UNIVERSITI PUTRA MALAYSIA

THE EFFECT OF ALOE VERA, VITAMIN C AND E ON TUMOUR MARKER ENZYMES IN

HEPATOCARCINOGENESIS

ABDAH MD AKIM

FSAS 1998 24

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THE EFFECT OF ALOE VERA, VITAMIN C AND E ON TUMOUR MARKER ENZYMES IN

HEPATOCARCINOGENESIS

By

ABDAH MD AKIM

Thesis Submitted in Fulfilment of the Requirements for the Degree of Master of Science in the

Faculty of Science and Environmental Studies, Universiti Putra Malaysia

February 1998

ACKNOWLEDGEMENTS

I would like to thank Associate Professor Dr. Nor Aripin Shamaan for his

enthusiasm, guidance and invaluable advice that had been given to me throughout

my project. I wish to express my deep appreciation to his wife, Professor Dr. Wan

Zurinah Wan Ngah from the Department of Biochemistry, Faculty of Medicine,

UKM, Kuala Lumpur for her advice. I would like to express my gratitude to

Associate Professor Dr. lohari Ramli and Associate Professor Dr. Mohd Arif Syed

for having to put in many long hours in reviewing my thesis. I am greatly indebted

to Puan Zanariah larien, Puan Rosnani Ismail, Puan Khairiah and other technologists

at the Department of Biochemistry in UKM for their assisstance and technical

advice. I am also thankful to my parents for their encouragement and moral support

in pursuing my dreams. To my baby daughter, for the incredible joy, gift and

privilege, she gives to me while I'm writing my thesis. Last but not least, to my

husband who is always there during the best and worst moments in my life.

ii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii LIST OF TABLES .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii LIST OF ABBREVIATIONS ......................................................................................... x ABSTRACT. . ............................................................................................................... XI ABSTRAK. . ............................................................................................................. XUI

CHAPTER

1 INTRODUCTION .................. ................................................................. 1

2 LITERATURE REVIEW ..... ......................... . ......................................... 3

Cancer in General. ................................................................................... 3 Liver Cancer . . .......................................................................................... 3 Causes of Liver Cancer . ....................... ................................................... 4 Multistage Carcinogenesis . ......... ........................... ............ ..................... 7 Carcinogen Metabolism . .. . ........................................ ......... , . . . . . . . . . . . . . . . . " .. 9 Electrophilic Theory .... ,', . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Classes of Chemical Carcinogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . 9 Terms in Metabolic Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 DNA Adduct Formation . ................................ , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 Diethylnitrosamine (DEN), . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2-Acetylaminofluorene .......................................................................... 13 Liver Models . ... ..................................................................................... 16 Cancer Prevention and Protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Vitamin C . ............................................................................................. 18 Chemical Nature and Structure . ................... ......................................... 18 Function in Cells . ....... ........................................................................... 19 Vitamin C and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Anticarcinogenic Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 Vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Chemical Nature and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Functions in Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Vitamin E and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Possible Anticarcinogenic Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Aloe vera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Chemical Components of Aloe vera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

iii

Functions of Aloe vera . ......................................................................... 29 Tumour Marker Enzymes ...................................................................... 29 Alkaline Phosphatase (ALP) . ................................................................ 31 Glutathione-S-Transferase (GST) . ........................................................ 33 Metabolism of Carcinogens by GST . .................................................... 36 Gamma-Glutamyl Transpeptidase (GGT) ............................................. 37 UDP-Glucuronyl Transferases (UDPGT) . ............................................ 38 Glutathione Peroxidase .......................................................................... 40 Glutathione Reductase ..................... ...................................................... 41 Glutathione Redox Cycle . ..................................................................... 42 Hypothesis ............................................................................................. 42 Objective of the Research ...................................................................... 43

3 METHODOLOGY ................................................................................ 44

Materials .................................................................................................... 44 Methods .......................................................................... , .......................... 44 Animal .... .............................................................................................. 44 Induction of Hepatocarcinogenesis ....................................................... 45 Partial Hepatectomy (PH) ..................................................................... 45 Aloe vera, Vitamin C and E Supplementation in Normal and Cancer-Induced Groups ............................................................. � ........... 46 The Cytosolic and Microsomal Fractions Preparations . ....................... 50 Alkaline Phosphatase (ALP) Assay . ............................................ ......... 50 Glutathione-S-Transfeqase (GST) Assay ............................................... 51 Gamma-Glutamyl 'Wtinsferase (GGT) Assay .......... ............................. 52 UDPGT Assay . ...................................................................................... 53 Glutathione Peroxidase Assay ............................................................... 54 Glutathione Reductase (GSH-Rx) Assay . ............................................. 55 Protein Determination . .......................................... ................................ 56 Statistical Analysis . ............................................................................... 56

4 RESULTS .............................................................................................. 58

The Effect of A V, VC, E and T Supplementation on Body and Liver Weights in Normal and Carcinogen-Induced Rats After 16 Weeks . .... 58 The Effect of AV, VC, E and T Supplementation on ALP Activities in Control Groups in 16 Weeks . ......... ............ ........................................... 59 The Effect of AV, VC, E and T Single Supplementation on ALP Activities in Hepatocarcinogen-Induced Groups in 16 Weeks . ............ 60 The Effect of A V, VC, E and T Supplementation on ALP Activities in Combined Supplementation in 16 Weeks . ............................................ 61

IV

The Effect of AV, VC, E and T Supplementation on GST Activities in Control Groups in 16 Weeks . .................................................. .............. 63 The Effect AV, VC, E and T Single Supplementation on GST Activities in Hepatocarcinogen-Induced Groups in 16 Weeks . . ........... ................. 64 The Effect of AV, VC, E and T Supplementation on GST Activities in Combined Supplementation in 16 Weeks . ............................. ...... ......... 65 The Effect of AV, VC, E and T Supplementation on GGT Activities in Control Groups in 16 Weeks . ................................................ ................ 66 The Effect of AV, VC, E and T Single Supplementation on GGT Activities in Hepatocarcinogen-Induced Groups in 16 Weeks . ............ 67 The Effect of A V, VC, E and T Supplementation on GGT Activities in Combined Supplementation in 16 Weeks . ........................ .................... 68 The Effect of AV, VC, E and T Supplementation on UDPGT Activities in Control Groups in 16 Weeks . ................. ........................................... 69 The Effect of AV, VC, E and T Single Supplementation on UDPGT Activities in Hepatocarcinogen-Induced Groups in 16 Weeks . ............ 70 The Effect of AV, VC, E and T Supplementation on UDPGT Activities in Combined Supplementation in 16 Weeks . ........................................ 71 The Effect of A V, YC, E and T Supplementation on GPx Activities in Control Groups in 16 Weeks . ...................................................... .......... 72 The Effect of AV, VC, E and T Single Supplementation on GPx Activities in Hepatocarcinogen-Induced Groups in 16 Weeks . ............ 73 The Effect of AV, VC, E and T Supplementation on GPx Activities in Combined Supplementation in 16 Weeks . ............................................ 74 The Effect of AV, VC, E and T Supplementation on GRx Activities in Control Groups in 16 Weeks . ....................... ........... .............................. 75 The Effect of AV, VC, E and T Single Supplementation on GRx Activities in Hepatocarcinogen-Induced Groups in 16 Weeks . ............ 76 The Effect of AV, YC, E and T Supplementation on GRx Activities in Combined Supplementation in 16 Weeks . .......................... .................. 77

5 DISCUSSION ....................................................................................... 79

The Effect ofHepatocarcinogens and Supplementation on Body Weight, Liver Weight and the Ratio LiverlBody Weight.. ........... ...................... 79 The Effect of AV, YC, E and T Supplementation on ALP Activities in Control and Hepatocarcinogen-Induced Groups in 16 Weeks .............. 80 The Effect of AV, VC, E and T Supplementation on GST Activities in Control and Hepatocarcinogen-Induced Groups in 16 Weeks .............. 81

The Effect of A Y, YC, E and T Supplementation on GGT Activities in Control and Hepatocarcinogen-Induced Groups in 16 Weeks .............. 83

v

The Effect of AV, VC, E and T Supplementation on UDPGT Activities in Control and Hepatocarcinogen-Induced Groups in 16 Weeks .......... 84 The Effect of A V, VC, E and T Supplementation on GPx Activities in Control and Hepatocarcinogen-Induced Groups in 16 Weeks .............. 85 The Effect of AV, VC, E and T Supplementation on GRx Activities in Control and Hepatocarcinogen-Induced Groups in 16 Weeks .............. 86 The Effect of Partial Hepatectomy on Normal Liver ............................ 87 General Discussion ................................................................................ 87 The Effect of Aloe vera Supplementation on Hepatocarcinogenesis .... 89 The Effect of Vitamin C Supplementation on Hepatocarcinogenesis ... 90 The Effect of Vitamin E Supplementation on Hepatocarcinogenesis ... 91 The Effect of Combined Supplementation on Hepatocarcinogenesis ... 91 The Effect of Antioxidants on Hepatocarcinogenesis ........................... 93 Possible Enzyme Induction ................................................................... 94 Host Susceptibility Factors .................................................................... 96 Recommendations for Future Research ................................................ 96

6 CONCLUSION ..................................................................................... 98

REFERENCES . ............................................................................................................ 99

APPENDICES

A-I The effect of A V, VC and E on body and liver weight after 1 6 weeks of treatment. ................ ................................................................................. I I I

A -2 The mean specific activity of ALP after 16 weeks treatment .................. 112 A-3 The mean specific activity of GST after 16 weeks treatment .................. 113 A-4 The mean specific activity of GGT after 16 weeks treatment ................. 114 A-5 The mean specific activity of UDPGT after 16 weeks treatment ............ 115 A-6 The mean specific activity of GPx after 16 weeks treatment.. ................. 116 A -7 The mean specific activity of GRx after 16 weeks treatment .................. 117 B-1 Reagents for Alkaline phosphatase assay ................................................. 118 B-2 Reagents for UDPGT assay ...................................................................... 119 B-3 Reagents for Glutathione-s-transferase assay ........................................... 120 B-4 Reagents for Gamma glutamyl transferase assay ..................................... 121 B-5 Reagents for Glutathione peroxidase assay .............................................. 122 B-6 Reagents for Glutathione reductase assay . ............................................... 123 B-7 Reagents for protein determination .......................................................... 124

VITA. . ............................................................................................................ 125

vi

LIST OF TABLES

Table Page

1 Causes of Cancer . ................................................................................. 6

2 Mechanisms of Cancer Chemoprotection by Antioxidants . ............... 17

3 Chemical Composition of Aloe vera . . ................. ................................ 28

4 Rat Cytosolic GST Subunits ............................................................... 35

5 Non Selenium-dependent GPx Activity Related to Protection from PUF A Peroxidation or Peroxide .................................. ..... . . ....... 41

6 Enzyme activities in supplementation of AY, YC, E and T in normal control and hepatocarcinogen-induced rats ........................ 78

Vll

LIST OF FIGURES

Figure Page

1 Pathway of carcinogen leading to cancer . . ............................................ 7

2 Multistage of cancer ................................................................................ 8

3 The two stages of 2-AAF being metabolic activated . .......................... 14

4 The different species of 2-AAF and DEN throughout the metabolic

activation . . ......................................................................................... 15

5 The oxidation reaction of 2-AAF and its products . . ............................ 15

6 Metabolism of vitamin C. . ................................................................... 1 9

7 Chemical structure of a -tocopherol. .................................................... 23

8 Chemical structure of y-tocotrienol. . ................................................... 23

9 The proposed location of vitamin E in cell membrane . . ...................... 24

1 0 The synergistic interaction between vitamin C and E . . ....................... 25

I I The involvement of phase I and II enzymes in carcinogenesis . . ......... 3 1

1 2 Glutathione conjugation and the formation of mercapturic acid . . ....... 36

1 3 Scheme for association of GGT with the membrane . . ......................... 38

1 4 The glutathione redox cycle . ................................................................ 42

1 5 Study protocol of Solt and Farber (1 976) with slight modification . .... 48

1 6 Summary diagram of the methods used to measure the specific activity of the tumour marker enzymes . . ................................ 49

17 ALP activities in the normal control and supplemented control groups . . ............................................................. 59

1 8 ALP activities in the control and hepatocarcinogen-induced groups with single supplementation . . .................................................. 60

1 9 ALP activities in the control and hepatocarcinogen-induced groups that received combined supplementation . ................................ 6 1

20 GST activities in the normal control and supplemented control groups . . ...................................................... 63

viii

2 1 GST activities in the control and hepatocarcinogen-induced groups with single supplementation . . .................................................. 64

22 GST activities in the control and hepatocarcinogen-induced groups that received combined supplementation . . ............................... 65

23 GGT activities in the normal control and supplemented control groups . . ............................................................. 66

24 GGT activities in the control and hepatocarcinogen-induced groups with single supplementation . . ........................ . . . ....................... 67

25 GGT activities in the control and hepatocarcinogen-induced groups that received combined supplementation . . ............................... 68

26 UDPGT activities in the normal control and supplemented control groups . . ............................................................. 69

27 UDPGT activities in the control and hepatocarcinogen-induced groups with single supplementation . . .................................................. 70

28 UDPGT activities in the control and hepatocarcinogen-induced groups that received combined supplementation . . . .............................. 71

29 GPx activities in the normal control and supplemented control groups . . ............................................................. 72

30 GPx activities in the control and hepatocarcinogen-induced groups with single supplementation . . .................................................. 73

31 GPx activities in the control and hepatocarcinogen-induced groups that received combined supplementation . . ............................... 74

32 GRx activities in the normal control and supplemented control groups . . ............................................................. 75

33 GRx activities in the control and hepatocarcinogen-induced groups with single supplementation . . .................................................. 76

34 GRx activities in the control and hepatocarcinogen-induced groups that received combined supplementation . . . .............................. 77

ix

AAF ALP BSA DEN DMBA DMN GGT GST GSH GPx GRx NADH NDEA UDPGT UV e.u. C AV VC E T CPH DA DAV DVC DE DT DEAV DEVC DTAV DTVC

LIST OF ABBREVIATIONS

2-Acetylaminofluorene Alkaline phosphatase Bovine Serum Albumin Diethylnitrosamine 7, 1 2 -dimetylben( a )antracene Dimetylnitrosamine Gamma-glutamyl transferase Glutathione-S-transferase Reduced glutathione Glutathione peroxidase Glutathione reductase Reduced nicotinamide adenine dinucleotide N -nitrosodietylamine Uridyl diphosphoglucuronyl transferase Ultra violet enzymes per unit Control Aloe vera Vitamin C y-Tocotrienol a-Tocopherol Control rats with partial hepactetomy DEN + AAF DEN + AAF + Aloe vera DEN + AAF + Vitamin C DEN + AAF + y-Tocotrienol DEN + AAF + a-Tocopherol DEN + AAF + y-Tocotrienol + Aloe vera DEN + AAF + y-Tocotrienol + Vitamin C DEN + AAF + a-Tocopherol + Aloe vera DEN + AAF + a-Tocopherol + Vitamin C

x

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

THE EFFECT OF ALOE VERA, VITAMIN C AND E ON TUMOUR MARKER ENZYMES IN HEPATOCARCINOGENESIS.

By

ABDAH MD. AKIM

February 1998

Chairman: Associate Professor Nor Aripin Shamaan, Ph.D.

Faculty: Science and Environmental Studies

The effects of Aloe vera (A V), vitamin C (VC), alpha-tocopherol (T) and

gamma-tocotrienol (E) on the activities of six tumour marker enzymes; alkaline

phosphatase (ALP), glutathione-S-transferase (GST), gamma-glutamyl transferase

(GGT), glutathione peroxidase (GPx), glutathione reductase (GRx) and uridyl

diphosphoglucuronyl transferase (UDPGT) in rat liver carcinogenesis were studied.

Liver cancer was induced by diethylnitrosamine (DEN) and 2 -acetylaminofluorene

(AAF) followed by partial hepatectomy. Ninety male rats (Rattus norwegicus, 120-

150g, 6-7 weeks old) were divided into 15 groups. Six groups comprised the normal

and supplemented-control groups. Hepatocarcinogenesis was induced in the other

nine groups. Four groups were supplemented with A V, VC, E and T respectively

and the other 4 groups were given TNC, T/AV, ENC and E/AV respectively. The

last group remained as the cancer-control group.

Xl

Aloe vera and vitamin C were administered ad libitum at doses of 0. 1 mg/L

water and 41 .7 mg/L water respectively, in both control and cancer groups. Alpha­

tocopherol and gamma-tocotrienol were administered at a dose of 34 mglkg diet and

30 mglkg diet, respectively. The rats were sacrificed by cervical dislocation after 16

weeks.

An increase (p<0.05) in all six tumour marker enzymes was observed in the

cancer-treated group compared to the normal-control group. Aloe vera

supplementation significantly (p<0.05) decreased the tumour marker enzyme

activities in cancer-induced liver compared to the cancer-control group. Gamma­

tocotrienol exerted a better effect than alpha-tocopherol in reducing the enzyme

activities in cancer-induced liver. Vitamin C significantly (p<0.05) decreased the

enzyme activities in GPx, GRx, GST and UDPGT. However, non of the

supplementations decreased the alkaline phosphatase activity in the liver cancer.

In the four cancer-induced groups that received combined supplementation,

the tumour enzyme marker activities were greatly reduced (p<0.05) compared to the

single supplementation. A combined supplementation of alpha-tocopherol and

vitamin C gave the best result compared to other combinations.

xii

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

KESAN ALOE VERA, VITAMIN C DAN E KE ATAS AKTIVITI ENZIM PENANDA TUMOR DALAM KANSER HEPAR.

Oleh

ABDAH MD AKIM

FEBRUARI 1998

Pengerusi: Profesor Madya Nor Aripin Shamaan, Ph.D.

Fakulti: Sains dan Pengajian Alam Sekitar

Kesan Aloe vera (A V), vitamin C (VC), alfa-tokoferol (T) dan gama-

tokotrienol (E) ke atas aktiviti enzim penanda tumor; alkaline fosfatase (ALP),

glutation-S-transferase (GS T), gama-glutamiltransferase (GG T), glutation perosidase

(GPx), glutation reduktase (GRx) dan uridil difosfoglukuronil transferase (UDPGT)

di dalam tikus teraruh hepatokarsinogenesis telah dikaji. Aruhan kanser ialah

melalui pemberian dietilnitrosamina (DEN) dan 0.02% 2-asetilaminofluorena (AAF)

diikuti hepatektomi separa. 90 tikus albino jantan (Rattus nowegicus, 120-150g,

berusia 6-7 minggu) telah dibahagikan kepada 1 5 kumpulan. 6 kumpulan adalah

untuk kawalan normal. Hepatokarsinogenesis telah diaruh di dalam 9 kumpulan di

mana 4 kumpulan diberi AV, VC, E dan T dan 4 kumpulan lagi diberi TNC, T/AV,

ENC dan E/AV. Kumpulan terakhir adalah kawalan kanser. Aloe vera dan vitamin

xiii

C diberi dengan dos 0.1 mg/L air dan 41.7 mg/L air secara ad libitum. Alfa­

tokoferol dan gama-tokotrienol diberi pada dos 34 mglkg diet dan 30 mglkg diet.

Semua tikus dibunuh secara dislokasi servikal selepas 16 minggu.

Kesemua aktiviti penanda tumor enzim didapati meningkat (p<O.05) pada

hepar kanser berbanding dengan kumpulan kawalan. Pada hepar kanser, Aloe vera

didapati mengur�gkan dengan signiftkan (p<0.05) semua aktiviti enzim kecuali

aktiviti alkalin fosfatase. Gama-tokotrienol menunjukkan kesan yang lebih ketara

daripada alfa-tokoferol dalam mengurangkan aktiviti enzim pada hepar kanser.

Vitamin C mengurangkan (p<O.05) aktiviti enzim GPx, GRx, GST dan UDPGT.

Kesemua empat kumpulan yang menerima suplementasi berkombinasi

mengurangkan (p<O.05) semua aktiviti enzim dengan lebih ketara berbanding dengan

kumpulan yang menerima hanya suplementasi tunggal. Kombinasi alfa-tokoferol

dan vitamin C memberi kesan perlindungan yang paling baik berbanding dengan

kombinasi lain.

xiv

CHAPTERl

INTRODUCTION

In some parts of the world especially tropical Africa and parts of Asia,

primary cancer of the liver is one of the commonest forms of malignant tumour. In

the Western world, alcohol is the most recognised cause whereas in Asia, the

possible role of chemical carcinogens such as aflatoxin has been identified (Alfm­

Slater and Kritchevsky, 1991). Diagnosis is difficult since there is no specific

symptoms; in all cases, there is enlargement of the liver. Chemotherapy and

radiotherapy has little effect to help alleviate the situation and removal of the

affected part of the liver may be feasible.

As the familiar proverb "prevention is better than cure", the prevention of this

disease is obviously the aim of all those who are concerned with health. A number

of studies had been done on various putative anticarcinogenic substances, including

the supplementation used in this study; Aloe vera, vitamin C, a-tocopherol and y­

tocotrienol.

Aloe vera, which is commercially used in skin care product, has only been

tested recently for its anticarcinogenic properties. Although vitamin C has been long

known to be a potential anticarcinogen, it gains less interest in this

2

subject because it is more commonly used in preventing scurvy and cold. The effect

of two types of vitamin E; a-tocopherol and y-tocotrienol, which are purified from

palm oil, on liver cancer had been extensively studied where y-tocotrienol possesses

a higher anti-cancer property than a-tocopherol (Ong et aI, 1994). There had been

individual studies on the effect of each supplementation previously (Beattie and

Sherlocks, 1976). Therefore, this study aims to examine the simultaneous effect of

the supplementations.

Measuring the known tumour marker enzymes; alkaline phosphatase (ALP),

uridyl diphosphoglucuronyl transferase (UDPGT), gamma glutamyl transferase

(GGT), glutathione-S-transferase (GST), glutathione reductase (GRx) and

glutathione peroxidase (GPx) will obviate the effect of these supplementations. Each

tumour marker enzyme used was known to increase its activity in cancer-induced

liver. However, liver ALP is not being used as extensively as the plasma ALP in

detecting cancer whereas GST and GGT are now the most used as markers. GPx and

GRx are involved in the glutathione redox cycle, which is the major endogenous

protective system against radical species.

Details on liver cancer, possible anticarcinogenic properties of Aloe vera,

vitamin C, a-tocopherol and y-tocotrienol and the tumour marker enzymes were

introduced in the literature review.. The hypotheses that vitamin C and E act as

anticarcinogens via their antioxidant properties with the elevation of the tumour

marker enzymes was tested. Therefore, the objective of this research was to evaluate

the activities of the tumour marker enzymes in Aloe vera, vitamin C, a-tocopherol

and y-tocotrienol-supplemented as well as their combination in liver cancer.

CHAPTER 2

LITERATURE REVIEW

Cancer in General.

Cancer may be considered as an alteration of the control mechanisms

involved in cell division and differentiation. It is a multi-step process which involves

chemical modification of cellular or genomic DNA (Arcos et ai, 1961). Once

modified, the affected DNA is replicated and transcribed differently from the normal

DNA. This hereditary transmittable modification (from neoplastic cells to daughter

cells) leads to a permanent and anarchic mUltiplication of transformed cells which

may be different from that of the normal cells. The carcinogenic process may be

initiated by several agents called initiators. Once carcinogenesis is initiated; the

second step, termed "promotion", may continue and fmally the third stage of the

process, called "progression", occurs. Carcinogens may act as initiators, promoters

or a combination of both (Boyland and Sydnor, 1963).

Liver Cancer.

Hepatocellular carcinoma is a common cancer in sub-Saharan Africa and

parts of Southeast Asia where liver cancer may account for 5-8% of all

3

4

deaths. The risk of getting hepatoma is high in Asia and is more common in men

compared to women (Laidlaw and Swendseid, 1991). Liver cancer is rare in western

countries where it accounts for only 2% of cancer cases in the United States annually.

The first type ofliver cancer is primary liver cancer. Secondary cancer occurs

when the liver becomes the seat of secondary deposits of cancers arising primarily in

other organs. Metastases in the liver often lead to enlargement of the organ with

discomfort or pain below the ribs on the right and in the flank. There may be

jaundice arising from interference with the drainage of bile. Other symptoms of liver

disease such as irritation of the skin, twitching and drowsiness could be detected

(Scott, 1 979).

Two forms of liver cancer have been distinguished. The first form is called

hepatoma, which originates in the cells of the liver cortex and forms ninety percent of

primary liver cancer. The second is cholangiomas, which line the hepatic ducts that

drain the bile from the liver and forms 5-10 percent of liver cancer (Manahan, 1989).

Rapid enlargement of the liver can also be diagnosed as cancer, though not

necessarily so. Others are pains under the ribs on the right side, loss of weight and

jaundice. Biopsy of the liver will confirm the precise diagnosis. Treatment such as

chemotherapy and radiotherapy is of little help and at present, liver transplantation

has been successful and considered as one of the best treatment available.

Causes of Liver Cancer.

The reason for the frequent occurrence of liver cancer is because the liver is

often the main target for chemically induced toxicities by carcinogens. Its

susceptibility may be due to several factors; firstly, the liver is the primary site for

5

biotransformation of xenobiotics. Secondly, the liver is the fIrst major site of

metabolism for carcinogens absorbed from the gastrointestinal tract, which is the

major route of absorption for most xenobiotics (Boyland and Sydnor, 1963).

Furthermore, the liver is strategically located between the intestinal tract and

the general circulation . Through the portal vein, the liver receives a large volume of

venous blood from the digestive tract while it drains blood through the hepatic veins.

Compounds absorbed from the intestines, including products of digestion as well as

toxicants pass through the liver to be metabolised. This renders it to be particularly

vulnerable to the toxic effects of metabolites (Hodgson and Levi, 1994). Specific

hepatocarcinogens that have been identified are alcohol, safrol, pyrrolizidine

alkaloids, cycasin, aflatoxin and several steroid hormones.

Cancer is the second leading cause of death after heart disease. From

epidemiological studies, about 35% of cancer is related to diet (Table 1 , Hodgson and

Levi, 1994). The diet probably accounts directly for liver cancer . Carcinogens, both

natural and synthetic may affect hormone levels, causing other cancers, e.g.,

chemicals in food components that alter oestrogen level in the breast and

endometrium may cause breast and uterine cancer whereas alteration in testosterone

level can cause prostate cancer.

Although the effect of dietary protein on hepatic tumour development has

been shown in several animal models (Emmelot and Muhlbock, 1964), no human

epidemiological studies has shown the associations between protein intake and liver

cancer. The hypothesis that liver cancer was related directly to kwashiorkor (protein­

calorie malnutrition) has now been discarded since the discovery of aflatoxin in

contaminated foods in Africa and Asia. Although aflatoxin ingestion has been

correlated closely with liver cancer (Alfm-Slater and Kritchevsky, 1991), the

6

hepatitis B virus also appears to be an important cofactor with carriers having a 200-

fold higher risk compared to non-carriers in Taiwan (Becker, 1975). Prevention is

possible through a reduction or elimination of aflatoxin in the diet and vaccination

against the hepatitis B virus; now being undertaken in Gambia (Brennan, 1977).

Table 1 Causes of Cancer.

Factors involved in cancer

Major Factors Best Estimates

Diet 35%

Tobacco 30%

Reproductive and sexual behavior 7%

Viruses 5%

Occupation 4%

Geophysical Factors 3 %

Alcohol 3%

Pollution 2%

Food Additives 1 %

Medicines 1 %

Industrial Products 1 %

Unknown ? (Source taken from Hodgson and Levi, 1 994).

The major factor in liver cancer in North America and Europe appears to be

caused by excessive alcohol ingestion (Greenberg and Harper, 1960). A threshold

effect has been suggested where liver cancer occurs only when the level of ingestion

is sufficient to cause alcoholic hepatitis and consequent cirrhosis (Harris, 1991). It

7

could be concluded that exceSSIve alcohol consumption is the major factor ill

Western countries and North America for cancer of the liver.

CARCINOGEN DETOXIFICATION / 1

PRODUCTS � METABOLISM

� '" ULTIMATE CARCINOGEN D N A

URINE,FECES

� REPLICATION M 0 /,IE � A --------.1

LABILE STABLE ADDUCTS ADDUCTS

� � REPAIR

URINE,FECES DNA + X CANCER

Figure 1 Pathway of carcinogen leading to cancer. (Source taken from Hodgson and Levi, 1994).

Multistage Carcinogenesis.

From animal and human studies, it is clear that carcinogenesis is a multistage

process (Harris, 1 991) . Carcinogenesis can be divided into at least three stages

termed initiation, promotion and progression (Figure 2). Generally, it is thought that

initiation is a genotoxic event, one in which the primary sequence of DNA is altered

while promotion is considered a non-genotoxic or epigenetic event (Dragan and Pitot,

1 992).

Initiation involves the exposure of normal cells to chemical or physical

carcinogens, which results in a permanent heritable change in the cell's genome. The

affected cell is called the initiated cell . The initiated cell may have a decreased

8

response to the inter- and intracellular signals that maintain normal tissue architecture

and homeostatic growth (Harris, 1991). The initiated cells may remain dormant until

exposed to a tumour promoter.

Tumour promotion results m the proliferation of initiated cells and

enhancement of genetic damage. The development of malignancy from a benign

tumour encompasses the third step of carcinogenesis termed progression. It involves

the survival and proliferation of cancer cells and the process of maintenance and

evolution of malignancy.

NORMAL CELL

INITIATION + CARCINOGENS

hours days

INITIATED CELL

t MUTATED CELL �PROMOTERS weeks

months PROMOTION years

+ TRANSFORMED CELL

I t PROGRESSION MALIGNANT CELL

l � years

TUMOUR

Figure 2 Multistage of cancer . (Source taken from Voiculet et aI, 1991) .

9

Carcinogen Metabolism.

Electrophilic Theory

In the metabolism of chemical carcinogens, the electrophilic theory states that

carcinogens are metabolically activated to electrophilic metabolites that are capable

of interacting with nucleophilic sites in DNA, RNA and protein (Iversen, 1 988).

Most of the chemical carcinogens are electrophilic reactants. The formation of

electrophiles generally involves a two-electron oxidation to a carbonium ion

catalysed by a cytochrome P 450-dependent mixed function oxidase. Most of the

radicals formed probably do not react with DNA. There are also compounds which

do not form electrophiles but undergo biotransformation and produce neoplasm, for

example saccharin and nitrilotriacetic acid. From this observation, two classes of

chemical carcinogens are distinguishable; genotoxic and epigenetic compounds

(Iversen, 1988).

Classes of Chemical Carcinogens.

Chemical carcinogens can be divided into two major categories; genotoxic

agents which produce alterations in the genetic material of the host (e.g. DEN) and

epigenetic (nongenotoxic) agents which do not alter the primary sequence of DNA

(for e.g. saccharin). The genotoxic agents possess initiator activity whereas the

epigenetic compounds possess promoter activity (Hodgson and Levi, 1994).

10

Genotoxic agents are further sub-divided into four categories (Iversen, 1988):

Direct-acting carcinogens which do not require metabolic activation to interact with

DNA (e.g. beta propiolactone).

Indirect-acting carcinogens which require metabolic activation (e.g. DEN).

Radiation which directly ionise DNA.

Inorganic agents such as arsenic which interfere with the enzymes associated with

DNA replication.

Terms in Metabolic Activation.

The terms parent, proximate and ultimate carcmogen were developed to

differentiate the action of carcinogens. A parent carcinogen is a compound that must

be metabolised in order to have carcinogenic activity. A proximate carcinogen is an

intermediate metabolite requiring further metabolism steps resulting in the ultimate

carcinogen which is the actual metabolite that covalently binds to the DNA

(Nakahara et ai, 1 972). Diethylnitrosamine (DEN) and 2-Acetylaminofluorene (2-

AAF) are examples of chemical carcinogens that require metabolic activation before

they exert their effect. After metabolic activation, both DEN and 2-AAF bind to

DNA to form DNA adducts. It is further suggested that if the DNA adduct is not

repaired before the cell replicates, a mutation may be permanent in the daughter cell

(Emmelot and Muhlbock, 1 964).