UNIVERSITI PUTRA MALAYSIA

EFFECT OF AZADIRACWTA INDICA EXTRACT ONHEPATOCARCINOGENESIS-INDUCED RATSMANAL MOHAMED

MANAL MOHAMED EL HASSAN TAHA

FPSK(M) 2007 3

3 0 HAY 2008

EFFECT OF AZADIRACWTA INDICA EXTRACT ON HEPATOCARCINOGENESIS-INDUCED RATS

MANAL MOHAMED ELHASSAN TAHA

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for the Degree of Master of

Science.

February 2007

DEDICATION

Specially dedicated to,

M y beloved parents, husband, sisters Manahil and Sarah, brother Ahmed, daughter Roa, supervisors

For their invaluable support, love, patience and intellectual stimulation.. . . . . . . . . . .

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science.

EFFECT OF AZADIRACHTA IADICA EXTRACT ON HEPATOCARCINOGENESIS-INDUCED RATS

MANAL MOHAMED ELHASSAN TAHA

February 2007

Chairman: Associate Professor Fauziah Othman, PhD

Faculty: Medicine and Health Sciences

The effects of 5% A. indica aqueous extract (AI), or more commonly as Neem, on

hepatocarcinogenesis induced Spraque-Dawley male rats were investigated.

Hepatocarcinogenesis was induced in rats by employing a two carcinogen

system: an intraperitoneal injection of 200 mg/kg diethyl nitrosamine (DEN) as

initiator; followed by 0.02% of 2-acetylaminofluorene (AAF) in rat chow for two

weeks to promote carcinogenesis. The rats were then left for two weeks to allow

hepatic preneoplastic lesions to occur. The plant extract was prepared in 5% w/v

in distilled water. Fresh leaves were collected, blended and mixed with distilled

water. Twenty male rats Spraque-Dawley weighmg 150-250g were acclimatized

for 1 week before use. The rats were divided into four groups of five rats each.

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The groups were: DENIAAF-iduced rats (C), DENIAAF-iduced rats treated with

5% A. indica (CAI), normal control rats (N) and normal rats treated with 5% A.

indica extract group (NAI). The rats in group N and NAI were not induced with

cancer however were injected once intraperitoneally with corn oil and act as

normal control. The plant extract was fed to CAI and NAI groups to study its

effects on both cancer and normal groups, respectively.

In this study several parameters were evaluated as means of determining the

effects of A1 on DENIAAF-induced hepatocarcinogenesis in rats. Body and liver

weight profiles, foremost, hepatic lesions were scored in rats induced with

DENIAAF cacinogens especially in the portal and lobular regions of the liver

sections examined for histology. Loss of normal cell organization was also

observed once the hepatocarcinogenesis was induced. In addition to histological

observations, the TUNEL Assay, liver antioxidant enzyme Glutathione S

transferase (GS-T), Glutathione Peroxidase (GPx) in the serum and liver, tumor

marker alpha-fetoprotein (AFP) in serum and molecular detection of AFP and

albumin genes expressions were conducted. The observation of the lesion

scoring have shown significant difference (p<0.05) between DENIAAF and

normal control groups (N, NAI). Histologically there were sigruficant changes in

the lesion scoring of the liver in portal and lobular region in DENIAAF induced

group (C) compared to the DENJAAF treated with A.indica (CAI). TUNEL assay

supported that there was more numbers of apoptotic cells in the liver of (CAI)

group compared to(C) group. The liver enzymes (GST & GPx) activity was

measured and the result for both glutathione Stransferase and glutathione

peroxidase were sigruficantly (p<0.05) higher in the (C) compared to the other

groups (CAI, N, NAI). This result revealed that A. indica extract could reduce the

activity of liver and serum GPx and GST enzymes of rats during

hepatocarcinogenesis. However, the results of body and liver weight profiles

showed that the CAI group was not significantly different (pN.05) from N, C

and NAI groups.

Alpha fetoprotein (AFP), a notable liver tumor marker, level was measured. The

DENIAAF induced group (C) showed the highest increase in AFP levels while

in CAI group illustrated significant (pc0.05) decrease in AFP level. There was no

significant (pM.05) difference between N, NAI and CAI group.

Mokdar detection of gene expression was done by RT-PCR for a-fetoprotein

and albumin specific genes. However, the expression of the AFP gene was

observed only in DEN/AAF induced group (C). Albumin gene expression was

observed in all the study groups C, N, NAI and CAI proving the hepatic nature

of the studied tissue and used as a housekeeping control gene in the RT-PCR

experiments.

As a conclusion, A. indica (Neem) has revealed a chemopreventive capability by

regressing the hepatacarcinogenesis induced by DENJAAF carcinogens. This

capability can be seen from the modulating effects of the plant in the biological

indicators used in this study which can encourage the researchers to consider

the A. indica (Neem) for further on mechanism and toxicology study.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains.

KESAN EKSTRAK AZADIRACHTA INDICA TERHADAP TIKUS ARUHAN HEPATOKARSINOGENESIS

Oleh

MANAL MOHAMED ELHASSAN TAHA

Februari 2007

Pengerusi: Profesor Madya Fauziah Othman, PhD

Fakulti: Perubatan dan Sains Kesihatan

Kesan 5% larutan ekstrak akuas A. indica (AI) atau lebih di kenali sebagai Neem

pda hepatokarsinogenesis terhadap tikus jantan Sprague-Dazoley telah dikaji.

Hepatokarsinogenesis telah diaruh pada tikus menggunakan dua sistem

karcinogen, melalui suntikan 200 mg/kg diethylnitrosamine (DEN) secara

intraperitoneal sebagai pencetus hepatocarcinogenesis dan diikuti dengan

memberikan makanan yang dicampurkan dengan 2-acetylaminofluorene (0.02%

AAF) sebagai 'promoter' hepatokarsinogenesis selama 2 minggu. Ekstrak

tumbuhan disediakan di dalam air suling pada kepekatan 5% w/v. Daun

tumbuhan tersebut dikumpul, dikisar dan dicampurkan bersama air. 20 tikus

jantan Spraque Dawley, 150g-250g diaklimatisasikan selama satu minggu

sebelum diujikaji. Tikus tersebut dibahagikan kepada empat kurnpulan dengan

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lima ekor tifus setiap kumpulan. Kurnpulan tersbut ahila: tikus yang diaruh

dengan DENIAAF (C), tikus yang diaruh dengan DENIAAF dirawat dengan 5%

A. indica (AI), tikus kewalan normal (N) dan tikus normal yang dirawat dengan

5% ekstark A. indica (NAI). Tikus kumpulan N dan NAI tidak diaruhkan dengan

kanser tetapi disuntik secara intraperitonial dengan minyak jagung dan

dijadikan sebagai kawalan. Ekstrak tumbuhan tersebut diberikan kepada

kumpulan CAI dan NAI untuk dikaji kesannya terhadap kumpulan kanser dan

normal.

Dalam kajian ini, beberapa parameter telah ditentukan sebagai purata untuk

menentukan kesan bagi A1 terhadap DENIAAF yang memyebabkan

hepatokarsinogenesis pada tikus. Profil berat dan hati, selain itu, kesan hati

telah dikira dalam tifus disuntik dengan bahan karsinogen DENIAAF terutama

dalamm kawasan portal dan lobular hati telah dikaji dalam pemeriksaan

histologi. Kehilangan organisasi pada sel normal juga telah ditihat apabila

karsinogenesis diaruh. Pemeriksaan histologi juga telah dijalankan

menggunakan asai TUNEL, enzim antioksida hati Glutathione Stransferase

(GST), Glutathione peroxidase (GPx) di dalam serum dan hati, penanda tumor

alpha-fetoprotein (AFP) di dalam serum dan pengesananan molekular AFP dan

ekspresi gen albumin juga telah dijalankan. Pemerhatian terhadap ujian skor

. . . Vll l

kesan menunjukkan perbezaan yang signifikan (p10.05) di antara DENIAAF

dan kumpulan kawalan normal (N, NAI). Pemeriksaan histologi menunjukkan

perubahan pada ujian skor kesan hati dalam kawasan portal pada kumpulan

aruhan DENIAAF (C) berbanding dengan DEN/AAF yang dirawat dengan A.

indica (CAI). Asai TUNEL mengukuhkan bahawa terdapat beberapa sel

apoptotic di dalam hati kumpulan CAI berbanding kurnpulan C. Aktiviti enzim

hati (GST & GPx) diukur dan keputusan bagi kedua glutathione Stransferase

and glutathione peroxidase adalah berbeza secara sigrufikan pada aras keertian

(pc0.05) di antara kumpulan kanser (C) dan kumpulan rawatan (CAI, N, NAI).

Keputusan ini menunjukkan A.indica mampu mengurangkan aktiviti enzim

hati dan serum GST dan GPx pada tikus semasa hepatokarsinogenesis. Walau

bagaimanapun keputusan profil berat jisim tubuh dan hati menunjukkan

kumpulan CAI tidak memberikan perbezaan yang signifikan pada aras keertian

(p > 0.05) daripada kumpulan N, C dan NAI.

Aras alpha fetoprotein (AFP) telah diukur sebagai penanda pertumbuhan sel

kanser hati. Kumpulan DENIAAF (C) menunjukkan peningkatan aras AFP yang

tertinggi, manakala kumpulan CAI menunjukkan penurunan yang signifikan

pada aras keertian (p < 0.05) pada aras AFP. Namun tiada perbezaan yang

signifikan (p > 0.05) di antara kumpulan N, NAI dan CAI.

Pengesahan secara molecular ke atas ekspresi gen telah dilakukan

menggunakan RT-PCR ke atas gen spesifik a-fetoprotein dan albumin. Tetapi ,

ekspresi gen AFP hanya dapat dilihat dalam kumpulan yang diaruh dengan

DENIAAF (C). Ekspresi gen albumin diperhatikan dalam kurnpulan kajian C, N,

NAI dan CAI yang membuktikan sifat semulajadi hepatic pada organ yang

dikaji dan pembersih gene kawalan dalam ujikaji RT-PCR.

Kesimpulannya, A. indica (Neem) telah dinyatakan sebagai kemopreventif

untuk penyakit hepatokarsinogenesis yang diaruhkan oleh bahan karsinogen

DENIAAF. Keupayaan ini boleh dilihat melalui kesan tumbuhan dalam

penunujuk biologi yang digunakan dalam kajian ini dimana akan

menggalakkan penyelidik A. indica (Neem) untuk mempertimbangkan

mekanisma dan toksikologi yang akan datang.

ACKNOWLEDGEMENTS

First my praise to Almighty Allah for giving me the power and will to complete

this study and peace be upon his final Prophet and Messenger Mohamed.

I would like to convey sincere gratitude to Associate Professor Dr. Fauziah

Othman the Chairman of my Supervisor Committee for her invaluable advice,

guidance, constant support and encouragement. I would like to extend my

grateful thanks and appreciation to the members of my Supervisory Committee

Associate Professor Dr. Patimah Ismail and Dr. Parichehr Hanachi for their

constructive suggestion, advice and support throughout the course of this study.

I gratefully acknowledge the "Malaysian Technical Co-operation Programme

(MTCP) for giving me this opportunity and for their financial support for my

Master programme.

I am gratefully thanking all the staff of Faculty of Medicine and Health Science

and Institute of Bioscience for their constant assistance and friendship.

It is worth to mention my colleagues and friends from Sudanese community in

UPM and Serdang area for their friendship and companion. Finally yet

importantly, I would like to extend my sincere appreciation to my husband

Siddig Ibrahim and my daughter Roa for their patience, sacrifices and moral

support during the course of the study.

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I certifiy that an Examination Committee has met on 26th February 2007 to conduct the final examination of Manal Mohamed Elhassan Taha on her Master of Sceince thesis entitled "Effect of Azadirachta indica Extract on Hepatocarcinogenesis-Induced Rats" 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 relevant degree. Members of the Examination Committee are as follows:

Abdul Hamid Abdul Rashid, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman)

Chong Pei Pei, PhD Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner)

Abdah Md. Akim, PhD Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner)

Azimatol Hawariah Lope Pihie, PhD Professor Faculty of Science and Technology Universiti Kebangsan Malaysia (External Examiner)

School of Graduate Studies Universiti Putra Malaysia

Date: '17 MAY 2007 ...

X l l l

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows:

Fauziah Othman, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman)

Patimah Ismail, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member)

Parichehr Hanachi, PhD Department of Biomedical Sciences Alzahra Research Centre Tehran, Iran (Member)

AINI IDERIS, PhD Professor/Dean School of Gradute Studies Universiti Putra Malaysia

Date: 14 JUNE 2007

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DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

MANAL MOHAMED ELHASSAN TAHA

Date: 17 July 2006

3 0 MAY 2008

TABLE OF CONTENTS

DEDICATION ABSTRACT ABSTRACT ACKNOWLEDGEMENTS APPROVAL DECLARATION TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

CHAPTER

INTRODUCTION

LITRETURE REVIEW 2.1 A. indica (Neem)

2.1.1 Elements in Aindica Leaves 2.1.2 A. indica as an Anti-cancer Agent 2.1.3 Lirnonoids as Anti-cancer Agent

2.2 Cancer 2.3 Liver

2.3.1 Histology of the Liver 2.4 Liver Cancer

2.4.1 Causes of Liver Cancer 2.4.2 Hepatocellular Carcinoma (HCC) 2.4.3 Hepatocarcinogenesis 2.4.4 Modeling Hepatocarcinogenesis 2.4.5 Introduction of Hepatocarcinogenesis

2.5 Chemical Carcinogens 2.5.1 Diethylnitrosamine (DEN) 2.5.2 Acetylaminoflourine AAF

2.6 Biotransformation Enzymes 2.6.1 Glutathione Stransferase (GST) 2.6.2 Glutathione Peroxidase (GPx)

2.7 Lesion Scoring 2.8 Apoptosis

Page ii iii vii xi xiii xv xvi xvii XX

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2.9 TUNEL Assay (Apoptosis Detection System) 2.10 Tumor Markers 2.9.1 Alpha Fetoprotein (AFP) 2.10.2 AFP Gene Expression 2.9.3 RT-PCR

MATERIAL AND METHODS 3.1 Materials and chemicals: 3.2 Preparation of 5% Neem leaves aqueous extract 3.3 Diethylnitrosamine (DEN) preparation 3.3 Acetylarninofluorene (AAF) preparation: 3.4 Animals

3.4.1 Pre-treatment 3.4.2 Treatment 3.4.3 Post treatment

3.5 Preparation of the cytosol 3.6 Enzyme assay

3.6.1 Glutathione S-transferase (GST) Assay 3.6.2 Glutathione Peroxidase Assay 3.6.3 Protein determination

3.7 Histological study 3.7.1 Light microscope 3.7.2 Mean lesion scoring

3.8 Alpha-feto protein Assay 3.8.1 The principle of the alpha fetoprotein (AFP) kit

3.9 Detection of Apoptotic Cells (TUNEL Assay) 3.10 Reverse Transcriptase Polymerase Chain Reaction

3.10.1 RNA Isolation 3.10.2 RT-PCR 3.11 Statistical Analysis

RESULTS 4.1 Body Weight Profile 4.2 Liver Weight and Relative Liver Weight 4.3 Biotransformation Enzymes 98

4.3.1 Gluatathione -S-Transferase (GST) Assay 4.3.2 Glutathione Peroxidase (GPx) Assay

4.4 Histology 4.5 Lesion Scoring 4.6 The Assay of Alpha-fetoprotein 4.7 Expression of AFP and albumin gene

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4.8 The Detection of Apoptosis (TUNEL assay)

5 DISCUSSION 118 5.1 The Effect of Heapatocarcinogens and Supplementation of A. indica on Body Weight, Liver Weight and the Ratio LiverIBody Weight 120 5.2 Detoxification Enzyme 121

5.2.1 The Effect Of A. Indica On Glutathione-STransferase Activities In DEN/AAF Induced Liver Cancer And Control Rats Groups. 122 5.2.2 The Influence of A. indica Oral Administration on Glutathione Peroxidase (GPx) Activity in Liver and Serum of DENIAAF Induced Liver Cancer. 1 24

5.3 Histology 127 5.3.1 Light Microscopy 127 5.3.2 Mean Lesion scoring 1 29

5.4 Alpha-fetoprotein 130 5.5 The Effect of A. indica on the Expression of AFP mRNA. 132 5.6 Apoptogenic Effects of A. indica 1 34

6 CONCLUSION REFERENCES APPENDICES BIODATA OF THE AUTHOR LIST OF PUBLICATIONS

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Table

LIST OF TABLES

Causes of death due to cancer among medically certified deaths in Malaysia.

Tissue Dehydration in the tissue processing

Colorization with Hematoxylin and Eosin (H&E) According to McManus (1960).

The effect of A. indica on the mean lesion scoring of rats treated with DEN and AAF and normal control groups.

Page

xix

LIST OF FIGURES

Figure Page

A. indica A. Juss

Structure of the AFP gene regulatory region.

Flow chart on the method of preparing the 5% aqueous 75

Neern leaves extract

Schematic representation of the animal treatment of cancer

group.

Schematic representation of the animal treatment of normal 80

group.

The effect of 5% A. indica aqueous extract on body weight 97 profile in control and cancer groups.

The effect of 5% Neem on liver weight and relative liver weight in control and cancer groups.

The effect of 5% Neem on G S activity in the liver and serum 100 of DEN and AAF-induced cancer rats.

The effect of 5% Neem on GPx activity in the liver and serum 102 of DEN and AAF-induced cancer rats.

Light micrograph of normal rat liver from the normal control 104

€Pup.

Light micrograph of 5% Neem treated rat liver from normal 104 group (NAI) over a period of 10 weeks.

Light Micrograph of liver cancer control group rats induced 105 by DENJAAF.

Light micrograph of liver section from DENIAAF induced 105 hepatocarcinogenesis rat supplemented with 5% Neem aqueous leaves extract.

Blood AFP concentration in the different group of rats.

Effect of A. indica aqueous extract on the expression of AFP 110 gene of DENIAAF induced rats.

Effect of A . indica aqueous extract on the expression 111 hepatocyte-specific gene for albumins.

Confocal micrograph of TUNEL assay of rat liver tissue 113 section of normal group.

Confocal micrograph illustrating liver of normal rats treated 114 with A.indica extract (NAI).

Confocal Microgarphs of TUNEL assay done for liver 115 sections from DENIAAF induced hepatocarcinogenesis rats.

Confocal migrogarphs of TUNEL assay done for liver 116 sections from DENIAAF induced hepatocarcinogenesis rats treated with 5% aqueous extract of A . indica leaves.

Graph showing the effect of 5% A.indica aqueous extract on 117 DEN /AAF induced cancer in rats using TUNEL assay.

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A.indica

Abs

OC

cDNA

Cm

CDNB

DEN

DNA

EDTA

FITC

g

GSH

GST

GSSG redudase

LIST OF ABBREVIATIONS

Alpha-fetoprotein

A . indica

Absorbance

Centigrade

Complementary DNA

Centimeter

Deoxyribonuecleic acid

Disodiurn Ethylene Diarninetetracetate

Fluorescein isothiocynate

Gram

Glutathione

Glutathione Stransf erase

Glutathione Peroxidase

Glutathione reductase

Hydrogen peroxide

Phosphoric acid

HCL

tr 1

mg

NaOH

NADPH

NMDA

Neem

PBS

RT-PCR

Hematoxylin and eosin

Hydrochloric acid

Potassium dihydrogen orthophosphate

Potassium dihydrogen orthophosphate

Kilogram

Botassium chloride

Mililitre

Minute

Microlitre

Miligram

Sodium hydroxide

Nicotinamide Adenine Dinucleotide Phosphate

N-dirnethyhitrosamine

A. indica

Sodium nitrate

Phosphate buffer saline

Basepair

Hydrogen ion concentration

Propidium iodide

Reverse Transcriptase Polymerase Chain Reaction

xxiii

RNA

RLUs

S.D

TdT

TUNEL

TBE

UPM

UV

Ribonucleic acid

Relative light units

Standard deviation

Terminal deoxynuclmtidyl transferase

Terminal deoxynuclmtydil transferase-mediated dUTP nick end-labeling Tris base EDTA

Universiti Putra Malaysia

Ultraviolet

xxiv