking. Indeed, exposure of human plasma to cigarette smoke resulted in the depletion of antioxidants such as ascorbic acid and billirubin (Frei et al., 1991).

Cigarette smoke contains numerous organic and metallic compounds emitted as gases and condensed tar particles, many of them being oxidants and prooxidants, capable of producing reactive oxygen species (ROS), thus enhancing lipid peroxidation (LP) in cell membranes (Preston, 1991). These chemically reactive oxygen species are known to be present or formed in cigarette smoke which may lead to modification of biological macromolecules (Liu et al., 1998). The relatively high levels of ROS in cigarette smoke can deplete antioxidants and initiate peroxidation of the polyunsaturated fatty acids (PUFAs) and modification of proteins and nucleic acids in biological systems (Smith and Fischer, 2001). Aside from smoke-borne organic radicals, cigarette smoking can also induce endogenous production of ROS including superoxide radical, hydrogen peroxide and hydroxyl radical in various cells (Naziroğlu, 2007). Exposure to cigarette smoke is also associated with increased oxidative DNA damage (Asami et al., 1996) and increased change of lipid profiles (Steinberg and Chait, 1998).

In addition, erythrocytes are extremely susceptible to oxidative damage induced by ROS because erythrocytes contain hemoglobin and PUFAs which can readily be peroxidized (Chung and Wood, 1971; Yilmaz et al., 1997). Lipid peroxidation causes injury to cell and intracellular membranes and may lead to cell destruction and subsequently cell death (Naziroğlu, 2007).

Flavonoids

Definitions, Biological Effects and Mechanisms of Actions

Flavonoids belong to a group of natural substances with variable phenolic structures and are found in fruits, vegetables, grains, barks, roots, stems, flowers, tea, and wine (Manach et al., 2004). These natural phenolic products were known for their beneficial effects on health long before flavonoids were isolated as the effective compounds. More than 4000 varieties of flavonoids have been identified, many of which are

20 Introduction responsible for the attractive colors of flowers, fruits and leaves (de Groot and Rauen, 1998).

Flavonoids are ubiquitous polyphenolic compounds comprised of several classes including flavonols, flavanones, flavanols, isoflavones and flavans (Manach et al., 2004). Many flavonoids possess anti- inflammatory, anti-viral, antioxidant and anticarcinogenic properties (Di Carlo et al., 1999; Middleton et al., 2000; Riboli and Norat, 2003). It is estimated that the average consumption of flavonoids in human diet is about 1 g per day (Verma et al., 1988; Stavric, 1994).

Biological effects of flavonoids

The great prevalence of flavonoids and anthocyanidines in the vegetal kingdom is not accidental; not only do they act as the coloured pigments of flowers but also as enzyme inhibitors, precursors of toxic substances, a defence against ultraviolet radiation exposure, chelating agents of metals noxious for plants, and reducing agents. In addition, flavonoids are involved in photosensitization and energy transfer, morphogenesis and sex determination, levels of respiration and photosynthesis, action of plant growth hormones and regulators (Firmin et al., 1986; Peters et al., 1986; Djordjevic et al., 1987; Zaat et al., 1987),as well as gene expression and behavior.

Through the food chain animals and humans ingest flavonoids. There is much data concerning a wide range of biological activities of these compounds in humans. For example, they were utilized in medicine as protection for vascular integrity (Beretz and Cazenave, 1988; Hongwei and Dongmin, 2009), as antiosteoporotic agents (Eaton-Evans, 1994, Bitto et al., 2008) and for their antihepatotoxic properties (Soike and Leng-Peschlow, 1987; Pandey et al., 2011). Furthermore, some flavonoids were examined for their activity in experimental tumor model systems both in vitro (Ye et al., 2012) and in vivo (Karna et al., 2011). Certain flavonoids were shown to inhibit the activity of enzymes such as aldose reductase (Iwu et al., 1990) and xanthine-oxidase (Arimboor et al., 2011). Flavonoids were also reported to act in the gastrointestinal tract as either antiulcer (Hamaishi et al., 2006), antispasmodic (Macêdo et al., 2011), antisecretory or antidiarrhoeal (Di Carlo et al., 1993) agents. Finally, some flavonoids were found to possess a good antinflammatory activity (Landolfi et al., 1984), that was related mainly to their inhibitory effect on the production of inflammatory mediators such as prostaglandins, leukotrienes (Meeran et al., 2009) or nitric oxide (Suzuki et al., 2009).

21 Introduction

Flavonoids are known to exhibit various biological effects such as inhibiting platelet aggregation, scavenging free radicals and preventing cell proliferation (Formica and Regelson, 1995). Furthermore, they have been proposed to exert beneficial effects in a multitude of disease states, including cancer, cardiovascular disease and neurodegenerative disorders (Middleton et al., 2000; Joseph et al., 2005). Flavonoids have been also described to have relaxing effects on the contractility of various smooth muscle, such as vascular smooth muscle (Chan et al., 2000; Morello et al., 2006), bladder (Dambros et al., 2005), vas deferens (Capasso and Mascolo, 2003), and uterus (Revuelta et al.,1997). In particular, in the gastrointestinal tract, flavonoids reduce smooth muscle cell contraction triggered by several spasmogens (Herrera et al., 1992; Ghayur et al., 2007), with a mechanism probably due to an interference with calcium movements through cell membranes (Capasso et al., 1991; Di Carlo et al., 1999). In addition, flavonoids have been shown to delay small and large intestinal transit in mice (Viswanathan et al., 1984; Di Carlo et al., 1993).

In addition to their antioxidant properties, flavonoids have been reported to exhibit other multiple biological effects, such as antiviral (Weber et al., 2003), antibacterial (Alvesalo et al., 2006), anti-inflammatory (Subarnas and Wagner, 2000; Widlansky et al., 2005), vasodilatory (Calderone et al., 2004), anticancer (Formica and Regelson, 1995), and anti-ischemic (Rump et al., 1995; Duthie et al., 2000; Mladěnka et al., 2010). Moreover, they are able to inhibit lipid peroxidation and improve increased capillary permeability and fragility (Valensi et al., 1996; Hubbard et al., 2004; Cirico and Omaye, 2006).

The different mechanisms by which flavonoids can prevent injury caused by free radicals include:

(1) Direct scavenging of reactive oxygen species (ROS) (Akhlaghi and Bandy, 2009), (2) Activation of antioxidant enzymes (Nijveldt et al., 2001), (3) Metal chelating activity (Ferrali et al., 1997), (4) Reduction of α-tocopheryl radicals (Hirano et al., 2001; Heim et al., 2002), (5) Inhibition of oxidases (Cos et al., 1998; Heim et al., 2002), (6) Mitigation of oxidative stress caused by nitric oxide (van Acker et al., 1995), (7) Increase in uric acid levels (Lotito and Frei, 2006),

22 Introduction

(8) Increase in antioxidant properties of low molecular antioxidants (Yeh et al., 2005).

Fig. (ii): Mechanisms of antioxidant effects of flavonoids (c.f. Akhlaghi and Bandy, 2009).

23 Introduction

Catechin

Fig (iii): Molecular structure of catechin (c.f. Min and Ebeler, 2008).

(+)-Catechin is a polyphenolic flavonoid from the group of catechins, which also include (_)-epicatechin, (_)-epigallocatechin, (_)- epicatechingallate, and (+)-gallocatechin. It is known to be present in green tea, black tea and other plant foods (Cook and Samman, 1996). Several epidemiological and in vitro studies suggest that catechins have beneficial effects on human health, serving to protect against congestive heart failure (Ishikawa et al., 1997), cancer (Yamanaka et al., 1997), myoglobinuric acute renal failure (Chander et al., 2003), and reduce the incidence of myocardial ischemia and the risk of ischemic heart disease mortality, due to their antioxidant activities (Arts et al., 2001).

Flavanols such as catechin and epicatechin undergo significant metabolism and conjugation during absorption in the small intestine and in the colon. In the small intestine these modifications lead primarily to the formation of glucuronide conjugates that are more polar than the parent flavanol and are marked for renal excretion. Other phase II processes lead to the production of O-methylated forms that have reduced antioxidant potential via the methylation of the B-ring catechol. Significant modification of flavanols also occurs in the colon where the resident microflora degrade them to smaller phenolic acids, some of which may be absorbed. Remaining compounds derived from falvonoid intake pass out in the feces. Cell, animal and human studies have confirmed such metabolism by the detection of flavanol metabolites in the circulation and tissues (Scalbert et al., 2002; Spencer, 2003).

Catechins have been reported to possess multiple properties such as prevention of cancer (Katiyar and Mukhtar, 1996), hypotensive effects

24 Introduction

(Henry and Stephen-Larson, 1984), antiviral properties (Nakayama et al., 1993), antioxidative properties (Yoshino et al., 1994), inhibition of plaque formation (Hattori et al., 1990), antiallergic potential (Kakegawa et al., 1985), and blood glucose-lowering effects (Matsumoto et al., 1993).

Additionally, tea catechins have been shown to affect lipid metabolism by reducing triglycerides and total cholesterol (Chan et al., 1999), stimulating lipid catabolism in the liver (Murase et al., 2002), and enhancing energy consumption (Dulloo et al., 2000). Available evidence indicates that long-term consumption of tea catechins may be beneficial for the suppression of high-fat diet-induced obesity by modulating lipid metabolism. Tea catechins could have a beneficial effect against lipid and glucose metabolism disorders implicated in type 2 diabetes, and could also reduce the risk of coronary disease (Crespy and Williamson, 2004). Traditionally, green tea was used to improve blood flow, eliminate alcohol and toxins, improve resistance to disease, relieve joint pain and to clear urine and improve its flow (Balentine et al., 1997). Research has focused a great deal on effects of green tea related to the prevention of cancer (Kavanagh et al., 2001) and cardiovascular diseases (Sueoka et al., 2001). Other beneficial effects of green tea catechins on inflammation (Dona et al., 2003), angiogenesis (Sartippour et al., 2002; Oak et al., 2005; Rodriguez et al., 2006), and oxidation (Osada et al., 2001) are emerging areas of interest.

Numerous in vivo animal studies have reported the anti-obesity effects of green tea catechins, in particular epigallocatechingallate (EGCG). Their findings comprise: reduction of fat mass (Choo, 2003; Klaus et al., 2005; Wolfram et al., 2005); reduction in triacylglycerides in hyperlipidemia models (Yang et al., 2001) as well as reduction of free fatty acids and total cholesterol (Ashida et al., 2004). Interestingly, green tea catechins in combination with endurance exercise promoted beta-oxidation and enhanced exercise capacity in mice (Shimotoyodome et al., 2005; Murase et al., 2006).

The green tea catechins (GTC) are strong antioxidants and free radical quenchers (Zhao et al., 2001). The flavan-3-ol structure makes them efficient scavengers of superoxide, singlet oxygen, nitric oxide, and peroxynitrite (Babu and Liu, 2008). They efficiently chelate free iron and copper ions that otherwise may catalyze free radical generation (Wang et al., 2007). They can up regulate antioxidant and other detoxifying enzymes (Khan et al., 1992; Chou et al., 2000) and protect DNA from oxidative damage (Luo et al., 2006). In animals and humans, oral intakes of GTC

25 Introduction decrease plasma biomarkers of oxidative stress and lipid peroxidation (Krahwinkel and Willershausen, 2000). GTC also demonstrate anti- inflammatory effects.

Pure (+)-catechin has been used to treat hepatitis since 1976 (Rauch, 1986). This compound has been shown to be an efficient immune stimulator, promoting activation of macrophages, cytotoxic-T- lymphocytes, and natural killer cells in mice in a dose-dependent manner (Rauch, 1986). Moreover, several clinical studies demonstrated the effectiveness of (+)-catechin in the treatment of viral hepatitis. One double-blind study showed a significant drop in antibodies to hepatitis B e antigen (HBeAg) in patients with HBeAg positive hepatitis B. Patients were given 1.5 g for two weeks, followed by 2.25 g for 14 weeks. HBeAg antibody titers decreased at least 50% in 31% of patients, and HBeAg completely disappeared in approximately 11%. The patient group responding best to the treatment had higher initial values of AST, ALT and gamma-globulin than the patients whose HBeAg titers remained unchanged. Mean values for these liver enzymes also fell significantly in the treatment group. The compound was reported to be well tolerated in this study, the only notable side-effect being a transient febrile reaction in 13 patients (Suzuki et al., 1986). Furthermore, in an animal model of viral hepatitis, pre-treatment with green tea extract significantly prevented increases in hepatic transaminases and alkaline phosphatase levels in a dose-related manner (Hayashi et al., 1992).

26 Introduction

Rutin

Fig (iv): Molecular structure of rutin (c.f. Gong et al., 2010).

In 1930 a new substance was isolated from oranges, which had been believed to be a member of a new class of vitamins, and was designated as vitamin P. It became then clear that this substance was the flavonoid rutin. A flurry of research began in an attempt to isolate the various individual flavonoids and to study the mechanism by which flavonoids act (Nijveldt et al., 2001).

Because of the hydrophilic character of its glycosides (rutin), only quercetin without a sugar group, i.e. the aglycon, was initially suggested to be taken up in the gastro-intestinal tract by passive diffusion (Kuhnau, 1976). However, a study with human ileostomy volunteers showed not only that quercetin glycosides can indeed be absorbed in the small intestine, but also that this absorption surpasses that of the aglycon by far, i.e. 52% of the glycosides was absorbed versus 24% of the aglycon (Hollman et al., 1995). After absorption, quercetin becomes metabolized in various organs including the small intestine, colon, liver and kidney (Hollman and Katan, 1997). Metabolites formed in the small intestine and liver are mainly the result of phase II metabolism by biotransformation enzymes and therefore include the methylated, sulphated and glucuronidated forms (Hollman and Katan, 1997; Day et al., 2000).

Rutin, is a natural flavone derivative, quercetin- 3-rhamnosylglucoside, which known for its anti-inflammatory and vasoactive properties (Ihme et al., 1996; Lindahl and Tagesson, 1997), diminishing capillary

27 Introduction permeability, exerting a vasoconstrictive effect on the peripheral blood vessels and inhibiting the mucosal content of platelet-activating factor (PAF) (Izzo et al., 1994). It has been reported that rutin prevents gastric mucosal ulceration in animal models including those induced by absolute ethanol (Pe´rez-Guerrero et al., 1994). This flavonoid is an important anti-lipoperoxidant agent (Negre´-Salvayre et al., 1991), and has also been found to be a strong scavenger of hydroxyl and superoxide radicals (Metodiewa et al., 1997, Quettier-Deleu et al., 2000). Both superoxide and hydroxyl radicals are involved in tissue injury through initiation of lipid peroxidation and disruption of the interstitial matrix (Hogg et al., 1992). Substances which are able to hinder their formation or to capture the free oxygen radicals formed are thus potential anti-ulcerogenic agents.

Rutin has several pharmacological properties, including antioxidant, anticarcinogenic, cytoprotective, antiplatelet, antithrombic, vasoprotective and cardioprotective activities (La Casa et al., 2000; Janbaz et al., 2002; Schwedhelm et al., 2003; Sheu et al., 2004; Trumbeckaite et al., 2006). In addition, rutin was identified as the major LDL antioxidant compound of mulberry in an in vitro study (Enkhmaa et al., 2005).There are also studies that show a dose–response effect in inhibiting low-density lipoprotein (LDL) peroxidation of rutin (Jiang et al., 2007). It also displayed anti-hypercholesterolaemic effects in an animal model in combination therapy with lovastatin (Ziaee et al., 2009). Moreover, rutin was found to be a neuroprotective agent (Pu et al., 2007). It showed anticonvulsant activities in pentylenetetrazol model in rat and mice (Nassiri-Asl et al., 2008) and has also ameliorated ischaemic reperfusion injury in the brain (Gupta et al., 2003). Rutin also showed antiulcer, vasodilator (Novakovic et al., 2006) and hepatoprotective activities (Janbaz et al., 2002), as well as antihyperglycemic action in diabetic rats (Kamalakkannan and Prince, 2006). These effects could be related to the antioxidative effects of rutin (Koda et al., 2008). It also suppressed microglial activation and pro-inflammatory cytokines (Koda et al., 2009).

Anti-inflammatory effects of rutin among flavonoids have been reported in several studies. Moreover, their role in the inhibition of nitric oxide (NO) production has also been discussed (Rotelli et al., 2003; Marzouk et al., 2007; Fang et al., 2008; Wang et al., 2008). In another study, rutin and quercetin were found to protect the cell membrane from lipid oxidation, and this effect was related to their antioxidant effects (Lopez- Revuelta et al., 2006).These antioxidative effects have also been shown to prevent the inactivation of α1-antitrypsin (Bouriche et al., 2007). Collectively, all of these mechanisms could be contributing to the cognition-enhancing effects of rutin.

28 Introduction

Rutin has been widely used in treating several diseases according to its pharmacological activities including antiallergic (Morimoto et al., 2003, Zwirtes de Oliveira et al., 2006), anti-inflammatory (Kim et al., 2004) and vasoactive, antitumour, antibacterial, antiviral (Kessler et al., 2003), and antiprotozoal properties (Calabro` et al., 2005). In addition, hypolipidaemic, cytoprotective (La Casa et al., 2000), antispasmodic and anticarcinogenic (Webster et al., 1996) activities of rutin have also been reported.

Studies, reported that rutin acts synergistically with vitamin C to support a healthy immune system (Petruzzellis et al., 2002). It was, also; reported to have a radioprotective action (Agrawal and Nagaratnam, 1981; Weiss and Landauer, 2003) and prevents the alterations in the haematological profile of rats following whole body γ-irradiation (Kanwar and Verma, 1992). In addition, rutin was proved to have a radioprotective effect against X-ray-induced chromosomal damage in mice (Benavente-García et al., 2002) and in human lymphocytes (Del Baño et al., 2006). In another study, rutin administration before induction of cerebral ischemia markedly attenuated the ischemia and reperfusion induced increase in cerebral infarct size (Gupta et al., 2003). Furthermore, rutin plays an important role as neuroprotective agent by improving the spatial memory impairment and attenuating the neuronal death induced by repeated cerebral ischemia in rats (Pu et al., 2007). Moreover, rutin was reported to reduce ischemia/reperfusion-induced cardiac dysfunction in isolated rat hearts (Lebeau et al., 2001).

29

AIM OF THE WORK

Aim of the Workِ

Ionizing radiation has been found to produce deleterious effects on the biological systems. The cellular damage induced by ionizing radiation is predominantly mediated through generation of ROS which when present in excess can react with certain components of the cell and cause serious systemic damage to various organs. The extent of the damage depends upon the total amount of energy absorbed, the time period, the dose rate of the exposure, and the particular organs exposed.

Cigarette smoking has been found to account for hundreds of thousands of premature deaths and chronic diseases annually. It is well established that cigarette smoking can increase the risk of COPD, cardiovascular diseases and several forms of cancer. Epidemiological evidence suggests that cigarette smoking is also deleterious some parts of the gastrointestinal tract (GIT). The toxicity of cigarette smoke is due to various oxidants and inducers of reactive oxygen species (ROS) that initiate, promote, or amplify oxidative damage. Free radicals or ROS induced by cigarette smoke are thought to be responsible for the induction of many diseases.

Damage produced by ionizing radiation and cigarette smoke may be inhibited by exogenous antioxidant supplementations. Natural products have been used by mankind to treat various disorders and offer an alternative to the synthetic compounds as they have been considered less toxic. The antioxidant effects of plants and herbs may be mediated through several mechanisms. Scavenging of free radicals and elevation of cellular antioxidant activity could be the leading mechanisms of protection by plants and herbs.

The present study was designed to investigate the possible protective effects of certain natural flavonoid products (catechin and rutin) and their possible mechanisms of action in guarding against the oxidative stress and inflammation induced by ionizing radiation and/or cigarette smoke extract in rats.

30 Aim of the Workِ

In order to achieve the goal of the present experiments, exposure to γ- radiation and treatment with CSE either alone or in combination were carried out and compared with a normal group of rats. Similar sets of animals were used to study the possible effects of the selected flavonoids catechin and rutin.

At the end of the experiment blood samples were withdrawn to be used for estimation of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), activities. The blood malondialdehyde (MDA), glutathione (GSH), nitric oxide (NO) and tumor necrosis factor – alpha (TNF- α) levels were also evaluated. Myeloperoxidase (MPO) activity was also estimated in the gastric mucosa.

31

MATERIAL AND METHODS

Material and Methods

Material:

Animals:

Male albino Wistar rats, each weighing 120-180g, were purchased from the animal breeding unit of National Research Centre, Giza, Egypt. They were housed under appropriate conditions of temperature (25°c±2), humidity (60%±5), and day-night cycle (12:12 h). The rats were allowed free access to water ad libitum and were fed a standard pellet diet. They were housed for at least one week before starting the experiment.

Drugs and Doses:

1-Catechin: (+) Catechin was obtained from Fluka, St.Gallen, Switzerland. It was dissolved in bi-distilled water and was used in a dose of 50 mg/kg (Rao and Vijayakumar, 2007).

2-Rutin: Rutin was obtained from Sigma, St.Louis, Mo, USA. It was suspended in bi-distilled water using 1% tween 80 and was used in a dose of 40 mg/kg (Florek et al., 2005).

Radiation Facilities:

Whole body γ-irradiation of rats was carried out at the National Centre for Radiation Research and Technology (NCRRT), Atomic Energy Authority, Egypt, using the Gamma Cell-40 biological irradiator furnished with a Caesium 137 source. Animals were irradiated at a radiation dose level of 0.5, 1.0, or 2.0 Gy delivered at a rate of 0.72 Gy/min.

Chemicals and Reagents: Chemicals, reagents, and kits used in this study and their sources are shown in the following two lists:

32 Material and Methods

List 1:

Kits Source Alanine transaminase kit Biodiagnostic,Egypt

Alkaline phosphatase kit Biodiagnostic,Egypt

Aspartate transaminase kit Biodiagnostic,Egypt

Lactate dehydrogenase kit Biomed, Egypt

Nitric oxide kit Biodiagnostic,Egypt

TNF-α (tumor necrosis R&D Systems,USA factor- α) kit

List 2:

Chemicals Source Absolute alcohol (HPLC) GCC diagnostic,UK grade n-Butanol(Analar) BDH chemicals,England Chloroform ADWIC,Egypt Diethyl ether ADWIC,Egypt Dimethyl sulfoxide Sigma-Aldrich,Germany (DMSO) Di-sodium hydrogen Sigma,USA phosphate 5,5’-dithiobis(2- Sigma, USA nitrobenzoic acid) Ethylene diamine tetraacetic Cambrian acid disodium salt (EDTA) chemicals,England Glutathione Sigma,USA Hexadecyl tetramethyl PARK Scientific ammonium bromide Limited,U.K. (HTAB) Horse radish peroxidase Sigma,USA Hydrogen peroxide ADWIC,Egypt

33 Material and Methods

Malondialdehyde standard (1, 1, 3, 3 tetraethoxy Sigma,USA propane) Fluka,Swizerland Meta-phosphoric acid ortho-phosphoric acid BDH chemicals,England Sodium chloride ADWIC,Egypt Sodium hydroxide ADWIC,Egypt Sulphuric acid ADWIC,Egypt Tween 80 Sigma,USA 3, 3′, 5, 5′-tetramethyl Amresco,USA benzidine(TMB) 2-Thiobarbituric acid Sigma, USA

N.B: All other chemicals are of analytical grade.

Experimental Design:

Rats were randomly classified into two main sets: the animals of the first set were normal while the animals of the other set were irradiated.

N.B.: The used CSE in both sets contains equal parts of ethanol and chloroform cigarette extracts.

The first set of rats was classified into groups each of 8 rats as follows:

Group (1): animals received saline and served as normal group.

Group (2): animals received catechin at a dose of 50mg/kg p.o for 3 successive days.

Group (3): animals received rutin at a dose of 40mg/kg p.o for 10 successive days.

Group (4): animals received cigarette smoke extract (CSE) at a dose of 5mg/kg p.o for 3 successive days.

The set of the irradiated rats was further classified into groups each of 8 rats as follows:

Group (1-3): animals were exposed to a single dose of whole body γ- rays of 0.5, 1.0, and 2.0 Gy respectively.

34 Material and Methods

Group (4-6): animals received cigarette smoke extract (CSE) at a dose of 5 mg/kg p.o for 3 successive days then exposed to whole body γ-rays at a dose level of 0.5, 1.0, and 2.0 Gy respectively.

Group (7): animals received catechin at a dose of 50mg/kg p.o and cigarette smoke extract (CSE) at a dose of 5 mg/kg p.o for 3 successive days then exposed to whole body γ-irradiation at a dose level of 2.0 Gy.

Group (8): animals received rutin at a dose of 40mg/kg p.o for 10 successive days, and cigarette smoke extract at a dose of 5 mg/kg p.o for 3 successive days starting from day 8 of rutin injection. After the last dose of rutin and cigarette smoke extract, animals were exposed to an acute dose of whole body γ-rays at a dose level of 2.0 Gy.

Sampling:

Blood Samples: Rats were anesthetized by exposure to diethyl ether, blood samples were collected by decapitation from the trunk and divided into 2 portions: The first portion of the blood was collected in a dry clean test tube containing heparin. Heparenized blood (0.1 ml) was used for the estimation of glutathione.

The other portion of the blood sample was incubated in a dry clean test tube at 37°c for 20 min then centrifuged at 3000 rpm for 15 min to separate serum to be used for determination of malondialdehyde (MDA), nitric oxide (NO), and tumor necrosis factor-α (TNF- α) levels and the activity of alanine transaminase (ALT), and aspartate transaminase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH).

Tissue Samples:

Preparation of Gastric Mucosa:

The stomach was excised, opened along the greater curvature, and rinsed thoroughly with normal saline to remove the attached debris. After the stomach was blotted dry the glandular mucosa was scraped with a glass slide on an ice-cold dish. The mucosal samples were stored at -20ºC until evaluation of myeloperoxidase (MPO) activity.

35 Material and Methods

Methods:

Preparation of Cigarette Smoke Extracts:

Cigarette smoke extracts were prepared according to the method of Ma, et al., (2000). 320 Cleopatra Super Star cigarettes (Eastern Company- Egypt), purchased from the local market were used in this study. Each cigarette contains 1mg nicotine and 15 mg tar. The substances in this cigarette smoke were extracted using a smoke perfusion system. Smoke from burning cigarettes was bubbled into series of bottles containing either 500 ml of chloroform (2 bottles)or 95% ethanol(4 bottles) respectively. The substances in smoke were thus absorbed in the solvents. The substances dissolved in ethanol were regarded as ethanol fraction. This fraction was preconcentrated by a rotary evaporator (Rotavapor Buchi-RE111,Switzerland with a water bath Buchi 461,Switzerland)connected to a cooling system(Selecta,Spain) to evaporate the excess ethanol. After evaporation, chloroform was added to the ethanol fraction to extract those substances soluble in chloroform. The remaining insoluble part was regarded as ethanol extract (ECE).All the chloroform soluble fractions were combined and concentrated again following the same procedures, and this was known as chloroform extract (CCE).Both ECE and CCE were finally prepared in 0.1% dimethyl sulfoxide (DMSO) solution before oral administration Shin et al., (2002a).

Fig. (v) : Apparatus used for the preparation of CSE

36 Material and Methods

Identification of Compounds from Cigarette Smoke Extracts:

Thin Layer Chromatography Method:

Ethanol extract was spotted onto silica gel precoated thin layer chromatography plate. Alkaloids were separated using a mobile phase consisting of chloroform-methanol-water-acetic acid (7:4:1:0.5), run of a thin-layer chromatography plate, and were detected with Dragendorff reagent. Chloroform extract was developed by a mobile phase composed of toluene-ethyl acetate (93:7). Terpenoids were identified by vanillin sulfuric acid and the phenolic compounds were shown by ammonia vapor (Chow, et al., 1997).

This method was carried out to identify the main constituents of the ethanolic and chloroformic smoke extracts. Alkaloids were identified in the first while terpenoids and phenolic compounds were identified in the second extract.

The Measured Parameters:

1-Determination of Serum Alanine Transaminase Activity:

Alanine transaminase activity was measured according to the method of Reitman and Frankel (1957), using a colourimetric kit.

Principle: ALT Alanine+ α-ketoglutarate pyruvate + Glutamate

The kito acid pyruvate formed is measured colourimetrically in its derivative form, 2,4-dinitrophenylhydrazone.

Reagents :

1- Standard pyruvate 2 mmol/L 2- Buffer substrate: Phosphate buffer PH = 7.5 100 mmol/L Alanine 200 mmol/L α-ketoglutarate 2 mmol/L 3- Colour Reagent: 2,4 dinitrophenylhydrazine 1 mmol/L

37 Material and Methods

4- Sodium hydroxide 0.4 N

Procedure:

1- Test tubes were labeled Tn for test samples and B for the reagent blank. 2- Reagent 2 (0.5 ml) was pippetted in test samples tubes Tn, followed by incubation for 5 minutes at 37ºC. 3- To test samples tubes Tn 0.1 ml of serum was added, mixed, and followed by incubation for 30 minutes at 37ºC. 4- Reagent 3 (0.5 ml) was added to tubes, followed by mixing and incubation for 20 minutes at room temperature. 5- Addition of 5 ml of reagent 4 to all tubes was done, followed by mixing, then standing at room temperature for 5 minutes. 6- The absorbance of samples (A sample) was read against distilled water as blank at 505 nm using spectrophotometer Unicam 5626 UV/VIS, England; the colour was stable for 1 hour.

Standard Curve:

1-Five test tubes were labeled from S1 to S5.Distilled water (0.1 ml) was added to each tube. 2-Reagent 2 was added in volumes of 0.5, 0.45, 0.4, 0.35, and 0.3 ml starting from tube labeled S1 to tube labeled S5. 3-Standard pyruvate (R1) was added in volumes of 0.05, 0.1, 0.15, and 0.2 ml from tube S2 to tube S5. 4-Reagent 3 (0.5 ml) was added to tubes, followed by mixing and incubation for 20 minutes at room temperature. 5- Addition of 5 ml of reagent 4 to all tubes was done, followed by mixing, then standing at room temperature for 5 minutes, then the absorbance was read against distilled water as blank at 505 nm using spectrophotometer, Unicam 5626 UV/VIS, England; the colour was stable for 1 hour.

Calculation:

Number of units/ml of ALT of serum sample was calculated using the standard curve.

38 Material and Methods

1.2

R2 = 0.9652 1.0

0.8

0.6

0.4

Absorbance at 505 nm 505 at Absorbance 0.2

0.0 0 20 40 60 80 100 120 140 160

Activity of ALT U/ml

Fig. (vi): Standard Curve of ALT

2-Determination of Serum Aspartate Transaminase Activity:

Aspartate transaminase activity was measured according to the method of Reitman and Frankel (1957), using a colourimtric kit.

Principle: AST

Aspartate+ α-ketoglutarate Oxaloacetate + Glutamate

The kito acid oxaloacetate formed is measured colourimetrically in its derivative form, 2,4-dinitrophenylhydrazone.

Reagents :

1- Standard pyruvate 2 mmol/L 2- Buffer substrate: Phosphate buffer PH = 7.5 100 mmol/L Aspartate 100 mmol/L α-ketoglutarate 2 mmol/L 3- Colour Reagent: 2,4 dinitrophenylhydrazine 1 mmol/L 4- Sodium hydroxide 0.4 N

39 Material and Methods

Procedure:

1- Test tubes were labeled Tn for test samples and B for the reagent blank. 2- Reagent 2 (0.5 ml) was pippetted in test samples tubes Tn, followed by incubation for 5 minutes at 37ºC. 3- To test samples tubes Tn 0.1 ml of serum was added, mixed, and followed by incubation for 60 minutes at 37ºC. 4- Reagent 3 (0.5 ml) was added to tubes, followed by mixing and incubation for 20 minutes at room temperature. 5- Addition of 5 ml of reagent 4 to all tubes was done, followed by mixing, then standing at room temperature for 5 minutes. 6- The absorbance of samples (A sample) was read against distilled water as blank at 505 nm using spectrophotometer Unicam 5626 UV/VIS, England,the colour was stable for 1 hour.

Standard Curve:

1-Five test tubes were labeled from S1 to S5.Distilled water (0.1 ml) was added to each tube. 2-Reagent 2 was added in volumes of 0.5, 0.45, 0.4, 0.35, and 0.3 ml starting from tube labeled S1 to tube labeled S5. 3-Standard pyruvate (R1) was added in volumes of 0.05, 0.1, 0.15, and 0.2 ml from tube S2 to tube S5. 4- Reagent 3 (0.5 ml) was added to tubes, followed by mixing, incubation for 20 minutes at room temperature. 5- Addition of 5 ml of reagent 4 to all tubes was done, followed by mixing, then standing at room temperature for 5 minutes, then the absorbance was read against distilled water as blank at 505 nm using spectrophotometer Unicam 5626 UV/VIS, England; the colour was stable for 1 hour.

Calculation:

Number of units/ml of AST of serum sample was calculated using the standard curve.

40 Material and Methods

1.2 R2 = 0.9629 1.0

0.8

0.6

0.4

Absorbance at 505 nm 505 at Absorbance 0.2

0.0 0 20 40 60 80 100 120 140 Activity of AST U/ml

Fig. (vii): Standard Curve of AST

3-Determination of Serum Alkaline Phosphatase Activity:

Alkaline phosphatase activity was measured in serum according to the method of Belfield and Goldberg (1971), using a colourimetric kit.

Principle: Alkaline phosphatase Phenyl phosphate Phenol + Phosphate PH = 10.0

The liberated phenol is measured colourimetrically in the presence of 4- aminophenazone and potassium ferricyanide.

Reagents :

1- Standard phenol 1.59 mmol/L 2- Buffer substrate: Buffer PH = 10.0 Phenyl phosphate 5 mmol/L 3- Enzyme inhibitor: EDTA: 100 mmol/L (Ethylene diamine tetra acetic acid) 4- Aminophenazone 50 mmol/L

41 Material and Methods

5- Colour Reagent Potassium ferricyanide 200 mmol/L

Procedure:

1- Test tubes were labeled Tn for test samples, S for the reference standard and B for the reagent blank. 2- Standard (25 µl) was pippetted in tube S and 25 µl of each serum sample was pippetted in tubes Tn. 3- To all labeled tubes, 0.5 ml of reagent 2 was added, followed by incubation for 20 minutes at 37ºC. 4- Reagent 3 (0.25 ml) was added to all tubes, followed by well mixing. 5- Addition of 0.25 ml of reagent 4 to all labeled tubes was done, followed by well mixing, then standing at room temperature in the dark for 10 minutes. 6- The absorbance of samples (A sample) and the standard (A standard) was read against the blank at 510 nm using spectrophotometer Unicam 5626 UV/VIS, England; the colour was stable for 1 hour.

Calculation: A Enzyme activity (IU/L) = sample X 100 A standard

4-Determination of Blood Glutathione (GSH) level:

Glutathione in blood was determined according to the method described by Beutler et al., (1963).

Principle:

The method depends on the reduction of 5,5`-dithio bis(2- nitrobenzoic acid) [Ellman’s reagent] by sulfhydryl (SH) moiety of GSH to form 2-nito,5-mercaptobenzoic acid which has stable yellow colour and can be measured colourimetrically at 412 nm. Prior precipitation of SH-containing proteins was carried out by meta-phosphoric acid.

Reagents:

1- Precipitating solution: meta-phosphoric acid 1.67 g, disodium EDTA 0.2 g and sodium chloride 30 g were dissolved in 100 ml of bidistilled water.

42 Material and Methods

2- Phosphate solution (0.3M): disodium hydrogen phosphate 42.59 g were dissolved in one litre of bidistilled water. 3- Ellman’s reagent (0.01 M): 5, 5′-dithiobis-(2-nitrobenzoic acid) 40 mg was dissolved in 100 ml 1% sodium citrate. 4- Serially diluted standard GSH solutions ranging from 0.0187 to 0.15 mg/l were prepared and used for construction of a standard curve.

Procedure:

1-An aliquot of 0.1 ml of blood sample was haemolysed by the addition of 0.9 ml bidistilled water. 2- To the haemolysate, 1.5 ml of the precipitating solution was added, mixed and allowed to stand for 5 minutes. 3-Centrifugation was carried out (3000 r.p.m. /15 min), then the supernatant (1 ml) was mixed with 4 ml phosphate solution and 0.5ml Ellman’s reagent. The absorbance was measured at 412 nm within 5 min using UV spectrophotometer Unicam 5626 UV/VIS, England.

Calculation:

The concentration of GSH in blood was calculated from the standard curve and was expressed as mg/ml.

0.8

2 0.7 R = 0.9412

0.6

0.5

0.4

0.3

0.2 Absorbance at 412 nm 412 at Absorbance

0.1

0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 GSH level (mg/ml)

Fig. (viii): Standard Curve of GSH

43 Material and Methods

5-Determination of Serum Malondialdehyde level:

Malondialdehyde was measured in serum according to the method of Uchiyama and Mihara(1978).

Principle:

The lipid peroxidation products were estimated in tissue homogenate by the determination of the level of thiobarbituric acid reactive substances (TBARS). The principle depends on colourimetric determination of a pink coloured product, resulting from the reaction of TBARS with thiobarbituric acid in acidic medium, at high temperature. The resultant colour product was extracted with n-butanol and measured at two wavelengths, 520 and 535 nm, to avoid interfering substances. The difference in the absorbance at the two wavelengths (∆A535-520) was used to calculate TBARS content using a standard curve of malondialdehyde (MDA).

Reagents:

1-Thiobarbituric acid (TBA): 0.6 % W/V aqueous solution in bidisttiled water 2-Ortho-phosphoric acid: 1% V/V aqueous solution bidisttiled water 3-n-Butanol 4-Standard malondialdehyde (1,1,3,3 tetramethoxypropane) serially diluted standard MDA solutions ranging from 5 to 30 nmol/l were used in constructing the standard curve.

Procedure:

1- Standard preparation: serial dilutions of standard MDA were prepared to set up a standard curve for malondialdehyde (5 – 30 nmol/ 0.5ml). 2- Test tubes were labeled Tn for test samples, Sn for the reference standards and B for the reagent blank. 3- Distilled water (0.5 ml) was pippetted in blank tube B, 0.5 ml of standard was pippetted in standard tube Sn, and 0.5 ml of each serum samples were pippetted in their tubes Tn. 4- To all labeled tubes, 1% ortho-phosphoric acid (3 ml) was added followed by shaking. 5- To the mixture, thiobarbituric acid 0.6% (1 ml) was added, mixed, and warmed for 45 minutes in a boiling water bath followed by rapid cooling,n-butyl alcohol (4 ml) was added and shaken.

44 Material and Methods

6- The butanol upper layer was separated by centrifugation at 3000 rpm for 15 minutes. 7- The absorbance of samples (A sample) and the standards (A standard) was read against the blank at 535 nm using spectrophotometer Unicam 5626 UV/VIS, England.

Calculation:

Malondialdehyde concentration in serum was determined as nmol/ml using malondialdehyde standard curve.

3.0

2.5 R2 = 0.9037

2.0

1.5

1.0

0.5 Absorbance at 535 nm

0.0 0 5 10 15 20 25 30 35 Concncentration of MDA nmol/0.5 ml

Fig. (ix): Standard Curve of MDA

6-Determination of Serum Nitric Oxide level:

Nitric oxide was measured in serum according to the method of Montgomery and Dymock (1961), using a colourimetric kit.

Principle:

In acid medium and in the presence of nitrite, the formed diazotise sulphanilamide and the product are coupled with N-(1-naphthyl) ethylenediamine. The resulting azo dye has a bright reddish-purple colour which can be measured at 540 nm.

45 Material and Methods

Reagents:

1- Standard sodium nitrite 50 µmol/L 2- Sulphanilamide 10 mmol/L 3- N-(1-naphthyl)-ethylenediamine (NEDA) 1 mmol/L

Procedure:

1- Test tubes were labeled Tn for test samples, Bn for samples' blanks, S for the reference standard and BS for the standard's blank. 2- Standard (0.1 ml) was pippetted in both standard tube S and standard blank tube BS, while 0.1 ml of each sample was pipetted in sample tubes Tn and blank tube Bn. 3- To all labeled tubes 1 ml of reagent 2 was added, and then well mixing was done. 4- After standing for 5 min, 0.1 ml of reagent 3 was added to the standard tube S and to all sample tubes Tn (excluded blank tubes), followed by well mixing and standing for another 5 minutes. 5- The absorbance of sample (A sample) was read against sample blank and the standard (A standard) was read against the standard blank at 540 nm using spectrophotometer Unicam 5626 UV/VIS, England, the colour was stable for many hours.

Antioxidants may interfere with the colour development reaction. Azide, ascorbic acid, dithiothreitol, and mercaptoethanol may interfere with the colour development when present at concentration as low as 100 µM. Alkyl amines, most sugars, lipids, or amino acids (except those containing thiol groups) did not interfere.

Calculation:

A Nitrite in serum (µmol/L) = sample X 50 (st. conc.) A standard

7-Determination of Serum Lactate Dehydrogenase (LDH) activity:

Lactate dehydrogenase in serum was determined according to the method of Gay et al., (1968), using a kinetic kit.

46 Material and Methods

Principle:

LDH specifically catalyses the oxidation of lactate to pyruvate with the subsequent reduction of NAD to NADH. The rate of NADH formation is proportional to LDH activity. The method described determines NADH absorbance increase per minute at 340 nm .

L-Lactate + NAD+ LDH Pyruvate + NADH+H+ Reagents:

Reagent (1): Phosphate buffer (pH 7.5) 50 mmol/l Pyruvate 0.6 mmol/l Azide 8 mmol/l Reagent (2): NADH > 0.18 mmol/l Sodium azide 8 mmol/l Working solution: was prepared by mixing five volumes of reagent (1) and one volume of reagent (2).

Procedure:

An aliquot of 1.0 ml of the working reagent was added into a cuvette for each sample at 37οC.Serum sample 20 µl was added into the cuvette and mixed gently. The initial absorbance was recorded after 30 seconds at 340 nm using UV spectrophotometer Unicam 5626 UV/VIS, England. Finally, the mean absorbance change per minute (∆A/min) was determined.

Calculations:

The LDH activity was calculated using the following formula:

U/L = 8095 x ∆A 340 nm/min

8- Determination of Myeloperoxidase (MPO) activity in Gastric Mucosa:

Myeloperoxidase activity in gastric mucosa was determined according to the method of Bradley et al., (1982) and Shin et al., (2002a).

47 Material and Methods

Principle:

Myeloperoxidase catalyzes the oxidation of electron donor (e.g. halides) by hydrogen peroxide to form coloured compound which can be measured spectrophotometrically.

Reagents:

1- Phosphate buffer (50 mM, PH 6) 2- Phosphate buffered saline (80 mM, PH 5.4) 3-Hexadecyl tetramethyl ammonium bromide (HTAB) (0.5%) 4-3, 3′, 5, 5′-tetramethyl benzidine(TMB) (2mM) 5-Hydrogen peroxide (0.3 M) 6- Sulphuric acid (0.2 M) 7- Serially diluted standard horse radish peroxidase solutions ranging from 0.003725 to 0.3725 unit/ml were prepared and used for construction of a standard curve.

Procedure:

1- Gastric mucosa (200 mg) was homogenised in 1.0 ml of phosphate buffer PH 6 containing 0.5 % HTAB in ice cold conditions. 2-Freeze thawing followed by sonication for 10 seconds were carried out for three times. 3-Samples were centrifuged at 12000 rpm for 30 minutes at 4οC. 4- Supernatant 50 µl was added to a mixture of 3, 3′, 5, 5′-tetramethyl benzidine(TMB) (2mM) 150 µl, hydrogen peroxide (0.3 M) 50 µl, and phosphate buffered saline (80 mM, PH 5.4) 250 µl, followed by incubation for 25 minute at 25οC. 5- The reaction was stopped by addition of sulphuric acid (0.2 M) 2.5 ml. 6-Absorbance was measured at 450 nm using UV spectrophotometer Unicam 5626 UV/VIS, England.

Calculation:

Number of units/ml of MPO of gastric mucosal sample was calculated using the standard curve.

48 Material and Methods

1.2 R2 = 0.8319 1.0

0.8

0.6

0.4

Absorbance at 450 nm Absorbance450 at 0.2

0.0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 Activity of MPO U/ml

Fig. (x): Standard Curve of MPO

9-Determination of Serum Tumor Necrosis Factor-α (TNF-α) level:

Principle:

ELISA Quantikine® R&D systems kits were used in the assay employing the quantitative sandwich enzyme immunoassay technique (Flick and Gifford, 1984).Where a monoclonal antibody specific for rat TNF-α has been pre-coated onto the inner surface of a microplate consisting of 96 wells. Standards, control and samples pipetted into the wells and any rat TNF-α present is bound to the immobilized antibody. Unbound substances are washed away, and then an enzyme-linked polycolonal antibody specific to rat TNF-α is added to the wells, which binds to the already formed antibody-TNF-α complex. Unbound excess is washed away; a substrate solution is added to the wells. The enzyme acts on the added substrate to yield a blue product that turns yellow upon the addition of a stop solution. The intensity of the final colour obtained is directly proportional to the amount of TNF-α bound in the initial step. The concentration is then calculated using a standard curve.

49 Material and Methods

Reagents:

1-Rat TNF-α Conjugate concentrate: twenty three fold concentrated solution (1 ml) containing polycolonal antibody against rat TNF-α conjugated to horse radish peroxidase, with preservatives. 2- Type 2 conjugate diluent: twenty three ml of diluent for diluting conjugate concentrate, with preservatives. 3- Rat TNF-α standard: recombinant rat TNF-α in a buffered protein base (1.6 ng/vial) with preservatives (lyophilized). 4- Rat TNF-α control: recombinant rat TNF-α in a buffered protein base with preservatives (lyophilized). The assay value of the control should be within the range specified on the control vial label. 5- Assay diluent RD1-41: buffered protein base with preservatives (12.5 ml). 6- Calibrator diluent RD5-17: buffered protein base with preservatives (21 ml/vial). 7- Wash buffer concentrate: twenty five fold concentrated solution of a buffered surfactant (50 ml), with preservatives. 8- Colour reagent A: stabilized hydrogen peroxide (12.5 ml). 9- Colour reagent B: stabilized chromogen (12.5 ml) (tetramethyl benzidine). 10- Substrate solution: prepared by mixing equal volumes of colouring reagents A and B, the mixture is used within 15 minutes of its preparation. 11- Stop solution: diluted hydrochloric acid solution (23 ml).

Procedure:

1- All reagents and samples were brought to room temperature before use. 2- Serial dilutions of standard rat TNF-α were prepared as instructed in the kit manual, the resulting solutions contained 12.5, 25, 50, 100, 200, 400 and 800 pg/ml rat TNF-α. 3- Lyophilized reagents were reconstituted and other reagents, working standards, control and samples were prepared according to the instructions of the kit manufacturer. 4- Assay Diluent (50 µl) was added to each well, followed by the addition of 50 µl of standard, control or sample per well. 5- Microplate was incubated for two hours at room temperature. 6- Aspiration of each well followed by washing using the "Wash Buffer" was repeated for 4 times. 7- Diluted rat TNF-α conjugate (100 µl) was added to each well, again the microplate was incubated at room temperature for two hours.

50 Material and Methods

8- The step of aspiration and washing was repeated 5 times. 9- 100 µl of "Substrate Solution" was added to each well. 10- Microplate was incubated for 30 minutes at room temperature while being protected from light. 11- Stop Solution (100 µl) was added to each well. 12- The optical density of each well was determined within 30 minutes after the addition of stop solution, using Dynatech® MR5000 (Guernsey, Channel Islands, Great Britain) ELISA microplate reader. The reader was set to 450 nm (correction wavelength is set at 540 nm; this was done to correct for the optical imperfections in the microplate).

Calculation:

The standard curve was constructed by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and the best fit curve was drawn through the points on the graph. Then, the concentration of TNF-α in each sample was obtained from the constructed standard curve.

5

4

R2 = 0.9765 3

2

Absorbance450at nm 1

0 0 200 400 600 800 1000 1200 1400 1600 TNF-α concentration (pg/ml)

Fig. (xi): Standard Curve of TNF-α

51 Material and Methods

Statistical Analysis:

Values are given as means ± standard error (S.E.) of the mean. Comparisons between different groups were carried out by one way analysis of variance (ANOVA) followed by Tukey-Kramer's multiple comparison test (Armitage and Berry, 1987). The differences were considered statistically significant at p < 0.05 (Dawson-Saunders and Trapp, 1990). Graph pad software instant (version 2) was used to carry out these statistical tests. The figures were drawn using Microsoft Excel program.

52

RESULTS

Results

Effects of catechin (50mg/kg) or rutin (40mg/kg) on serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gastric myeloperoxidase (MPO) activities and levels of blood glutathione (GSH), serum malondialdehyde (MDA), nitric oxide (NO) and tumor necrosis factor – alpha (TNF- α) in normal rats:

Results are shown in Table (1):

The normal activities of serum ALT, AST, ALP, LDH and gastric MPO were 16.56 U/ml, 114.2 U/ml, 123.4 U/L, 1.51 U/L and 1.37 U/g tissue respectively.

The normal blood level of GSH, MDA, NO and TNF-α were 12.84 mg/ml, 3.52 nmol/ml, 2.68 µmol/L and 72.1 pg/ml respectively.

Administration of either catechin (50 mg/kg) or rutin (40 mg/kg) daily for 3 and 10 days respectively to normal rats did not alter the normal values.

53 Results

Table (1) :Effects of catechin (50mg/kg) or rutin (40mg/kg) on serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gastric myeloperoxidase (MPO) activities and levels of blood glutathione (GSH), serum malondialdehyde (MDA), nitric oxide (NO) and tumor necrosis factor – alpha (TNF- α) in normal rats.

Parameters ALT AST ALP GSH MDA NO LDH MPO TNF-α (U/ml) (U/ml) (U/L) (mg/ml) (nmol/ml) (µmol/L) (U/ml) (U/g tissue) (pg/ml)

Groups

Normal 16.56 ± 0.70 114.20 ± 2.18 123.40 ± 7.28 12.84 ± 0.71 3.52 ± 0.06 2.68 ± 0.06 1.51 ± 0.10 1.37 ± 0.07 72.10 ± 1.94

54

Catechin 19.45 ± 0.83 111.40 ± 1.74 167.30 ± 5.75 12.63 ± 0.61 3.53 ± 0.07 2.44 ± 0.20 1.34 ± 0.09 1.61 ± 0.10 68.64 ±2.59 (50mg/kg)

Rutin 19.83 ± 0.40 116.30 ± 3.10 140.00 ± 3.81 10.51 ± 0.59 3.77 ± 0.03 2.47 ± 0.14 1.38 ± 0.06 1.39 ± 0.06 72.54 ± 1.72 (40mg/kg)

Three groups each of 8 rats were used; the first group received saline and served as normal group. The other two groups were either orally injected with catechin for 3 days or rutin for 10 days. All values are expressed as mean ± SE.

Results

Effects of catechin (50mg/kg) or rutin (40mg/kg) on serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) in irradiated normal and cigarette smoke extract treated rats:

The results demonstrated in Tables (2a & b) and illustrated in Figures (1a& b) showed that irradiation of rats at dose levels 1 Gy or 2Gy induced a significant rise in serum ALT activity which amounted to 195% & 200% of the normal values, respectively . On the other hand, administration of CSE alone or followed by whole body γ-irradiation at dose levels of 0.5 Gy, 1 Gy or 2Gy resulted in a significant increment in the enzyme activity to about 137% , 138%, 262% & 346 % of the values observed in normal rats, respectively .

Results of the current study revealed that administration of catechin (50 mg/kg) and rutin (40 mg/kg) daily for 3 and 10 days respectively to the irradiated rats (2Gy) pretreated with CSE (5 mg/kg) significantly attenuated the rise in ALT activity to about 214% & 146% of the values recorded in normal rats, respectively.

The results demonstrated in Tables (2a& b) and illustrated in Figures (2a& b) showed that irradiation of rats at dose levels 1 Gy or 2Gy induced a significant rise in serum AST activity which amounted to 127% & 152% of the normal values, respectively . On the other hand, administration of CSE alone or followed by whole body γ -irradiation at dose levels of 0.5 Gy , 1 Gy or 2Gy resulted in a significant increment in the enzyme activity to about 153% ,150% , 170% & 188% of the values observed in normal rats, respectively .

Results of the current study revealed that administration of catechin (50 mg/kg) and rutin (40 mg/kg) daily for 3 and 10 days respectively to the irradiated rats (2 Gy) pretreated with CSE (5 mg/kg) significantly attenuated the rise in AST activity to about 157% & 124% of the values recorded in normal rats, respectively.

The results demonstrated in Tables (2a& b) and illustrated in Figures (3a& b) showed that irradiation of rats at dose levels 0.5 Gy, 1 Gy or 2Gy induced a significant rise in serum ALP activity which amounted to 216%, 259% & 440% of the normal values, respectively. On the other hand, administration of CSE alone or followed by whole body γ-irradiation at dose levels of 0.5 Gy, 1 Gy or 2Gy

55 Results resulted in a significant increment in the enzyme activity to about 250%, 412%, 490% & 530% of the values observed in normal rats, respectively.

Results of the present study showed that administration of catechin (50 mg/kg) and rutin (40 mg/kg) daily for 3 and 10 days respectively to the irradiated rats (2 Gy) pretreated with CSE (5 mg/kg) significantly attenuated the rise in ALP activity to about 175% & 284% of the values recorded in normal rats, respectively.

56 Results

Table (2a): Effect of CSE (5mg/kg) administration on serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) activities in whole body gamma irradiated rats (at dose levels of 0.5 Gy, 1 Gy or 2 Gy).

Parameters ALT AST ALP (U/ml) (U/ml) (U/L) Groups Normal 16.56 ± 0.70 114.20 ± 2.18 123.40 ± 7.28 (saline) Irradiated # $ # @ $ * # $ 0.5 Gy 18.32 ± 0.95 117.10 ± 3.30 266.90± 7.06 Irradiated * @ $ * # @ $ * # $ 1.0 Gy 32.27± 1.34 145.10 ± 2.56 319.60 ± 16.51 Irradiated * * * 2.0 Gy 33.06 ± 1.09 173.20 ± 3.76 543.40 ± 17.31 CSE (5mg/kg) * # * * # 22.66 ± 0.90 175.20 ± 4.19 306.10 ± 20.37 CSE+ 0.5 Gy * # $ * $ * @ $ 22.8 ± 0.73 171.00 ± 4.54 508.70± 17.53 CSE+ 1.0Gy * # @ $ * # @ $ * @ 43.37 ± 1.92 194.50 ± 3.80 604.50 ± 20.81 CSE+ 2.0 Gy * # @ * # @ * # @ 57.31 ± 1.26 214.70 ± 3.75 654.20 ± 17.55

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of (0.5, 1 or 2 Gy). Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 or 2 Gy.

All values are expressed as mean ± SE. (n=8) *: significantly different from normal at p < 0.05. #: significantly different from 2Gy at p < 0.05. @: significantly different from CSE at p < 0.05. $: significantly different from CSE + 2Gy at p < 0.05

57 Results

Table (2b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) activities in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Parameters ALT AST ALP (U/ml) (U/ml) (U/L) Groups Normal 16.56 ± 0.70 114.20 ± 2.18 123.40 ± 7.28 (saline) Irradiated * * * 2.0 Gy 33.06 ± 1.09 173.20 ± 3.76 543.40 ± 17.31 CSE (5mg/kg) * # * * # 22.66 ± 0.90 175.20 ± 4.19 306.10 ± 20.37 CSE+ 2.0 Gy * # @ * # @ * # @ 57.31 ± 1.26 214.70 ± 3.75 654.20 ± 17.55 Catechin * @ $ * $ * # @ $ (50mg/kg) + 35.51 ±1.04 189.20 ± 3.11 216.50 ± 7.84 CSE+ 2.0 Gy Rutin (40mg/kg) * # $ * # @ $ * # $ + CSE + 2.0 Gy 24.14 ± 1.02 142.00 ± 4.35 350.60 ± 11.25

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as mean ± SE. (n=8) *: significantly different from normal at p < 0.05. #: significantly different from 2Gy at p < 0.05. @: significantly different from CSE at p < 0.05. $: significantly different from CSE + 2Gy at p < 0.05.

58 Results

400 * # @ normal * 350 0.5 Gy # 1 Gy @ 300 2 Gy $ * CSE 250 @ * * CSE+ 0.5 Gy $ # * CSE+1 Gy 200 # # $ CSE+ 2 Gy

(%of (%of normal) 150 $ ALT activity (IU/ml) ALT activity 100

50

0 1

Figure (1a): Effect of CSE (5 mg/kg) administration, gamma irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination on serum activity of ALT (IU/ml) in rats.

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of 0.5, 1 & 2 Gy. Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

59 Results

400 * # @ Normal 350 * 2.0 Gy @ 300 $ CSE 250 * * CSE + 2.0 Gy * # 200 Catechin+CSE+2.0 Gy # $

(%of (%of normal) Rutin+CSE+2.0Gy

150 ALT activity (IU/ml) ALT activity

100

50

0 1

Figure (1b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on serum activity of (ALT) (IU/ml) in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

60 Results

* # @ 200 * # normal * @ $ 0.5 Gy 180 * # * * $ 1 Gy 160 @ 2 Gy # $ CSE 140 @ CSE+0.5 Gy $ 120 CSE+1 Gy CSE+2 Gy 100

(%of (%of normal) 80

AST activity (IU/ml) AST activity 60 40

20

0 1

Figure (2a): Effect of CSE (5 mg/kg) administration, gamma irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination on serum activity of (AST) (IU/ml) in rats.

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of 0.5, 1 & 2 Gy. Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

61 Results

200 * # @ Normal

180 * $ * 2.0 Gy * * # 160 CSE @ 140 $ CSE+2.0 Gy Catechin+CSE+2.0 Gy 120 Rutin+CSE+2.0 Gy 100

(%of (%of normal) 80

AST activity (IU/ml) AST activity 60

40

20

0 1

Figure (2b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on serum activity of (AST) (IU/ml) in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

62 Results

600 *# @ normal * @ 500 * 0.5 Gy 1 GY * @ $ 2 Gy 400 CSE * # $ * # CSE+0.5 Gy 300 CSE+1 Gy * # $

CSE+2 Gy (% of normal)

ALP activity (U/L) ALP activity 200

100

0 1

Figure (3a): Effect of CSE (5 mg/kg) administration, gamma irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination on serum activity of (ALP) (U/L) in rats.

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of 0.5, 1 & 2 Gy. Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05.

63 Results

600 * # @ Normal 2.0 Gy 500 * CSE CSE+2.0 Gy 400 Catechin+CSE+2.0 Gy * Rutin+CSE+2.0 Gy * # # * # $ 300 @

$

(% of normal) ALP activity (U/L) ALP activity 200

100

0 1

Figure (3b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on serum activity of (ALP) (U/L) in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

64 Results

Effects of catechin (50mg/kg) or rutin (40mg/kg) on levels of blood glutathione (GSH), serum malondialdehyde (MDA), nitric oxide (NO) in irradiated normal and cigarette smoke treated rats:

The results shown in Tables (3a& b) and illustrated in Figures (5a& b) showed that irradiation of rats at dose levels 1 Gy or 2Gy induced a significant decrease in blood GSH level which amounted to 66% & 36% of the normal values, respectively. On the other hand, administration of CSE alone or followed by whole body γ-irradiation at dose levels of 0.5 Gy, 1 Gy or 2Gy resulted in a further decrease in GSH level to about 42%, 37%, 39% & 27% of the values observed in normal rats, respectively.

Results of the current study revealed that administration of catechin (50mg/kg) or rutin (40mg/kg) for 3 or 10 days respectively before whole body gamma irradiation (2 Gy) and CSE (5mg/kg) administration resulted in slight increase in GSH level to about 55% & 34% of the values recorded in normal rats, respectively.

The results demonstrated in Tables (3a& b) and illustrated in Figures (4a& b) showed that irradiation of rats at dose levels 1 Gy or 2Gy induced a significant rise in serum MDA level which amounted to 147% & 243% of the normal values, respectively. On the other hand, administration of CSE alone or followed by whole body γ-irradiation at dose levels of 0.5 Gy, 1 Gy or 2Gy resulted in a significant increment in MDA level to about 158%, 202%, 245% & 362% of the values observed in normal rats, respectively.

Results of the present study revealed that administration of catechin (50mg/kg) or rutin (40mg/kg) for 3 or 10 days respectively before whole body gamma irradiation (2 Gy) and CSE (5mg/kg) administration significantly attenuated the rise in MDA level to about 214% & 186% of the values recorded in normal rats, respectively.

The results demonstrated in Tables (3a& b) and illustrated in Figures (6a& b) showed that irradiation of rats at dose level of 2Gy induced a significant rise in serum NO level which amounted to 224% of the normal values. On the other hand, administration of CSE alone or followed by whole body γ-irradiation at dose levels

65 Results of 0.5 Gy, 1 Gy or 2Gy resulted in a significant increment in NO level to about 349%, 366%, 551% & 605% of the values observed in normal rats, respectively.

Results of the present study revealed that the administration of catechin (50mg/kg) or rutin (40mg/kg) for 3 or 10 days respectively before whole body gamma irradiation (2 Gy) and CSE (5mg/kg) administration significantly attenuated the rise in NO level to about 450% & 357% of the values recorded in normal rats, respectively.

66 Results

Table (3a): Effect of CSE (5mg/kg) administration on malondialdehyde (MDA), nitric oxide (NO) serum levels and glutathione (GSH) blood level in whole body gamma irradiated rats (at dose levels of 0.5 Gy, 1 Gy or 2 Gy).

Parameters MDA GSH NO (nmol/ml) (mg/ml) (μmol/L) Groups Normal 3.52 ± 0.06 12.84 ± 0.71 2.68 ± 0.06 (saline) Irradiated # @ $ # @ $ # @ $ 0.5 Gy 3.36 ± 0.03 11.02 ± 0.70 2.36 ± 0.13 Irradiated * # $ * # @ $ # @ $ 1.0 Gy 5.16 ± 0.09 8.49 ± 0.74 3.83 ± 0.13 Irradiated * * * 2.0 Gy 8.55 ± 0.19 4.65 ± 0.28 5.99 ± 0.31 CSE (5mg/kg) * # * * # 5.57 ± 0.13 5.41 ± 0.39 9.33 ± 0.40 CSE+ 0.5 Gy * # @ * * # $ 7.11 ± 0.12 4.79 ± 0.47 9.79 ± 0.27 CSE+ 1.0Gy * @ * * # @ 8.64 ± 0.22 5.00 ± 0.27 14.73 ± 0.29 CSE+ 2.0 Gy * # @ * * # @ 12.76 ± 0.31 3.47 ± 0.27 16.17± 0.68

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of (0.5, 1 & 2 Gy). Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as mean ± SE. (n=8) *: significantly different from normal at p < 0.05. #: significantly different from 2Gy at p < 0.05. @: significantly different from CSE at p < 0.05. $: significantly different from CSE + 2Gy at p < 0.05.

67 Results

Table (3b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on malondialdehyde (MDA), nitric oxide (NO) serum levels and glutathione (GSH) blood level in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Parameters MDA GSH NO (nmol/ml) (mg/ml) (μmol/L) Groups Normal 3.52 ± 0.59 12.84 ± 0.71 2.68 ± 0.06 (saline) Irradiated * * * 2.0 Gy 8.55 ± 0.19 4.65 ± 0.28 5.99 ± 0.31 CSE (5mg/kg) * # * * # 5.57 ± 0.13 5.41 ± 0.39 9.33 ± 0.40 CSE+ 2.0 Gy * # @ * * # @ 12.76 ± 0.31 3.47 ± 0.27 16.17± 0.68 Catechin * # @ $ * $ * # @ $ (50mg/kg) + 7.55 ± 0.38 7.04 ± 0.16 12.04 ±0.44 CSE+ 2.0 Gy Rutin (40mg/kg) * # @ $ * * # $ + CSE + 2.0 Gy 6.56 ± 0.18 4.37 ± 0.32 9.55 ±0.49

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as mean ± SE. (n=8) *: significantly different from normal at p < 0.05. #: significantly different from 2Gy at p < 0.05. @: significantly different from CSE at p < 0.05. $: significantly different from CSE + 2Gy at p < 0.05.

68 Results

400 * # @ normal 350 0.5 Gy * 1 Gy * 300 @ 2 Gy * # # $ @ CSE 250 $ CSE+0.5 Gy * * CSE+1 Gy 200 # # $ CSE+2 Gy # (%of (%of normal) 150 @

$ MDA level MDA (nmol/ml) 100

50

0 1

Figure (4a): Effect of CSE (5 mg/kg) administration, gamma irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination on serum level of (MDA) (nmol/ml) in rats.

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of 0.5, 1 & 2 Gy. Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05.

69 Results

400 * # @ Normal

350 2 Gy CSE 300 *# * @ * CSE+2 Gy # 250 @ Catechin+CSE+2 Gy $ 200 * Rutin+CSE+2 Gy #

(% of normal) 150 MDA level(nmol/ml)MDA 100

50

0 1

Figure (4b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on serum level of (MDA) (nmol/ml) in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

70 Results

120 normal 0.5 Gy # 1 Gy 100 @ $ 2 Gy * # CSE 80 @ CSE+0.5 Gy $ CSE+1 Gy 60 CSE+2 Gy * * * (%of (%of normal) * 40

* GSH blood level (mg/ml)

20

0

Figure (5a): Effect of CSE (5 mg/kg) administration, gamma irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination on blood level of (GSH) (mg/ml) in rats.

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of 0.5, 1 & 2 Gy. Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05.

71 Results

120 Normal 2 Gy 100 CSE CSE+2.0 Gy 80 * Catechin+CSE+2.0 Gy $ Rutin+CSE+2.0 Gy 60

(% of normal) * * *

40 * GSH GSH blood level (mg/ml)

20

0 1

Figure (5b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on blood level of (GSH) (mg/ml) in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

72 Results

* # 700 * # @ Normal @ 0.5Gy 600 1.0Gy 2.0Gy 500 * # @ $ CSE * 400 # CSE+0.5Gy CSE+1.0Gy # CSE+2.0Gy 300 @ * (%of (%of normal) # $ NO level NO (µmol/L) @ 200

100

0 1

Figure (6a): Effect of CSE (5 mg/kg) administration, gamma irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination on serum level of nitric oxide (NO) (µmol/L) in rats.

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of 0.5, 1 & 2 Gy. Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05.

73 Results

700 * # @ Normal 600 *# 2 Gy @ $ CSE 500 * CSE+2 Gy * # Catechin+CSE+2 GY 400 # $ Rutin+CSE+2 GY

300 *

(% of normal) NO level NO (µmol/L) 200

100

0 1

Figure (6b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on serum level of (NO) (µmol/L) in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

74 Results

Effects of catechin (50mg/kg) or rutin (40mg/kg) on serum lactate dehydrogenase (LDH) activity in irradiated normal and cigarette smoke treated rats:

The results demonstrated in Tables (4a& b) and illustrated in Figures (7a& b) showed that irradiation of rats at dose levels 0.5Gy, 1Gy or 2Gy induced a significant rise in serum LDH activity which amounted to 148%, 187% & 221% of the normal values, respectively . On the other hand, administration of CSE alone or followed by whole body γ-irradiation at dose levels of 0.5 Gy , 1 Gy or 2Gy resulted in a significant increment in the enzyme activity to about 155% , 170% , 223% & 223 % of the values observed in normal rats, respectively.

Results of the current study indicated that administration of each of catechin (50mg/kg) or rutin (40mg/kg) for 3 or 10 days respectively to the irradiated rats (2 Gy) pre-treated with CSE (5mg/kg) significantly attenuated the rise in LDH activity to about 117% & 207% of the values recorded in normal rats, respectively.

75 Results

Table (4a): Effect of CSE (5mg/kg) administration on serum lactate dehydrogenase (LDH) activity in whole body gamma irradiated rats (at dose levels of 0.5 Gy, 1 Gy or 2 Gy).

LDH Groups (U/ml) Normal 1.51 ± 0.10 (saline) Irradiated 0.5 Gy * # $ 2.24 ± 0.09 Irradiated 1.0 Gy * # @ $ 2.826 ± 0.03 Irradiated 2.0 Gy * 3.35 ± 0.09 CSE (5mg/kg) * # 2.34 ± 0.06 CSE+ 0.5 Gy * # $ 2.57 ± 0.07 CSE+ 1.0Gy * @ 3.38 ± 0.09 CSE+ 2.0 Gy * @ 3.37 ± 0.07

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of (0.5, 1 & 2 Gy). Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as mean ± SE. (n=8) *: significantly different from normal at p < 0.05. #: significantly different from 2Gy at p < 0.05. @: significantly different from CSE at p < 0.05. $: significantly different from CSE + 2Gy at p < 0.05.

76 Results

Table (4b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on serum lactate dehydrogenase (LDH) activity in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Groups LDH (U/ml)

Normal 1.51 ± 0.10 (saline) Irradiated 2.0 Gy * 3.35 ± 0.09 CSE (5 mg/kg) * # 2.34 ± 0.06 CSE+ 2.0 Gy * @ 3.37 ± 0.07 Catechin (50mg/kg)+ CSE+ 2.0 Gy # @ $ 1.77 ± 0.04 Rutin (40mg/kg) + CSE+ 2.0 Gy * @ 3.14 ± 0.05

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as mean ± SE. (n=8) *: significantly different from normal at p < 0.05. #: significantly different from 2Gy at p < 0.05. @: significantly different from CSE at p < 0.05. $: significantly different from CSE + 2Gy at p < 0.05.

77 Results

250 * * * * @ @ normal # @ 0.5 Gy * $ 200 * * # 1 Gy # # $ 2 Gy $ CSE 150 CSE+0.5 Gy CSE+1 Gy CSE+2 Gy

(%of (%of normal) 100 LDH activity(U/ml) LDH

50

0 1

Figure (7a): Effect of CSE (5 mg/kg) administration, gamma irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination on serum activity of (LDH) (U/ml) in rats.

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of 0.5, 1 & 2 Gy. Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

78 Results

250 * * @ normal * @ 2 GY 200 CSE * # # CSE+2 Gy @ 150 $ Catechin +CSE+2 GY

Rutin+CSE+2 GY

(% of normal) 100 LDH activity(U/ml) LDH

50

0 1

Figure (7b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on serum activity of (LDH) (U/ml) in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

79 Results

Effects of catechin (50mg/kg) or rutin (40mg/kg) on gastric myeloperoxidase (MPO) activity in irradiated normal and cigarette smoke treated rats:

The results shown in Tables (5a& b) Figures (8a& b) revealed that irradiation of rats at dose levels 0.5 Gy, 1 Gy or 2Gy induced a significant rise in gastric MPO activity which amounted to 170 % , 216% & 253 % of the normal values, respectively . On the other hand, administration of CSE alone or followed by whole body γ - irradiation at dose levels of 0.5 Gy , 1 Gy or 2Gy resulted in a significant increment in the enzyme activity to about 343 % , 515 % , 524 % & 672 % of the values observed in normal rats, respectively.

Result of the present study showed that administration of each of catechin (50mg/kg) or rutin (40mg/kg) for 3 or 10 days respectively to the irradiated rats (2 Gy) pretreated with CSE (5mg/kg) significantly attenuated the rise in MPO activity to about 198 % & 186 % of the values recorded in normal rats, respectively.

80 Results

Table (5a): Effect of CSE (5mg/kg) administration on the activity of myeloperoxidase (MPO) in gastric mucosa in whole body gamma irradiated rats (at dose levels of 0.5 Gy, 1 Gy or 2 Gy).

Groups MPO (U/ g tissue) Normal 1.37 ± 0.07 (saline) Irradiated 0.5 Gy * # @ $ 2.33 ± 0.06 Irradiated 1.0 Gy * @ $ 2.95 ± 0.12 Irradiated 2.0 Gy * 3.46 ±0.07 CSE (5mg/kg) * # 4.69 ± 0. 22 CSE+ 0.5 Gy * # @ $ 7.04± 0.13 CSE+ 1.0Gy * # @ $ 7.16 ± 0.16 CSE+ 2.0 Gy * # @ 9.19 ± 0.26

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of (0.5, 1 & 2 Gy). Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as mean ± SE. (n=8) *: significantly different from normal at p < 0.05. #: significantly different from 2Gy at p < 0.05. @: significantly different from CSE at p < 0.05. $: significantly different from CSE + 2Gy at p < 0.05.

81 Results

Table (5b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on the activity of myeloperoxidase (MPO) in gastric mucosa in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Groups MPO (U/ g tissue) Normal 1.37 ± 0.07 (saline) Irradiated 2.0 Gy * 3.46 ±0.07 CSE (5mg/kg) * # 4.69 ± 0. 22 CSE+ 2.0 Gy * # @ 9.19 ± 0.26 Catechin (50mg/kg)+ CSE+ 2.0 Gy * # @ $ 2.705± 0.1661 Rutin (40mg/kg) + CSE+ 2.0 Gy * # @ $ 2.546 ± 0.0636

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as mean ± SE. (n=8) *: significantly different from normal at p < 0.05. #: significantly different from 2Gy at p < 0.05. @: significantly different from CSE at p < 0.05. $: significantly different from CSE + 2Gy at p < 0.05.

82 Results

800 normal * # @ 0.5 Gy 700 * # 1 Gy * # @ 600 @ $ $ 2 Gy CSE 500 * # CSE+0.5 Gy $ CSE+1 Gy 400 * CSE+2 Gy @ (% of normal) 300 * * # $ @ $ MPO(U/g tissue) activity 200

100

0 1

Figure (8a): Effect of CSE (5 mg/kg) administration, gamma irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination on serum activity of (MPO) (U/g tissue) in rats.

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of 0.5, 1 & 2 Gy. Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

83 Results

800 * # Normal 700 @ 2 Gy

600 CSE CSE+2 GY 500 * Catechin+CSE+2 Gy * 400 # # * Rutin+CSE+2 Gy * @ #

(% of normal) 300 $ @

$ MPO activity(U/g tissue) MPO activity(U/g 200

100

0 1

Figure (8b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on mucosal activity of (MPO) (U/g tissue) in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

84 Results

Effects of catechin (50mg/kg) or rutin (40mg/kg) on serum tumor necrosis factor-alpha (TNF-α) in irradiated normal and cigarette smoke treated rats:

The results shown in Tables (6a& b) Figures (9a& b) revealed that irradiation of rats at dose levels 1 Gy or 2Gy induced a significant rise in serum TNF-α level which amounted to 130 % & 150 % of the normal values of , respectively . On the other hand, administration of CSE alone or followed by whole body γ -irradiation at dose levels of 0.5 Gy , 1 Gy or 2Gy resulted in a significant increment in TNF- α level to about 156 % , 139 % , 176 % & 212 % of the values observed in normal rats, respectively .

Result of the present work showed that the administration of each of catechin (50mg/kg) or rutin (40mg/kg) for 3 or 10 days respectively to the irradiated rats pretreated with CSE (5mg/kg) significantly attenuated the rise in TNF-α level to about 140 % & 133 % of the values recorded in normal rats, respectively.

85 Results

Table (6a): Effect of CSE (5mg/kg) administration on serum tumor necrosis factor-alpha (TNF-α) level in whole body gamma irradiated rats (at dose levels of 0.5 Gy, 1 Gy or 2 Gy).

TNF-α Groups (pg/ml) Normal 72.10 ± 1.94 (saline) Irradiated 0.5 Gy # @ $ 70.93 ± 2.27 Irradiated 1.0 Gy * # @ $ 93.24 ± 1.26 Irradiated 2.0 Gy * 107.70 ± 2.38 CSE (5mg/kg) * 112.80 ± 2.20 CSE+ 0.5 Gy * @ $ 100.00 ± 2.15 CSE+ 1.0Gy * # @ $ 126.90 ± 3.05 CSE+ 2.0 Gy * # @ 152.90 ± 3.02

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of (0.5, 1 & 2 Gy). Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as mean ± SE. (n=8) *: significantly different from normal at p < 0.05. #: significantly different from 2Gy at p < 0.05. @: significantly different from CSE at p < 0.05. $: significantly different from CSE + 2Gy at p < 0.05.

86 Results

Table (6b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on serum tumor necrosis factor-alpha (TNF-α) level in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

TNF-α Groups (pg/ml) Normal 72.10 ± 1.94 (saline) Irradiated 2.0 Gy * 107.70 ± 2.38 CSE (5mg/kg) * 112.80 ± 2.20 CSE+ 2.0 Gy * # @ 152.90 ± 3.02 Catechin (50mg/kg)+ CSE+ 2.0 Gy * @ $ 101.0± 3.176 Rutin (40mg/kg) + CSE+ 2.0 Gy * # @ $ 95.71 ±0.7577

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as mean ± SE. (n=8) *: significantly different from normal at p < 0.05 #: significantly different from 2Gy at p < 0.05. @: significantly different from CSE at p < 0.05. $: significantly different from CSE + 2Gy at p < 0.05.

87 Results

250 * # normal @$ 0.5 Gy * # 1 Gy @$ 2 Gy 200 * # CSE * * @ * @ CSE+0.5 Gy # $ CSE+1 Gy 150 @ $ CSE+2 GY

(%of (%of normal) 100 TNF-alpha level (pg/ml)TNF-alpha

50

0 1

Figure (9a): Effect of CSE (5 mg/kg) administration, gamma irradiation (0.5 Gy, 1 Gy or 2 Gy) or their combination on serum level of (TNF-α) (pg/ml) in rats.

Eight groups each of eight rats were used. Three groups were whole body gamma irradiated at a dose level of 0.5, 1 & 2 Gy. Another two groups were injected with saline or CSE (5mg/kg) to serve as normal and control groups. The last three groups received CSE and were exposed to whole body gamma radiation at a dose level of 0.5, 1 & 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE + 2Gy at p < 0.05.

88 Results

250 * # normal @ 2 Gy 200 * CSE * @ * # * @ $ $ CSE+2 Gy 150 Catechin+CSE+2Gy

Rutin+CSE+2Gy

(%of (%of normal) 100 TNF-alpha level (pg/ml)TNF-alpha 50

0 1

Figure (9b): Effects of catechin (50mg/kg) or rutin (40 mg/kg) on serum level of (TNF-α) (pg/ml) in whole body gamma irradiated rats (2 Gy) pretreated with CSE (5mg/kg).

Six groups each of eight rats were used; the first group was injected with saline and served as normal. Another two groups were either injected with CSE (5mg/kg) or exposed to whole body gamma radiation at a dose level of 2 Gy. The fourth group was injected with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy. The last two groups were given either catechin (50mg/kg) or rutin (40mg/kg) with CSE (5mg/kg) then exposed to whole body gamma radiation at a dose level of 2 Gy.

All values are expressed as % mean. *: significantly different from normal at p<0.05. #: significantly different from 2 Gy at p<0.05. @: significantly different from CSE at p<0.05. $: significantly different from CSE+ 2 Gy at p<0.05.

89

DISCUSSION

Discussion

In the present study, exposure of rats to acute dose (1 and 2 Gy) of γ- radiation induced a significant increase in the activities of serum ALT, AST and ALP. Furthermore, serum LDH was also significantly increased by exposure to 2 Gy γ-radiation. The increase of ALT and AST is in agreement with the findings of El-Ghazaly and Ramadan (1996) who reported that rats irradiated at 3 or 6 Gy γ -rays, showed similar increase in the activities of both enzymes. This increase by exposure to radiation may be due to the damage of cellular membranes of hepatocytes, which in turn leads to an increase in the permeability of cell membranes and facilitates the passage of cytoplasmic enzymes outside the cells leading to the increase in serum aminotransferase activities (Khamis and Roushdy, 1991).

The increase in the alkaline phosphatase activities of γ-irradiated rats observed in the current study was similar to the increase in liver phosphatase enzymes observed earlier (Bharatiya and Khan, 1983; Khan et al., 1984) after irradiation with various doses. Radiation-induced cell death may be a possible reason for increased activity of acid phosphatase (ACP) and alkaline phosphatase (ALP) (Soyal el al., 2007). The increase of ALP activity in blood serum can be attributed to the possible release of this enzyme from different tissues associated with the obstruction of the blood stream to the liver (Khamis and Roushdy, 1991).

Transaminases are considered as sensitive markers of hepatotoxicity and any alterations in their levels are taken as an index of hepatocellular damage (Wroblewski, 1959). Pradeep el al. (2008) reported that exposure of rats to γ-radiation at a dose of 1, 3 and 5 Gy leads to a marked elevation in the activities of serum AST ALT, ALP, and LDH. This might be due to the release of these enzymes from the cytoplasm into the blood circulation rapidly after rupture of the plasma membrane and cellular damage (Sallie et al., 1991). This fact is further supported by the observed decrease in the levels of these enzymes in the liver tissues.

Moreover, γ-irradiation of rats with cesium- 137 source was accompanied by elevated activities of AST and ALP and increased levels of urea and creatinine, as well as decreased contents of total proteins, albumin, and total globulins in serum indicating liver and kidney injury (El-Missiry et al., 2007). Similar results were observed in other studies (Bhatia and Manda, 2004) using high-energy radiation from cobalt source.

Ionizing radiation is toxic to organisms because it induces deleterious structural changes in essential macromolecules. These changes can be the

90 Discussion result of direct interaction with radiation. However, it is generally considered that, because of the abundance of water in living organisms, a more important mechanism is the interaction with free radicals formed by photolysis of water (Navarro et al., 1997).

Multiple biological effects of γ-radiation were induced through direct interaction with DNA or production of activated free radical species from water (Chung et al., 2001). Ionizing radiation causes oxidative damage to tissues within an extremely short period, and possible protection against it would require the rapid transfer of antioxidants to the sensitive sites in cells (El-Missiry et al., 2007).

It is well known that the molecule most often reported to be damaged by ionizing radiation is DNA. About 60–70% of cellular DNA damage produced by ionizing radiation is caused by OH•, which is considered as the most damaging of all free radicals generated in organisms (Ward , 1988).

It is suggested that oxidative stress is linked to the organ damage following exposure to ionizing radiation. Increased levels of MDA (an index of lipid peroxidation), and NO associated with reduced level of GSH were observed in the current study in γ-irradiated rats. These results are in accordance with the results of Şener et al. (2006a) who found that tissue MDA was increased in the lung, liver, kidney and ileum of whole-body irradiated rats, indicating the presence of radiation-induced oxidative damage. Peroxidation of membrane lipids can disrupt membrane fluidity and cell compartmentation, which may contribute to impaired cellular function and necrosis (Reiter et al., 2001).

Moreover, the study of Adaramoye, (2010) showed an increase of serum levels of MDA, urea and activities of ALT, AST and decrease of GSH level and activities of GST, CAT and SOD in rats exposed to 5 Gy γ-radiation.

Increased level of MDA may be due to the attack of free radicals on the fatty acid component of membrane lipids (Kamat et al., 2000), leading to lipid peroxidation and finally resulting in cell death. Lipid peroxidation can cause severe impairment of membrane function by increasing membrane permeability and membrane protein oxidation (Logani and Davies, 1980; Mathews et al., 1994; Valko et al., 2007).

GSH, a well-known antioxidant, provides major protection in oxidative injury by participating in the cellular system of defence against oxidative

91 Discussion damage (Ross, 1988). GSH plays a key role in protecting cells against electrophiles and free radicals (Sies, 1986). This is due to the nucleophilicity of the SH group and to the high reaction rate of thiols with free radicals (Jaeschke, 1990). Radiation resistance of many cells is associated with high intracellular levels of GSH (Mitchell, and Russo, 1987; Meister, 1991; Estrela et al,. 1995). Cells containing low levels of GSH were found to be much more sensitive to the effect of irradiation than controls (Meister, 1991). GSH can act directly as a free radical scavenger by neutralizing OH•, or indirectly by repairing initial damage to macromolecules inflicted by OH• (Sies, 1986). There is ample evidence that thiols protect molecules from radiation injury, mainly by hydrogen donation, which can restore damaged molecules to their original state (Willson, 1983). Indeed, GSH is essential in the maintenance of enzyme SH groups in the proper redox state (Ericksson et al., 1974). Moreover, GSH is a substrate or a cofactor for a number of protective enzymes, such as GSH peroxidase and the GSH S-transferases (Sies, 1986).

The recorded decrease in GSH level accompanied by increase in blood MDA and NO levels post irradiation in the present experiments are in agreement with those recorded in other studies (Koc et al., 2003; Samarth and Kumar, 2003; Bhatia and Jain, 2004). Under normal conditions the inherent defense system, including glutathione and the antioxidant enzymes, protect against oxidative damage.

The present decrease in GSH levels may be due to its consumption during the oxidative stress induced by irradiation (Şener et al., 2006a). This could be due to an enhanced utilization of the antioxidant system as an attempt to detoxify the free radicals generated by radiation. The decrease in reduced glutathione by irradiation could be attributed to oxidation of the sulphydryl group of GSH resulting from the decrease in glutathione reductase and the enzyme which reduces the oxidized glutathione (GSSG) into a reduced form using NADPH as a source of reducing equivalent (Flohe and Gunzler, 1976).

The present results showed that whole body γ-irradiation of rats enhanced the formation of NO. Similar results have been reported by Voevodskaya and Vanin, (1992) and Gorbunov et al. (2000). γ- irradiation may enhance endogenous NO biosynthesis in liver, intestine, lung, kidney, brain, spleen or heart of the animals, presumably by facilitating the entry of Ca2+ ions into the membrane as well as the cytosol of NO-producing cells through irradiation induced membrane lesions (Gorbunov et al., 2000).

92 Discussion

In the present work, elevated MPO activity in the gastric mucosa following exposure to whole body γ-radiation, indicated that radiation- induced oxidative injury in the tissue involves the contribution of neutrophil accumulation. The present observation is in agreement with the finding of Şener et al. (2006a,b) who suggested that myeloperoxidase activity in tissue would be increased after whole body irradiation. Another study by Erbil et al. (1998) reported that irradiation of rats caused enteritis and elevation of mucosal MPO. Moreover, Swantek et al. (2000) observed that whole body gamma irradiation induced ROS which activate neutrophil, increase tissue MPO activity and can induce or exacerbate tissue injury.

MPO is an essential enzyme for normal neutrophil function. It is a heme enzyme that uses the superoxide and hydrogen peroxide generated by the neutrophil oxidative burst to produce hypochlorous acid and other reactive oxidants, and when neutrophils are stimulated by various stimulants, MPO increases like other cellular tissue-damaging substances (Valko et al., 2006 & 2007).

Exposure to ionizing radiation in the present study resulted in increased serum TNF-α, indicating the role of this cytokine in irradiation-induced toxicity. This finding is in harmony with results of other studies of Şener et al. (2006a,b).

Tissue damage and repair initiated by irradiation are associated with the production of important biological mediators, including cytokines (Rubin et al., 1995), which perpetuate the inflammatory and fibrogenic processes associated with irradiation injury (Rubin et al., 1992).

Moreover, tissue macrophages and monocytes respond to any noxious event by secreting cytokines such as interleukin-1 and TNF-α (Schwacha 2003, Enomoto et al. 2004).

Furthermore, Ran et al. (2007) showed that serum levels of TNF-α and IL-6 were increased significantly after 12-Gy γ-irradiation followed by 30% body surface burn in the rat. Similarly, the study of Shah et al. (2009) showed that the production of TNF-α and IL-6 was elevated significantly after radiation injury combined with severe sepsis.

Cigarette smoke in the present experiments caused the elevation of ALT, AST, ALP, and LDH serum activities. Also, CSE caused the elevation of mucosal MPO activity, and finally, the elevation of MDA,

93 Discussion

NO, and TNF-α serum levels. On the other hand, it has reduced the level of blood GSH.

Cigarette smoke has been identified as a major risk factor for liver and kidney-related diseases. It has the capacity to produce a highly diffusible ROS which cause oxidative damage in vital organs. The damage to the organs by cigarette smoke is evidenced by the elevation of biomarkers in serum (Vanisree and Sudha, 2006). In this study, it was observed that significant elevation in serum AST, ALT and ALP activities was recorded in cigarette smoke-treated rats. These are the important indices for the diagnosis of hepatic dysfunction and this indicates the damage of cells, cellular leakage and loss of functional integrity of cell membrane in the liver.

Similar results have been reported by Pekmez el al. (2007) who observed a significant increase in the activity of ALT, AST, SOD and GSH-Px and levels of total bilirubin and MDA in rats exposed to cigarette smoke compared with those of the controls. The elevations of these markers proved that cigarette smoke induced oxidative damages in liver. The damage caused by cigarette smoke is generally associated with free radicals (Ramesh et al., 2010).

Watanabe et al. (1995) reported a significant decrease in liver weight and increased lipid peroxidation in the liver in cigarette smoke exposed rats. In the smoke treated group, the AST, gamma glutamyl transpeptidase (gamma-GTP), total bilirubin and LDH values were significantly higher than those in the control group. These results suggested that cigarette smoking might induce liver injury by enhancing lipid peroxidation.

The impact of smoking on various liver disorders has been extrapolated from experimental studies .It has been suggested that tobacco induced liver injury is ascribed to oxidative stress associated with lipid peroxidation (Husain et al., 2001).

Chronic nicotine ingestion was found to increase activities of marker enzymes, LDH and ALP, which may be attributed to lung and liver damage (Muthukumaran et al., 2008). Rawson and Peto(1990) have reported that the high LDH activity indicated interstitial fibrosis of the lung. It has been documented that serum ALP activity was elevated during pulmonary fibrosis and alcoholic hepatitis (Capelli et al., 1997; Rukkumani et al., 2003).

94 Discussion

Cigarette smoke has been implicated as a major risk factor in many diseases such as pulmonary and cardiovascular pathologies. The adverse action of the CS is due to presence of a large variety of compounds like nicotine, benzo (a) pyrene, oxidants, and free radicals that could initiate, promote and/or amplify oxidative damage. Although, CS is a major determinant of diseases related to oxidative stress, the inter-individual variations in incidence and extent of these diseases are many (Riboli et al., 1996).

Cigarette smoke includes both reactive aldehydes and a large number of oxidants that contain about 1014 ROS molecules per puff. In addition to these reactants, nicotine in cigarettes is also oxidized and generates ROS in the body (Pryor and Stone, 1993).

The free radical species in cigarette smoke include not only oxygen free radicals but also reactive nitrogen species (Pryor and Stone, 1993). Cigarette smoke is also known to contain a large quantity of reactive aldehydes (Stedman, 1968). All of these compounds are suggested to react with protein thiols and presumably with small thiols such as glutathione (Eiserich et al., 1995).

Because cigarette smoke is a complex mixture of numerous reactive substances, it can elicit complicated physiological and pathological responses including inflammatory-immune system activation. Therefore, oxidative damage of cell components can result not only from a direct reaction with reactive substances in the smoke, but also from the smoke- induced secondary events such as activation and infiltration of phagocytes into the lung (Park et al., 1998).

Similarly, exposure of body cells to cigarette smoke extracts leads to tissue oxidative stress by the increased production of ROS (Zhang et al., 2006).

Significant increase in serum MDA level and decrease in blood GSH level were observed in CSE treated rats in the present study. This observation is consistent with the study of Ramesh et al. (2010), who also found significant increase in MDA and decrease in GSH in liver and kidney of cigarette smoke exposed rats. Similar results were found in human smokers in the study of Tanriverdi et al. (2006). Also, Bingol et al. (1999) reported that chronic smoking causes peroxidation reactions in both plasma and erythrocytes which leads to increased MDA levels.

95 Discussion

Cigarette smoke has been reported to generate lipid peroxidation in tissues (Cigremis et al., 2004). The damage to the tissues is the site of lipid peroxidation, which subsequently leads to cell injury and cell death that disrupts membrane structure and function. GSH depletion is due to the destruction of free radicals and enhanced utilization during detoxification of toxic components released from cigarette smoke. This observation is consistent with earlier reports (Cigremis et al., 2004, Anbarasi et al., 2006, Ramesh el al., 2008).

Furthermore, Joshi et al. (1988) suggested that depletion of GSH levels by cigarette smoke could be due to either oxidation to GSSG, protein- SSG, conjugation with electrophilic components of cigarette smoke, or markedly inhibited synthesis (Larsson et al., 1983). One of the major pathways of GSH utilization is the enzymatic conjugation with electrophilic components (Jakoby and Habig. 1980). Cigarette smoke components are known to react with GSH (Sato et al., 1962).

Moreover, decreased GSH were reported in the study of Muthukumaran el al. (2008), who suggested that the decreased level of circulation and tissue GSH in nicotine-treated rats may be due to enhanced utilization during detoxification of nicotine.

The present work showed an increase in nitric oxide serum level of cigarette smoke treated rats. This finding is in harmony with the study of Wright et al. (1999) who reported an increase in iNOS with CS exposure. Furthermore, Muthukumaran el al. (2008) also reported an increased level of nitric oxide (NO) in circulation, lung, liver and kidney of nicotine treated rats. CS exposure could promote more superoxide and NO production by attenuating SOD activity and increasing iNOS activity, respectively (Guo et al., 1999). In contrast, a reduction in nitrite levels was reported by Hoyt et al. (2003) together with a reduction in NO synthase (iNOS) mRNA and protein expression after cigarette smoke exposure.

The effects of cigarette smoke upon the NO pathways and NOS iso enzymes are controversial and may vary according to the disease, model or location of the NOS. For example, while exhaled NO has been shown to be decreased in humans after acute cigarette exposure, iNOS mRNA expression increased in the lungs of rats exposed to cigarette smoke, while nNOS showed a longer term increase in both transcription and translation (Kharitonov et al., 1995, Wright et al., 1999, Chang et al., 2001, Yates et al., 2001). Cigarette smoke has been shown, however, to

96 Discussion cause a reduction in nitrite concentration and iNOS expression in a murine lung epithelial cell line in vitro (Hoyt et al., 2003).

In contrast, Comhair et al. (2000) showed no change in iNOS expression in airway cells from healthy subjects exposed to cigarette smoke. The effects of cigarette smoke on NOS in the vasculature have shown a reduction in eNOS in the pulmonary vessels in vitro and in vivo (Su et al., 1998, Barbera et al., 2001), while vascular intimal thickening and up–regulated iNOS has been described in mice (Anazawa et al., 2004). These seemingly contradictory effects probably explained in part by the different tissue situations and also by variation in the constituents of the cigarette smoke (Wei et al., 2005).

Nicotine has been recognized to result in oxidative stress by inducing the generation of ROS by various mechanisms (Yildiz et al., 1998). These ROS in turn are capable of initiating and promoting oxidative damage in the form of lipid peroxidation (Kovacic and Cooksy, 2005). Peroxidized lipids are considered to be important biological markers as they may have a major role in the development of cancer, heart disease and brain dysfunction as well as in the aging process. Lipid peroxidation to cellular membranes produces marked alterations in molecular organization of lipids resulting in increased membrane permeability and leakage of cytoplasmic markers into circulation (Mason et al., 1997).

There is an increase of MPO activity in gastric mucosa of CSE treated rats reported in the present study which is in agreement with the results of Guo et al. 1999, who found an increase of colonic MPO in rats exposed to CS, suggesting that CS exposure was able to aggravate neutrophil infiltration and inflammatory damages in the colonic tissue. Cigremis et al. (2006) found that MPO activity was significantly increased in the kidney tissues of CS and ethanol treated rats. Similar alterations in MPO activity have also been reported in other studies such as ethanol and cigarette smoke-induced pancreatic injury (Hartwig et al., 2000), and gastric mucosa damage (Chow et al., 1997, Shin et al., 2002a).

However, Chow et al. (1998), and Nguyen et al. (2001), found that ethanol or cigarette smoke alone did not increase MPO activity in the gastric mucosa of rats and in neutrophils of human. However, combined administration of ethanol and cigarette smoke to the rats significantly increased MPO activity of gastric mucosa (Chow et al., 1998). Discrepancy among these studies could be due to the differences in the doses administered and the duration of the experiments (Cigremis et al., 2006).

97 Discussion

The current study showed an increase in TNF-α in serum of CSE treated rats which is in line with the results of Petrescu et al. (2010) and Barbieri et al. (2011), who observed elevated levels of TNF-α and IL-1β in the serum of smokers compared with non smokers.

Furthermore, Xu et al. (2011) found that CS directly acts on alveolar macrophages to synergize secretion of IL-1β and TNF-α in response to LPS. The high levels of TNF-α and IL-8 in the lungs of smoking rats, demonstrated that heavy smoking inuced emphysema and airway inflammation in the lungs of rats (Bai et al., 2012). In addition, several studies have associated smoking risks with changes in the expression of these cytokines (Hart et al., 2008; Hou et al., 2009) emphasizing that short-term exposure to cigarette smoke in vivo is sufficient to increase IL-1β and/or TNF- α (Castro et al., 2004; Barbieri and Weksler, 2007).

Exposure of CSE treated rats to γ-radiation in the present study showed various changes of the measured parameters. Increase of serum activities of ALT, AST, and ALP was recorded compared to both the irradiated and CSE treated rats. Serum activity of LDH revealed similar increase but when compared only to CSE treated rats.

The present blood levels of MDA and NO increased in the CSE treated rats by exposure to γ-rays compared to both γ-irradiated and CSE treated rats. However GSH level was unaffected compared to either γ-radiation or CSE treatment.

Mucosal MPO activity and blood TNF-α level increased in a similar pattern when compared to both γ-irradiated and CSE treated rats.

One can suggest that since γ-irradiation and CSE treatment are considered among the risk factors that induce many cellular injury, their combined effects are expected to be higher than the effects produced by each of them alone. However it seems that previous studies about the effects of their combination are not available.

Treatment with catechin prior to CSE administration and γ-irradiation in the current study resulted in significant reduction of liver enzymes. These findings are in accordance with the results in the study of Fang, (1992) who found a protective effect of catechin on carbon tetrachloride (CCl4) and galactosamine-induced cytotoxicity in cultured rat hepatocytes which appear in the reduction of ALT, AST and LDH activities.

98 Discussion

Moreover, catechin resulted in less LDH, AST and ALT leakage from cultured hepatocytes, and less morphological alterations by hepatotoxins, suggesting that catechin may act by stabilizing plasma membrane against toxic insults (Davila et al., 1989). Kobayashi et al. (2010) found that administration of green tea catechin lowered activities of ALT, AST, ALP and LDH in serum and decreased MDA content in hepatic tissue in rats.

Administration of rutin prior to CSE treated and γ-irradiated rats showed a hepatoprotective effects which appear in lowering the liver enzymes. This finding is in harmony with the study of Acquaviva et al. (2009) that found that treatment with rutin in rats subjected to I/R injury produced a significant reduction of plasma ALT, AST activities and lipid peroxides level. Rutin was found to protect DNA from fragmentation, suggesting that rutin provides protection in post ischemic reperfusion injury by participating in the cellular defense systems against oxidative damage. In addition, rutin is an efficient antioxidant, free radical scavenger and has been shown to quench the hydroxyl radical, superoxide anion radical, and peroxynitrite anion (Russo et al., 2000). Furthermore, rutin was reported to prevent changes of ALT, AST and LDH in the serum, liver and heart of streptozotocin (STZ)-diabetic rats, indicating the protective effect of rutin against the hepatic and cardiac toxicity caused by STZ. These results suggest that rutin can inhibit the progression of liver and heart dysfunction in STZ-induced diabetic rats (Fernandes et al., 2010) On the same line, rutin was also found to produce hepatoprotective effect against paracetamol-induced liver toxicity (Janbaz el al., 2002).

The current results showed a protective effect of catechin against oxidative stress produced by administration of cigarette smoke extract and exposure to gamma irradiation. This protection is manifested by reduction in levels of MDA and NO and elevation of glutathione level in catechin treated rats compared to rats treated with CSE and exposed to gamma radiation. These findings are in accordance with the study of Chander et al. (2003) who found a marked reduction in renal oxidative stress coupled with significant improvement in renal function as well as renal morphology by catechin which indicated that the protective effect of catechin is based on free radical scavenging activity. Moreover, catechin is reported to directly scavenge oxy free radical species (Nanjo et al., 1999), decrease the accumulation of lipid peroxides in plasma and significantly reduce the consumption of α-tocopherol by repairing tocopheryl radical (Sano et al., 1995), and protection of the hydrophilic

99 Discussion antioxidant ascorbate, which also repairs this radical (Rice-Evans et al., 1996; Skrzydlewska et al., 2002a).

It is well established that catechins contain free radical scavenging properties and act as biological antioxidants. It has been demonstrated that they can scavenge both superoxide and hydroxyl radicals (Ruch et al., 1989; Kashima, 1999; Zhao et al., 2001), as well as the peroxyl radicals (Sang et al., 2003), NO (Kelly et al., 2001), carbon- center free radicals, singlet oxygen and lipid free radicals (Zhao et al., 2001). Catechins chelate metal ions such as copper and iron to form inactive complexes and prevent the generation of potentially damaging free radicals (Guo et al., 1996; Seeram and Nair, 2002).

Another mechanism by which catechins scavenge free radicals is by forming stable semiquinone free radicals, thus, preventing the deaminating ability of free radicals (Guo et al., 1996). In addition, after the oxidation of catechins, due to their reaction with free radicals, a dimerized product is formed, which has been shown to have increased superoxide scavenging and iron-chelating potential (Yoshino et al., 1999). The prevention of damage by catechins against free radicals is effective because catechins can inhibit the ROS-induced damage from a wide array of initiators. These include hydrogen peroxide (Ruch et al., 1989; Grinberg et al., 1997), iron (Grinberg et al., 1997; Seeram and Nair, 2002), and radiolysis (Anderson et al., 2001). Furthermore, catechins have shown increased antioxidant effects compared to other antioxidants, such as vitamins C and E (Zhao et al., 1989).

In addition, catechins can protect different cells from lipid peroxidation and DNA deamination induced by oxidative stress. This is evidenced by the fact that green tea extract can decrease lipid peroxidation markers in the liver, serum and brain, including lipid hydroperoxides, and malondialdehyde in rats (Skrzydlewska et al., 2002b).

The results of the current work have demonstrated the guarding effect of rutin against oxidative stress caused by γ-irradiation and CSE administration by decreasing MDA and NO levels and slightly increasing GSH level in rats. Similar results were obtained by Korkmaz and Kolankaya, (2010) who found that pretreatment of ischemic/reperfusion induced renal injury rats with rutin significantly attenuated renal dysfunction, reduced elevated MDA levels, and restored the depleted SOD activity and GSH levels. The study of Shenbagam and Nalini, 2011) reported that supplementation of rutin along with ethanol significantly decreased the levels of liver marker enzymes, lipid

100 Discussion peroxidation and significantly elevated the activities of liver SOD, CAT, GSH, glutathione peroxidase, vitamins C and E when compared to untreated ethanol supplemented rats.

Rutin possesses a unique ability to inhibit free radical processes in cells at three different stages: an initiation (by the interaction with superoxide ion), the formation of hydroxyl or radicals (by chelating iron ions) and lipid peroxidation (by reacting with lipid peroxy radicals). Lipid peroxidation inhibition is explained by both chelating and antioxidative properties of rutin, as inhibition was observed at flavonoid concentrations much smaller than the concentrations of ferrous ions (Afanas'ev et al., 1989).

The results of the current study showed the anti-inflammatory action of catechin which appeared in the reduction of MPO activity and TNF-α level in rats treated with CSE and irradiated with γ-rays. The study of Guruvayoorappan and Kuttan, (2008) suggested that (+)-catechin could significantly inhibit nitrite and TNF-α production in lipopolysaccharide (LPS) stimulated macrophages. Khalatbary and Ahmadvand, (2011), reported a significant decrease in MPO activity and attenuation of TNF-α, IL-1β, and iNOS expression in traumatized spinal cord rats treated with epigallocatechin gallate (EGCG). Another study by Katiyar and Mukhtar, (2001) found that topical application of EGCG cream inhibited UV-B-induced infiltration of leukocytes as evidenced by decreasing MPO activity.

The anti-inflammatory effects of catechins may be due to their scavenging of NO and reduction of NO synthase (NOS) activity (Chan et al., 1995; Suzuki et al., 2004). Furthermore, NO and peroxynitrite can be directly scavenged by catechins and green tea extract with EGCG being the most effective (Paquay et al., 2000). However, catechins have varying effects on the three different isoforms of NOS. The neuronal NOS (nNOS) isoform of NOS produces toxic effects through NO, and so catechin inhibition of nNOS may be a mechanism through which catechins are anti-inflammatory. In addition, in mouse peritoneal cells, nNOS activity was inhibited by EGCG after stimulation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) (Chan et al., 1997). Evidence also exists that the inhibition of inducible NOS (iNOS) may also be a mechanism behind the anti-inflammatory effects of catechins.

Inhibition of iNOS by catechins appears not to be through a direct mechanism but by preventing inhibitor κB disappearance, which inhibits nuclear factor κB (NF-κB) from binding to the promoter of the iNOS

101 Discussion gene thereby inactivating it (Lin and Lin, 1997). However, Tedeschi et al. (2004) showed that green tea extract did not inhibit iNOS by reducing NF-κB but down-regulated DNA binding activity of the transcription factor signal transducer and activator of transcription-1. In contrast, another model of wound healing showed that ECG improved the quality of scarring by inducing iNOS and cyclooxygenase (COX)-2, which were originally thought to be exclusively proinflammatory enzymes as showed in the study of Kapoor et al. (2004). However, this can be explained by the fact that NO derived from iNOS is vital to the wound healing process (an angiogenic-dependent process) and can enhance angiogenesis by inducing vascular endothelial growth factor (Frank et al., 1999).

The third isoform of NOS, endothelial NOS (eNOS), is a vasodilator- inducing enzyme, and its modulation may directly contribute to the anti- inflammatory effects of catechins. When EGCG was administered to rat aortic rings, dose-dependent vasorelaxation occurred simultaneously with eNOS activity induction (Lorenz et al., 2004). The mechanism may be that eNOS contains an antioxidant response element (ARE) on its promoter and green tea polyphenols can bind to the ARE and activate eNOS (Yu et al., 1997).

The results of the present study reveal that rutin possesses anti- inflammatory potential as reflected by the reduction in the elevated serum TNF-α level and MPO activity of irradiated rats treated with CSE. Rutin was reported to decrease gene expressions and production of the proinflammatory cytokines TNF-α and IL-1β levels in stimulated human mast cells (Park et al. 2008). In addition, Abd-El-Fattah et al. (2010) reported that supplementation with rutin possesses anti-inflammatory potential as reflected by the reduction in the elevated serum TNF-α and IL-1β levels of irradiated rats subjected to I/R.

Shin et al. (2002b) reported that treatments of oesophagitis rats with rutin and harmaline inhibited lipid peroxidation, and myeloperoxidase (MPO) in the oesophagus in comparison with untreated rats, suggesting that rutin and harmaline may have beneficial protective effects against reflux oesophagitis by the inhibition of gastric acid secretion, oxidative stress, inflammatory cytokine production (i.e. IL-1beta), and intracellular calcium mobilization in PMNs in rats. Moreover, Abdel-Raheem, (2010) found that Pre-treatment with rutin protected gastric tissues against indomethacin-induced gastropathy as demonstrated from reduction in the ulcer index, attenuation of histopathological changes and amelioration of the altered oxidative stress and biochemical parameters. This protective mechanism is probably through inhibiting neutrophil infiltration,

102 Discussion suppression of oxidative stress generation and replenishing nitrite/nitrate levels.

The protective activities of catechin and rutin in the γ-irradiated and CSE treated animals indicated that, they may have a possible preventive value in the inhibition of tissue damage including oxidative stress and inflammation. Thus, these drugs could have the potential to modulate the damage induced by combined γ-radiation exposure and cigarette smoke. Further clinical investigations are required to assess the beneficial health effects of these drugs and hence support their use.

103

SUMMARY AND CONCLUSIONS

Summary & Conclusions

The present work was performed to study the effect of the natural antioxidants catechin and rutin on some of the biochemical changes induced in rats by whole body γ-irradiation and/or cigarette smoke, in an attempt to protect or minimize the damage that has resulted from exposure to γ- radiation, treatment with cigarette smoke extract (CSE) or both.

Male albino Wistar rats have been used. Irradiation of animals was performed using the gamma Cell-40 biological irradiator furnished with Caesium137 source.

Rats were randomly classified into 2 batches, one of which was subjected to irradiation and the other was kept non-irradiated. The non- irradiated batch was further subclassified into 4 groups. One group served as normal, while the other three groups were given either CSE (5mg/kg p.o.) for 3 days, catechin (50mg/kg p.o.) for 3 days, or rutin (40mg/kg p.o.) for 10 days. The batch subjected to radiation was further subclassified into groups, being exposed to radiation dose levels of 0.5, 1 or 2 Gy respectively. Three other groups were given CSE for three days then exposed to γ- radiation at dose level of 0.5, 1, or 2 Gy. Two other rat groups were treated either with catechin or rutin and received CSE then γ-irradiated at dose level of 2 Gy.

Animals were sacrificed 24 hours after radiation exposure and blood samples were collected and gastric mucosa was scraped, weighed, homogenized and stored at −20 °C until biochemical analysis. Blood GSH, MDA and nitric oxide levels were determined as indicators of oxidative stress, in addition; serum activities of ALT, AST, ALP and LDH were also measured, and inflammatory mediators such as MPO activity in gastric mucosa and serum TNF-α level were also estimated.

Exposure to acute doses of γ-radiation or administration of cigarette smoke extract (CSE) were found to exert an oxidative stress due to generation of ROS, which are characterized by elevation in MDA and nitric oxide levels and depletion in reduced glutathione, as well as elevation in MPO activity and level of TNF-α. The results also showed that activities of ALT, AST, ALP, and LDH were increased. Damaging effects were significantly increased in animals exposed to gamma irradiation combined with administration of cigarette smoke extract in comparison to irradiation or administration of cigarette smoke extract only.

104 Summary & Conclusions

Pretreatment with catechin or rutin has protected the tissues from oxidative stress induced by irradiation through protection against elevation in MDA and nitric oxide and had prevented the depletion in reduced glutathione. Moreover, they protected against inflammatory reaction induced by irradiation through protection against TNF-α elevation and had inhibited neutrophil infiltration characterized by prevention of increased mucosal MPO activity. This observed protective effect of catechin or rutin was mostly because they are belonging to flavonoids which have shown cytoprotective properties. Also, catechin and rutin caused significant decrease in transaminases, alkaline phosphatase, and lactate dehydrogenase activities. These effects could be due to their ability to scavenge or neutralize free radicals, increase the levels of enzymatic and non-enzymatic antioxidants, chelate some metal ions involved in oxidative stress reactions.

Based on the present findings it could be concluded that:

І- Gamma irradiation or cigarette smoke:

1- Caused oxidative stress that resulted in lipid peroxidation characterized by elevation in serum level of MDA and NO and depletion of the reduced glutathione.

2- Resulted in inflammatory reaction as indicated by the increment in serum level of TNF-α and increased tissue activity of MPO.

3- Increased the activity of serum transaminases, alkaline phosphatase and lactate dehydrogenase.

II- Gamma irradiation and cigarette smoke: Exposure of rats to an acute radiation dose of 2Gy treated with cigarette smoke extract resulted in a more pronounced damage than that induced by the 2Gy radiation dose or cigarette smoke extract alone.

III- Pre-treatment with catechin or rutin showed protection against oxidative stress characterized by:

1- A marked reduction in the level of lipid peroxidation. Catechin and rutin pre-treatment also protected against elevation of NO, and prevented tissue depletion of reduced glutathione.

105 Summary & Conclusions

2- Catechin and rutin have shown an anti-inflammatory activity as marked by the suppression of serum level of TNF-α, as well as, the prevention of neutrophil infiltration characterized by decreased tissue MPO activity.

3- Catechin and rutin showed protective effects against radiation and cigarette smoke induced changes in AST, ALT and ALP activities.

Based on the above mentioned findings, catechin and rutin could have the potential to safely and effectively modulate the damage induced by radiation exposure in cigarette smoke-treated animals. Further clinical investigations are required to assess the beneficial health effects of these agents during radiotherapy and hence support their use for smokers.

106

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159

ARABIC SUMMARY

الولخص العرتٖ

ٗشتتروه ُتتلا الثؽتتس التتٔ ثيا تتح ذتت ش٘ر تعط هعتتاثاخ األكستت ج كتتاذ٘ن٘ي ّيّذتت٘ي( فتتٖ تعتتط الرغ٘راخ ًر٘عح ذعرض ذكْي العرذاى لإلشعاع العاهٖ. ّثيا ح ذ ش٘ر كت ه هٌِوت ا فتٖ ذمل٘ته أّ الؽواٗح هي اٙشاي العايج لوسرخلص ثخاى السعائرفٖ العرذاى الوشععح.

ّل ذن إ رخ ام خل٘ح الرشع٘ع الثْ٘لْظٖ فٖ اول٘ح الرشع٘ع العاهٖ للعرذاى ّالرتٔ ذؽرتْٓ التٔ ٘سْٗم-٧٣١ كوص يإشعاع يئ٘سٖ.

ّلتت ذتتن ذمستت٘ن العتترذاى إلتتٔ هعوْاتتاخ ذرنتتْى كتته هعوْاتتح هتتي ٨ ظتترذاى ّ ُتتلٍ الوعوْاتتاخ ذشتتروه التتٔ هعوْاتتح ظتتاتؽح غ٘تتر هشتتععح ّ اْلعتتد فمتتػ توؽلتتْ هلؽتتٔ ّ هعوتتْار٘ي ذتتن االظِوا تواثذٖ كاذ٘ن٘ي ّيّذ٘ي ّهعوْاح أخرٓ ذن ااؽاؤُتا هسترخلص ثختاى الستعائر كتلل شالز هعوْااخ ذن ذعرظِا إلٔ أشعح ظاها تعرااخ٥٫٥ ٫٥ّ٧٫٥ّ ٢ظرإ ّشالز هعوْاتاخ أخرٓ ذن ااؽاؤُا هسرخلص ثخاى السعائر لثه ذعرظِا ألشعح ظاهتا تعراتاخ ٥٫٥ ٫٥ّ٧٫٥ّ ٢ظرإ ّ هعوْار٘ي أخ٘رذ٘ي ذن ااؽاؤُوا كاذ٘ن٘ي أّ يّذ٘ي شن هسرخلص ثخاى الستعائر ل ثته ذعرظِوا ألشعح ظاها تعراح٫٥ ٢ظرإ.

تع هرّي ٢٤ ااح هي الرشع٘ع ذن أخل اٌ٘اخ هي هصه ال م ّالغشاء الوخاؼٖ للوع ج ّفؽصِن لم٘اش هسرْٓ هالًْ إ-ال ُاٗ ّ ظلْذاشْ٘ى الوخرس ّ هسرْٓ اّكس٘ الٌ٘ررٗ NO) ك ل٘ه الٔ اإلظِاث الر كس ٓ( فٖ ال م ّ ل٘اش ًشاغ إًسٗواخ الٌالالخ األهٌ٘٘ح ALT, AST) ّإًسٗن الفْ فاذاز الملْٕ (ALP) ّإًسٗن الكراخ ثِٗ٘ يّظٌ٘از(LDH) كلل إًسٗن ه٘٘لْت٘رّكس٘ ازفٖ الغشاء الوخاؼٖ للوع ج تاإلظافح الٔ ل٘اش أؼ ّ ائػ اإللرِاتاخ فٔ هصه ال م ُّْ ااهه الْيم الٌخرٕ– ألفا (TNF-α).

ّ ل أثٓ الرعرض لإلشعاع أّ هسرخلص ثخاى السعائر إلٔ زٗاثج اإلظِاث الر كس ٓ ًر٘عح إًراض العلّيالؽرج لألكسع٘ي ّ الٌ٘ررّظ٘ي ّ ذل ٗشوه زٗاثج هلؽْظح فتٔ هسترْ ٓ هالًْت إ-ال ُاٗت ّ أّكستت٘ الٌ٘ررٗتت ّ ًمصتتا فتتٔ هستترْٓ ظلْذتتاشْ٘ى الوخرتتس ّكتتلل زٗتتاثج هلؽْظتتح فتتٔ ًشتتاغ ه٘٘لْت٘رّكس٘ از ّكلل زٗاثج هلؽْظتح فتٔ هسترْٓ ّ تػ٘ اإللرِاب.كوتا أظِترخ الٌرتائط زٗتاثج هلؽْظح فٖ ًشاغ إًسٗواخ الٌالالخ األهٌ٘٘ح ALT,AST)ّ إًسٗن الفْ فاذاز الملتْٕ (ALP) ّإًسٗن الكراخ ثِٗ٘ يّظٌ٘از (LDH). ّ ل لؼْظ زٗتاثج الرت ش٘ر العتاي فتٖ العتر ذاى الوعرظتح ألشعح ظاها ّالوعؽاج هسترخلص ثختاى الستعائر اٌتَ فتٖ الوعوتْار ٘ي الوشتععح اّ الرتٖ أاؽ٘تد هسرخلص السعائر كال الٔ ؼ ج.

ّل ذث٘ي أى الوعالعح ت ٕ هي كاذ٘ن٘ي أّ يّذ٘ي ذتدثٓ إلتٔ الْلاٗتح هتي ذت ش٘ر اإل شتعاع الوتدٗي أّ هسرخلص ثخاى السعائرؼ٘س أثٓ إلٔ الؽواٗح هتي زٗتاثج هسترْٓ هتالْى ثإ-ال ُاٗت ّ أّكست٘ الٌ٘ررٗ ّ ًمص هسرْٓ ظلْذاشْ٘ى الوخرس ّكلل السٗاثج فٔ ًشاغ ه٘٘لْت٘رّكس٘ از ّ السٗاثج فتٔ هسترْٓ ّ تػ٘ اإللرِتا ب فتٔ كته هتي العترذاى الوشتعع ح ّ الرتٖ ٗونتي إيظااِتا إلتٔ لت يج هعاثاخ االكس ج الٔ هٌع العلّي الؽرج هي الر ش٘ر العاي فتٔ االًستعح ّ ذلت ٳلًروائِوتا ٳلتٔ هعوْاح الفالفًْْٗ اخ الرٖ ذعوه الٔ هٌاُعح العتلّي الؽترج ّ تتلل ذؽوتٖ غشتاء الخل٘تح هتي أكستت ج التت ُْى. تاٳلظتتافح التتٔ أى كتتاذ٘ن٘ي ّ يّذتت٘ي تتثثا اًخفاظتتا فتتٖ ًشتتاغ إًسٗوتتاخ الٌتتالالخ األهٌ٘٘تتح ALT,AST) ّ إًتتسٗن الفْ تتفاذاز الملتتْٕ (ALP) ّإًتتسٗن الكرتتاخ ثِٗ٘تت يّظٌ٘از .(LDH)

٧1 الولخص العرتٖ

ل ذنْى ُلٍ اٙشاي ًر٘عح للمت ي ج التٔ المعتاء التٔ أّ هعاثلتح العتلّي الؽترجز ّزٗتاثج هسترْٗاخ هعاثاخ األكس ج األًسٗو٘تح ّغ٘تر األًسٗو٘تح كتلل ذصثتػ٘ هسترْٕ ّ تػ٘ األلرِتاب ااهته التْيم الٌخرٕ-ألفا ( فٖ الوصه .

وفى ضوء نتائج هزه الذساسة ، أمكن استخالص اآلتى:

٧- أثٓ الرعرض لالشعاع الودٗي أّ هسرخلص ثخاى السعائر إلٔ زٗاثج اإلظِاث الر كس ٓ ّ ذل ٗشوه زٗاثج هلؽْظح فٔ هسرْٓ هالْى ثإ-ال ُاٗ ّ الٌ٘ررٗد ّاًخفاظا فٔ هسرْٓ ظلْذتاشْ٘ى الوخرس ّكتلل زٗتاثج هلؽْظتح فتٔ ًشتاغ ه٘٘لْت٘رّكس٘ ازًّشتاغ إًسٗوتاخ خالٌتالال األهٌ٘٘تح ّ الفْ فاذاز الملْٕ ّ الكراخ ثِٗ٘ يّظٌ٘از ّ زٗاثج فٔ هسرْْٓ ػ٘ اإللرِاب فٖ هصه ال م.

٢- أثٓ ذعرض العرذاى لٳلشعاع تعراح ٢ ظرإ هع ذٌاّ هسرخلص ثخاى الستعائرالٔ زٗتاثج فٖ الرلف ذسٗ اي الٌاذط تسثة ذعرض العرذاى لٳلشعاع أّذٌاّ هسترخلص الستعائر كت ه التٔ ؼ ج.

٣-أظِتترالعالض الوستتثك تنتتاذ٘ن٘ي أّ يّذتت٘ي ؼواٗتتح ظتت اٳلظِتتاث الر كستت ٕ ذو٘تتسخ تتتٌمص فتتٖ هسرْٓ ال ُْى فْق الودكس ج كتلل ؼوتد ظت ايذفتاع هسترْٓ اّكست٘ الٌ٘ررٗت تاٳل ظتافح التٔ هٌع اًخفاض هسرْٓ ظلْذاشْ٘ى الوخرس فٖ ال م.

٤- أظِتتر كتتاذ٘ن٘ي ّيّذتت٘ي ًشتتاؼا هعتتاثا لٳل لرِتتاب هتتي ختتال ذخفتت٘ط هستترْٓ ااهتته التتْيم الٌخرٕ-ألفا فٖ هصه ال م ًّشاغ ه٘٘لْت٘رّكس٘ از فٖ الغشاء الوخاؼٖ للوع ج.

٥- الرتتتت ش٘ر الْلتتتتائٖ لنتتتتاذ٘ن٘ي ّ يّذتتتت٘ي ظتتتت الرعتتتترض لٳلشتتتتعاع ّ ثختتتتاى الستتتتعائر ّ التتتتلٕ أظِرذغ٘تتراخ فتتٖ إًسٗوتتاخ الٌتتالالخ األهٌ٘٘تتح ّالفْ تتفاذاز الملتتْٕ يتوتتا ٗرظتتع التتٔ لتت يج هرعتت ث الفٌْ٘ الوعاثج لألكس ج الٔ كثػ ظواغ لا ش ت ْ ا ي ث ا ل ع ا ه ؽ ت ح ه و ت ا ٗ و ٌ ت ع ذ ل ت ف ا أل ً س ت ع ح ّ ه ت ا ذ ؽ ر ْ ٗ ت َ ّهٌع خرّض اٳلًسٗواخ للوصه.

استنادا الى النتائج السابق ركشها فٳى ذفااالخ كاذ٘ن٘ي ّ يّذ٘ي الوعاثج لٳللرِاب ّ ل يذِوا الٔ ذمل٘ه اٳلظِاث الر كس ٕ ْٗظػ ل٘ورِوا الوؽرولح فٖ هٌع ذلف األًسعح ًر٘عح ذفااالخ العلّيالؽرج.ّتالرالٖ ٗوني ا رعوا كاذ٘ن٘ي ّ يّذ٘ي لم يذِوا الٔ زٗاثج فعال٘ح ّ أهاى العالض تاٳلشعاع هع ا رعوا ثخاى السعائر ّلني ُلا ٗرؽلة إظراء الوسٗ هي األتؽاز اي الفْائ الصؽ٘ح لِوا .

2 ٢

التأثٍر الضار لدخان السجائر فً الجرذان المشععة بأشعة جاما:الوقاٌة المحتملة لبعض المىتجات الطبٍعٍة

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تحت إشساف

أ.د./ سىاء عبد الباقً قىاوي أ.د./ مىى عبد اللطٍف الغزالً أصتبذ الفبزهبكولوجى أصتبذ الفبزهبكولوجى قضن األدويت والضووم زئيش الوسكز القوهي لبحوث و تكنولوجيب اإلشعبع كليت الصيدلت- جبهعت القبهسة هيئت الطبقت الرزيت - هصس

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