Kurdistan Regional Government Ministry of Higher Education and Scientific Research University of Sulaimani/College of Veterinary Medicine Department of Histopathology

EGFR protein expression after UVB radiation of mouse skin utilizing IHC technique, evaluation of total antioxidant status and assessing affectivity of antioxidants on EGFR expression. A thesis Submitted to the Council of the College of Veterinary Medicine As partial fulfillment of the degree of Master of Science in Pathology

By Azad Kareem Saeed B. V. M.S

Supervised By Assistant Professor Dr. Nabil Salmo M. B., Ch. B., MSc. Department of Pathology and Forensic Pathology School of Medicine

1432 (Hijri)

2011 (A. D.)

Committee Certification

ii

‫[سُورَةُ البَقَرة]‬

‫[سُورَةُ البَقَرة]‬

‫‪iii‬‬

Acknowledgement Thanks to God for giving me the patience and strength to achieve this study. I would like to express my deep sense of gratitude and humble regards to my supervisor, Assistant professor Dr. Nabil Salmo, Department of Pathology, School of Medicine, University of Sulaimani, who introduced me to scientific research and for his guidance during the course of this study and for his patience, support and encouragement. I wish to express my deep gratitude to the Dean, Professor Dr. Aumed Othman for permitting me to carry out this study and to use the facilities of this College. My deepest thanks and gratitudes are due to Dr. Rzgar R. Sulaiman, former manager of the Veterinary Teaching Hospital and all of the staff of Veterinary Teaching Hospital for their support and help in carrying out my work. Special thanks are due to Dr. Shilan M. Salih, for her support and encouragement throughout this study. I would also like to express my special thanks and appreciation to my graduate colleague, Dr. Snur M. Hassan for her help and kind cooperation throughout the period of this work. My deepest thanks and gratitudes are due to Dr. Newroz A. Tahir in the College of Agriculture for helping me with statistical analysis. I also express my profound sense of gratitude to all the doctors and technicians in the Histopathology Department of Shoresh Hospital for their cooperation and assistance, especially Dr. Michael Hughson and Miss Wesen A. Ali for their helpful assistance and generous support during my research study. Last, but not the least, I am thankful to all those who helped me directly or indirectly in carrying out this study.

iv

Abstract Background: UVB is the most damaging component of sunlight, has been shown to be much more carcinogenic than UVA; UVBR has been shown to induce morphologic, biochemical and genetic damage in skin keratinocytes, which lead to early neoplastic progression in this tissue. Skin exposure to UVBR generates reactive oxygen species (ROS) in excessive quantities that quickly overwhelm tissue antioxidants. Antioxidant molecules in the skin interact with ROS or their by-products to either eliminate or to minimize their deleterious effects. Chronic exposure to UV irradiation induces skin cancer, in part, through epigenetic mechanisms that result in dysregulation of cell proliferation. UV irradiation also rapidly activates the epidermal growth factor receptor (EGFR), which in turn will activate the proliferation of many cell types including keratinocytes of the skin because of its highly mitogenic properties. Aims of this study: The purposes of this study are to demonstrate the EGFR protein expression in keratinocytes after UVB exposure by application of IHC, to see the effect of antioxidants on EGFR expression in mouse skin upon UVB exposure and to compare the total antioxidant status (TAS) in the blood in control, exposure and treatment groups. Materials and methods: Forty mice were used in this experiment and were divided into 3 groups; 10 of which were considered as control group (not exposed and not treated with antioxidants); 15 of which were considered as exposure group (exposed to UVB light only) and 15 of which were considered as treatment group (exposed to UVB light and treated with antioxidants). Mice in treatment group were treated with antioxidants (vitamin E and selenium) in a dose of 4µl/1gm of body weight through S.C injection. The mice were then treated 3 days/week and exposed to UVB light 5 days/week throughout the experiment period of 3 months. Mice from both groups (exposure and treatment groups) were exposed to UVB light for 20 minutes. At the end of the experiment, incisional biopsies were taken. Sections were fixed, processed and embedded in paraffin blocks and three sections of 5µm thickness were taken from each paraffin embedded tissue block. The first section was mounted on an ordinary slide for H&E staining for detection of any histological lesions. The second section was for Giemsa stain for mast cells counting while the third section was mounted on positively charged slide, then proceeding with the process of immunohistochemistry staining following the protocol that was supplied with the kit of anti-EGFR (Super Sensitive™ Polymer-HRP IHC Detection System, Biogenex). Before taking biopsies, for TAS analysis 0.5ml of blood sample was collected from each mouse. Each blood sample was collected into an eppendorf tube, the serum was collected into another eppendorf tube and stored in the freezer until analysis. Results: The TAS was measured for each mouse’s serum; in the control group the mean value was equal to 1.201mmol/l, as there is no previous published study, this measurement was regarded as normal in the mouse. In exposure group the mean value decreased to 0.87mmol/l, but the treatment group the mean value elevated to 1.309mmol/l. There was a statistically significant relation between the effect of UVB on decreasing TAS level in

v

Abstract exposure group and the control group with a P-value of 0.0001 (highly significant), while there was no significant relation between treatment group and control group. Immunohistochemical staining was implemented to reveal the scores of EGFR expression in all cases, including 15 cases of seborrheic keratosis (SK) in exposure group and one case of SK in the treatment group, while the remaining 14 cases showed various degrees of epidermal hyperplasia which were also examined through IHC for EGFR. The highest score of EGFR expression in exposure group was 3+ with a frequency of 8 (53%) and the lowest score of EGFR expression was 2+ which was 7 (43%). The highest score of EGFR expression in the treatment group was 0 with a frequency of 6 (40%) and 2+ in 6 cases (40%) and the lowest score of EGFR expression was 1+ with a frequency of 3 (20%). There was a statistically significant relation between the effect of UVB on increasing number of apoptotic bodies in exposure group with a P-value of 0.0001 (highly significant), while apoptotic bodies decreased in treatment group with a P-value of 0.0001 (highly significant). There was a statistically significant relation between the effect of UVB on the increasing number of mast cells in exposure group with a P-value of 0.0001(highly significant), while mast cells decreased in treatment group with a P-value of 0.0001 (highly significant). Conclusions: UVB is the causative agent which induces SK in mice. EGFR expression is related to UVB irradiation in mice. Chronic UVB irradiation decreases the TAS level. Parenteral administration of antioxidants effectively reduces UVB-induced SK in mice, reduced EGFR expression, kept the TAS level within a normal range in most cases, decreased apoptotic bodies and mast cells. Recommendations: EGFR inhibitors can be used against skin tumor development. Administration of other types of antioxidants to reduce other types of tumors, even skin tumors and further studies will be needed to document this. Further studies on the relation between SK with EGFR and other genes such as; P53, Bcl-2 and FGFR3 are required to further explore and understand their role in determining unknown causative agents of SK development.

vi

Table of Contents No.

Subject

Page No.

CHAPTER 1

INTRODUCTION

1.1

Introduction

1

1.2

Aim of this study

2

CHAPTER 2

LITERATURE REVIEW

2.1

Normal skin histology

3

Introduction

3

Light

4

2.2.1

UV light

4

2.2.2

Ozone layer

6

2.2.3

Effects of UV radiation

6

2.2.4

How can UVB damage cells?

8

Epidermal growth factor receptor

8

2.3.1

Introduction

8

2.3.2

EGFR ligands

9

2.3.3

Architecture of the EGFR

12

2.3.4

Chromosomal location

13

2.3.5

Physiological role of EGFR

13

2.3.6

Pathological role of EGFR

13

2.3.7

UV irradiation and EGFR

15

Free radicals

16

2.4.1

Introduction

16

2.4.2

Roles of free radicals

18

2.4.3

Source of free radicals

19

2.4.4

Relation between oxidative stress and carcinogenesis

19

2.4.5

Production of ROS by UV radiation in skin

23

2.4.6

Metabolism of free radicals

23

Antioxidants

23

Introduction

23

Immunohistochemistry

33

2.6.1

Introduction

33

2.6.2

IHC methods

34

2.1.1 2.2

2.3

2.4

2.5 2.5.1 2.6

vii

Table of Contents 2.6.3

Applications of IHC

35

CHAPTER 3

MATERIALS AND METHODS

3.1

Animal model

36

3.1.1

Treatment of mice with antioxidants

36

3.1.2

UVB exposure

36

3.1.3

Blood collection and storage

37

Materials

37

3.2.1

Equipments

37

3.2.2

Reagents and solutions

38

Methods

38

3.3.1

Sample preparation

38

3.3.2

Immunostaining method

38

Slide interpretation

39

3.4.1

Immunohistochemical scoring

40

3.4.2

Giemsa staining

40

3.5

Total antioxidant status assay

40

3.6

Statistical analysis

41

CHAPTER 4

RESULTS

4.1

Macroscopical and microscopical findings

42

4.1.1

Non-irradiated control group

42

4.1.2

Irradiated (exposure) group

42

4.1.3

Treatment group

42

Total number of apoptotic bodies in 10 high power fields.

53

4.2.1

Effect of chronic UVB irradiation on total number of apoptotic bodies in exposure group.

53

4.2.2

Effects of antioxidants on total number of apoptotic bodies in chronically irradiated UVB in treatment group.

54

4.2.3

Difference in total number of apoptotic bodies in exposure and treatment groups.

55

Mean number of mast cells in 1 high power field.

57

4.3.1

Effect of chronic UVB irradiation on mean number of mast cells in exposure group.

57

4.3.2

Effect of antioxidants on mean number of mast cells in chronic UVB irradiation in treatment group.

57

3.2

3.3

3.4

4.2

4.3

viii

Table of Contents 4.3.3

Difference in mean number of mast cells in exposure and treatment groups.

58

4.3.4

Correlation between apoptotic bodies and mast cells.

60

Results of immunohistochemical scoring of EGFR expression.

63

4.4.1

Effect of UVB on frequency of EGFR expression scores and their percentages in exposure group.

64

4.4.2

Effect of antioxidants on frequency of EGFR expression score and their percentages in treatment group.

66

4.4.3

Difference in EGFR expression scores and their percentages in exposure and treatment groups.

67

Results of total antioxidant status measurement.

69

4.5.1

TAS measurement in control group.

69

4.5.2

TAS measurement in exposure group.

69

4.5.3

TAS measurement in treatment group.

71

4.5.4

Difference in TAS mean values among control, exposure and treatment groups.

72

Pearson’s correlation test.

73

4.6.1

Pearson’s correlation test for exposure group.

73

4.6.2

Pearson’s correlation test for treatment group.

74

DISCUSSION

76

Conclusions

80

Recommendations

80

REFERENCES

81

4.4

4.5

4.6

CHAPTER 5

ix

List of Tables Table No. 2-1 2-2 4-1 4-2 4-3 4-4 4-5 4-6 4-7 4-8 4-9 4-10 4-11 4-12 4-13

Title Types of free radicals. Function of selenium containing proteins. Skin lesions of exposure group after 3 months of exposing to UVB irradiation. Epidermal thickness in treatment group. Difference in total number of apoptotic bodies in exposure and treatment groups. Differences in the mean number of mast cells between exposure and treatment groups. Correlation between total number of apoptotic bodies and mean number of mast cells in exposure group. Correlation between total number of apoptotic bodies and the mean number of mast cells in treatment group. Correlation between mast cells and apoptotic bodies in exposure group to mast cells and apoptotic bodies in treatment group. Scores for EGFR protein expression in exposure group. Scores for EGFR protein expression in treatment group. Scores for EGFR protein expression in both exposure group and treatment group. Difference in TAS mean values in control, exposure and treatment groups. Pearson’s correlation for exposure group. Pearson’s correlation test for treatment group.

x

Page No. 17 31 52 53 55 58 61 62 63 66 67 67 72 74 75

List of Figures Figure No. 2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 2-9 2-10 2-11 2-12 2-13 2-14 2-15 2-16 2-17 2-18 2-19 4-1 4-2 4-3 4-4 4-5 4-6 4-7 4-8 4-9 4-10 4-11 4-12 4-13 4-14 4-15 4-16

4-17 4-18

Title Histological view of skin. Electromagnetic spectrum with expanded UV region. A model for induction of skin cancer by UV. Schematic diagram of the EGFR and downstream signaling pathway. Schematic diagram showing the set of interactions in the model of EGFR signalling, endocytosis and down-regulation. Schematic representation of domains of the epidermal growth factor receptor sequence. Mechanisms through which the UV-induced activation of EGFR contribute to skin tumorigenesis. Molecules that react with free radicals are in turn converted into free radicals. Mechanisms for producing free radicals and oxidative damage. Oxidative stress interacts with all three stages of the cancer process. Lipid peroxidation refers to the oxidative degradation of lipids. Protein oxidation by free radicals. DNA single-strand breaks are the most common damage inflicted by ROS. Oxidative stress produces a number of pathological conditions. Antioxidant neutralizing a free radical. Mechanisms of oxidative stress. Interacting network of non-enzymatic antioxidants. Neutralizing of free radicals by enzymatic antioxidants. Vitamin E prevents lipid peroxidation. Normal mouse skin appearance, after making a window. Normal mouse skin histology, unexposed to radiation (H&E X100). Different gross lesions in exposure group. A mouse from exposure group with eye damage. Microscopical appearance of seborrheic keratosis, acanthotic type (H&E X40). Microscopical appearance of seborrheic keratosis, acanthotic type (H&E X100). Squamous eddies; squamous cells resembling eddy currents in a stream (H&E X400). Black arrows showing sunburn cells or apoptotic bodies (H&E X400). (A): Moderate dermal inflammation and (B): Severe dermal inflammation (H&E X400). (A): Dermal mast cells (H&E X400) and (B): Dermal mast cells (Giemsa stain X400). Variable gross lesions in treatment group. (A): A mouse in control group, (B): A mouse in treatment group and (C): A mouse in exposure group. Mild epidermal hyperplasia (H&E X400). Moderate epidermal hyperplasia (H&E X400). Severe epidermal hyperplasia (H&E X400). (A): Normal epidermis (2-3 layers), (B): Mild epidermal hyperplasia of 4-6 layers, (C): Moderate epidermal hyperplasia of 7-9 layers and (D): Severe epidermal hyperplasia more than 10 layers (H&E X100). Pie chart showing the percentage degrees of epidermal thickness in treatment group. (A): Dermal mast cells in treatment group and (B): Dermal mast cells in

xi

Page No. 4 6 8 11 12 13 16 17 18 19 20 21 22 22 23 25 26 27 29 43 43 44 44 45 45 46 46 47 47 48 48 49 49 50 50

51 51

List of Figures

4-19 4-20 4-21

4-22 4-23 4-24 4-25 4-26 4-27 4-28 4-29 4-30 4-31 4-32 4-33 4-34 4-35 4-36 4-37 4-38 4-39 4-40 4-41 4-42

exposure group (Giemsa stain X100). Column chart showing the total number of apoptotic bodies of each case in exposure group. Column chart showing effects of antioxidants on total number of apoptotic bodies in treatment group. White arrows showing the differences in apoptotic body numbers between 2 different fields; (A): Exposure group and (B): Severe case of treatment group (H&E X400). Column chart showing the apoptotic bodies mean of both exposure and treatment groups. Column chart showing the effect of chronic UVB irradiation on mean number of mast cells in exposure group. Effect of antioxidants on mean number of mast cells in treatment group. Differences in the number of mast cells between two different fields; (A): treatment group and (B): exposure group (Giemsa stain X400). Column chart showing difference in mean number of mast cells in exposure and treatment groups. Column chart showing correlation between total number of apoptotic bodies and mean number of mast cells in exposure groups. Column chart showing correlation between total number of apoptotic bodies and the mean number of mast cells in treatment group. EGFR Score 0; no stain (X400). EGFR score 1+; weak stain (X400). EGFR score 2+; moderate stain (X400). EGFR score 3+; strong stain (X400). Pie chart showing the effect of UVB on frequencies of EGFR expression scores and their percentages in exposure group. Pie chart showing effect of the UVB on frequencies of EGFR expression scores and their percentages in treatment group. Column chart showing the effects of UVB on frequencies of EGFR expression scores in exposure group and treatment group. EGFR scores: (A): Score 0, (B): Score 1+, (C): Score 2+ and (D): Score 3+ (X400). Column chart showing the TAS measurement in normal mice (control group). Column chart showing TAS measurement in exposure group. An exposed sample showing increased bluish-green color. Column chart showing TAS measurement in treatment group. A treatment sample showing reduced bluish-green color. Column chart showing the difference of TAS mean values among different groups.

xii

54 55 56

56 57 58 59 60 61 62 64 64 65 65 66 67 68 68 69 70 70 71 72 73

List of Abbreviations 1

O2

Singlet oxygen

6-4P A1, A2, ∆A

6-4 photoproducts Absorbance

ABC

Avidin biotin complex

ABTS

(2,2'-Azino-di-[3-ethylbenzthiazoline sulphonate])

Akt

Serine/threonine protein kinase

ATP

Adenosine triphosphate

Bax

Bcl2–associated X protein

Bcl-2

B-cell lymphoma 2

CAT

Catalase

CFCs

Chlorofluorocarbons

CHS

Contact hypersensitivity

cis-UCA

cis-urocanic acid

CPD

Cyclobutane pyrimidine dimers

CR1

Cysteine-rich domain 1

CR2

Cysteine-rich domain 2

CT

Carboxy terminal

DAB

Diaminobenzidine

DNA

Deoxyribonucleic acid

ECM

Extracellular matrix

EGF

Epidermal growth factor

EGFR

Epidermal growth factor receptor

EGFRI

Epidermal growth factor receptor inhibitor

ERK1

Extracellular regulated kinase 1

ERK2

Extracellular regulated kinase 2

Fe+2

Ferrous

Fe

+3

Ferric

FGFR3

Fibroblast growth factor receptor 3

GDP

Guanosine diphosphate

GJIC

Gap junctional intercellular communication

GPx

Glutathione peroxidase

GR

Glutathione reductase

Grb2

Growth factor receptor-bound protein 2

GSH

Glutathione

GSSG

Oxidized glutathione

GTP

Guanosine triphosphate

H&E

Haematoxylin and Eosin

H2O2

Hydrogen peroxide

H2Se

Selenide

HIV

Human immunodeficiency virus

xiii

List of Abbreviations HOCl

Hypochlorous acid

HPF

High power field

HRP

Horse radish peroxidase

IAPs

Inhibitor of apoptosis proteins

IgG

Immunoglobulin gamma

IHC

Immunohistochemistry

IL-10

Interleukin-10

IL-1α

Interleukin-1 alpha

JAK

Janus kinase

JM

Juxtamembrane domain

JNK

C-jun NH2-terminal kinase

KDa

Kilodalton

L1

Ligand binding domain 1

L2

Ligand binding domain 2

LAB

Labeled avidin biotin

LPO

Lipid peroxide

LSAB

Labeled strept avidin biotin

mAbs

Monoclonal antibodies

MAPK

Mitogen activated protein kinase

mCRC

Metastatic colorectal cancer

MED

Minimal erythemal dose

Na2SeO3

Selenite

Na2SeO4

Selenate

NO

Nitric oxide

NO2

Nitric dioxide

NSCLC

Non small cell lung cancer

O2

.

Superoxide anion

OH.

Hydroxyl radical

ONOO

Peroxynitrite

P38

P38 protein kinase

P53

Protein 53

PAP

Peroxidase anti-peroxidase

PI3K

Phosphatidylinositol-3-kinase

PtdIns (3, 4, 5)P3 PtdIns (4, 5)P2 PUFA

Phosphatidylinositol (3, 4, 5) triphosphate Phosphatidylinositol (4, 5) bisphosphate Poly unsaturated fatty acid

Raf

Serine/threonine-specific protein kinases

Ras

Rat Sarcoma

RDA

Recommended daily allowance

RNS

Reactive nitrogen species

xiv

List of Abbreviations RO

Alkoxy

ROO

Peroxy radicals

ROOH

Organic hydroperoxide

ROS

Reactive oxygen species

rpm

Round per minute

RTK

Receptor tyrosine kinase

SBCs

Sunburn cells

SCCHN

Squamous cell carcinoma of the head and neck

Se

Selenium

SH2

Src Homology 2

SHC

SHC-transforming protein

SK

Seborrheic keratosis

SOD

Superoxide dismutase

SOS

Son of Sevenless

STAT

Signal transducer and activator of transcription or Signal transduction and transcription

TAS

Total antioxidant status

TGF-α

Transforming growth factor-alpha

TKI

Tyrosine kinase inhibitor

TNF-α

Tumor necrosis factor-alpha

trans-UCA

trans-urocanic acid

UV

Ultraviolet

UVA

Ultraviolet type A

UVB

Ultraviolet type B

UVBR

Ultraviolet B radiation

UVC

Ultraviolet type C

UVR

Ultraviolet radiation

VEGF

Vascular endothelial growth factor

xv

Chapter one

Introduction CHAPTER ONE INTRODUCTION

1.1: Introduction Chronic exposure to ultraviolet (UV) radiation leads to the development of non-melanoma skin cancer which is an increasingly prevalent disease that comprises approximately half of all diagnosed cancers in the United States (Pence et al., 1994; Thomas-Ahner et al., 2007). One of the results of UV irradiation in areas that have thinner ozone layers is an increasing rate of skin cancer (Huang et al., 1996). Ultraviolet B (UVB) is a minor component of sunlight reaching to the earth surface and is experimentally demonstrated to be the most effective light to induce skin cancer in animals. UVB radiation is a complete carcinogen, being able to initiate, promote and advance the development of skin cancer (Sluyter and Halliday, 2000; Ichihashi et al., 2003; Gottipati et al., 2008). Indeed, murine skin cancers induced by repeated exposure to UV radiation provides an excellent model system for investigating the molecular mechanisms of UV carcinogenesis. Experiments with animal models, particularly the mouse have already yielded a lot of data on how skin tumor development depends on dose, time and wavelength of the UV radiation (Dumaz et al., 1997). UV causes both DNA damage and epigenetic effects in response to DNA damage (El-Abaseri et al., 2005). The epigenetic effects of UV radiation (UVR) include changes in signaling and gene expression that regulate cell proliferation and survival in the short-term and contribute to skin cancer development in the longterm (El-Abaseri et al., 2006). Exposure to UV induces a number of pathological changes initiated in mammalian skin, including erythema, edema, epidermal hyperplasia, sunburn cell formation, immune suppression and changes in expression of numerous genes associated with proliferation, differentiation and eventually skin cancer development (Ley and Reeve, 1997; Fischer et al., 2003; Svobodova et al., 2003). UV exposure results in the rapid activation of epidermal growth factor receptor (EGFR) by a reactive oxygen intermediate-mediated mechanism. In response to UV-induced activation, EGFR increases cell proliferation, suppresses cell death (apoptosis), augments and accelerates epidermal hyperplasia. EGFR has been implicated previously in mouse skin carcinogenesis, because genetic ablation of the receptor reduces skin tumor growth (El-Abaseri et al., 2005; El-Abaseri et al., 2006; El-Abaseri and Hansen, 2007). EGFR overexpression is demonstrated in 30-100% of various solid tumors (Bianchini et al., 2008). Skin exposure to UV radiation generates reactive oxygen species (ROS) in excessive quantities that quickly overwhelm tissue antioxidants and other oxidant-degrading pathways. Parenteral administration of various antioxidants may reverse UVB-induced changes in cell cycle profile and cell cycle regulatory proteins (Bickers and Athar, 2006). Antioxidants are substances that neutralize free radicals or their actions. Vitamin E and selenium appear to act synergistically, which function as a chain-breaking antioxidant and prevent the propagation of free radical reactions in all cells in the body (Devasagayam et al., 2004; Bickers and Athar, 2006; Ramoutar and Brumaghim, 2010). Immunohistochemistry refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues. Expression of EGFR protein is visualized using a monoclonal antibody recognizing EGFR protein by immunohistochemistry protocol (GamboaDominguez et al., 2004; Ramos-Vara, 2005).

1

Chapter one

Introduction

1.2: Aims of this study 1.

To demonstrate the EGFR protein expression in keratinocytes after UVB exposure by application of IHC.

2.

To see the effect of antioxidants on EGFR expression in mouse skin upon UVB exposure.

3.

To compare the total antioxidant status (TAS) in control, exposure and treatment groups.

2

Chapter two

Literature review CHAPTER TWO LITERATUTE REVIEW

2.1: Normal skin histology: 2.1.1: Introduction The skin is the largest organ of the body and is approximately one sixth of the total body weight. It has four major functions including protection, sensation, thermoregulation and metabolic activity. It varies over regions of the body in thickness, color and presence of appendages such as hairs, glands and nails (Urmacher, 1990; Young and Heath, 2002; Topping et al., 2006; Gartner and Hiatt, 2007). Skin is composed of three layers (Figure 2-1): A. Epidermis: The epidermis is a stratified keratinized squamous epithelium that dynamically renews itself but maintains its normal thickness through the process of desquamation. The majority of cells in the epidermis are keratinocytes (90- 95%). The rest of the epidermal cells are non-keratinocytes (5-10%) and they include melanocytes, Langerhans cells and Merkel cells. The following five morphologically distinct layers of the epidermis can be identified. From the inner to the outer layer: 1.

Stratum basale (Germinativum).

2.

Stratum spinosum (Prickle-cell layer).

3.

Stratum granulosum.

4.

Stratum lucidum (In thick skin only).

5.

Stratum corneum (Young and Heath, 2002; Eroschenko, 2005; Gartner and Hiatt, 2007; Mills, 2007).

B. Dermis: The dermis is the connective tissue which supports the epidermis and binds it to the subcutaneous tissue (hypodermis). It contains two layers with rather indistinct boundaries; the outermost papillary layer and the deeper reticular layer. The thin papillary layer is composed of loose connective tissue, fibroblasts and other connective tissue cells, such as mast cells and macrophages. The reticular layer is thicker, composed of irregular dense connective tissue and therefore has more fibers and fewer cells than does the papillary layer (Junqueira and Carneiro, 2003; Eroschenko, 2005). C. Hypodermis: The hypodermis layer consists of loose connective tissue that binds the skin loosely to the subjacent organs, making it possible for the skin to slide over them. When the hypodermis is infiltrated by many adipocytes, this layer is then called the panniculus adiposus (Banks, 1993; Junqueira and Carneiro, 2003). Mouse skin histology: The mouse skin consists of thin epidermis (2-3 layers), dermis and hypodermis. The mouse skin has no eccrine sweat glands; these are located in its footpad only. The mouse does not normally have rete ridges where

3

Chapter two

Literature review

the lower aspect of the epidermis forms ridges of cells that extend into the dermis. Rete ridge-like structures become prominent when mouse skin heals following ulceration (Hedrich and Bullock, 2004; Atlas of Laboratory Mouse Histology, 2011).

Figure (2-1): Histological view of skin (http://www.lab.anhb.uwa.edu.au/mb140/CorePages/Integumentary/Integum.htm, 2011).

2.2: Light Early scientists described electromagnetic radiation or “light” as having a “wave nature”. This explained the behavior of light being bent by a lens or dispersed into various colors (spectrum) by a prism or by water droplets forming a rainbow. The energy of the sun reaching the earth is known as electromagnetic radiation, which consisted of the many forms of energy that are recognized as visible light, infrared, ultraviolet, X-rays, etc. These terms simply define different portions of electromagnetic radiation with which we associate specific phenomena such as sight (light), heat (infrared) and medical examinations (X-rays). They are all portions of the electromagnetic radiation that differ from one another in “energy”. The more energetic regions of the spectrum are at smaller or shorter wavelengths (Gibson, 2011).

2.2.1: UV light Sunlight is composed of a continuous spectrum of electromagnetic radiation that is divided into three main regions of wavelengths; ultraviolet, visible and infrared (Soehnge et al., 1997). UV light belongs to the non-ionizing part of the electromagnetic spectrum and ranges between 200nm and 400nm; 200nm has been chosen arbitrarily as the boundary between non-ionizing and ionizing radiation (Clydesdale et al., 2001; Svobodova et al., 2003; IARC, 2006). UV levels will be highest around noon on a clear sunny day. UV levels will also be its highest near surfaces that reflect sunlight, such as snow or sand (Earth gauge, 2011).

4

Chapter two

Literature review

UV radiation is further divided into three sections (Figure 2-2), each of which has distinct biological effects (Jablonski and Chaplin, 2000; Lucas et al., 2006; Ashawat et al., 2007): a.

UVC (200-290nm).

b.

UVB (290-320nm).

c.

UVA (320-400nm).

Ultraviolet A: UVA has the longest wavelength in contrast to the other types of UV. It penetrates deep into the epidermis and dermis of the skin (Svobodova et al., 2003; Zeman, 2009). Most of us are exposed to large amounts of UVA throughout our lives. UVA rays account for up to 95% of the UV radiation reaching the earth’s surface. Although they are less intense than UVB, they pose relatively equal intensity during daylight hours throughout the year and can penetrate clouds and glass (Navy Environmental Health Center, 1992; Skin Cancer Information, 2011). Ultraviolet B: UVB is the most damaging component of sunlight (Maeda et al., 2005). Its intensity varies by season, location and time of day. The most significant amount of UVB hits the United States between 10am and 4pm from April to October each year. Most of the UVB absorbed by ozone layer, only 1-5% reaches our planet. However, UVB does not pass through glass in contrast to UVA (Zeman, 2009; Canadian Centre for Occupational Health and Safety, 2011; Skin Cancer Information, 2011). Furthermore, UVB is more genotoxic and about 1000 time more capable of causing sunburn than UVA. UVB is less penetrating and acts mainly in the epidermal basal cell layer of the skin (Svobodova et al., 2006). Ultraviolet C: UVC has the shortest wavelength; it is the most energetic and has the greatest potential for biological damage to all forms of life, even with only very short exposures. It is highly mutagenic and toxic. It is absorbed by proteins and nucleic acids and is extremely damaging to the skin, however, it is completely absorbed by the ozone layer and does not reach the ground and it is frequently used in germicidal lamps to destroy bacteria and other organisms (Svobodova et al., 2006; Zeman, 2009; Centers for Disease Control and Prevention, 2011).

5

Chapter two

Literature review

Figure (2-2): Electromagnetic spectrum with expanded UV region (Goldman, 2005).

2.2.2: Ozone layer The ozone layer is a natural shield in the stratosphere, which absorbs most of the harmful UV radiation before it reaches the earth’s surface, but its thickness varies depending on the time of year and the changing weather patterns (Environmental Protection Agency, 2006). Ozone depletion, observed since 1985, is due to environmental pollutants like chlorofluorocarbons (CFCs), a long lived chemicals used in coolants in refrigerators, air conditioners, foam-blowing agents and solvents (Narbutt et al., 2005). It has been calculated that a 10% reduction in ozone layer would raise the incidence of melanoma by 10%, basal cell carcinoma cases by 20% and squamous cell carcinoma by 50% (Amerio et al., 2009).

2.2.3: Effects of UV radiation Exposure to UV radiations can have both positive and negative effects. The ultimate effects depend primarily on the amount of exposure an individual receives (Schwab and Siekmann, 2002): I.

Positive effects (Schwab and Siekmann, 2002; Akram and Rubock, 2005):

A. The formation of vitamin D is the most significant of the positive effects, which is used in the prevention of rickets. B. Light and UV radiations are also considered to have positive effects for therapeutic purposes, i.e., in treating skin disease. C. Ultraviolet radiation is widely used for killing microorganisms.

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Negative effects: The negative effects are due to too much exposure to UV radiation. It can impact both the skin and the

eyes; the severity on which depend on the duration, intensity and wavelength of exposure (Schwab and Siekmann, 2002; Akram and Rubock, 2005). A. Skin damage (Schwab and Siekmann, 2002; Mudgil et al., 2003; Narbutt et al., 2005): 1.

Sunburn (erythema): Inflammation of the skin that disappears after several days. This results in pigmentation (the tan) and a thickening of the top-most epidermal (stratum corneum) skin layers which in turn increases the body’s resistance against a renewed burn.

2.

Aging of the skin: Frequently repeated or long-term exposure to UV radiation can make the skin dry, leathery, rough, slack and cause wrinkling.

3.

Skin cancer: UV is a well known etiologic agent for skin cancer through DNA damage, damage to the repair system and immunosuppression. Skin cancer is the most common form of cancer in the United States. There are more than 1 million skin cancers diagnosed annually. There are three primary types of skin cancers: basal cell carcinoma, squamous cell carcinoma and melanoma.

4.

Phototoxic reactions (photo allergy): The combination of UV radiation and certain chemicals (e.g.: particular medications and cosmetics) can cause toxic reactions and trigger an allergic reaction.

B. Eye damage (Schwab and Siekmann, 2002): 1.

Inflammation of the cornea (keratitis), photo-conjunctivitis: UV radiations can destroy the outermost cells of the cornea and/or the conjunctiva. This phenomenon is known to mountain climbers as “snow blindness” and to welders as “flashing”. The damage is felt for six to eight hours after the exposure in the form of pain in the eyes. The condition heals completely after one or two days.

2.

Clouding of the lens (cataracts): Among other causes, long-term exposure of the eyes to UV radiation can lead to an irreversible clouding of the lens tissue. This is particularly true for people who are often outdoors (farmers, seamen). The condition primarily affects the elderly. These may develop age-related cataracts after decades of exposure to sunlight.

C. Suppression of the immune system: UV radiation alters the body’s immune responses and makes it more susceptible to infections. Many genes and several viruses, from Herpes simplex to Human immunodeficiency virus (HIV), are activated by ultraviolet radiation. Ultraviolet radiation also decreases the effectiveness of vaccines (Dresbach and Brown, 2008). UVB can induce immune suppression at both local and systemic levels. The systemic suppression results from the induction of suppressor T cells, either by damaged Langerhans cells or inflammatory macrophages which enter the skin following UV exposure. In addition, UV irradiation can also convert normal skin chromophores into agents that are immunosuppressive, such as the conversion of trans-urocanic acid to cisurocanic acid (Figure 2-3). This mediates immunosuppression through alteration in tumor antigen presentation by Langerhans cells and release of cytokines such as Interleukin-10 (IL-10), tumor necrosis factor-alpha (TNFα) and Interleukin-1 alpha (IL-1α) (Soehnge et al., 1997; IARC, 2006; Welsh et al., 2008).

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2.2.4: How can UVB damage cells? The most cytotoxic and mutagenic wave band among types of solar radiation corresponds to UVB light (Ichihashi et al., 2003). UVB radiation can cause cell damage through different mechanisms. UVB is a complete carcinogen that is absorbed by DNA and can directly damage DNA. This damage, induced by UVB irradiation, typically includes the formation of cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4P). If repair mechanisms fail to restore genomic integrity, mutations are likely to occur and persist through subsequent cell divisions (Liebler and Burr, 2000; IARC, 2006; Kumari et al., 2008). UVB is known to upregulate gene expression through intracellular signal transduction pathways, which may contribute to developing skin cancer at the tumor promotion stage (Ichihashi et al., 2003).

Figure (2-3): A model for induction of skin cancer by UV (Soehnge et al., 1997).

2.3 : Epidermal growth factor receptor 2.3.1: Introduction Epidermal growth factor receptor (EGFR) was the first receptor tyrosine kinase (RTK) to be discovered. Most of the principles and paradigms of RTK activation were first established for EGFR (Nair, 2005). EGFR, also known as HER-1/ErbB1 belongs to the ErbB family of RTKs, which includes three other members, namely,

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ErbB2/HER-2, ErbB3/HER-3 and ErbB4/HER-4 (Teraishi et al., 2005; Scaltriti and Baselga, 2006; Olsen et al., 2007). EGFR is a single transmembrane glycoprotein of approximately 170 kDa that was discovered in the mid-1970s as a binding site for epidermal growth factor (EGF) (Lee et al., 2004; Rampaul et al., 2005; ElAbaseri and Hansen, 2007). The EGFR molecule consists of the following (Rowinsky, 2004): o

Extracellular or ligand-binding domain: The portion of the protein located outside the cell that contains the site where binding to growth factors such as epidermal growth factor (EGF) or transforming growth factoralpha (TGF-α).

o

Transmembrane domain: The portion of the receptor located inside the cell membrane that anchors the receptors in the cell membrane.

o

Intracellular domain: The portion of the receptor that projects into the interior of the cell. The intracellular portion is responsible for transferring signals to other proteins inside the cell.

2.3.2: EGFR ligands EGF, acting through its receptor EGFR, is a potent mitogen for epidermal keratinocytes (Rundhaug and Fischer, 2010). EGF is now known to be part of a larger family of ligands for ErbB receptors. These growth factors are of three types: those that bind to EGFR including EGF, amphiregulin and TGF-α; those that bind to both EGFR and ErbB4 including betacellulin, epiregulin and heparin-binding EGF-like growth factor and those that bind to ErbB3 and ErbB4 including heregulins (Harris et al., 2003; El-Abaseri and Hansen, 2007; Siwak et al., 2010). EGFR becomes activated by receptor overexpression (frequent in cancer) as well as ligand-dependent and ligand-independent mechanisms (Scaltriti and Baselga, 2006). Binding of ligands, lead to homo and heterodimerization of the receptors (either with a second EGFR or with another member of the ErbB family). Dimerization, in the case of EGFR, leads to autophosphorylation of specific tyrosine residues in the intracellular tyrosine kinase domain. These phosphorylated tyrosine residues serve as docking sites for other kinases, including the mitogen activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI3K/Akt) pathway, modulating gene transcription and protein translation. Activation of the ErbB receptor family triggers a number of different responses including mitogenesis, suppression of apoptosis, cellular motility, angiogenesis and differentiation (Lynch et al., 2007; Olsen et al., 2007; Song et al., 2008). Several modes of indirect EGFR (ligand-independent receptor) activation have been described. Stimulation of EGFR phosphorylation occurs after treatment with unphysiological stimuli, including hyperosmolarity, oxidative stress, mechanical stress, UV light and γ-irradiation (Goldkorn et al., 1997; Scaltriti and Baselga, 2006). This effect has been predominantly attributed to the inactivation of phosphatases that antagonize the intrinsic receptor kinase activity, thereby shifting the equilibrium of basal autophosphorylation and dephosphorylation towards the activated state (Kedar et al., 2002; Oksvold et al., 2002). Cellular signaling generally involves protein-protein interactions and enzymatic activities involved in the cellular response to a signal (Blinov et al., 2006). Intracellular signaling occurs via several pathways (Figure 2-4):

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1. Ras/Raf/MAPK: This is a critically important route that regulates cell proliferation and survival. Following EGFR phosphorylation, the complex formed by the adaptor proteins Grb2 and Son of Sevenless (SOS) binds directly, or through association with the adaptor molecule SHC, to specific docking sites on the receptor. This interaction leads to a conformational modification of SOS, now able to recruit Ras-GDP, resulting in Ras activation (RasGTP). Ras-GTP activates Raf-1 that, through intermediate steps, phosphorylates the mitogen-activated protein kinases; extracellular regulated kinases 1 and 2 (ERK1 and ERK2) (Henson and Gibson, 2006; Scaltriti and Baselga, 2006). Activated MAPKs are imported into the nucleus where they phosphorylate specific transcription factors involved in cell proliferation and increased transcription of Bcl-2 family members and inhibitor of apoptosis proteins (IAPs), thereby promoting cell survival (Hill and Treisman, 1995; Gaestel, 2006; Henson and Gibson, 2006). 2. PI3K/Akt pathway: This pathway is involved in cell growth, apoptosis resistance, invasion and migration (Vivanco and Sawyers, 2002; Shaw and Cantley, 2006). EGF promotes cell survival through the activation of PI3K/Akt signaling. EGF triggers the recruitment of PI3K to activated ErbB receptors, which is mediated by the binding of SH2 domains in PI3Kto the phosphorylated tyrosine residues. The catalytic subunit of PI3K in turn phosphorylates phosphatidylinositol (4,5) bisphosphate (PtdIns (4,5) P2) leading to the formation of PtdIns (3,4,5) P3. PI3K can also activate Ras, resulting in the activation of ERK signaling, thereby facilitating cross-talk between survival pathways. A key downstream effector of PtdIns (3,4,5) P3 is Akt (PKB). Akt promotes cell survival through the transcription of anti-apoptotic proteins. Collectively, these processes all promote cell growth and survival in response to EGF (Lizcano and Alessi 2002; Asnaghi et al., 2004; Henson and Gibson 2006). 3. JAK/STAT pathway: Another signaling cascade initiated by EGF is the JAK/STAT pathway, which is also implicated in cell survival responses. JAK phosphorylates STAT proteins localized at the plasma membrane. This leads to the translocation of STAT proteins to the nucleus where they activate the transcription of genes associated with cell survival (Kisseleva et al., 2002; Henson and Gibson 2006).

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Figure (2-4): Schematic diagram of the EGFR and downstream signaling pathway (Rocha-Lima et al., 2007). Besides sending signals to downstream effectors, the activated EGFR will also initialise endocytosis; internalized receptors are either degraded in lysosomes or translocated to the nucleus, where they act as transcription factors or co-regulators of gene transactivators (Figure 2-5) (Casey et al., 2007; Siafaca et al., 2007).

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Figure (2-5): Schematic diagram showing the set of interactions in the model of EGFR signalling, endocytosis and downregulation (Casey et al., 2007).

2.3.3: Architecture of the EGFR The EGFR is synthesized from a 1210-residue polypeptide precursor; after cleavage of the N-terminal sequence, a 1186-residue protein is inserted into the cell membrane. Over 20% of the receptor’s 170-kDa mass is N-linked glycosylation and this is required for translocation of the EGFR to the cell surface and subsequent acquisition of function (Jorissen et al., 2003; Nair, 2005). Overexpression of the EGFR or altered glycosylation can reveal peptide epitopes suitable for antibody therapies. The sequence can be categorized into a number of domains as shown in figure 2-6 (Johns et al., 2002; Jorissen et al., 2003). The EGFR extracellular portion (or ectodomain) consists of four domains referred to as L1, CR1, L2 and CR2 domains (Garrett et al., 1998). Ligand binds between the L1 and L2 domains of the EGFR. The CR1 and CR2 domains consist of a number of small molecules, each appearing to be held together by one or two disulphide bonds. When the receptor dimerizes, loops from CR1 make contact with each other (Garrett et al., 2002; Ogiso et al., 2002; Nair, 2005).

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The juxtamembrane region appears to have a number of regulatory functions like downregulation and ligand-dependent internalization events (Yamabhai and Anderson, 2002). The carboxy-terminal domain of the EGFR contains tyrosine residues where phosphorylation modulates EGFR mediated signal transduction (Jorissen et al., 2003).

Figure (2-6): Schematic representation of domains of the epidermal growth factor receptor sequence (Jorissen et al., 2003).

2.3.4: Chromosomal location The gene responsible for producing EGFR proteins was assigned to human chromosome 7, whereas in mouse EGFR protein is located in chromosome 11 (Wang et al., 1993; Klysik and Singer, 2005).

2.3.5: Physiological role of EGFR Normal body cells grow, divide and die in an orderly fashion. As part of this process, cells receive signals from their external environment in the form of chemical messengers specifically EGFR, which is activated when the naturally occurring ligands EGF or TGF-α bind to the extracellular domain, this binding triggers internal cellular signal that stimulate cell growth (Rowinsky, 2004). RTKs as well as cytoplasmic protein tyrosine kinases play a dominant role to control normal cellular processes during embryonic development and to regulate many metabolic and physiological processes in a variety of tissues and organs (Nair, 2005). In naïve skin, EGFR stimulates proliferation, regulates differentiation and promotes cell survival. Upon trauma to the skin, the receptor increases cell adhesion and the migration of keratinocytes during wound reepithelialization (El-Abaseri and Hansen, 2007). Mice lacking EGFR die soon after birth, exhibiting impairment of epithelial cell development in various organs, including the skin, lung and gastrointestinal tract (Siafaca et al., 2007).

2.3.6: Pathological role of EGFR The EGFR is proposed to participate in the pathogenesis or maintenance of a number of cancers of epithelial origin. This supposition is based on the common finding that numbers of EGFR ligands are elevated in tumors or that the EGFR itself is overexpressed, amplified, or constitutively activated by ligand interaction or mutation (Hansen et al., 2000). EGFR overexpression has been reported in a variety of premalignant lesions as well as epithelial malignancies, including lung, prostate, gastric, breast, colon, pancreatic, head and neck cancer. The EGFR is expressed approximately 100 times more than the normal number of EGFR found on the surface of the normal cell. EGFR is dysregulated in various tumor types and its overexpression has been correlated with disease

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progression, poor prognosis and a reduced sensitivity to chemotherapy. EGFR overexpression, is demonstrated in 30-100% of various solid tumors (Levitzki, 2003; Zhang, 2005; Lynch et al., 2007; Bianchini et al., 2008). Not surprisingly, since cell proliferation is necessary for tumor promotion, EGFR signaling plays an important role in skin carcinogenesis (Rundhaug and Fischer, 2010). EGFR has been implicated previously in mouse skin carcinogenesis, because genetic ablation of the receptor reduces skin tumor growth (Dlugosz et al., 1997). Activation of the EGFR promotes multiple properties of neoplastic cells, including proliferation, migration, angiogenesis, stromal invasion and resistance to apoptosis (Nair, 2005; Rocha-Lima et al., 2007; Patel et al., 2009). Hyperplasia: Hyperplasia (hypergenesis) means increase in number of cells/proliferation of cells. It may result in the gross enlargement of an organ and the term is sometimes mixed with benign neoplasia/benign tumor. Hyperplasia is a common preneoplastic response to stimulus. Microscopically cells resemble normal cells but are increased in numbers. Sometimes cells may also increase in size. Hyperplasia is different from hypertrophy in that the adaptive cell change in the latter is an increase in cell size, whereas the former involves an increase in the number of cells. Hyperplasia is an adaptive response in cells capable of replication, whereas hypertrophy occurs when cells are incapable of dividing (Carlton and McGavin, 1995; Jones et al., 1997; Kumar et al., 2007; McGavin and Zachary, 2007). Hyperplasia is considered to be a physiological (normal) response to a specific stimulus and the cells of a hyperplastic growth remain subject to normal regulatory control mechanisms, this stands in contrast to neoplasia (the process underlying cancer and benign tumors) in which genetically abnormal cells proliferate in a non physiological manner which is unresponsive to normal stimuli (Cotran et al., 1999). Hyperplasia may be due to any number of causes, including increased demand (for example, proliferation of basal layer of epidermis to compensate skin loss), chronic inflammatory response, hormonal dysfunctions, or compensation for damage or disease elsewhere. Hyperplasia may be harmless and occurs in a particular tissue. An example of a normal hyperplastic response would be the growth and multiplication of milksecreting glandular cells in the breast as a response to pregnancy, thus preparing for future breast feeding (Antonio and Gonyea, 1994; Jones et al., 1997). Cell migration: Cell migration is important in many biological processes such as embryogenesis, angiogenesis, inflammatory reactions and wound repair. These processes are thought to be regulated by interactions with other cells, cytokines and extracellular matrix (ECM) proteins. EGF has been reported to be a chemoattractant for different epithelial cells such as keratinocytes, intestinal cells, corneal cells and liver epithelial cells (Palmer et al., 1999). Apoptosis: Cell death can be divided into two classes, apoptosis and necrosis. Apoptosis has come to be used synonymously with the phrase “programmed cell death” as it is a cell-intrinsic mechanism for suicide regulated by a variety of cellular signaling pathways. For cell death to be classified as apoptotic, nuclear condensation and fragmentation, cleavage of chromosomal DNA into internucleosomal fragments and packaging of the deceased cell into apoptotic bodies without plasma membrane breakdown must be observed. Apoptotic bodies are

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recognized and removed by phagocytic cells and thus apoptosis is also notable for the absence of inflammation around the dying cell (Edinger and Thompson, 2004). Angiogenesis: Angiogenesis is the physiological process involving the growth of new blood vessels from pre-existing vessels (Sun et al., 2007; Penn, 2008). Angiogenesis is a critical and obligatory component of promotion, progression and metastasis of solid cancers, most of which are of epithelial origin (Lu and Jiang, 2001). The main steps that occur in angiogenesis from pre-existing vessels are listed below (Kumar et al., 2007): •

Vasodilation in response to nitric oxide and increased permeability of the pre-existing vessel induced by vascular endothelial growth factor (VEGF).



Migration of endothelial cells toward the area of tissue injury.



Proliferation of endothelial cells just behind the leading front of migrating cells.



Inhibition of endothelial cell proliferation and remodeling into capillary tubes.



Recruitment of periendothelial cells (pericytes for small capillaries and smooth muscle cells for larger vessels) to form the mature vessel.

Metastasis: Metastasis refers to the transfer of malignant cells from one site to another not directly connected with it (Rubin and Strayer, 2008). Metastasis occurs by one of three basic routes: 1- Direct exfoliation of tumor cells from a primary neoplasm into a body cavity with subsequent implantation and growth of cells on mesothelial surface, a process referred to as seeding. 2- Invasion of lymphatics and transport of the tumor cells as emboli in the lymph. 3- Direct invasion of blood vessels with dissemination of the neoplastic emboli via blood stream (Jones et al., 1997). Tumor cells require proteolytic degradation of the extracellular matrix for each step of tumorigenesis. These events include the ability of malignant cells to invade surrounding stroma by degrading the basement membrane and extracellular matrix components, such as collagen, laminin, fibronectin and proteoglycan (Cvejić et al., 2000).

2.3.7: UV irradiation and EGFR The increased incidence of skin tumors following UV exposure is believed to stem from the combined influences of DNA damage and its epigenetic effects. These effects include changes in signaling and gene expression that regulate cell proliferation and survival in the short-term and contribute to skin cancer development in the long-term (El-Abaseri et al., 2006). EGFR is rapidly activated following exposure to UV, this is a result of both the induction of EGFR ligands and through reactive oxygen species mediated inactivation of the cytoplasmic protein tyrosine phosphatases that maintain low basal levels of phosphorylated EGFR (ElAbaseri et al., 2005; El-Abaseri and Hansen, 2007), thus blocking deactivation, altering receptor internalization

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and degradation and increasing EGFR ligand expression at a later point. UV exposure therefore results in both a short lived and immediate effect as well as a more delayed and prolonged activation of EGFR (El-Abaseri et al., 2006). Upon UV-induced activation of the receptor, EGFR increases proliferation, suppresses apoptosis, augments hyperplasia and increases UV-induced skin tumorigenesis (Figure 2-7) (El-Abaseri et al., 2006; ElAbaseri and Hansen, 2007).

Figure (2-7): Mechanisms through which the UV-induced activation of EGFR contribute to skin tumorigenesis (El-Abaseri and Hansen, 2007).

2.4: Free radicals 2.4.1: Introduction Free radicals can be defined as molecules or molecular fragments containing one or more unpaired electrons. The presence of unpaired electrons usually confers a considerable degree of reactivity upon a free radical. Those radicals derived from oxygen represent the most important class of species generated in living systems (Valko et al., 2004; Zadak et al., 2009). In addition, free radicals initiate autocatalytic reactions; molecules that react with free radicals are in turn converted into free radicals (Figure 2-8), further propagating the damage chain (Kumar et al., 2007).

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Figure (2-8): Molecules that react with free radicals are in turn converted into free radicals (www.healthy-antiaging-for-life.com/free-radicals.html, 2011). Reactive oxygen species (ROS) comprise a number of active metabolites including hydroxyl radical (OH・), superoxide anion (O2・-) and peroxyl radical and hydrogen peroxide (H2O2). Reactive nitrogen species (RNS), such as nitric oxide (NO) and nitric dioxide (NO2), are also generated (Table 2-1) (Sander et al., 2004). ROS and RNS: (i) are generated during irradiation by UV light, by X-rays and by γ-rays; (ii) are products of metal-catalyzed reactions; (iii) are present as pollutants in the atmosphere; (iv) are produced by neutrophils and macrophages during inflammation; (v) are by-products of mitochondria-catalyzed electron transport reactions and other mechanisms (Figure 2-9) (Valko et al., 2006). Table (2-1): Types of free radicals (Lobo et al., 2010). Free radicals

Description

O2- superoxide anion

One-electron reduction state of O2, formed in many autoxidation reactions and by the electron transport chain. Rather unreactive but can release Fe2+ from iron-sulfer proteins and ferritin. Undergoes stimulation to form H2O2 spontaneously or by ezymatic catalysis and is a precursor for metal-catalyzed OH formation.

H2O2

hydrogen

Two-electron reduction state, formed by dismutation of O2- or by direct reduction of

peroxide

O2. Lipid soluble and thus able to diffuse across membranes.

OH. hydroxyl radical

Three-electron reduction state, formed by Fenton reaction and decomposition of peroxynitrite. Extremely reactive, will attack most cellular components.

ROOH,

organic

Formed by radical reactions with cellular components such as lipids and nucleobases.

hydroperoxide RO, alkoxy and ROO,

Oxygn centered organic radicals. Lipid forms participate in lipid peroxidation

peroxy radicals

reactions. Produced in the presence of oxygen by radical addition to double bonds or hydrogen abstraction.

HOCl,

hypochlorous

Formed from H2O2 by myeloperoxidase. Lipid soluble and highly reactive. Will

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ONOO-, peroxynitrite

Formed in a rapid reaction between O2- and NO. Lipid soluble and similar in reactivity to hypochlorous acid. Protonation forms peroxynitrous acid, which can undergo homolytic cleavage to form hydroxyl radical and nitrogen dioxide.

Figure (2-9): Mechanisms for producing free radicals and oxidative damage (Young and Woodside, 2001).

2.4.2 : Roles of free radicals ROS and RNS are known to play a dual role in biological systems, as they can be either harmful or beneficial to living systems (Valko et al., 2004; Ramoutar and Brumaghim, 2010). The beneficial effects of ROS occur at low/moderate concentrations and involve physiological roles in cellular responses to noxious, for example, in defense against infectious agents and in the function of a number of cellular signaling systems. One further beneficial example of ROS at low/moderate concentrations is the induction of a mitogenic response. The harmful effect of free radicals causing potential biological damage is termed oxidative stress and nitrosative stress (Valko et al., 2007). Oxidative stress occurs when this critical balance is disrupted due to depletion of antioxidants or excess accumulation of ROS, or both. That is, when antioxidants are depleted and/or if the formation of ROS increases beyond the ability of the defenses to cope, then oxidative stress and its detrimental consequences ensue (Klaunig et al., 1998; Scandalios, 2005). The delicate balance between beneficial and harmful effects of free radicals is a very important aspect of living organisms and is achieved by mechanisms called “redox regulation”. The process of “redox regulation” protects living organisms from various oxidative stresses and maintains “redox homeostasis” by controlling the redox status in vivo (Dr¨oge, 2002).

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2.4.3: Source of free radicals ROS can be produced both endogenously and exogenously (Klaunig et al., 1998). Free radicals are produced endogenously during cellular metabolism (endogenous sources include mitochondria, cytochrome P450 metabolism, peroxisomes and inflammatory cell activation). Their production may be greatly enhanced by exogenous factors like environment pollutants, drugs, radiation and pathogens (Figure 2-9) (Das et al., 2000; Inoue et al., 2003; Scandalios, 2005). ROS are byproducts of aerobic life. Although ROS normally account for approximately 1% of the oxidant load in aerobes, these levels can reach up to 17% during times of oxidative stress and in humans; increased levels of ROS have been associated with cancer, atherosclerosis and diabetes (Pelle et al., 2003). Transition metals: Most metal ions, particularly copper and iron, are essential enzyme cofactors. In its free form these ions in biological systems can facilitate transfer of electrons to susceptible macromolecules such as proteins, lipids and DNA. It is important for organisms that free transition metals in biological fluids are restricted to very low levels (Figure 2-9). Exposure to redox-active transition metals is controlled primarily through the action of specific chelating proteins such as ceruloplasmin or transferrin (Zadak et al., 2009).

2.4.4: Relation between oxidative stress and carcinogenesis ROS may be involved in carcinogenesis through two possible mechanisms (Figure 2-10): the induction of gene mutations that result from cell injury and the effects on signal transduction and transcription factors. Cellular targets affected by oxidative stress include DNA, phospholipids, proteins and carbohydrates on the cell membrane (Noda and Wakasugi, 2001).

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Figure (2-10): Oxidative stress interacts with all three stages of the cancer process (Klaunig et al., 1998). At high concentrations, ROS can be important mediators of damage to cell structures, nucleic acids, lipids and proteins (Valko et al., 2007). 1. Lipid peroxidation: Double bonds in membrane polyunsaturated lipids are vulnerable to attack by ROS (Kumar et al., 2007). The hydroxyl radical removes a hydrogen atom from the unsaturated fatty acids of membrane phospholipids, a process that forms a free lipid radical. The lipid radical, in turn, reacts with molecular oxygen and forms a lipid peroxide radical. This peroxide radical can, in turn, function as an initiator, removing another hydrogen atom from a second unsaturated fatty acid. A lipid peroxide and a new lipid radical result and a chain reaction is initiated. Lipid peroxides are unstable and break down into smaller molecules. The destruction of the unsaturated fatty acids of phospholipids results in a loss of membrane integrity (Figure2-11) (Rubin and Strayer, 2008).

Figure (2-11): Lipid peroxidation refers to the oxidative degradation of lipids (Clark, 2008).

2. Protein interactions: Hydroxyl radicals may also attack proteins. The sulfur-containing amino acids cysteine and methionine, as well as arginine, histidine and proline, are especially vulnerable to attack by OH・. As a result of oxidative damage, proteins undergo fragmentation, cross-linking, aggregation and eventually degredation (Figure 2-12) (Rubin and Strayer, 2008). ROS have been implicated as second messenger in regulating gene expression. It has been shown that oxidative stress can modulate the activity of protein kinases, which in turn phosphorylate a wide range of cellular proteins (Clair et al., 2005).

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Figure (2-12): Protein oxidation by free radicals. (a) Hydrogen atom elimination at the α-carbon of the protein backbone, resulting in backbone fragmentation, or (b) hydrogen atom elimination at side chains, resulting in different products, including peroxides, alcohols and carbonyls. As with lipid perioxidation, protein peroxides are unstable and propagate further reactions (Clark, 2008).

3. DNA damage: The hydroxyl 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 & Gutteridge, 1999). DNA is an important target of the hydroxyl radical. A variety of structural alterations include strand breaks, modified bases and cross-links between strands. In most cases, the integrity of the genome can be reconstituted by the various DNA repair pathway. However, if oxidative damage to the DNA is sufficiently extensive, the cell dies (Figure 213) (Rubin and Strayer, 2008). Oxidized and injured DNA have the potential to induce genetic mutation. It is apparent that some telomere genes are highly susceptible to mutation in the presence of free radicals and it is known that tumor suppressor genes such as p53 and cell cycle-related genes may suffer DNA damage (Noda and Wakasugi, 2001).

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Figure (2-13): DNA single-strand breaks are the most common damage inflicted by ROS (Clark, 2008). The excess ROS can damage cellular components inhibiting their normal function. Because of this, oxidative stress has been implicated in a number of human diseases as well as in the ageing human process (Figure 2-14) (Chang et al., 2007; Valko et al., 2007).

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Figure (2-14): Oxidative stress produces a number of pathological conditions (www.antioxidants-healthguide.com/oxidative-stress.html, 2011).

2.4.5: Production of ROS by UV radiation in skin Skin is very susceptible to UV radiation, skin exposure to UV induces extensive generation of ROS. These can cause oxidative damage to organic compounds. Such injuries result in a number of harmful effects; disturbed cell metabolism, morphological and ultrastructural changes, attack on the regulation pathways and alterations in the differentiation, proliferation and apoptosis of skin cells (Svobodova et al., 2003; MasatsujiKato et al., 2005; Black et al., 2008). UVB induces cell cycle alterations in epidermal keratinocytes similar to those evoked by ROS. In addition, parenteral administration of various antioxidants may reverse UVB induced changes in the cell cycle profile and cell cycle regulatory proteins. Similarly, both UVB and ROS induce apoptosis in keratinocytes by altering mitochondrial membrane permeability (Bickers and Athar, 2006).

2.4.6: Metabolism of free radicals ROS are natural and inseparable part of metabolism. In skin, they are constantly generated in keratinocytes and fibroblasts and are rapidly removed by enzymatic and non-enzymatic antioxidants; this maintains the pro-oxidant/antioxidant balance, thus resulting in cell and tissue stabilization (Inal et al., 2001; Svobodova et al., 2003; Sander et al., 2004). The excess of free radicals results in a cascade of events mediating a progressive deterioration of a cellular structure and function and this can lead to the differentiation of neoplasic tissues (Afaq and Mukhtar, 2002). It has been reported that ROS/RNS induce various types of oxidative DNA lesions that are thought to be important for the initiation stage in carcinogenesis (Nishigori et al., 2004). Antioxidant molecules in the skin interact with ROS or their by-products to either eliminate or to minimize their deleterious effects (Bickers and Athar, 2006).

2.5: Antioxidants 2.5.1: Introduction An antioxidant is a molecule capable of inhibiting the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions that damage cells (Sies, 1997). Exposure to free radicals from a variety of sources has led organisms to develop a series of defence mechanisms (Cadenas, 1997). Defence mechanisms against free radical-induced oxidative stress involve (Valko et al., 2007): o

Preventative mechanisms.

o

Repair mechanisms.

o

Physical defences.

o

Antioxidant defences. Antioxidants effectively neutralize free radicals, for example, by giving them the electron they so

desperately seek (Figure 2-15), but without becoming an active free radical in themselves, thus break the chain

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of reactions. Antioxidants therefore, play an important role in maintaining the health and integrity of all the different types of cells within the body. In fact the balance between free radicals production and antioxidants is thought to be strongly related to lifespan (Marlin and Dunnett, 2007).

Figure (2-15) : Antioxidant neutralizing a free radical (Kris Health Blog, 2011). A variety of proteins and enzymes synthesized in the body may function as antioxidants (Basu et al., 1999); such substances include enzymatic antioxidant defences which further includes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). Non-enzymatic antioxidants are represented by ascorbic acid (Vitamin C), tocopherol (Vitamin E), glutathione (GSH) and other antioxidants. Under normal conditions, there is a balance between both the activities and the intracellular levels of these antioxidants (Figure 2-16). This balance is essential for the survival of organisms and their health (Mates et al., 1999; McCall and Frei, 1999; Masatsuji-Kato et al., 2005; Valko et al., 2007).

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Figure (2-16): Mechanisms of oxidative stress (Nuttall et al., 1999). A good antioxidant should (Valko et al., 2006): 1.

Quench free radicals.

2.

Chelate redox metals.

3.

Interact with (regenerate) other antioxidants within the “antioxidant network”.

4.

Have a positive effect on gene expression.

5.

Be readily absorbed.

6.

Have a concentration in tissues and biofluids at a physiologically relevant level.

7.

Work in both the aqueous and/or membrane domains. Certain antioxidants are able to regenerate other antioxidants and thus restore their original function.

This process is called an “antioxidant network” (Sies et al., 2005). The redox cycles of vitamins E and C form such an antioxidant network. The capacity to regenerate one antioxidant by another is driven by the redox potentials of the [Red/Ox] couple (Figure2-17). There is a link between increased levels of ROS and disturbed activities of enzymatic and non-enzymatic antioxidants in tumor cells (Valko et al., 2006).

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Figure (2-17): Interacting network of non-enzymatic antioxidants (Pinnell, 2003). Enzymatic antioxidant levels and non-enzymatic antioxidant levels in the cell can be decreased through modification in gene expression, decreased in their uptake in the diet, or can be overloaded in ROS production, which creates a net increase in the amount of oxygen free radicals present in the cell (Klaunig et al., 1998). Enzymatic antioxidants protect by directly scavenging superoxide radicals and hydrogen peroxide, converting them to less reactive species. SODs catalyze the dismutation of O 2•− to H2O2 and CAT and peroxidases reduce H2O2 to 2H2O (Figure 2-18). The similarity between the SOD and CAT reactions is that each is an oxidationreduction in which the substrate, O2•− for SOD and H2O2 for CAT, is both reductant and oxidant, whereas different reductants are required for the peroxidases, depending on their specificities (Scandalios, 2005).

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Figure (2-18): Neutralizing of free radicals by enzymatic antioxidants (www.benbest.com/lifeext/aging.html, 2011). Effects of cell type on antioxidant levels: Antioxidant enzyme defences vary depending on skin cell type. Fibroblasts have more catalase, peroxidase and SOD activity than either keratinocytes or melanocytes. Melanocytes were found to have the smallest amounts of the antioxidants measured and may rely on the radical scavenging ability of melanin rather than other antioxidant enzymes. However, melanin is stored in membrane-bound cytoplasmic vesicles and may not be available to freely scavenge non-melanosomal toxic oxygen species; therefore, melanin would not be protective against cell membrane or cytoplasmic insults, which would be a disadvantage during acute UV exposure (Jurkiewicz, 1995). Ultraviolet radiation effects on antioxidants: The skin naturally relies on antioxidants to protect it from oxidant stress generated by sunlight and pollution (Thiele et al., 2000). Both enzymatic and non-enzymatic antioxidant defences form a protective network against reactive forms of oxygen produced by UV radiation in the skin. Antioxidants are found to inhibit UV-induced lipid peroxidation and reduce chronic cutaneous damage by scavenging ROS (Jurkiewicz, 1995). Antioxidant enzymes may be depleted or inactivated during UV radiation exposure resulting in lowered defence capabilities. Depending upon what oxidative products are produced, antioxidants may be affected to varying degrees. For example, although glutathione peroxidase is quite resistant to oxidative damage by hydrogen peroxide, it is quite vulnerable to depletion by hydroperoxides, which is produced by UV radiationinduced lipid peroxidation in the skin (Jurkiewicz, 1995). Enzyme activities in human skin are higher in epidermis than dermis; catalase is especially high. When skin fibroblasts were irradiated with UVA, catalase activity was preferentially destroyed, superoxide dismutase

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activity was diminished, but glutathione peroxidase and glutathione reductases were virtually unchanged. Similar results were seen when murine skin was irradiated with solar irradiation (Pinnell, 2003). Low molecular weight, non-enzymatic antioxidants include ascorbic acid in the fluid phase, glutathione in the cellular compartment, vitamin E in membranes and ubiquinol in mitochondria. On a molar basis, ascorbic acid is the predominant antioxidant in skin; its concentration is 15-fold greater than glutathione, 200-fold greater than vitamin E and 1000-fold greater than ubiquinol/ubiquinone. Concentrations of antioxidants are higher in epidermis than dermis; 6-fold for ascorbic acid and glutathione and 2-fold for vitamin E and ubiquinol/ubiquinone. Solar-simulated irradiation of murine skin, reduced levels of non-enzymatic antioxidants. Ubiquinol/ubiquinone and glutathione were most sensitive; while α-tocopherol and ascorbic acid were less sensitive (Pinnell, 2003). Patients with actinic keratosis and basal cell carcinoma have significantly decreased plasma levels of ascorbic acid, α-tocopherol and glutathione (Vural et al., 1999). Antioxidant supplementation: Supplementation of skin with various antioxidants may compensate for UV radiation induced depletion thereby preventing free radical damage. Topical application and dietary supplementation of certain antioxidants were found to decrease erythema following acute UV exposure and delay the onset of tumor formation in chronically exposed animals. Topical application of ascorbate or α- tocopherol was found to be most effective against short-term UV radiation-induced lipid peroxidation products in the skin, whereas application of glutathione, superoxide dismutase and catalase were found to have minimal to no protective effect in the system examined. Dietary supplementation of antioxidants, though found to be less effective than topical application due to the length of time required to reach optimal concentrations in the skin, are also found to protect against UV radiation-induced damage. These antioxidant studies provide strong circumstantial evidence of a significant role for antioxidants in protecting skin against UV radiation-induced damage (Jurkiewicz, 1995). A. Vitamin E: Vitamin E is the most important fat-soluble, membrane-bound antioxidant in the body. Several forms of vitamin E exist in nature (Burke et al., 2000). Vitamin E is a term that encompasses a group of potent, fatsoluble, chain-breaking antioxidants. Structural analyses have revealed that molecules having vitamin E antioxidant activity include four tocopherols (α, β, γ, δ) and four tocotrienols (α, β, γ, δ) (Brigelius-Flohe and Traber, 1999; Valko et al., 2006). The molecules consist of a hydrophobic prenyl tail that inserts into membranes and a polar chromanol head group exposed to the membrane surface. Tocopherols and tocotrienols differ only in their prenyl tails. Tocopherols have linear, saturated tails whereas tocotrienols have a nonlinear unsaturated tail. Human beings use predominantly α-tocopherol because a specific α-tocopherol transfer protein selectively transfers α-tocopherol into lipoproteins (Pinnell, 2003). Synthetic vitamin E, designated dl-α-tocopherol, is the less expensive cousin of the naturally occurring form, d-α- tocopherol. The natural form of the vitamin is synthesized only by plants and is found predominantly in plant oils. Vitamin E (tocopherol) is also present in high amounts within the chloroplast and therefore the leaves of most plants. In contrast, the tocotrienols are synthesized and found in the germ and bran sections of the plant. The fat-soluble property of vitamin E allows it to be stored within fatty tissue of animals and humans,

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thus, a diet that includes meat supplies additional vitamin E. However, the amount of vitamin E obtained in a meat inclusive diet is less than the amount supplied by plant sources (Groff et al., 1995). Serum concentrations of vitamin E (α-tocopherol) depend on the liver, which takes up the nutrient after the various forms are absorbed from the small intestine. The liver preferentially resecretes only α-tocopherol via the hepatic α-tocopherol transfer protein (Traber, 2006). The liver metabolizes and excretes the other vitamin E forms (Traber, 2007). As a result, blood and cellular concentrations of other forms of vitamin E are lower than those of α-tocopherol and have been the subject of little research (Dietrich et al., 2006; Sen et al., 2006). Vitamin E is the major antioxidant involved in maintaining cell membrane integrity (Einstein et al., 1994). Vitamin E has been suggested to also play an important role in the functioning of the immune system and is believed to be important for normal growth and muscle function (Marlin and Dunnett, 2007). One form, αtocopherol, is the most abundant form in nature, has the highest biological activity based on fetal resorption assays and reverses vitamin E deficiency symptoms in humans (Brigelius-Flohe and Traber, 1999). The major antioxidant function of vitamin E is to prevent lipid peroxidation, when ROS attacks membrane lipids, a peroxyl radical may form to create more peroxyl radicals, resulting in a chain reaction which may threaten the structural integrity of the membrane. Tocopherols and tocotrienols scavenge the peroxyl radical, ending the chain reaction (Figure 2-19). Vitamin E may also quench singlet oxygen. Once oxidized, vitamin E can be regenerated back to its reduced form by ascorbic acid, allowing it to be reactivated without creating a new membrane structure (Figure 2-17). The relative antioxidant activities of tocopherol in lipid systems is α > β > γ > δ (Pinnell, 2003).

Figure (2-19): Vitamin E prevents lipid peroxidation (Elkashef and Wyatt , 1999).

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The antioxidant activity of vitamin E has persuaded many groups to study its ability to prevent chronic diseases, especially those believed to have an oxidative stress component such as cardiovascular diseases, atherosclerosis and cancer (Brigelius-Flohe and Traber, 1999). Vitamin E is especially abundant in stratum corneum, delivered there in sebum. Its concentration is highest at the lower levels of the stratum corneum, with a decreasing gradient outward. The stratum corneum is the outermost defence of the body and first to absorb the oxidative stress of sunlight and pollution. Vitamin E is depleted in the process and in the absence of co-antioxidants, is unable to be regenerated. Vitamin E is important for protecting the lipid structures of the stratum corneum and for protecting stratum corneum proteins from oxidation. The lipophilic nature of vitamin E makes it attractive for application to and absorption into skin (Pinnell, 2003). Topical application of tocopherol has been shown to decrease the incidence of UV-induced skin cancer in mice. Vitamin E provides protection against UV-induced skin photodamage through a combination of antioxidant and UV absorptive properties (Jacobson et al., 2005). Topical α-tocopherol protected rabbit skin against UV-induced erythema, mouse skin against UV-induced lipid peroxidation, mice against UV-induced photoaging changes, mice against UV immunosuppression and mice against UV photo carcinogenesis (Pinnell, 2003). Topical application of α-tocopherol on mouse skin inhibits the formation of cyclobutane pyrimidine photoproducts. However, topically applied α-tocopherol is rapidly depleted by UVB radiation in a dosedependent manner (Liebler and Burr 2000) as vitamin E in the skin can absorb UV light and generate the tocopheryl radical (Jacobson et al., 2005). Commercially available, vitamin E supplements usually contain only α-tocopherol, provided either unesterified or usually as the ester of acetate, succinate, or nicotinate. Supplements can contain either the natural d-α-tocopherol or the synthetic dl-α-tocopherol (Brigelius-Flohe and Traber, 1999). Vitamin E function is similar to that of a selenium containing enzyme and it is thus able to protect animals against selenium defeciency (and vice versa). The most common product used in diet supplementation is α-tocopherol acetate, which is often given in combination with selenium (Einstein et al., 1994). B. Selenium: Selenium is an essential micronutrient required for at least 2 types of enzymes involved in defence against oxidative stress in mammals (Table 2-2). These enzymes, namely glutathione peroxidase and thioredoxin reductase, represent significant portion of the cell’s total defence against oxidative stress and are vital to maintaining a stable redox balance in the cell (Pinnell, 2003). The status of essential microelement selenium is primarily determined by its food intake. The low selenium levels in food chain elements correspond to its low levels in people. Selenium levels in plasma/serum in European countries range from 63-110μg/l, whereas selenium status in the Slovak population is at the bottom of this scale. Optimum activity of this antioxidant enzyme is achieved in serum/plasma at selenium (Se) levels between 90-100μg/l (Mrázová et al., 2009).

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Deficiency of selenium produced experimentally in animals resulted in abnormalities such as defective growth, hepatic necrosis, myocardial degeneration and muscular dystrophy in sheep, cattle, chickens and horses. In humans, it is well recognized that selenium plays a crucial role in various physiological processes and its altered level has a direct impact on our health, leading to the development of diseases (Saxena and Jaiswal, 2007). Table (2-2): Function of selenium containing proteins (Saxena and Jaiswal, 2007). Selenium containing proteins and their functions No.

Name

Function

1

Glutathione peroxidase

- An antioxidant enzyme decomposes H2O2 and other hydroperoxides. - Maintains intracellular redox milieu. - Replenishes a number of crucial antioxidants e.g., vitamin E and C from their oxidized state. -Forms a structural protein and shields the developing sperm cells.

2

Thioredoxin reductase

- Provides protection to skin from free radicals. - Protein thiol redox regulation. -Vitamin C recycling and DNAsynthesis.

3

Iodothyronine deiodinases

- Synthesis of active thyroid hormone.

4

Selenoprotein 1R

-Provide protection against free radical mediated oxidative stress.

5

Selenoprotein P

- Functions as an antioxidant. - In the transport of selenium. -Protection against Hepatitis B virus X protein induced lipid peroxidation.

6

Selenoprotein S

-Regulation of cellular redox balance.

Biological forms: Selenium occurs in both inorganic and organic forms. Among the inorganic forms (i.e., selenates (Na2SeO4), selenides (H2Se) and selenite (Na2SeO3)); the selenide form is more frequently found in the food supply. These selenates and selenites are reduced to selenides in the liver with dimethyl and trimethyl selenide as the end products. The organic form includes selenomethionine and selenocysteine and is found in plants and animals respectively (Marlin and Dunnett, 2007; Saxena and Jaiswal, 2007; Ramoutar and Brumaghim, 2010). Sources of selenium: Selenium is an important micronutrient for both humans and animals. It’s obtained through diets including cereals, grains, nuts, vegetables, meat and seafood (Ramoutar and Brumaghim, 2010). Plant foods are the major sources of selenium in most countries throughout the world. The amount of selenium present in the plant material depends upon the concentration of selenium in the soil of that region as it varies by region, whereas dairy products, fruits and vegetables are relatively poor sources of selenium (Saxena and Jaiswal, 2007). Absorption: At high concentrations, naturally occurring seleno-amino acids and soluble selenium salts are readily absorbed.

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Seleno-amino acids are more quickly absorbed than selenite and selenate, which are in turn more quickly absorbed than selenides and elemental selenium.

2.

The small intestine is the primary absorption site; no absorption takes place in the stomach or rumen.

3.

Absorbed selenium is transported by plasma proteins (Osweiler, 1996).

Metabolism: a)

Selenium is metabolized by both reduction and methylation.

b) Selenium may form a complex with mercury and proteins that reduces the availability of the complex component (Osweiler, 1996). Deposition: 1.

A selenium-protein-cadmium complex increases tissue deposition but reduce toxicity.

2.

Because of its availability to replace sulfur in amino acids, selenium has a high affinity for hair and epithelial structures (e.g., hooves and horns) (Osweiler, 1996).

Excretion: o

Urine is the major route of excretion in monogastric animals, but ruminants have significant amounts in the feces as well.

o

Normally, a very small amount of selenium is excreted in bile. Arsenic enhances biliary excretion of selenium.

o

Methylated selenium is volatile and may be excreted by the lungs (Osweiler, 1996). The recommended daily allowance (RDA) for selenium ranges from 55µg/day to an upper limit of 350-

400µg/day and daily intake comes from dietary supplementation and foods rich in this mineral. Although selenium toxicity has been observed for supplementation greater than 400µg/day, it is important to note that some studies conducted with a selenium intake ranging from 750-850µg/day (0.01mg/kg) reported no signs of selenium toxicity in humans. Animal studies reported selenium toxicity within 12 hours upon supplementation of 2mg/kg selenium (Ramoutar and Brumaghim, 2010). There are at least 30 selenoproteins that have been identified in mammals and it has been estimated that humans have about 25 selenoproteins. The functional roles of some of these selenoproteins are still not fully understood (Jackson and Combs, 2008; Tinggi, 2008). The selenoenzyme glutathione peroxidase (GPx) can reduce and detoxify H2O2 as well as various organic hydroperoxides at the expense of glutathione (GSH) to form oxidized glutathione (GSSG). GSH is naturally regenerated from GSSG by the catalyst glutathione reductase (GR) (Jurkiewicz, 1995). In selenoenzymes, the selenium is present as selenocysteine and a specific and elaborate system exists for its incorporation into these proteins. The activity of selenoenzymes can be increased by selenium supplementation. Several cellular studies have demonstrated the protective effects of selenium for UV-induced damage including cytotoxicity, DNA oxidation, DNA damage, IL-10 expressions and lipid peroxidation. Oral sodium selenite protected hairless mice against UV-induced erythema and subsequent pigmentation. Oral selenium protected mice against UV-induced skin cancer, although an oral trial in human beings, failed to protect against basal or squamous cell carcinoma. Topical selenomethionine protected mice against UV-induced erythema and skin cancer. In human beings, topical selenomethionine increased the minimal erythema dose in a dose-responsive fashion (Pinnell, 2003). Selenium has sparing effect on vitamin E. As a component of GSH and GPx, selenium helps to destroy peroxides and thereby reduces the peroxidation of PUFA (poly unsaturated fatty acid) of lipid membrane which,

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in turn, reduces the vitamin E requirement for the maintenance of membrane integrity. In addition, GSH and GPx also play a central role in co-ordinating the synergism of various antioxidants (vitamin C and E) (Sen, 1995; Chatterjea and Shinde, 2002). Current researches emphasize the combined effect of vitamin E and selenium in amelioration of depleted level of antioxidant enzymes and are thought to be a key factor in disease prevention. In connection with this, it was demonstrated that combined vitamin E and Se deficiency is associated with alteration in the expression level of genes encoding for proteins involved in inflammation and acute phase response (Fischer et al., 2001). It also observed that combined vitamin E and Se supplementation decreases the peroxidative tissue damage by promoting the antioxidative defence systems in the kidney of rats (Beytut et al., 2004). C. Total antioxidant status (TAS): Total antioxidant status (TAS) assays, measure the capacity of biological samples only under defined conditions prescribed by the given method using different oxidants in each case (Apak et al., 2007). The total antioxidant status is used to determine the level of oxidative stress, an increased formation of reactive oxygen species leads to increased oxidative stress and decreased antioxidant capacity (Okuonghae et al., 2011). Most of the commercially available kits for total antioxidants estimation are based on the principle of supression of ABTS (2,2'-Azino-di-[3-ethylbenzthiazoline sulphonate]) radical cation formation by antioxidant in the serum sample (Gupta et al., 2009). TAS kit has been widely used to determine in vivo free radical trapping power in patients with various disease conditions. In relation with several diseases, plasma or serum TAS was consistently decreased in patients with cancer, hypertension, osteoporosis and psoriasis, but was not significantly changed in patients with acute myocardial infarction (Kwak and Yoon, 2007).

2.6: Immunohistochemistry 2.6.1: Introduction The immunohistochemistry (IHC) is a combination of immunologic and chemical reactions visualized with a photonic microscope (Ramos-Vara et al., 2008). Immunohistochemistry refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues. It takes its name from “immune,” in reference to antibodies used in the procedure and “histo” meaning tissue (Ramos-Vara, 2005). IHC is a key tool for the analysis of localization of target molecules within tissues. It is used routinely for almost every aspect of modern biomedical research. Technical ease of use, rapidity and reliability usually determine the techniques utilized in academic or medical settings. Most importantly, immunohistochemistry performed on real tissues offer no simple and direct way to compare the specificity of an antibody for related molecules (Levin, 2004; Goldstein et al., 2007). Immunohistochemical technique has equipped the histopathologist with the tools needed to tackle the most common diagnostic problems in tumor pathology especially the characterization of the undifferentiated or poorly differentiated malignant tumors, whether primary or metastatic. No other method, during the past fifty years, has had such a major impact on histopathology (Delellis and Dayal, 1987; Chan, 2000; Coindre, 2003). The publication of a paper by Coons et al. in 1941, describing an immunofluorescence technique for detecting cellular antigens in tissue sections, marked the beginning of IHC. Since then, IHC has become a

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valuable tool in both diagnosis and research of infectious and neoplastic diseases in humans and animals (Ramos-Vara, 2005). Simply defined, IHC is the study of antigen to antibody interactions and how these reactions are visualized in tissues. The primary antibody is applied to the tissue, where the antigen is (or is suspected to be) present. The antigen is made up of a combination of several proteins in a specific sequence and conformation. The site on the antigen where the antibody binds is referred to as the “epitope” and is made up of generally 5-16 amino acids, which may represent a small percentage of the length of the total antigen. One antigen may have multiple “epitopes” (antibody binding sites). Each binding site is given a different name, referred to the antibody clone name. The antibody will bind to the epitope and the detection system used will allow for visualization of this antibody-antigen reaction (Elias, 2003). IHC provides the most direct method for identifying both the cellular and subcellular distribution of protein and can provide a relatively rapid indication of gene expression or protein distribution (Mello and Fire, 1995).

2.6.2: IHC methods An antigen-antibody interaction can be visualized using following methods: 1-Direct method is a one-step staining method and involves a labeled antibody (Different labels have been used, including fluorochromes, enzymes, colloidal gold and biotin) reacting directly with the antigen in tissue sections. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity is lower due to little signal amplification and is less commonly used than indirect methods. 2- Indirect method involves an unlabeled primary antibody (first layer) that binds to the target antigen and a labeled secondary antibody (second layer) that reacts with the primary antibody. The secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. This method is more sensitive than direct detection strategies because of signal amplification; due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to the fluorescent or enzyme receptor (Carson, 1997; Ramos-Vara, 2005 and Mashhood, 2008). A- Avidin-biotin complex (ABC) method: ABC method is standard IHC method and one widely used technique for immunhistochemical staining. Avidin is a large glycoprotein, which can be labeled with peroxidase or fluorescent and has a very high affinity for biotin. The technique involves three layers. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is a complex of avidin-biotin peroxidase linked with appropriate label. The peroxidase is then developed by the DAB or other substrate to produce different colorimetric end products. B- Labeled avidin-biotin (LAB) or labeled streptavidin-biotin (LSAB) method: Streptavidin, derived from Streptococcus avidini, is a recent innovation for substitution of avidin. LSAB is technically similar to standard ABC method. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is enzyme-streptavidin conjugates (HRP-Streptavidin or AP-Streptavidin) to replace the complex of avidin-biotin peroxidase. A recent report suggests that LSAB method is about 5-10 times more sensitive than standard ABC method.

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C- Peroxidase–antiperoxidase (PAP) method: PAP method is a further development of the indirect technique and it involves a third layer which is a rabbit antibody to peroxidase, coupled with peroxidase to make a very stable peroxidase anti-peroxidase complex. The sensitivity is about 100-1000 times higher since the peroxidase molecule is not chemically conjugated to the anti IgG but immunologically bound and loses none of its enzyme activity. D- Polymeric methods: Polymeric Methods are based on dextran polymer technology and new method of polymerizing enzymes and attaching these polymers to antibody (Boenisch, 2001; Ramos-Vara, 2005 and Chen et al., 2010).

2.6.3: Applications of IHC 1.

Diagnosis of tumors of uncertain histogenesis.

2.

To identify abnormal protein deposits within cells.

3.

IHC is widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.

4.

IHC is a highly sensitive and specific method, especially advantageous as a diagnostic tool for infectious diseases in animals.

5.

Categorization of leukemia and lymphomas.

6.

Identifying the origin and type of secondary deposits.

7.

Identification of hormone receptors, which are of prognostic value as estrogen, progestron and HER-2/neu in breast cancer to determine the mode of treatment (Levin, 2004; Tuffaha and Muin, 2008).

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CHAPTER THREE MATERIALS AND METHODS 3.1: Animal model A prospective study was conducted from October 22, 2010 to February 30, 2011, at two different locations. The first part of the study was housing, exposure and treatment of mice; carried out at the Veterinary Medicine Teaching Hospital and the second part which included fixation, processing, paraffin block, sectioning and staining by conventional H&E stain, immunohistochemistry technique and serum analysis carried out in the histopathology laboratory and chemical laboratory of Shoresh Hospital in Sulaimani Governorate. Mice from Mus musculus species, BALB/c strain, each of which weighed 20-40g, of both sexes with approximately the same age and were obtained from the animal house of the University of Sulaimani, School of Pharmacy, there were acclimatized for 7 days before starting the experiment and fed with poultry pellet standard ration, bread, milk and cucumber with drinking water. Forty mice were used in this experiment and were divided into 3 groups; 10 of which were considered as control group (not exposed and not treated with antioxidants); 15 of which were considered as exposure group (exposed to UVB light only) and 15 of which were considered as treatment group (exposed to UVB light and treated with antioxidants).

3.1.1: Treatment of mice with antioxidants Mice, 3-4 weeks of age, were housed in climate quarters (25°C), with a 12/12 hours light/dark cycle under a white fluorescent light. The antioxidants used in this study were as an injectable solution (vitamin E and selenium) in a dose of 4µl/1g of body weight. The mice were treated with antioxidants one week before exposure in a 3-day treatment for that week, after which the mice were treated 3 days/week and exposed to UVB light 5 days/week throughout the experiment period of 3 months. Antioxidants were injectable solution (vitamin E as dlα-tocopheryl acetate and selenium as sodium selenite). Each mouse was treated with a 100µl/S.C injection of antioxidants (in a single dose). 3.1.2: UVB exposure The mice were subjected to UVB irradiation with a calculated power of 53mj/sec using a Lamp of 312nm wavelength, 15 watts; Vilber-Lourmat-France. Mice from both groups (exposure and treatment group) were exposed to UVB light for 20 minutes. This was done after making a window by shaving the mouse’s back skin (2X5cm). At the end of the experiment, incisional biopsies were taken from shaved area of the control, exposure and treatment groups. All animals were anesthetized using general anesthetic drug (xylazine-ketamine for mouse: 0.1ml/10g of body weight) as recommended dose intraperitonially (In a sterile 10ml bottle with a rubber stopper, mix 1ml of ketamine (100mg/ml) + 0.1ml of xylazine (100mg/ml) + 8.9ml of sterile water for the injection). Biopsies were taken, then tissue samples were fixed in 10% formalin, processed and embedded in paraffin blocks.

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3.1.3: Blood collection and storage Before taking biopsies, for TAS analysis, 0.5ml of blood sample was collected from each animal in the control, exposure and treatment groups via cardiac puncture. Each blood sample was placed into an Eppendorf tube, incubated in an incubator at 37°C for 20 minutes. Serum was obtained after centrifugation at 5000rpm for 5 minutes. The serum was collected into another Eppendorf tube and stored in the freezer until analysis.

3.2: Materials 3.2.1: Equipments 1.

Ultraviolet lamp (312nm wavelength, 15 watts; Vilber-Lourmat-France).

2.

Sakura rotary microtome (Acuu-Cut SRM200-Japan).

3.

Spectrophotometer (Apeal PD-303-Japan).

4.

Centrifuge (SIGMA 1-14, Germany).

5.

Cuvettes.

6.

Micropipettes.

7.

Eppendorf tubes.

8.

Rack for Eppendorf tubes.

9.

Water bath.

10. Oven. 11. Light microscope (Olympus 6V20 whal-Japan). 12. Incubator and humid chamber. 13. Sensitive balance. 14. Positively charged slides (Fisherbrand-U.S.A) 15. Ordinary glass slides. 16. Glass staining jars, Coplin jars. 17. Slide holder. 18. Cover slips. 19. Wash bottles. 20. Cylinders and flasks. 21. Filter paper. 22. Pap pen-Dako. 23. Absorbent wipes. 24. Ordinary syringes. 25. Insulin syringes. 26. Timer (stop watch). 27. Surgical gloves.

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3.2.2: Reagents and solutions 1.

Antioxidants (Erfan-Darw Company Ltd-Iran) 50ml (selenium as sodium selenite 0.5mg/ml and vitamin E as dl-α-tocopheryl acetate 50mg/ml).

2.

Randox kit “Randox Laboratories Ltd, U.K” (R1: Buffer 1X100ml, R2: Chromogen 5X10ml, R3: Substrate 2X5ml, Cal: Standard 5X1ml).

3.

Distilled water.

4.

Xylazine (Ceva sante animale-France).

5.

Ketamine (Holden medica-India).

6.

Ethanol (Absolute, 90% and 70%).

7.

Xylene.

8.

Giemsa stain (R1 “Giemsa stock solution” 100ml and R2 “buffer solution” 100ml).

9.

Counter stain (Mayer’s Haematoxylin).

10. Mounting medium (Dixtrene polysteren xylene “DPX”). 11. Mouse monoclonal antibody (AM207-5ME)-clone E30, species mouse, Ig class-IgG1 (6ml). 12. Super Sensitive™ Polymer-HRP IHC Detection System: 

Peroxide block (1X6ml).



Power block (1X6ml).

13. Biogenex wash buffer (concentrated Tris-buffered saline solution, PH 7.6). 14. Pepsin 3-Pack kit (3 Vials Lyopphilized pepsin powder + 3X5ml Pepsin reconstitution buffer).

3.3: Methods 3.3.1: Sample preparation Three sections of 5µm thickness were taken from each paraffin embedded tissue block. The first section was mounted on an ordinary slide for H&E staining for the detection of any histological lesions. The second section was for Giemsa stain for the counting of mast cells while the third section was mounted on positively charged slide, then proceeding with the process of immunohistochemistry staining following the protocol that was supplied with the kit of anti-EGFR (Super Sensitive™ Polymer-HRP IHC Detection System).

3.3.2: Immunostaining method Immunostaining method was done by using kits from Biogenex as per the manufacturer’s instructions. A. Procedure: 1.

Formalin fixed-paraffin embedded sections were cut into 5µm thickness for obtaining optimum resolution, then placed on positively charged slides to be stained.

2.

The sections were baked in the oven (over night at 56°C), then dewaxed in xylene (for 5-10 minutes).

3.

The sections were rehydrated using graded alcohol (ethanol) in descending concentrations to water:

a.

Absolute ethanol for 10 minutes.

b.

90% ethanol for 5 minutes.

c.

70% ethanol for 5 minutes.

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Materials and methods

The sections washed under running tap water for 5 minutes, then placed in 3 changes of wash buffer, 2 minutes each.

5.

The slides were tapped off and the area around the specimen wiped to remove any remaining liquid and section encircled with pap pen.

6.

Few drops of previously prepared digestive enzyme pretreatment solution (1 vial lyophilized pepsin + 1 vial of reconstitution buffer which were already refrigerated and allowed to come to room temperature) were applied to cover the specimen and incubated in a humid chamber for about 5 minutes at room temperature, then rinsed with wash buffer 3 times, 2 minutes each.

7.

Few drops with peroxide block were applied to cover the specimen and incubated in a humid chamber for about 10 minutes at room temperature, then rinsed with wash buffer 3 times, 2 minutes each.

8.

Few drops with power block were applied to cover the specimen and incubated in a humid chamber for about 10 minutes at room temperature. Blot the slides without washing.

9.

Few drops of primary antibody (Mouse monoclonal anti-EGFR) were applied to cover the specimens and incubated for 2 hours in a humid chamber at room temperature, then rinsed with wash buffer 3 times, 2 minutes each.

10. Few drops with super enhancer reagent were applied to cover the specimen and incubated in a humid chamber for 20 minutes at room temperature, then rinsed with wash buffer 3 times, 2 minutes each. 11. Few drops of previously prepared substrate-chromogen solution (1 drop of chromogen + 1ml of substrate) were applied to cover the specimens and incubated for 30 minutes in a humid chamber at room temperature, then rinsed with wash buffer 3 times, 2 minutes each. 12. Few drops of substrate solution (SS label polymer HRP) were applied to cover the specimens and incubated for 10 minutes in a humid chamber at room temperature, then washed under running tap water for 5 minutes. 13. The slides were immersed in a bath of aqueous haematoxylin for less than 1 minute, then rinsed gently under running tap water. 14. The slides were dehydrated consequatively by dipping in a glass jars containing the following: o

70% ethanol for 5 minutes.

o

90% ethanol for 5 minutes.

o

Absolute ethanol for 10 minutes.

o

Xylene for 5-10 minutes.

15. Slides were mounted using mounting medium (DPX) and covered with cover slips and left to dry. B. Results: EGFR staining

brown

Back ground

blue

3.4: Slide interpretation The H&E stained slides of the exposure and treatment groups, biopsies were thoroughly examined for any histological lesions or changes in epidermis and dermis by two independent pathologists.

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3.4.1: Immunohistochemical scoring EGFR immunostaining was evaluated by two pathologists. The staining was categorized as either positive or negative based on two parameters: (1) The percentage of positive cells was scored on a scale of 0-3: 0 = 0%, 1 = <25%, 2 = 26-50%, 3 = >50% positive cells and (2) The strength of staining intensity was scored on a scale of 0-3: 0 = negative, 1 = weak or light staining, 2 = moderate staining, 3 = intense or strong staining. The EGFR protein expression was classified into 4 point scales: 0; no staining, 1+; <25% positive cells for weak staining, 2+; 26-50% positive cells for moderate staining, 3+; >50% positive cells for strong staining.

3.4.2: Giemsa staining 1. Reagents: o

R1 (Giemsa stock solution).

o

R2 (buffer solution, PH: 6.8).

2. Procedure: Cut paraffin embedded sections into 5µm thickness and place on glass slides for staining. Dewax specimens which are placed in xylene container and placed in the oven adjusted to a temperature above the melting point of paraffin for 5-10 minutes. Rehydrate the sections using graded alcohol (ethanol) in descending concentrations to water: 1) Absolute ethanol for 10 minutes. 2) 90% ethanol for 5 minutes. 3) 70% ethanol for 5 minutes. 4) Slides were washed under tap water for 2 minutes. 15 drops of R1 (Giemsa stock solution) were added on each slide for 1 minute. 30 drops of R2 (buffer solution) were added, then left until the color changed to metallic shine. Slides were washed under tap water for 2 minutes, then dried up by their placement in the oven for 10-15 minutes. Put slides in xylene for 2 minutes. Slides were mounted by DPX, covered by cover slips and left to dry. 3. Results: Mast cells Background

violet blue

3.5: Total antioxidant status assay Total antioxidants were measured by the ABTS method using kits from Randox as per the manufacturer’s instructions. Using this method, metmyoglobin (peroxidase) present in the choromogen provided in the kit reacts with H2O2 to form ferrylmyoglobin, a free radical species. The chromogen also contains ABTS which reacts with ferrylmyoglobin to produce a radical cation which has blue-green color and can be measured at 600 nm by spectrophotometer. Antioxidants present in the added serum cause suppression of this color production, proportional to their concentration. Calibration of the assay was done using 6-hydroxy-2, 5, 7, 8-

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tetra-methylchroman-2-carboxylic acid. The results were expressed in mmol/l. 20µl sample and 1ml of chromogen was required for the assay. 1) Reagents:  R1 (phosphate buffer saline 80mmol/l, PH 7.4) 1X100ml.  R2 (Chromogen: metmyoglobin 6.1µmol/l and ABTS® 610µmol/l) 5X10ml.  R3 (Substrate: hydrogen peroxide “H2O2”) 2X5ml.  CAL. (Standard: 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid). 2) Procedure (spectrophotometeric): Wavelength: 600nm. Cuvette: 1cm light path. Temperature: 37°C. Measurement: Against air (empty cuvette).

Pipette into Cuvette: Reagent

standard

sample

blank Distilled water

20µl

-

-

standard

-

20µl

-

sample

-

-

20µl

Chromogen (R2)

1ml

1ml

1ml

Mix well, incubate to bring to temperature and read initial absorbance (A1). Add: Substrate (R3)

200µl

200µl

200µl

Mix well and start timer simultaneously. Read absorbance after exactly 3 minutes (A2). A2-A1 = ∆A of sample/standard/blank. Calculation: Total antioxidant status: Factor= concentration

of standard

(∆Ablank - ∆Astandard)

mmol/l= factor X (∆Ablank - ∆Asample).

3.6: Statistical analysis The data obtained from our observations were analyzed using ANOVA, Duncan's test and Pearson’s Correlation.

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4.1: Macroscopical and microscopical findings 4.1.1: Non-irradiated control group (10 cases) Grossly, skin of mice appeared normal (Figure 4-1). Histologically, skin sections revealed a normal epidermis which comprised of 2-3 layers and the dermis showed normal skin appendages. This is observed in figure 4-2.

4.1.2: Irradiated (exposure) group (15 cases) The gross pathological examination of exposure group appeared to have slightly raised lesions (protrusion above the skin surface). The lesion measured only a few mms in diameter, occasionally reaching 2cms with variable color, from light to black, most of them were tan or brown, rough, of friable consistency with loss of hair in some regions, hemorrhagic spots, some have a smooth surface but characteristically show keratotic plugs (Figure 4-3). The mice showed evidence of keratitis and conjunctivitis, some of which became blind, due to UVB irradiation for a long period (Figure 4-4). Irradiated group: UVB-induced epidermal tumor development, which was benign seborrheic keratosis (SK), acanthotic type, was witnessed in all cases with variable characteristics like acanthosis, hyperkeratosis and papillomatosis, however, all 15 cases showed squamous eddies (Table 4-1). Microscopical examination of acanthotic SK showed thickening of epidermal layers resulting from basaloid cell proliferation, presence of epidermal cysts filled with keratin (horn cyst), which is a common feature of SK. Some of these cysts resulted from enfolding of the epidermis and are called pseudohorn cysts, with the presence of squamous eddies (Figure 4-5,6,7). Epidermis contained cells with basophilic nuclei and deep eosinophilic cytoplasm, regarded as sunburn cells (SBCs) or apoptotic bodies (Figure 4-8). The dermal changes showed infiltration of mononuclear inflammatory cells including macrophages and lymphocytes (Figure 4-9). The dermis showed an increasing number of typical inflammatory mast cells, which had basophilic granules in lower and upper dermis but especially in upper dermis surrounding blood capillaries (Figure 4-10).

4.1.3: Treatment group (15 cases) Treatment group with antioxidants: Gross lesions were also variable according to the severity of the lesions. Grossly, UVB-induced hyperplasia had thickening of skin of two-three folds than normal skin and was examined by hand touching (palpation), with loss of hair in some regions, particularly in those cases that had moderate and severe lesions (Figure 4-11). Comparison for gross appearance among three groups is shown in figure 4-12. Microscopical examination showed an increase in the number of epidermal layers and were classified according to the number of layers which led to the thickening of the entire epidermis and regarded as; mild = 4-6 layers (Figure 4-13), moderate = 7-9 layers (Figure 4-14), severe = >10 layers (Figure 4-15). In 15 cases of treatment group, 9 cases showed mild hyperplasia, 3 moderate hyperplasia and 3 cases showed severe hyperplasia and in one of them acanthotic SK was observed (Table 4-2) (Figure 4-16,17). Epidermis also contained cells with basophilic nuclei and deep eosinophilic cytoplasm, regarded as SBCs (apoptotic bodies). The dermal changes in treatment group also showed infiltration of mononuclear inflammatory cells including

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macrophages and lymphocytes (Figure 4-9). The dermis showed decreasing number of inflammatory mast cells in comparison to exposure group (Figure 4-18).

Figure (4-1): Normal mouse skin appearance, after making a window.

Figure (4-2): Normal mouse skin histology, unexposed to radiation (H&E X100).

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Figure (4-3): Different gross lesions in exposure group; (A): Skin was thickened (green arrows) showing 2 small elevated masses, (B): Showing large numbers of friable crusts and (C): Crusts were sloughed.

Figure (4-4): A mouse from exposure group with eye damage.

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Figure (4-5): Microscopical appearance of seborrheic keratosis, acanthotic type (H&E X40).

Figure (4-6): Microscopical appearance of seborrheic keratosis, acanthotic type (H&E X100).

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Figure (4-7): Squamous eddies; squamous cells resembling eddy currents in a stream (H&E X400).

Figure (4-8): Black arrows showing sunburn cells or apoptotic bodies (H&E X400).

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Figure (4-9): (A): Moderate dermal inflammation and (B): Severe dermal inflammation (H&E X400).

Figure (4-10): (A): Dermal mast cells (H&E X400) and (B): Dermal mast cells (Giemsa stain X400).

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Figure (4-11): Variable gross lesions in treatment group; (A): Thin skin appeared as normal, while in (B) and (C) 2-3 folds skin thickness was felt through palpation.

Figure (4-12): (A): A mouse in control group, (B): A mouse in treatment group and (C): A mouse in exposure group.

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Figure (4-13): Mild epidermal hyperplasia (H&E X400).

Figure (4-14): Moderate epidermal hyperplasia (H&E X400).

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Figure (4-15): Severe epidermal hyperplasia (H&E X400).

Figure (4-16): (A): Normal epidermis (2-3 layers), (B): Mild epidermal hyperplasia of 4-6 layers, (C): Moderate epidermal hyperplasia of 7-9 layers and (D): Severe epidermal hyperplasia more than 10 layers (H&E X100).

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Figure (4-17): Pie chart showing the percentage degrees of epidermal thickness in the treatment group.

Figure (4-18): (A): Dermal mast cells in the treatment group and (B): Dermal mast cells in the exposure group (Giemsa stain X100).

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Table (4-1): Skin lesions of exposure group after 3 months of exposing to UVB irradiation. Case no.

Skin lesions Seborrheic keratosis (acanthotic type).

Squamous eddies

Acanthosis

Hyperkeratosis

Papillomatosis

1

Severe

Severe

Diffuse

Yes

2

Severe

Moderate

Diffuse

Yes

3

Severe

Moderate

Multifocal

Yes

4

Severe

Moderate

Multifocal

Yes

5

Severe

Mild

Focal

Yes

6

Severe

Mild

Focal

Yes

7

Severe

Severe

Multifocal

Yes

8

Moderate

Mild

Focal

Yes

9

Severe

Moderate

Multifocal

Yes

10

Severe

Moderate

Multifocal

Yes

11

Moderate

Mild

Focal

Yes

12

Moderate

Mild

Focal

Yes

13

Moderate

Mild

Focal

Yes

14

Moderate

Mild

Multifocal

Yes

15

Severe

Moderate

Multifocal

Yes

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Table (4-2): Epidermal thickness in treatment group. Case no.

Skin lesions Epidermal thickness (hyperplasia)

1

Mild

2

Moderate

3

Moderate

4

Mild

5

Mild

6

Mild

7

Moderate

8

Mild

9

Severe

10

Mild

11

Mild

12

Mild

13

Mild

14

Severe

15

Severe (SK)

4.2: Total number of apoptotic bodies in 10 high power fields 4.2.1: Effect of chronic UVB irradiation on total number of apoptotic bodies in exposure group The results of this study revealed strong effects of chronic UVB irradiation on apoptotic bodies in seborrheic keratosis i.e., the number of apoptotic bodies were increased with a range of 5-17 and mean number of 11.6/10HPF in chronically irradiated mice that led to the development of epidermal seborrheic keratosis (Figure 4-19).

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Figure (4-19): Column chart showing the total number of apoptotic bodies of each case in exposure group.

4.2.2: Effect of antioxidants on total number of apoptotic bodies in chronically irradiated UVB in treatment group According to the results of this study, there was a strong effect of antioxidants by decreasing the number of apoptotic bodies in the treatment group with chronic UVB irradiation. For example, there was a decreased number of apoptotic bodies in the treatment group with a range of 1-10 and mean number of 3.467/10HPF (Figure 4-20).

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Figure (4-20): Column chart showing effects of antioxidants on total number of apoptotic bodies in treatment group.

4.2.3: Difference in total number of apoptotic bodies in exposure and treatment groups In the present study, the results revealed that there was a strong effect of antioxidants in reducing the effect of chronic UVB irradiation of tumor development in treatment group by decreasing the numbers of cell damage, thus the apoptotic bodies were decreased with a range of 1-10 and mean number of 3.467/10HPF, while apoptotic bodies were increased in exposure group with a range of 5-17 and mean number of 11.6/10HPF (Figure 4-21,22), with a P-value of 0.0001 (according to F test) and with A-B symbols (according to Duncan’s test), which indicated a highly significant difference of both groups (Table 4-3). In mean number of apoptotic bodies and its role in reducing tumor development by antioxidants administration in treatment group and enhanced tumor development due to effect of chronic UVB irradiation in exposure group. Table (4-3): Difference in mean number of apoptotic bodies in exposure and treatment groups. Groups

Mean/10HPF

Exposure group

11.6 A

Treatment group

3.467 B

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Figure (4-21): White arrows showing the differences in apoptotic body numbers between 2 different fields; (A): Exposure group and (B): Severe case of treatment group (H&E X400).

Figure (4-22): Column chart showing the apoptotic bodies mean of both exposure and treatment groups.

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4.3: Mean number of mast cells in 1 high power field 4.3.1: Effect of chronic UVB irradiation on mean number of mast cells in exposure group When mast cells in 10HPF were counted in upper dermis in each case of exposure group, the result showed that there was a strong effect of UVB on mast cells, i.e. chronic UVB irradiation increased the mean number of mast cells which indicated a high prevalence of immunosuppression and development of tumor. For example, the mean number of mast cells were increased with a range of 18-26 and the mean number of 21.533/1HPF (Figure 4-23).

Figure (4-23): Column chart showing the effect of chronic UVB irradiation on the mean number of mast cells in exposure group.

4.3.2: Effect of antioxidants on mean number of mast cells in chronic UVB irradiation in treatment group Mast cells in 10HPF were counted in upper dermis in each case of treatment group, the results showed that there was a strong effect of antioxidants in reducing the mean number of mast cells and increasing immunity despite chronic UVB irradiation. For example, the mean number of mast cells were decreased with a range of 11-19 and the mean number of 14.067/1HPF (Figure 4-24).

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Figure (4-24): Effect of antioxidants on mean number of mast cells in treatment group.

4.3.3: Difference in mean number of mast cells in exposure and treatment groups Results revealed that there was a strong effect of antioxidants in reducing the effect of chronic UVB irradiation in the treatment group by decreasing the number of mast cells and UVB enhancing immunosuppression by increasing the number of mast cells in exposure group (Figure 4-25,26). For example, mast cells number was decreased in the treatment group with a range of 11-19 and mean number of 14.067/1HPF, while mast cells increased in the exposure group with a range number of 18-26 and mean number of 21.533/1HPF, with a P-value of 0.0001(according to F test) and with A-B symbols (according to Duncan’s test) which indicated a highly significant effect of antioxidants in reducing mast cells and UVB increased mast cells for exposure group (Table 4-4).

Table (4-4): Differences in the mean number of mast cells between exposure and treatment groups. Groups Exposure group

Mean/1HPF 21.533 A

Treatment group

14.067 B

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Figure (4-25): Differences in the number of mast cells between two different fields; (A): treatment group and (B): exposure group (Giemsa stain X400).

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Figure (4-26): Column chart showing the difference in mean number of mast cells in exposure and treatment groups.

4.3.4: Correlation between apoptotic bodies and mast cells 

Correlation between total number of apoptotic bodies and mean number of mast cells in exposure groups: Results of this study showed a correlation between the total number of apoptotic bodies in 10HPF and

the mean number of mast cells in 1HPF in chronic UVB irradiation in exposure groups (Figure 4-27), i.e., the increasing number of apoptotic bodies is related to the increasing number of mast cells, according to Pearson's correlation coefficient test (Table 4-5).

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Figure (4-27): Column chart showing correlation between total number of apoptotic bodies and mean number of mast cells in exposure groups. Table (4-5): Correlation between total number of apoptotic bodies and mean number of mast cells in exposure group. Pearson’s Correlation

Exposure group Mast cells-exposure

Mast cells-exposure

1.00

Apoptotic bodies-exposure

0.08

Apoptotic bodies-exposure

1.00

Correlation is significant at the 0.01 level (2-tailed). Conclusion: At the level of significance Alpha = 0.01 the decision is to reject the null hypothesis of absence of correlation. In other words, the correlation is significant.



Correlation between total number of apoptotic bodies and mean number of mast cells in treatment group: Results showed a strong correlation between the total number of apoptotic bodies in 10HPF and the

mean number of mast cells in 1HPF in the treatment group (Figure 4-28), i.e., the decreasing number of apoptotic bodies were related to the decreasing number of mast cells in antioxidants administration group with chronic UVB irradiation. According to Pearson's correlation coefficient test there is a highly significant correlation between decreasing number of mast cells with decreasing number of apoptotic bodies related to the effect of antioxidants in the treatment group (Table 4-6).

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Figure (4-28): Column chart showing the correlation between total number of apoptotic bodies and the mean number of mast cells in the treatment group.

Table (4-6): Correlation between total number of apoptotic bodies and the mean number of mast cells in treatment group. Pearson’s Correlation

Treatment group Mast cells-treatment

Mast cells-treatment

1.00

Apoptotic bodies-treatment

0.672**

Apoptotic bodies-treatment

1.00

**.Correlation is significant at the 0.01 level (2-tailed). Conclusion: At the level of significance Alpha = 0.01 the decision is to reject the null hypothesis of absence of correlation. In other words, the correlation is significant.



Correlation between mast cells and apoptotic bodies in exposure group to mast cells and apoptotic bodies in treatment group: Pearson’s correlation coefficient test was used to determine the relationship among mast cells and

apoptotic bodies, as shown in table 4-7. This test revealed that mast cells in exposure group had a significant relationship to apoptotic bodies of the same group by the same direction, while it was significant for mast cells in treatment group in opposite direction, i.e., increasing mast cells in exposure group meant decreasing mast cells in treatment group and vice versa, but mast cells of exposure group had no relationship to apoptotic bodies in treatment group. Apoptotic bodies in exposure group were related to mast cells in treatment group by opposite

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direction, while it was significant to apoptotic bodies in treatment group by the same direction. Mast cells in treatment group are highly significant to apoptotic bodies within the same group by the same direction.

Table (4-7): Correlation between mast cells and apoptotic bodies in exposure group to mast cells and apoptotic bodies in treatment group. Pearson’s Correlation

Exposure group Mast cells-exposure

Treatment group Apoptotic

bodies-

Mast cells-treatment

exposure

treatment

cells-

1.00

Apoptotic bodies-

0.08

1.00

-0.18

-0.25

1.00

0.00

0.22

0.672**

Mast

Apoptotic bodies-

exposure

exposure Mast

cells-

treatment Apoptotic bodies-

1.00

treatment **. Correlation is significant at the 0.01 level (2-tailed).

4.4: Results of immunohistochemical scoring of EGFR expression Immunohistochemical staining of EGFR in exposure and treatment groups showed membranous stain accumulation of EGFR protein and recognized as brown discoloration of the keratinocytes membrane. The EGFR protein expression was classified into 4 point scales: 0; no staining (Figure 4-29), 1+; <25% positive cells for weak staining (Figure 4-30), 2+; 26-50% positive cells for moderate staining (Figure 4-31), 3+; >50% positive cells for strong staining (Figure 4-32).

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Figure (4-29): EGFR Score 0; no stain (X400).

Figure (4-30): EGFR score 1+; weak stain (X400).

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Figure (4-31): EGFR score 2+; moderate stain (X400).

Figure (4-32): EGFR score 3+; strong stain (X400). 4.4.1: Effect of UVB on frequencies of EGFR expression scores and their percentages in exposure group

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EGFR expression was scored as 0, 1+, 2+ and 3+ (Table 4-8). The most frequent score was 3+ with a frequency of 8 (53%), followed by a score of 2+ with a frequency of 7 (47%) (Figure 4-33). This indicated a strong effect of chronic UVB irradiation on EGFR expression in epidermal SK.

Table (4-8): Scores for EGFR protein expression in exposure group. Scores for EGFR expression Group

Score 0

Score 1+

Score 2+

Score 3+

Exposure group

0

0

7

8

Figure (4-33): Pie chart showing the effect of UVB on frequencies of EGFR expression scores and their percentages in exposure group.

4.4.2: Effect of antioxidants on frequencies of EGFR expression scores and their percentages in treatment group The most frequent scores were 0 with a frequency of 6 (40%) and a score 2+ with a frequency of 6 (40%), followed by 1+ with a frequency of 3 (20%), for score 3+, no case was reported (Table 4-9), which proved the effect of antioxidants in reducing EGFR expression in the treatment group by chronic UVB irradiation (Figure 4-34).

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Table (4-9): Scores for EGFR protein expression in treatment group. Scores for EGFR expression Group

Score 0

Score 1+

Score 2+

Score 3+

Treatment group

6

3

6

0

Figure (4-34): Pie chart showing the effect of the UVB on frequencies of EGFR expression scores and their percentages in the treatment group.

4.4.3: Difference in EGFR expression scores and their percentages in exposure and treatment groups Results showed a remarkable effect of UVB in the exposure and treatment groups as shown in table 410. This proved that antioxidants had a highly effective role in reducing EGFR expression and reducing the effect of chronic UVB irradiation in treatment group, while in exposure group the EGFR expression was increased by UVB irradiation (Figure 4-35,36).

Table (4-10): Scores for EGFR protein expression in both exposure group and treatment group. Scores for EGFR expression Groups

Score 0

Score 1+

Score 2+

Score 3+

Exposure group

0

0

7

8

Treatment group

6

3

6

0

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Figure (4-35): Column chart showing the effects of UVB on frequencies of EGFR expression scores in exposure group and the treatment group.

Figure (4-36): EGFR scores: (A): Score 0, (B): Score 1+, (C): Score 2+ and (D): Score 3+ (X400).

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4.5: Results of total antioxidant status measurement 4.5.1: TAS measurement in control group Total antioxidant status (TAS) was measured in 10 mice, set as a baseline for comparison with other groups (exposure and treatment group). The range number of TAS value in control group was 1.098-1.371 with a mean of 1.201mmol/l (Figure 4-37).

Figure (4-37): Column chart showing the TAS measurement in normal mice (control group).

4.5.2: TAS measurement in exposure group TAS was measured in all cases of exposure group, which revealed a remarkable decrease in TAS measurement due to the effect of UVB exposure. The range number of TAS value in exposure group was 0.721.132 with a mean of 0.87mmol/l (Figure 4-38,39).

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Figure (4-38): Column chart showing TAS measurement in exposure group.

Figure (4-39): An exposed sample showing increased bluish-green color.

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4.5.3: TAS measurement in treatment group Antioxidants administration in treatment group played an important role by keeping TAS within normal or slightly elevated value of TAS in most cases of treatment group, especially those which showed mild epidermal thickness, while in some cases that had moderate and severe epidermal thickness, TAS was decreased (Figure 4-40,41). The range number of TAS value in the treatment group was (0.9-1.746) with a mean number of 1.309mmol/l.

Figure (4-40): Column chart showing TAS measurement in treatment group.

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Figure (4-41): A treatment sample showing reduced bluish-green color.

4.5.4: Difference in TAS mean values among control, exposure and treatment groups Results revealed that there was a strong effect of antioxidants on TAS and acted to reduce the effect of chronic UVB irradiation in treatment group, while in exposure group TAS was decreased due to the effect of chronic UVB irradiation. TAS mean value in control group was equal to 1.201mmol/l, which was used for comparison between exposure and treatment group (Figure 4-42), with a P-value of 0.0001(according to F test) and with A-B symbols (according to Duncan’s test). This indicated a highly significant relation between TAS value in exposure group and TAS value in the control group, while there was no significant relation between TAS value in treatment group and TAS value in the control group (Table 4-11). Table (4-11): Difference in TAS mean values in control, exposure and treatment groups. Groups TAS Mean values Treatment group

1.309 A

Control group

1.201 A

Exposure group

0.87 B

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Figure (4-42): Column chart showing the difference of TAS mean values among different groups.

4.6: Pearson’s correlation test 4.6.1: Pearson’s correlation test for exposure group Pearson’s correlation coefficient test revealed a relationship among all observations in exposure group (Table 4-12). TAS had a significant effect on acanthosis by the same direction, but it had more significant effect on hyperkeratosis and papillomatosis by opposite direction. There was a significant effect of TAS on EGFR expression scores by the same direction. Furthermore, TAS had a highly significant effect on mast cells and apoptotic bodies by opposite direction. The relationship among three characteristics of SK (acanthosis, hyperkeratosis and papillomatosis) were highly significant by the same direction. Scores of EGFR expression had a significant effect for both acanthosis and hyperkeratosis by the same direction, while for papillomatosis by opposite direction. Apoptotic bodies had a significant effect on acanthosis and scores for EGFR expression by opposite direction.

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Table (4-12): Pearson’s correlation for exposure group. Pearson’s Correlation

Exposure group

TAS

Acanthosi s

Hyperkertosis

Papillomatosis

Scores for EGFR expression

Mast cells

TAS

1.00

Acanthosis

0.08

1.00

Hyperkertosis

-0.37

0.674**

1.00

Papillomatosis

-0.38

0.555*

0.795**

1.00

Scores for EGFR expression

0.45

0.47

0.13

-0.17

1.00

Mast cells

-0.641*

0.04

0.22

0.09

-0.12

1.00

Apoptotic bodies

-0.41

-0.35

-0.08

0.01

-0.39

0.08

Apoptoti c bodies

1.00

*. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed).

4.6.2: Pearson’s correlation test for treatment group Pearson’s correlation coefficient test revealed strong relationship among all observations in treatment group (Table 4-13). TAS had a highly significant effect on skin lesion (epidermal hyperplasia), scores for EGFR expression, mast cells and apoptotic bodies by opposite direction. Skin lesions had a highly significant effect with scores for EGFR expression, mast cells and apoptotic bodies by the same directions. Scores for EGFR expression were also highly significant with mast cells and apoptotic bodies. Further, mast cells are highly significant to apoptotic bodies within the same group.

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Table (4-13): Pearson’s correlation test for treatment group. Pearson’s Correlation

Treatment group TAS

Skin lesion (epidermal hyperplasia)

Scores for EGFR expression

Mast cells

TAS

1.00

Skin lesion (epidermal hyperplasia)

-0.916**

1.00

Scores for EGFR expression

-0.790**

0.839**

1.00

Mast cells

-0.858**

0.940**

0.774**

1.00

Apoptotic bodies

-0.710**

0.572*

0.533*

0.672**

**. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed).

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Apoptotic bodies

1.00

Chapter five

Discussion CHAPTER FIVE DISCUSSION

In recent years, the incidence of various diseases and disorders related to solar ultraviolet radiation (UVR) have alarmingly increased and continued to grow. Chronic exposure of mammalian skin to UVR induces a number of pathological responses, including the development of erythema, edema, sunburn cell formation, epidermal hyperplasia, immune suppression, DNA damage and photoaging. These alterations are directly or indirectly involved in the development of skin cancer (Clydesdale et al., 2001; Ouhtit and Ananthaswamy, 2001; Svobodová et al., 2003). Experimental and epidemiological studies have drawn attention to sunlight as the most important environmental risk factor in the development of skin cancer (Amerio et al., 2009). Experimental studies using laboratory animals have demonstrated that the UVR component of sunlight, the UVB, is responsible for solar carcinogenesis. Mice are the experimental animals most susceptible to UVR carcinogenesis. Upon appropriate UVR exposure, mice develop a variety of epidermal and dermal tumors, some of which mirror human skin tumors (Kusewitt et al., 1991; Dwivedi et al., 2006; Väkevä, 2006). Skin carcinogenesis experiments with animal models, particularly mice, have already yielded a lot of data on how skin tumor development depends on dose, time and wavelength of the UVR (Dumaz et al., 1997). UV-induced activation of EGFR upregulates several MAPK and PI3K/Akt signaling pathways that control epidermal cell division and cell death, processes whose deregulation is critical during tumorigenesis (ElAbaseri et al., 2005). Several reports exist describing how UV light can activate the EGFR, hence activating the Akt and MAPK pathway. This is thought to be mediated through: i) increased expression of EGFR ligands and ii) inactivation of receptor-associated phosphatases and iii) altered internalization and degradation (Olsen et al., 2007). EGFR has been implicated previously in mouse skin carcinogenesis, because genetic ablation (knock-out mice) of the receptor reduces skin tumor growth (Dlugosz et al., 1997). Upon UV-induced activation of the receptor, EGFR increases proliferation, suppresses apoptosis, augments hyperplasia and increases UV-induced skin tumorigenesis (Nair, 2005; El-Abaseri and Hansen, 2007; Rocha-Lima et al., 2007). Seborrheic keratosis (SK) is a benign and common epidermal tumor that occurs most frequently in middle-aged or older people. It arises on the trunk, although the extremities, the head and neck may also be involved. Men and women are affected equally by SK and the incidence increases with age (Lee et al., 2001; Luba et al., 2003; Kumar et al., 2007; Kim et al., 2009). The etiology of SK is unknown (Lee et al., 2001; Rubin, 2001; Bhuiyan, 2007; Rubin and Strayer, 2008). Recently, activating fibroblast growth factor receptor 3 (FGFR3) mutations in human epidermis were shown to be involved in the development of SK (Hafner et al., 2007). SK is often appearing to be “stuck on” the surface of the skin. SK varies in color, from tan to brown and usually has a well-circumscribed border. Most lesions have a rough surface and usually range in size from 2mm to 3cm in diameter, but can be larger (Luba et al., 2003). SK is a combined hyperplasia of epidermis and supporting papillary connective tissue. Six histologic types were identified; hyperkeratotic, acanthotic, adenoid or reticulated, clonal, irritated and melanoacanthoma are distinguished. All types of SK have in common acanthosis, papillomatosis and hyperkeratosis (Bhuiyan, 2007; Mandinova et al., 2009). The vertical diameter (tumor thickness) of SK varies considerably. Flat (initial) SK frequently shows gradual vertical growth within

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years. However, malignant transformation is a very rare event in SK (Hafner et al., 2007). SK is composed of sheets of small cells that most resemble monotonous basal cells of the normal epidermis. Hyperkeratosis occurs at the surface and the presence of small keratin-filled cysts (horn cysts) and down-growth of keratin into the main tumor mass (pseudo-horn cysts) are known characteristics (Kumar et al., 2007). A previous study revealed that expression levels of EGFR were higher in SK group than that of normal control group “in human” (Cheng et al., 2001). In this study, the animal models were mice, one significant finding of this study showed that a highly significant association between chronic UVB irradiation and development of SK in exposure group, which indicated that UVB with a long duration initiated and promoted tumor growth and it was a major fact to induce benign SK. This is in disagreement with previous studies in which they described the etiology of SK as unknown (Lee et al., 2001; Rubin, 2001; Bhuiyan, 2007; Rubin and Strayer, 2008), but in agreement with the study of Haw et al., (2009). This study was the only research on UVB as a causative agent in inducing SK; therefore, our study was totally new in proving that UVB was an etiological factor in development of SK in mice. The current research work, EGFR expression increased in SK by chronic UVB irradiation in exposure group and this meant that EGFR had an important role in promoting SK development after initiation by UVB irradiation which is in agreement with the findings of Cheng et al., (2001). Acute UVR induces apoptosis involving p53 and the Fas-Fas ligand pathway, chronic exposure results in disruption of apoptosis regulation leading to abnormal proliferation of keratinocytes containing damaged DNA, accumulation of p53 mutations and loss of Fas-Fas ligand interactions, all of which contribute to carcinogenesis (Svobodova et al., 2006). Oxidative stress occurs when this critical balance is disrupted due to depletion of antioxidants or excess accumulation of ROS, or both. That is, when antioxidants are depleted and/or if the formation of ROS increases beyond the ability of the defenses to cope, then oxidative stress and its detrimental consequences ensue (Klaunig et al., 1998; Scandalios, 2005; Thomas-Ahner et al., 2007). There is a close relationship between apoptosis and cancer and many studies have reported this relationship (Ma et al., 2005). Increase, decrease or even absence of apoptosis play a major role in the development of various diseases (Kumar and Jugdutt, 2003). Apoptosis was highly expressed in the areas of squamous differentiation of irritated SK, but only mildly increased in the other varieties of SK. These datas support the hypothesis that apoptosis has a role in the squamous differentiation of irritated SK as demonstrated by Pesce and Scalora’s 2000 study. A moderate increase is observed in the rates of apoptosis in all varieties of SK compared to normal skin (Balin, 2009). The rates of apoptosis in SK are not significantly different from normal skin (Bowen et al., 2004). The study findings revealed that apoptosis was increased in numbers in SK; this is in line with Pesce and Scaloraet (2000) and Balin (2009), but in disagreement with Bowen et al. (2004). This finding referred to the generation of excess levels of ROS, which is important for activation of internal cell programs for cell suicide (apoptosis) that are in turn important protection mechanisms that kill cancer cells (Weijl et al., 1997; Kuipers and Lafleur, 1998). UVB irradiation of the skin not only induces DNA lesions but also suppresses the cutaneous immune system, enabling UV-induced tumors to escape immune destruction (Grimbaldeston et al., 2000; Welsh et al.,

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2008; Amerio et al., 2009). Mice of different strains differ in their susceptibility to UVBR induced immunosuppression (Yoshikawa and Streilein, 1990; Noonan and Hoffman, 1994). There is evidence that humans with increased incidence of nonmelanoma skin tumors have decreased contact hypersensitivity (CHS) responses to haptens after UVB exposure (Streilein et al., 1994). Mast cells, which have recently emerged as immune regulatory cells, are particularly important in UV-induced immune suppression. Chronic UVB irradiation increases the numbers and size of mast cells in a hairless mouse (Kligman and Murphy, 1996; Hart et al., 2001; Chacón-Salinas et al., 2011). Mast cells have been found to play a critical role in the suppression of immune reactions, which does not only produce inhibitory cytokines. Hence, mast cell infiltration into tumor may possibly remodel tumor microenvironment and profoundly influence tumor behavior by participating and regulating inflammatory and immune reactions. However, although some studies have shown that mast cells promote tumor angiogenesis and tumor growth because of their properties as inflammatory cells, the roles of mast cells in tumor progression have been incompletely understood so far (Huang et al., 2008). In this study, we revealed that there was an increase in mast cell numbers in exposure group due to the effect of chronic UVBR and this was in agreement with the findings of Kligman and Murphy (1996) and Chacón-Salinas et al. (2011). This serves as an indicator to a strong functional link between UVB-induced immunosuppression and the development of at least some forms of skin tumor (Hart et al., 2001). UVB enhanced the increase of the number of mast cells and the development of SK in all mice in the exposure group which indicated a strong relation between mast cells and SK development (Table 4-12). No previous studies have mentioned the increasing number of mast cells in SK. Hence, up to our knowledge this is the first study to document this finding. Several chronical diseases, including cancer, have been associated with oxidative stress produced through either an increased free radical generation and/or a decreased antioxidant level in the target cells and tissues (Klaunig et al., 1998; Bickers and Athar, 2006; Marlin and Dunnett, 2007). Antioxidant enzymes may be depleted or inactivated during UVR exposure resulting in lowered defense capabilities. Depending upon what oxidative products are produced, antioxidants may be affected to varying degrees (Jurkiewicz, 1995). Some experimental studies have reported that topical or oral antioxidant administration, particularly vitamin E and selenium, may lead to the regression of skin tumor. Topical α-tocopherol protected mouse skin against UV-induced lipid peroxidation (Lopez-Torres et al., 1998), mice against UV-induced photoaging changes (Bissett et al., 1990; Jurkiewicz et al., 1995), mice against UV immunosuppression (Gensler and Magdaleno, 1991; Yuen and Halliday, 1997; Steenvoorden and Beijersbergen, 1999; Burke et al., 2000) and mice against UV photocarcinogenesis (Gensler and Magdaleno, 1991; Burke et al., 2000; Jacobson et al., 2005). Several cellular studies have demonstrated the protective effects of selenium for UV-induced damage including DNA oxidation (Stewart et al., 1996), DNA damage (Emonet-Piccardi et al., 1998), interleukin 10 expressions (Rafferty, 2002) and lipid peroxidation (Moysan et al., 1995). Oral sodium selenite protected hairless mice against UV-induced erythema and subsequent pigmentation (Thorling et al., 1983). Oral selenium protected mice against UV-induced skin cancer (Pence et al., 1994). Topical selenomethionine was found to protect mice against UV-induced erythema and skin cancer (Burke et al., 1992).

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In present study, we analyzed TAS values in all groups (control, exposure and treatment) and revealed that TAS measurement decreased in exposure group due to chronic irradiation with UVB which was increased generation of free radicals and decreased antioxidant level in the body; as a result SK was developed. This finding was in agreement with previous studies in which antioxidant molecules in the skin interact with ROS or their by-products to either eliminate or to minimize their deleterious effects (Jurkiewicz, 1995; Klaunig et al., 1998; Bickers and Athar, 2006; Marlin and Dunnett, 2007). While in treatment group TAS level measurements in most cases were within normal range which meant that administration of antioxidants reduced the effects of chronic UVB irradiation by neutralization of generated free radicals and antioxidants also reduced the development of SK in only 1 case out of 15. This means the supplementation of skin with various antioxidants may compensate for UVR induced depletion and thereby prevent free radical damage. No previous studies have mentioned anything about TAS analysis of mice and the effect of antioxidants on SK, and hence, these results up to our knowledge were the first of its kind which showed the role of antioxidants in reducing SK development in mice. In this study we demonstrated that antioxidants (vitamin E and selenium) were effective in reducing tumor development in treatment group by chronic UVB irradiation (3 months) and showed the reduction of activation of EGFR by antioxidants; reduced apoptosis and reduced mast cell infiltration which played a role in tumorigenesis. This was in agreement with the previous studies of Gensler and Magdaleno (1991), Yuen and Halliday (1997), Steenvoorden and Beijersbergen (1999), Burke et al. (2000) and Neeraj et al. (2011). The result showed a highly significant relation between TAS and EGFR expression (Table 4-12, 13). No previous studies have mentioned any clue about effect of antioxidants (vitamin E and selenium) on SK development and thus our result up to our knowledge was the first to show such a finding. In present findings, we revealed that the decreased number of apoptotic bodies in the treatment group was due to the action of antioxidants, which led to decrease in number of apoptotic bodies when compared to exposure group and showed a highly significant relation (P-value 0.0001), thus the number of tumor development also decreased. Decrease in the number of apoptotic bodies was one of antioxidants effect which also reduced tumorigenesis in the treatment group despite chronic UVB irradiation. In this study, antioxidants reduced apoptosis and this was in line with Weijl et al. (1997), Kuipers and Lafleur (1998), Zeisel (2004), Blumenthal et al. (2000) and Neeraj et al. (2011). No previous studies have ever published or documented the effect of antioxidants on apoptotic bodies in SK. In present study, we showed a highly significant effect of antioxidants (Table 4-13) in reducing the number of inflammatory mast cells in chronic UVB irradiation in treatment group when compared to exposure group (Table 4-12) and this was due to the effect of antioxidants against immunosuppression by mast cells and decreased its role in immunosuppression in SK development. There is no previous research about the role of antioxidants on the decreasing the number of mast cells. This study up to our knowledge is the first to prove it. The present study found that there was a highly significant correlation between mast cells and apoptotic bodies in exposure group. The apoptotic bodies and mast cells were increased, but in treatment group, apoptotic bodies and mast cells were decreased (P-value 0.0001). This is related to effect of antioxidants in treatment

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group. No previous studies mentioned anything about this correlation, but in this study it was successfully proven.

Conclusions: 1.

UVB is the causative agent which induces SK in mice.

2.

EGFR expression is related to UVB irradiation in mice.

3.

UVB generated ROS can potentially play a significant role in the pathogenesis of UVB-induced epidermal SK in mice.

4.

EGFR expression increased in SK.

5.

Parenteral administration of antioxidants effectively reduced UVB-induced SK.

6.

Antioxidants reduced EGFR expression, epidermal changes, tumor development, mast cells and apoptotic bodies.

7.

UVB triggers dermal mast cell proliferation.

8.

TAS measurement was declined as a result, chronic UVB irradiation in exposure group.

9.

Administration of antioxidants, keeps TAS level measurement within normal range in most cases within treatment group.

Recommendations: 1.

EGFR inhibitors can be used against skin tumor development.

2.

Administration of other types of antioxidants to reduce other types of tumors, including skin tumors and further studies are needed to document this.

3.

Further studies on the relation between SK with EGFR and other genes such as; P53, Bcl-2 and FGFR3 are required to further understand their role in determining unknown causative agents of SK development.

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