UNIVERSITI PUTRA MALAYSIA

STUDIES ON THE EFFECT OF BENZO(a)PYRENE IN RATS

THORIA ALI ABDUL HAMEED DRAGH

FPV 2001 15

STUDEIES ON TIm EFFECT OF BENZO(a)PYRENE IN RATS

By

THORIA ALI ABDUL HAMEED DRAGH

Thesis Submitted in Fulfilment of the Requirement for the Degree of Master of Veterinary Science ill the Faculty of Veterinary Medicine

UlliYcrsiti Putra Malaysia

March 2001

DEDICATION

To my 'wonderful family

II

A bstract o f thcs is presented to the Senate o f Un ivers it i Putra Malay�i a in fu l fi l ment o f the requ i lement for the degree of Master of Veterinary Science

STUDIES ON THE EFFECT OF BENZO(a)pYRENE IN RATS

By

THORIA ALI ABDUL HAMEED DRAGH

March 2001

Chairman: Dr. NoonJiIl Mohamed Mustapba

Faculty: Veterinary Med icine

B enzo(a)pyrene [B(a)P] is a carcinogcn found in h igh concentrations during the

Malaysian haze episodes . In v icw of developing strategies in al leviating

de leterious effects of haze in humans, the effect of acute and chronic exposu re to

B( a)P was stud i ed in rats.

In the acute exposure studies, an eval uation of apoptosis fol lowing

treatment with B (a)P was assessed. The B (a)P treated rats recei ved 24 ng ( l3 1lL)

of B(a)P inst i l led i ntratraeheal l y, whi le s im i lar volumes of trieapryl i n (Tr) was

admin i stered to rats from the Tr group. Rats not receiv ing any treatmcnt served as

controls . An asscsc;mcnt of apoptos is was made on haematoxyl in and eoc;in

iii

(H&E) sta ined h istologic sections, term inal d eoxynuclcot idal transfcrase­

med iated d U TP-bioti lJ n ick end labe l ing (TUNEL) analys is and DNA l addcri n g

of lung samplcs. Rats in the D(a)P group were k i l l ed a t I , 2 , 4, 8, 16, and 24

hours post-instillation (p. i .) and rats frolll the control and Tr groups were only

k i l led 24 hours p.i.

Apoptos is at d i fferent stages was found in the pneumocytc and bronch ia l

epithel i um of B(a)P-treated rats k i l led 8, 16 and 24 hours p . i . Th is was a lso

confirmed pos itivc by TUNEL analys is and DNA laddering.

In the chronic exposure studies, changes in the lung of D(a)P-induced rats

during a three month period encompassing p53 express ion, pro l i ferating ce l l

nuclear antigen (rCNA) expression, immune response (IgA, IgG l evels and

a lveolar macrophage act ivity), levels of glutathioll s-transferase (GST) marker

enzyme and the effect of raw gar l ic as an anti-tumour agent were stud ied.

Rats from the control, B (a)P and Tr group were daily fed on a comm ercial

basa l diet while rats [rom the garlic (G) and [B(a)P+GJ group were fed the basal

rati on conta in ing gar l ic incorporated at the rate equ ivalent to an i ntake o f

8 0mg/kg bodyweight/rat/day.

The resu l ts showed growth d isturbances in pneumocytes and bronchia l

epithe l i um of rats from the B(a)P group. Apoptosis was d etected in fou r rats from

the B (a)p-l G group. The PCNA pos i tive areas were on ly fOllnd in hyperplast ic

IV

areas i n the lungs of rats from the B(a)P-treated group. In addit ion, rats treated

with I3(a)P and B(a)P+G had lower levels of IgA, IgG, alveolar l11acrophages

act iv i t ies and gl utath ione S-transferase in the lung.

In conclus ion, e ither short or long term exposures to I3(a)P produce

detr imental changes to lungs of rats and garl ic has great potential in a l l eviat ing

the chron ic effects.

v

Abstrak tesis telah dikcmukan kepada Senat Universiti Putra Malaysia scbagai memenuhi kcpcrIuan untuk Ijalah Master Sains Vetcrinar

KAJIAN KEATAS KESAN BENZO(a) PIREN PADA TIKUS

Oleh

TIIORIA ALI ABDUL HAMEED DRAGII

March 2001

Pcngcrusi: Dr. Nom-din Mohamed Mustapha

Fakulti: Pcmbatan Veterinar

Kepekatan bahan karsinogen, benzo(a)piren [B(a)P] yang tinggi telah dikcsan paela

episod jerebu eli Malaysia. Kajian mengenai kesan akut dan kronik pcneleelahan

kepada B(a)P telah dijalankan ke alas tikus bagi meraneang strategi mcngurang

kesan merbahaya terhadap manusia.

Dalam kajian pendedahan akut, penilian terhadap apoptosis seear'a histologi,

analisis pelabelan potongan hujung dUTP-biotin deoksinukleotid pcrantaraan

transferase (TUNEL) dan tangga DNA pada Iavaj bronkiol-alvcolus. Tikus dari

kumpulan B(a)P l11cnerima sebanyak 24 ng ( l3�lJ) B(a)P yang dibcn seeara

VI

intratrakea, manakala isipadu tricaprylin err) yang sama telah cliberi kcpacla tikus

clad kumpulan Tr. TiIms yang ticlak mcnerima apa-apa rawatan bertindak sebagai

kawalan. Tikus clari kumpulan B(a)P telah clibunuh pacla 1, 2, 4, 8, 16 clan 24 jam

pasca-pemberian (p.i) manakaJa tikus claripacla kumpulan lain hanya dibunuh scJcpas

24 p.i.

Apoptosis pacla peJbagai peringkat tclah clilihat pacla pneumosit clan cpiLelium

bronkiol tikus clari kumpulan B(a)P yang dibunuh selepas 8 , 16, clan 24 jam p.i.

Uj ian TUNEL clan tangga DNA memberikan keputusan yang sama.

Pacla kajian penclcclahan kronik, perubahan pacla paru-panl tikus clikaji 3

bulan selepas menerima rawatan dengan B(a)P merangkumi p53, antigen

pemproliferatan nuklias sel (PCNA), gerakbalas nuun, aras enzim petunjuk clan

kesan bawang putih mcntah sebagai anti-tumor.

Tikus clari kumpulan kawalan, B(a)P clan Tr dibcri makan makanan komersial

manakala tikus claripada kumpulan bawang (G) dan B(a)P+G menerima makanan

dengan penambahan bawang putih mentah pacla kadar 80 mg/kg beratllikus/hari.

Keputusan kajian menunjukan ganguan tumbesaran pada pneull10sit dan

cpitelium hronkiol pada tikus dari kumpulan B(a)P. Apoptosis telah dikcsan pada

empat ekor tikus clad kumpulan B(a)P+G. Tinclakbalas positif PCNA hanya cliclapati

Vll

pad a kawasan hipcrplasia el i paru-paru tilcus dari kumpulan B(a)P sahaja. Tambahan

lagi, paru-pam tikus yang diberi B(a)P dan B(a)P+G mcmpunyai aras Igi\, IgG,

aktiviti makro[aj alvcolus clan gilitation S-tranfcrase yang rcnclah.

Sebagai rlll11l1San, pencledahan akut atau kronik B(a)P mcnjana pcrubahan

membabayakan pada paru-paru tikus dan bawang putih 111cmpunyai potcnsi bcsar

lIntuk mcngurangi kcsan kronik B(a)P.

VIll

ACKNOWLEDGEMENTS

A l l praise to Alm ighty Al lah, the Merc i ful and the Benevolent. I Iad it not

been due to l I is ,vi I I and favour, the completion of th is study wou ld not have becn

possib le .

I would l ike to express l11y s incere gratefu lness and appreciation to l11y

supervisor, Dr. NOOl"d in Mohamed Mustapha, who has devoted much of h is t ime

for invaluable guidallce, advice, supervis ion , and support throughout thc course

of th is study.

I wish to express my s incere gratitude to the Dean of the Faculty o f

Veterinary Medicine, Professor Dato' Dr. Sheikh Omar Abdul Rahman who i s

a lso m y co-supervisor, for h is i nvaluable advice, suppoli and continuos

encouragcment towards the completion of this work.

S incere thanks are also due to my other co-supervisors, Dr. Mohd Azm i

Mohel Li la who has granted the use of h is laboratory that have enl ightened and

i m proved this study.

I would l ike to express my gratitude to Dr. Daud Ahmad Israf A l i who has

unselfishly provided fac i l i t ies in h is l aboratory during the course o f th is study.

ix

I would also l i ke to express my heartfelt grati tude to Dr. Panayiot is

Loukopoulos from the School of Veterinary Science, Univers ity of Queens land,

Austra l ia for h i s ass istance in immunoh istochem i stry techniques.

I have also been very fortunate in receiving ass istance from a n umber of

my col leagues and fi·iends . Many of them went out of their way to assist me and i t

wou ld not be poss ib le to name a l l of them . However, I wou ld l ike to thank Ms .

Abeer Hasan Sahtout, Mr. lsam Qudss iah, Puan Zun ita Zakaria and Puall .

Hazi l awat i Hannah.

I am deepl y indebted to the staff members of the post-mortem laboratory

and Mr. Fauzi Che Yuso[ who were involved e ither d i rectly or indi rectly 111

sharing their knowledge and skil led assistance during th is study.

Continuous moral support and encouragement fi·om l11y mother in- law

during my study i s deeply appreciated .

Grateful thanks are a l so conveyed to my supportive husband, Dr.

Muthafar H. AI-Haddawi and ch i ldren Zeinab, Hussain and Ridah who have long

put up with my l ate n ights and weekends at the office so that r cou ld complete

th is work.

x

I ceri i fy that an Examination Comm ittce met on 16th March 200 1 to conduct the final examination of Thoria A l i Abdul Hamced Dragh on her Master o f Veterinary Science thes is ent i tled "Stud ies o n The Effect of Benzo(a)Pyrene i n Rats" i n accordance with Univers it i Pertanian Malays ia (Higher Degree) Act 1980 and Un ivers it i Pertanian Malaysia (Higher Degree) Regulations 1981. The Comm ittee recommends that the cand idate be awarded the relevant degree. Members of tile Examination Committee are as fo llow:

Daud Ahmad Israf Ali, DVM., MS. , Ph .D Associate Professor Insti tute of B i oscience Univers i t i Putra Malaysia (Chairman)

NOOl'd i li Mohamad Mustapha, DVM, MS., Ph .D Associate Professor Faculty of Veterinary Medicine Univers it i Putra Malaysia (Member)

Dato' Sheikh Omar Abdul Rahman, B .V.Sc., M.V.Se. , M.R.C.V.S Professor Faculty of Veterinary Med icine Univers it i Putra Malaysia (Member)

Mohd Azmi Mohd Lila, DVM, MBA, Ph.D Associate Professor Faculty of Veteri nary Med icine Ull ivers it i Putra Malays ia (Member)

Q GHAZJ.\U MOHA YIDIN, Ph.D

Profe.sor Deputy Dean of Graduate School, Univers it i Putra Malays ia

Date: 0 4 MAY 2001

XI

This thesis submitted to the senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Master of Veterinary Sciences.

AINI IDERIS, Ph.D, Professor Dean of Graduate School, Universiti Putra Malaysia

Date:

r hereby declare that tile thesis is based on my origi nal work accept for quotations and citations wh ich have been duly acknowledged. I a lso declare that it has not been previously or concurrently subm itted for any other degree at UPM or other inst i tutions.

Thoria Ali Abdu l Hameed Dragh, BYM&S

Date: If· S· :ltJo I

xiii

TABLE OF CONTENTS

DEDICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. III

ABSTRAKT............................................................ VI

ACKNOWLEDGEMENTS ....................................................... IX

APPROVAL SHEETS....................... ........................................ XI

LIST OF TABLES. . ................................. ................................. XVI

LIST OF PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi i LIST or ABBREVIATIONS ...................................................... XIX

CHAPTER Page

I INTRODUCTION . . . .... . .. . . . . . . . . . . . . . . . . . . . . .... . ... . . . . . . . . . . . . .. . . .

n LITERATURE REVIEW................................................ 4 A i r pol lution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Haze Episodes in Malays ia . . . . . . . . . . . . . . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . 7 Bcnzo(a)Pyrene . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 8

Physical and Chemical Properties of Benzo(a)Pyrene . . . . . . . . .. , 8 Metabol ism and Toxicity of Benzo(a)Pyrcne . . . . .. . . . . . . . . . . . . . . . 8 Benzo(a)Pyrene as Immunosuppressor..... . . . ... . . . . . . . . . . . . . . . . . . . I I Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . 13 Benzo(a)Pyrene as Carcinogen . . . . . ... . . . . . . . . . . . . . . . ..... .... ....... 14 Type of Cancer Induced by Benzo(a)Pyrene. . . . . . . . . . . . . . . .. . . ... . . 1 7

Strategies i n al leviat ing BCllzo(a)Pyrenc-induced iqjury . . . . . . . . . . . . 1 9 Garl ic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . 1 9

Constituents and Beneficial Effect of Garl ic (Al l ium ,Sativum) . . . 20 Anticarcinogen ic and Antitumor potential of garlic . . .. . . . . .. . . . . . 22 Toxicity of Garlic . ... . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . ... . .. . . . . . ... . . . 26

III ACUTE EXPOSURE TO BENZO(a)PYRENE I ntroductioll. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Materials and Methods. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 29 Animals and Management . . ... . . ... ........ . . ................. .. . . 29 InoculuI11 . . . . . .. . . . . ... . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Pathology . . . . . . . . . . . . ,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Detection of DNA Ladclering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Detection of p53 Tumour Supprcsser Gene. . . . . . . . . . . . . . . . . . . . 34 Results .. . ............................................................ ...... 36 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

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IV CHRONIC EXPOSURE TO B ENZO(a)PYRENE Introduct ion . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Ani lllals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Jnoculunl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Garl i c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . .. 46 Experimenfal Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 6 Immunoh istochcmistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 47 Immullology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Glutathion S trnsferase Estimntion in Lung Tissue . . . . .. . . . . . . . . . 5 1

Rcsu l ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Cl in ical S igns . . . . . . . . . . 0 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Iml11unohistochem istry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 60 G lutaliI ion S-transferase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1

D iscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

V GENERAL DISCUSSION AND CONCLUSIONS . . . . . . . . . . . . . . . . . . 70

REFERENCES . . .. . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

BIODA TA OF THE AUTHOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

xv

LIST OF TABLES

Tahle Page

1 . The Major and Important Trace F lavour Compounds Found in Garl i c . . . . . . . . . . . . . . . . . . . . . . . . . ....... ........... . .. . ... ............... ......... .... 20

2. Th e Experimental Design of Acute Exposur e to B enzo(a)Pyrcne in Rats . . . . 3 0

3 . The Experimental Des ign of Chronic Exposure to Oenzo(a)Pyrene i n Rats . . 47

4 . The level of (00) TgA and IgG in the l ung lavage of rats at necropsy (Mean±SO) . .. . . .. . . . . .. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

5. The level (00) of IgG in the serum of rats at necropsy (Mean±SD) . . . . . . . 60

6 . The alveolar macrophage activ i t ies in the l ung lavage of rats (J\1can±S I)) . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1

7. The activity (00) of GST in l ung lavage of rats at necropsy (Mean±SD) . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... .... ........... 62

xvi

LIST 0 F PLATES

Plate Page

Photom icrograph , lung, rat from the B(a)P group k i l led 8 hrs p . i . Note apoptotic cel l (arrow head) w i t h chromati n condensat ion and compact ion of cytoplasm by development of vacuoles. I r&E x 5 00 . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

2 Photom icrograph , lung, rat from the B (a)P group k i l led 1 G hrs p. i. Note apoptotic ce l l (arrow head) with peripheral and margination of chromati n along the nuelear membrane. H&E x 500 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 3 7

3 Photom icrogroph, lung, rat from the B (a)P group k i l led 1 6 1m p . i . Note the apoptotic cel l (arrow heads) yel lowish green in colour and the normal cel ls red i n colour (arrow). F luoresce i n sta in x 500 . . . . . . . . . .. . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 8

4 Agarose (l %) gel electrophores i s of peR products of DNA of rats inoculated with B (a)P and control , lanes 1 and 2 are amp l ified DNA of rats k i l led 2 and 4 hrs p . i . , Lane 3 is a molecu lar weight marker ( 1 OObp), lane 4 represents a DNA of a rat k i l led 1 6 and 24 Ill'S p . i , l ane 5 represents a DNA of rats k i l led 8 h I's p . i . and lane 6 represents a DNA o f rats from control group . . . . . . . . . . . . . . . . .. . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 39

5 Photomicrograph, lung, fi'0l11 the B (a)P treated group k i l led 24 hrs p i . Note the faint b l ue sta in ing of the nucle i ind icating the absence of p53 prote in . (H&E x 500) . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 39

6 Photograph, l ung of rat from the B (a)P group k i l l ed 90 days p . i . Note the presence of p in -point wh it ish nodu les (arrow) . . . . . . . . . . . . . . . . . . . . '" . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . 52

7 Photograph, lung of rat from the B (a)P group k i l led 90 days p . i . Note the presence of haemorrhagic spots on the right apica l lobe (arro\v) . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 53

8 Photom icrograph, l ung, from the garl ic group k i l l ed at necropsy. Note hyperplas ia of bronchia l associ ated lymphoid t issue (BALT) with extens ion of lymphoid tissue toward the epithel ia l l in ing of bronchus. H&E x 3 70 . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 54

xvii

9 Photomicrograph, l ung, from the B(a)P group kil led at necropsy. Note pl O l iferation of mononuclear ce l l s (predominantly lymphocytes and macrophages) in lamina propria of trachea. I -I&E x 370 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

1 0 Photomicrograph, lung, fi'om the B(a)P group kil led at necropsy. Note increase in a lveolar wal l th ickening d ue to epithe l isation (arrow) . H&E x 1 40 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . 56

I I Photomicrograph, lung, from the B(a)P group kil l ed at necropsy. Note large prol iferated polygonal cel l s with pale and vacuolated cytoplasm. I I&E x 370 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

1 2 Photomicrograph , lung, from the B (a)P group kil l ed at necropsy. Note presence of large cel l s with scanty acidophil ic cytoplasm and some of which appeared with two nuclei (arrow). H&E x 3 70 . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . ... . . . . . . . . . . . . . . . . 57

13 Photomicrograph, lung, fi'om the B (a)P group k illed at necropsy. Note presence of c luster of cuboidal ce l ls forming g landu lar structure (arrow head). The area surrounding these cel ls was infi l trated with mononuclear ce l l (predominantly lymphocytes). l-I&E x 370 . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

14 Photomicrograph, l ung, from the B (a)P group kil led at necropsy. Note the peNA positive cel l s with yel lowish brown nuclei (arrow heads) and those of negative cel ls were faint b l ue (arrow) . J-I&E x 1 00 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

xviii

AFC Am0

ANP I3(a)P BALT bp I3-PI3S BPDE Cx DAB DAS DCNB DNA DNT ELISA g G GST H&E hr IgA IgG IgM IL KCI M MNU ng

p.1 PAIls PBS

PCNA PCR PM RBC TNF-a TUNEL

List of Ahbreviations

Antibody-forming cells Alveolar macrophage a-naphthoOavn Benzo(a)pyrene Bronchial associated lymphoid tissue I3ase pair Bovine srrum-phosphate buffered solution Bcnzo( a)pyrcne-7,8-diol-9, 1 O-epox ide Control 3' 3' diaminobenzidine-tetrahydrochloride Diallyl sulfide 1,2 dichloro-4nitrobenzene Deoxyribonuclie acid Dermonecrotic toxin Enzyme linked imll1unosorbant assay Grams Garlic Glutathion S-transferase Hematoxylin and eosin Hour Immunoglobulin A Immunoglobulin G Immunoglobulin M Interleukin Potassium chloride Molar Methylnitrosurea Nano-gram Post instillation Polycyclic aromati c hydrocarbons Phosphate buffered solution Proliferating cell nuclear antigen Polymerase chain reaction Particulate maHer Red blood cells Tumor necrosis factor-alpha Terminal deoxynucleotidal transferase-mediated DUTP-biotin nick end labeling Microgram M icroli ter

XIX

CHAPTER I

INTRODUCTION

Ai rborne pol lutants produce a wide variety of harmfu l effects on humans,

animals and p lants. Chem ica ls are released due to coa l combust ion, gas i ficat ion,

em iss ions, b iomass burning, coal and tobacco smokc that may i nteract with

d iffercnt m etabo l i c systems in human t issucs and cel l and eventua l l y cause cancer

(Tokiwa et 01., 1 998). Among these chem i ca ls is a group of mu l t i-ring hydrogen

and carbon compounds known as polycycl i c aromatic hydrocarbons (PAl Is) ,

wh ich are com monly found in the emiss ions from burncd plant and petroleum

products. Deteriorat ing a i r qual i ty duc to an increase in PAH concentration w i l l

cause i ncreasing adversc effects o n human heal th . (Om land c f 01., 1 994; 1 lock ef

01., 1997 ; Scarlett et 01., 1 996) One or the most studied of the PAHs i s

benzo(a)pyrel1c [B(a)P], wh i ch i s an ub iqu i tous envi ronmental po l l utant present

in coal tar, cigarette smoke and b iomass burning (Yang et al., 1 997) .

In heavi ly pol luted reg ions, i t has been found that a ir pol l ut ion can cause

congenital anomal ies especi a l ly those who are exposed to h igh leve ls of PMIO,

pollutant particles of less than 10 micrometers in diameters. The DNA damage by

PAHs has bcen reported (Leadon et 01., 1 995) to be due to adduction of thal can

lead to m utations and increase the probabil ity of developing cancer (Nakanish i el

01., 1 997).

2

Studies in animals have shown that dermal expo')ure to B(a)P and P/\II

can cause skin cancer. EfTects from breathing or ingestion were also reported to

d raw a connection with other kinds of cancer. Anim al stud ics have shown B(a)P

to be teratogenic, embryotoxic, and m utagenic (Lu ct al., 1 986; Ncsnow e{ al.,

1 998a). Other an imal tests have shown that exposure to D(a)P may cause

reproduction el i rticu lty (Zenzes c{ al., 1999).

Although acute toxic i ty appears to be low for humans, sub-chronic and

chronic toxic effects exist. Some PAHs such as B (a)P are known potent

immul1osuppressors (Wh ite and Holsapple, 1 984; Thomas et al., 1 987).

There is a growing concern about the heallh effects of air pol l ution in the

world (Scarlett e{ al., 1996). Many epidemiological and experimental stud ies

have been conducted to examine the effect of a ir pol lu tion 011 health (Dockery

and Pope, 1 994; Pope et al., 1995 ; Scarlett ef al., 1996).

In Malaysia there has been considerable concern about the effect of

pollution aris ing from haze. In 1997, Ma laysia and other Southeast Asia countries

h ave been exposed to a severe haze episode. However, 110 studies h ave exam ined

the effect of air pol lut ion on l ung function in human or animals .

The a im of this study was to determine the effect o f haze on health by

lIsmg rats as models for humans. L ikewise, it is also a imed at evaluating

3

envirolllllcntal pol lu tion causcd by hnzc and thc rclationship bctwecn thc

conccntration of B(a)P dUl ing thc haze episodc ancl thc outcome of lung injury.

Thc objectives o[th is study wcre:

i . to dctcrmine thc manner o f cell dcath during acute exposure to l3(a)l>

i i . to determ inc the development and type of tumour induced b y B ( a)P

i i i . to detcrminc the sensitive and rel iable indicators o f tumours induced by

B(a)P

IV. to assess the e fficacy of garl i c in allev iat ing B(a)P induced injury

CHAPTER II

LITERATURE REVIEW

Air Pollution

Undoubtedly, over decades air pollution is a problem of growing national

and i nternational i nterest. Air pol lution can be defined as any atmospheric

cond i t ion when excess ive substances are present above normal ambient Icvcls to

produce measurable effects on man , an imals, vegetation or materia l s . Substances

refer to any natura l or man-made chemical e lements or compounds capab le of

being airborne. These substances may exist in the gaseous, l iquid or solid states

i n the atmosphere (Painter, 1 974) .

The earl iest po l lutants noted in the atmosphere were probably of natural

or ig in . Man, p lants, an imals, and the act of nature woul d contribute to pol l ution

(Ottar, 1987).

The orig in of air pol lut ion var ies between smoke, fumes, ashes and gases

fwm vo lcanoes and forest fires, sand and dust from wind storms in arid region,

fog i n humid , l ow-lying areas and natura l trepan hazes from p ine trees i n

mountainous regions (Ross , 1972).

4

5

Haze is an atmospheric phenomenon caused by the presence or liny

particulate suspended in air. These particulate, which are molecular in size,

scatter and absorb sunlight resulting in diminished visibility giving the

atmosphere a characteristic opalescent appearance (1lasscll1 ('( 01., 1998).

Particulate matter (PM) which is produced by the burning of wood, diesel

and other fuels by agriculture activities and industrial processes is made up of

asb, smoke, soot, dust, fibers and liquid droplets. Apart from impairing ambient

visibility, the smaller PM can be inhaled and over an extended period of time can

injure the respiratory system.

Particulate maHer can be defined as any material that can remain in the

atmosphere or gas stream at standard conditions in the solid or liquid states. The

term PM 10 and Pivh 5 are primary particulate referred to during the occurrence of

an air pollution. The PM,o is a particle with an aerodynamic diameter less than or

equal to I O�lm and PM25 has an aerodynamic diameter less than 2.5�lm. These

particles represent health hazard, enhance atmospheric chemica! react ions, and

environmental response as well as negatively affecting aesthetic appearances

(HeiI, 1998).

Despite a poorly understood mechanism, epidemiological studies have

documented the association between fine particulate air pol/ution especially

particles of the size of PM 10 or less with the development of pulmonary diseases.

Illhalable particles with a diameter over I O�lIn arc predominantly deposited in the