universiti putra malaysia … of induced rat mammary gland tumor and the antitumor effect of...

UNIVERSITI PUTRA MALAYSIA

CHARACTERISATION OF INDUCED RAT MAMMARY GLAND TUMOR AND THE ANTITUMOR EFFECT OF RECOMBINANT

HUMAN ERYTHROPOIETIN AND TAMOXIFEN

SAIRAH ABDUL KARIM

IB 2009 12

CHARACTERISATION OF INDUCED RAT MAMMARY GLAND TUMOR AND THE

ANTITUMOR EFFECT OF RECOMBINANT HUMAN ERYTHROPOIETIN AND TAMOXIFEN

SAIRAH ABDUL KARIM

DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA

2009

CHARACTERISATION OF INDUCED RAT MAMMARY GLAND TUMOR AND THE ANTITUMOR EFFECT OF RECOMBINANT HUMAN

ERYTHROPOIETIN AND TAMOXIFEN

By

SAIRAH ABDUL KARIM

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in fulfillment of the Requirements for the Degree of Doctor of

Philosophy

2009

ii

DEDICATION

To my parents, Mr. Abdul Karim Jabar and Mrs. Noli

Othman…………………………..

To my beloved husband Mohd. Fadhil Ahmad and my lovely children,

Faiz Isqandar and Aleya Yasmin who bring me great happiness……….

iii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy

CHARACTERISATION OF INDUCED RAT MAMMARY GLAND TUMOR AND THE ANTITUMOR EFFECT OF RECOMBINANT HUMAN ERYHTROPOIETIN

AND TAMOXIFEN

By

SAIRAH ABDUL KARIM

2008

Chairman : Professor Rasedee Abdullah, PhD Faculty : Institute of Bioscience Breast cancer is the most common cancer and the incidence and mortality rate

had remained high. In Malaysia alone, breast cancers accounted for 31% of all

new cancer cases and are among the most fatal cancers. Since breast cancers

are complex diseases, there is no single marker that is both sensitive and

specific for early detection of the disease. The present study was undertaken to

characterize rat mammary gland tumors as a model for breast cancers and to

determine parameters that could be used as early tumor markers. The study

also undertook to determine the effect of recombinant human erythropoietin

(rHuEPO) and Tamoxifen on the rat mammary gland tumor.

In the first part of the study serum biochemical parameters, angiogenic factors

and tumor markers, tumor histopathology and ultrastructure and expression of

estrogen (ER) and erythropoietin receptors (EPOR) were determined. Twenty

female Sprague-Dawley rats, aged six to seven weeks were divided into two

iv

groups of 10 rats per group. The rats were treated intragastrically, the first

group with 20 mg 7,12-dimethylbenz(a)anthracene (DMBA) per rat to induce

mammary tumor development and the second group with 1 mL 0.9% normal

saline and served as the control. The animals were palpated weekly for tumor

mass and sacrificed two weeks after tumor occurrence. Blood was withdrawn

through cardiac puncture before tumor induction and weekly thereafter. Serum

biochemical parameters analysed were alkaline phosphatase (ALP), aspartate

aminotransferase (AST), alanine transferase (ALT), lactate dehydrogenase

(LDH), creatinine kinase (CK), glucose, blood urea nitrogen (BUN) and

creatinine by a chemistry analyser using standard diagnostic kits. Serum tumor

markers,namely α-fetoprotein (AFP) and CA15-3 were analysed using

automated immunoassay analyser, while the angiogenic factors, matrix

metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF)

were determined by the ELISA technique. Tumor tissues excised from

sacrificed animals were subjected to histopathological analysis and ER-α and

EPOR determination through immunohistochemistry (IHC). Tumor ultrastructure

was examined by transmission electron microscopy (TEM).

The results showed higher ALT, ALP and AST concentrations in the DMBA-

treated than the control group reflecting abnormal liver function. Pronounced

increases in serum LDH were also observed in the DMBA-treated group.

Angiogenic factor estimations showed that serum MMP-2 levels remained high

throughout the study and seemed to have played a greater role than VEGF in

v

early stage tumorigenesis. Serum tumor markers, AFP and CA 15.3 were not

detected in either the treated or control rats. Histopathological analysis showed

features typical of neoplastic cells which were enlarged nuclei, conspicuous

nucleoli nuclear pleomorphism, high nuclear to cytoplasma ratio,

hyperchromasia, and epithelial cell and stroma hyperplasia. These features are

similar to that found in breast cancers. Immunohistochemical analysis showed

that ER-α and EPOR were present in the DMBA-induced rat mammary tumor,

which also resemble human breast cancers. Transmission electron microscopy

analysis of the tumor demonstrated the co-existence of apoptosis, necrosis and

aponecrosis which may be used in the determination of mammary gland tumor

and breast cancer development in the early stages.

In conclusion, the combination of serum liver-related enzymes, serum MMP-2

and histological changes, ER-α and EPOR expression, evidences of apoptosis,

necrosis and aponecrosis may form the panel for screening and determination

of early mammary gland tumors in high risk cancer patients.

The second phase of the study involved the development of a xenograft-

induced mammary gland tumor model in rats as a substitute for the

conventional drug-induced method. The xenograft-induced mammary gland

tumor seems to be reliable and can produce tumors within a short period. The

xenograft model was then used to evaluate the effects of rHuEPO, Tamoxifen

and Tamoxifen-rHuEPO combination on mammary gland tumor growth and

vi

angiogenesis. In this study, 24 rats were divided into four groups of six rats

each. Each rat was treated orally: Group 1 with 60 IU rHuEPO; Group 2 with 20

mg Tamoxifen; Group 3 with a combination of 20 mg Tamoxifen and 60 IU

rHuEPO; Group 4 with 1 mL 0.9% normal saline and served as the control. The

results showed that rHuEPO did not promote mammary tumor growth and in

fact may enhance the cytotoxicity of Tamoxifen through the stimulation of

proapoptotic and antiproliferative effects. This study suggests that rHuEPO

treatment in cancer patients may not only be beneficial for the alleviation of

anemia due to the disease, but also augments the effect of certain

chemotherapeutic drugs used in the treatment of cancers.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Doktor Falsafah

PENCIRIAN TUMOR KELENJAR MAMA TIKUS TERARUH DAN KESAN ANTITUMOR ERITROPOIETIN MANUSIA REKOMBINAN DAN TAMOXIFEN

Oleh

SAIRAH ABDUL KARIM

2008

Pengerusi : Profesor Rasedee Abdullah, PhD

vii

Fakulti : Institut Biosains Kanser payudara merupakan kanser yang sangat biasa berlaku dan kadar

insidens dan kematiannya kekal tinggi. Di Malaysia sahaja, kanser payudara

menyumbang sebanyak 31% kepada kes baru kanser dan merupakan kanser

yang paling banyak menyebabkan kematian. Memandangkan kanser payudara

adalah penyakit yang kompleks, tiada satu petanda tunggal yang peka dan

khusus untuk pengesanan awal penyakit ini. Kajian ini telah diambil untuk

mencirikan tumor kelenjar mama tikus sebagai model untuk kanser payudara

and menentukan parameter yang boleh digunakan sebagai petanda awal

tumor. Kajian ini juga telah dijalankan bagi menentukan kesan eritropoietin

manusia rekombinan (rHuEPO) dan Tamoxifen terhadap tumor kelenjar mama

tikus.

Di dalam bahagian pertama kajian, parameter biokimia serum, faktor

angiogenesis dan petanda tumor, histopatologi tumor dan ultrastruktur dan

pernyataan reseptor estrogen (ER) dan eritropoietin (EPOR) telah ditentukan.

Di dalam kajian ini, 20 ekor tikus betina Sprague-Dawley, berumur 6 hingga 7

minggu telah dibahagikan kepada dua kumpulan terdiri daripada 10 ekor tikus

setiap kumpulan. Tikus-tikus ini diperlakukan secara intragaster, kumpulan

pertama dengan 20 mg 7,12-dimetilbenz(a)antrasin (DMBA) setiap ekor untuk

mengaruhkan pembentukan tumor dan kumpulan kedua diberi 1 mL 0.9%

larutan salina normal dan bertindak sebagai kumpulan kawalan. Haiwan

dipalpatkan setiap minggu untuk pengesanan tumor dan dimusnahkan dua

viii

minggu selepas kejadian tumor. Darah diambil secara tusukan jantung sebelum

pembentukan tumor diaruhkan dan setiap minggu seterusnya. Parameter

biokimia serum yang dianalisis ialah alkalin fosfatase (ALP), aspartat

aminotransefase (AST), alanin transferase (ALT), laktat dehidrogenase (LDH),

glukosa, urea nitrogen darah (BUN) dan kreatinin secara penganalisis kimia

dan kit diagnostik standard. Petanda tumor serum, α-fetoprotein (AFP) dan CA

15.3 telah dianalisis menggunakan penganalisis immunoassai automatik,

sementara faktor angiogenesis, matrik metalloproteinase-2 (MMP-2) dan faktor

pertumbuhan endotelium vesel (VEGF) telah ditentukan menggunakan teknik

ELISA. Pada tisu tumor yang diambil dari haiwan yang dimusnahkan, dilakukan

analisis histopatologi dan penentuan reseptor ER-α dan EPOR secara

immunohistokimia (IHC). Ultrastruktur tumor telah diperiksa menggunakan

mikroskop elektron transmisi (TEM).

Keputusan kajian menunjukkan kepekatan ALT, ALP dan AST lebih tinggi

dalam tikus kumpulan DMBA-diperlaku berbanding kumpulan kawalan,

mencerminkan keabnormalan fungsi hati. Peningkatan nyata LDH serum

diperhatikan dalam kumpulan DMBA-diperlaku. Penganggaran faktor

angiogenesis menunjukkan bahawa paras MMP-2 serum kekal tinggi

sepanjang kajian dan nampaknya memain peranan lebih besar daripada VEGF

pada peringkat awal tumorigenesis. Petanda tumor serum, AFP dan CA15.3

tidak dikesan samada dalam tikus tumor kelenjar mama DMBA-teraruh atau

kawalan. Analisis histopatologi menunjukkan sifat yang tipikal untuk sel

neoplasia seperti nukleus besar, pleomorfisma nukleus ketara, nisbah nuklear

ix

kepada sitoplasma tinggi, hiperkromasia dan hiperplasia sel epithelium dan

stroma. Semua sifat ini menyerupai apa yang terdapat pada kanser payudara.

Analisis immunohistokimia menunjukkan kehadiran ER-α dan EPOR pada

tumor kelenjar mama DMBA-teraruh yang juga menyerupai kanser payudara

manusia. Analisis elektron mikroskop transmisi terhadap tumor menunjukkan

pewujudan bersama apoptosis, nekrosis dan aponekrosis yang mungkin boleh

diguna dalam penentuan perkembangan tumor kelenjar mama dan kanser

payudara peringkat awal.

Sebagai kesimpulan, gabungan enzim berkaitan hati, MMP-2 serum dan

perubahan histologi, pernyataan ER-α dan EPOR, bukti apoptosis, nekrosis dan

aponekrosis mungkin boleh jadi panel bagi penyaringan dan penentuan untuk

peringkat awal tumor kelenjar mama bagi pesakit-pesakit berisiko tinggi untuk

kanser.

Fasa kedua kajian ini melibatkan pembentukan tumor kelenjar mama xenograf-

teraruh pada tikus sebagai ganti kepada kaedah drug-teraruh biasa. Tumor

kelenjar mama xenograf-teraruh adalah stabil dan penghasilan tumor berlaku

dalam masa yang singkat. Model xenograf ini seterusnya digunakan untuk

menilai kesan rHuEPO, Tamoxifen dan gabungan Tamoxifen-rHuEPO terhadap

pertumbuhan tumor kelenjar mama dan angiogenesis. Dalam kajian ini, 24 ekor

tikus dibahagikan kepada empat kumpulan terdiri daripada enam ekor setiap

kumpulan. Setiap ekor tikus diperlakukan secara oral: Kumpulan 1 diberi 60 IU

x

rHuEPO; Kumpulan 2 diberi 20 mg Tamoxifen; Kumpulan 3 diberi gabungan 20

mg Tamoxifen dan 60 IU rHuEPO; Kumpulan 4 diberi 1 mL 0.9% larutan salina

normal dan bertindak sebagai kawalan. Keputusan kajian menunjukkan

rHuEPO tidak menggalakan pertumbuhan tumor kelenjar mama malah mungkin

meningkatkan lagi kesitotoksikan Tamoxifen melalui perangsangan kesan

proapoptosis dan antiproliferatif. Kajian ini mencadangkan rawatan rHuEPO

bagi pesakit kanser bukan hanya berfaedah untuk meringankan anemia

disebabkan penyakit tersebut, tetapi juga memperkuatkan kesan kesitotoksikan

beberapa drug kemoterapi tertentu yang diguna dalam rawatan kanser.

xi

ACKNOWLEDGEMENTS

I would sincerely like to extend my greatest and deepest appreciation to my

main supervisor, Prof. Dr. Rasedee Abdullah for his guidance, advice and

encouragement which he unselfishly gave throughout the course of this study. I

would also like to thank to my co-supervisors, Prof. Dato Dr. Sheikh Omar

Abdul Rahman and Assoc. Prof. Dr. Rozita Rosli for their suggestions and

support.

Sincere gratitude and appreciation are forwarded to Dr. Hazilawati Hamzah,

Mrs. Fauziah Nordin, Mr. Halmi Othman, Mr. Abdullah Misron, staff in

Hematology Laboratory of Faculty of Veterinary Medicine for their technical

assistance during this study. Thanks also forwarded to staff in Electron

Microscopy and Imaging Unit, Institute of Bioscience (IBS) and to all my friends

in UPM.

Last but not least, I would like to extend my extreme gratitude to my sporting

and understanding loving husband, Mr. Mohd. Fadhil Ahmad, my inspired

children, Faiz Isqandar and Aleya Yasmin, my supportive parents and family

members for their constant support and encouragement.

xii

I certify that an Examination Committee has met on 13 August 2009 to conduct the final examination of Sairah Abdul Karim on her Doctor of Philosophy thesis entitled Characterisation of Induced Rat Mammary Gland Tumor and the Antitumor Effect of Recombinant Human Erythropoietin and Tamoxifen in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the Doctor of Philosophy. Members of the Examination Committee are as follows: Mohd Hair Mohd Bejo, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Abdul Rani Bahaman, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Examiner 1) Mohd Ali Rajion, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Examiner 2) Yasmin Hanum, PhD Associate Professor, Universiti Kebangsaan Malaysia External Examiner, _____________________ Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date :

xiii

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows: Rasedee Abdullah, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Dato’ Sheikh Omar Abdul Rahman Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Rozita Rosli, PhD Associate Professor Faculty of Medicine and Health Science Universiti Putra Malaysia (Member) ______________________ AINI IDERIS, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia Date:

xiv

DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledge. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institutions. ______________________________ SAIRAH ABDUL KARIM Date: 25 November 2008

xv

TABLE OF CONTENTS

Page DEDICATION ii ABSTRACT iii ABSTRAK vii ACKNOWLEDGEMENTS xi APPROVAL xii DECLARATION xiv LIST OF FIGURES xvii LIST OF TABLES xx LIST OF ABBREVIATIONS xxi CHAPTER

I INTRODUCTION 1

II LITERATURE REVIEW 3 Breast Cancer Overview 3 Introduction 3 Human Mammary Gland Tumors 5 Predisposition

Hormones Genes

6 6 7

Screening and Early Detection Imaging Tumor Markers Current Techniques in Breast Cancer Detection Enzymes Linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR) Immunohistochemistry Chromosome Analysis Comparative Genomic Analysis Fluorescence In Situ Hybridization (FISH) Proteomics Serum Biochemistry

9 10 11 14 14

14 15 15 16 17 18

Management of Breast Cancer Surgery Chemotherapy Drug Delivery Targeted Therapy

19 19 20 22 22

Tumor Angiogenesis Vascular Endothelial Growth Factor Matrix Metalloproteinases

24 26 27

Prognosis and Predictive Factors of Breast Cancer 28

xvi

Cell Death In Cancer Apoptosis Differences between Apoptosis and Necrosis

30 30 34

Erythropoietin Therapy in Cancer-Related Anemia 35 Relevant of Animal Models for Cancer Research 40 Animal Models for Human Diseases

Transgenic Animal Models Animal Ethics in Research Rodent Mammary Gland Tumor

40 42 43 44

III SERUM ENZYME AND METABOLITE

CONCENTRATIONS DURING DMBA-INDUCED MAMMARY TUMORGENESIS IN FEMALE SPRAGUE-DAWLEY RATS

45

Introduction 45 Materials and Methods 46 Animals and Management 46 Tumor Induction 46 Body Weight and Tumor Occurrence 47 Blood Collection and Serum Samples 47 Serum Enzymes and Metabolites Analysis 47 Statistical Analysis 48 Results 49 Discussion 60

IV SERUM ANGIOGENIC FACTOR AND TUMOR MARKER CONCENTRATIONS IN FEMALE SPRAGUE-DAWLEY RATS DURING TUMORGENESIS

67

Introduction 67 Materials and Methods 68 Animals and Management 68 Blood Collection and Serum Samples 68 Serum Angiogenic Factor Analysis 68 Serum Tumor Markers Analysis 69 Results 70 Discussion 73

V HISTOPATHOLOGY AND EXPRESSION OF ESTROGEN AND ERYTHROPOIETIN RECEPTORS ON DMBA-INDUCED MAMMARY TUMOR IN FEMALE SPRAGUE- DAWLEY RATS

77

Introduction 77 Materials and Methods 78 Animals and Management 78 Tumour Measurement and Histopathological

xvii

Analysis 78 Immunohistochemical Staining for ER-α 81 Immunohistochemical Staining for EPOR 81 Results 83 Discussion 85

VI ULTRASTRUCTURE OF EARLY STAGE MAMMARY TUMOR IN FEMALE SPRAGUE-DAWLEY RAT

104 Introduction 104 Materials and Methods 105 Animals and Management 105 Transmission Electron Microscopy Procedures 105 Morphometric Analysis 105 Results 107 Discussion 116

VII EFFECTS OF rHuEPO TOWARDS THE TUMOR GROWTH AND ANGIOGENESIS IN MAMMARY TUMOR XENOGRAFT MODELS

122 Introduction 122 Materials and Methods 123 Animals Management 123 Xenograft Technique for Mammary Tumor

Induction

123 Preparation of rHuEPO 123 Preparation of Tamoxifen

Experimental Designs Tumor Mass Measurement Blood Collection VEGF and MMP-2 Analysis Histopathological Analysis Results

124 124 124 125 125 125 127

Discussion 142

VIII GENERAL DISCUSSION AND CONCLUSION 142

REFERENCES 148 APPENDICES 176 BIODATA OF THE AUTHOR 184

xix

LIST OF FIGURES

Figure Page

1 Weekly changes in animal body weight in DMBA-treated and control rats.

54

2 Weekly changes in serum ALT of DMBA-treated and

control rats. 56

3 Weekly changes in serum ALP of DMBA-treated and

control rats. 57

4 Weekly changes in serum AST of DMBA-treated and

control rats. 58

5 Weekly changes in serum LDH of DMBA-treated and

control rats. 59

6 Weekly changes in serum glucose in DMBA-treated

and control rats. 61

7 Weekly changes in BUN of DMBA-treated and control

rats. 62

8 Weekly changes of serum creatinine of DMBA-treated

and control rats. 63

9 Mammary tumor tissues dissected from female

Sprague-Dawley rat 2 week after detection. The tumor showed a nodule appearance.

10 Mammary tumor section from a female Sprague-

Dawley rat with grade 1 mitotic activity (arrows) (H&E,x1000)


Dawley rat with moderate nuclear pleomorphism (grade II) (H&E, x1000).


Dawley rat with grade II tubule formation (10-75%) (H&E, x400)

xx

13 Mammary tumour section from a female Sprague-Dawley rat with mild hyperplasia (H&E, x400)

14 Mammary tumour section from a female Sprague-

Dawley rat with focal necrosis (H&E, x200)

15 Normal mammary gland tissue of Sprague-Dawley rat

showing single layer of epithelial cells surrounding each tubules (H&E, x200)

16 Immunohistochemical staining of ER-α in normal

mammary gland tissue shows negative immunostaining in the epithelial cells of the tubules (DAB)

17 Immunohistochemical staining of ER-α in the

mammary tumor from a female Sprague-Dawley rat showing strong intensity immunostaining (brown) (DAB)

18 Immunohistochemical staining of ER-α in the

mammary tumor from a female Sprague-Dawley rat showing medium intensity immunostaining (brown) (DAB)

19 Immunohistochemical staining of EPOR in the normal

mammary gland tissue from a female Sprague-Dawley rat showing nuclear staining in several cells (arrows) and classified as level 1 (DAB, x400)

20 Immunohistochemical staining of EPOR in the

mammary tumor from a female Sprague-Dawley rat showing homogenous nuclear staining (DAB)


mammary tumor from a female Sprague-Dawley rat showing strong nuclear staining of tumor cells with granular appearance (arrow) (DAB)


mammary tumor from a female Sprague-Dawley rat showing cell with cytoplasmic EPOR staining (arrow) and granular or non-homogenous appearance in most of the tumor cells (DAB)

xxi

23 Mammary tumor tissue from female rat showing cells pleomorphism with perinuclear chromatin condensation suggesting the cell undergoing apoptosis and the presence of multiple nucleoli. The amorphous dense masses (arrows) are necrotic cells.

24 Mammary tumor tissue from female rat showing

aponecrotic degeneration (red arrow). The cytoplasm is scanty and contained very few organelles, variable-sized vacuoles (yellow arrow) and disrupted mitochondria. The nucleus still intact and the nuclear envelope convoluted and irregular. Heterochromatin is prominent at the perinuclear region (green arrow).

25 Mammary tumor tissue section from female rat shows

swelling cell with relatively large nucleus and a prominent nucleolus (N). The cytoplasm contains numerous variable sized of vacuoles and defective cristae of mitochondria are noted (arrows).


bizarre nuclear shape with an indentation and the cytoplasm contains numerous cisternae rough endoplasmic reticulum (rER) (arrows).

27 Mammary tumor tissue from female rat showing a cell

with very distinctive multiple nucleoli (N) and indented nuclear membrane (arrows).


transverse section of eosinophil which can be recognized by the presence electron dense-stripe crystals with cytoplasmic projection (arrows) or pseudopodias.

29 Mammary tumor tissue from female rat showing a

plasma cell (centre) with eccentrically-positioned nucleus and very extensive rough endoplasmic reticulum (rER) in the cytoplasm. Mast cell (left) is characterised by the presence of large dense granules.

30 A female Sprague-Dawley rat with mammary tumor

induced by xenograft technique. Note the tumor is localized at the thoracic region (arrow)

xxii

31 Rate of mammary tumor progression in control rats and rats treated with Tamoxifen, rHuEPO and Tamoxifen-rHuEPO combination.

32 Serum MMP-2 concentration in control rats and rats

treated with Tamoxifen, rHuEPO and Tamoxifen-rHuEPO combination.

33 Mammary tumor section from control group showing

a mitotic figure (arrow) (H&E, x400)

34 Mammary tumor section from the rHuEPO group

showing several mitotic figures (arrow) (H&E, x600)

xx

LIST OF PLATES

Plate Page

1 Mammary tumor tissues dissected from female Sprague-Dawley rat 2 week after detection. The tumor showed a nodule appearance.

88


Dawley rat with grade 1 mitotic activity (arrows) (H&E,x1000)

90


Dawley rat with moderate nuclear pleomorphism (grade II) (H&E, x1000).

90


Dawley rat with grade II tubule formation (10-75%) (H&E, x400)

91


Dawley rat with mild hyperplasia (H&E, x400) 91


Dawley rat with focal necrosis (H&E, x200) 92

7 Normal mammary gland tissue of Sprague-Dawley rat

showing single layer of epithelial cells surrounding each tubules (H&E, x200)

92

8 Immunohistochemical staining of ER-α in normal

mammary gland tissue shows negative immunostaining in the epithelial cells of the tubules (DAB)

95

9 Immunohistochemical staining of ER-α in the mammary

tumor from a female Sprague-Dawley rat showing strong intensity immunostaining (brown) (DAB)

95

10 Immunohistochemical staining of ER-α in the mammary

tumor from a female Sprague-Dawley rat showing medium intensity immunostaining (brown) (DAB)

96

11 Immunohistochemical staining of EPOR in the normal

mammary gland tissue from a female Sprague-Dawley rat showing nuclear staining in several cells (arrows) and classified as level 1 (DAB, x400)

98

xxi


mammary tumor from a female Sprague-Dawley rat showing homogenous nuclear staining (DAB)

98


mammary tumor from a female Sprague-Dawley rat showing strong nuclear staining of tumor cells with granular appearance (arrow) (DAB)

99


mammary tumor from a female Sprague-Dawley rat showing cell with cytoplasmic EPOR staining (arrow) and granular or non-homogenous appearance in most of the tumor cells (DAB)

99

15 Mammary tumor tissue from female rat showing cells

pleomorphism with perinuclear chromatin condensation suggesting the cell undergoing apoptosis and the presence of multiple nucleoli. The amorphous dense masses (arrows) are necrotic cells.

114


aponecrotic degeneration (red arrow). The cytoplasm is scanty and contained very few organelles, variable-sized vacuoles (yellow arrow) and disrupted mitochondria. The nucleus still intact and the nuclear envelope convoluted and irregular. Heterochromatin is prominent at the perinuclear region (green arrow).

115

17 Mammary tumor tissue section from female rat shows

swelling cell with relatively large nucleus and a prominent nucleolus (N). The cytoplasm contains numerous variable sized of vacuoles and defective cristae of mitochondria are noted (arrows).

116

18 Mammary tumor tissue from female rat showing bizarre

nuclear shape with an indentation and the cytoplasm contains numerous cisternae rough endoplasmic reticulum (rER) (arrows).

117

19 Mammary tumor tissue from female rat showing a cell

with very distinctive multiple nucleoli (N) and indented nuclear membrane (arrows).

118

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