Biochemical effects of ethanol on oreochromis mossambicus (Peters)

(1)

BIOCHEMICAL EFFECTS OF ETHANOL ON OREOCHROMIS MOSSAMBICUS (PETERS)

<25/¢w>wuAmu¢ed¢@

COCHIN UNIVERSITY UF SCIENCE AND TECHNULUGY

[IUCTUH OF PHILOSOPHY In

BIOCHEMISTRY

UNDER THE FACULTY OF MARINE SCIENCES

939

SMITHA.V.BHANU

Reg. No 2'/es

DEPARTMENT OF MARINE BIOLOGY, MICROBIOLOGY AND BIOCHEMISTRY

SCHOOL OF MARINE SCIENCES

COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY COCHIN-682016, KERALA, INDIA

NOVEMBER 2009

(2)

***

cgad, ¥=1l=¥=Ir#*#

5 O

(3)

Department oi'.\-'larine Biology. .\"licrol>iolo;.;y and Bioclicntistry School <>i'M;u'i11e Sciences

Cochin I711i\-'e1'sity oi‘ Science anti Teclinology

liiltv ztrls ."\\t‘llllt‘. ('t>t'hii| - li;“%‘_’lllfi

Dr. Babu Philip Professor

/_ ‘U Q

@erl1ftra1e

This is to certify that the thesis entitled “BIOCHEMICAL EFFECTS OF ETHANOL ON OREOCHROMIS MOSSAMBIC US (PETERS)” is an authentic

record of the research work carried out by Ms. Smitha. V. Bhanu under my

supervision and guidance in the Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology, in partial fulﬁllment of the requirements for the degree of Doctor of philosophy in Biochemistry ot Cochin University of Science and Technology, and no part there of has been presented for the award of any other degree, diploma or associateship in any

university.

‘

/ / / a t

L /

Koc/it'~682016 Dr. BABU PHILIP

Wovem 6er 2009

(4)

DECLARATION

l hereby declare that the thesis entitled “BIOCHEMICAL EFFECTS OF ETHANOL ON OREOCHROMIS MOSSAMBICUS (PETERS)” is a genuine

record of research work done by me under the supervision and guidance of Prof. Dr. Babu Philip, Department of Marine Biology, Microbiology and

Biochemistry, Cochin University of Science and Technology. The work presented in this thesis has not been submitted for any other degree or diploma earlier.

/oiiidldir Q W

Smitha. V. Bhanu Kocﬁi -68201 6

Wovem 6er 2009

(5)

Acknowledgement

I express my sincere and deqaest gratitude to my research guide Dr. Babu Philip, Professor; Department of Mmne Biology, Mcrobiology and Bioch emistiy ﬁat the motivation, afi‘ectionate treatment, constant support and Valuable guidance I received from him. I/Vithout his valuable suggestions and intellectual inputs, this thesis Would not have seen the light of the day. [acknowledge him with immense gratitude.

I am grateful to Dr. Ram Mohan, Director and Dean, School of Manne Sciences, CUBA T, forgiving me constant encouragement and support.

I remain thankful to Dr. K. I Damodaran, Former Director, School of Maiine

Sciences, C USA Y, for allowing me to utililze the facilities of the school of marine Sciences.

l express my thankfulness to Dr. A. 1/. Saramma, Reader (Former Head), Department ofMai1ne Biology, l\licrobiology and Biochemistiy; for the encouragement, support and also providing me With the necessaiy facilities.

I am thanltﬁil to Prof Dr. Aneykutty foseph, Head, Department ofManne Biology,

lwicrobiology and Bio ch emilstiy.

l put into Words my gratitude towards Dr. Rosamma Philip, DI‘. S. Bijoy Nandaii and Dr. Mohamed Hatha A./1, faculty members, Department of Marine Biology, lllicrobiology and Biochemistry; for their support and motivation.

l am greatly indebted to my dear teacher Dr. Sudha. G. Menon, Head, Department of Biochemistiy, Kongunadu Arts and Science College, Coimbatore, for her friendly and open mind-set which inspired me a lot.

l am gzateful to my teacher Elamathi, A/ﬂ>/1111?» helping me to build a strong foundation in

biochemistry during my under graduate mys. l individually thank Di: Dayanandan, Dr.

Lakshminaiayztnan, Dr. Bhaskar, Di: Rani, Dr. Surya vathana and Dr. Mohandas for their excellent classes on biochemistry during my under graduate and post graduate peiiod Which helped me to

establish a {inn grasp on the subject: ‘

I remain thankﬁzl to Di: lwohandas, Emeritus Professor; NC’/1A1-L Cochin, forgiving me constant encouragement; support and valuable suggestions during the course of the stuay

I extend my heartfelt thaizlcs to my Valiachan Mr. Govindankutty Menon, Retd CUSA T, Suresh Kumar, Reyil, Salim, Dirya knshnan and fehosheeba P. Mathews for their help rendered during the research period

I record my gratefulness to Dr. C Chantﬂiha, Retd Piinc:tpalS'aentist; Cl\ﬂ”_R.L Cochin for her Well Wishes and intelligent suggestions which aided me a lot throughout research phase

(6)

l)r. Balachandian, Retd Saentist; l\UO Cochin, and Di: Puiushan, Former Dean, collqe of Fisheries Panangad are specially ackno Wledged Ear their enthusiastic aizirude to Wards my Work.

I drank Dr: fose, College ofFishene$ Panangad for pro viding me With healthy specimens of Tilapia Whenever] Was in need of them.

Sincere thanks to Dr. KC George, Retd. Scientist, C'l\ﬂ3R1: Cochin and Dr. Ajith l\7aml>iar, Pathologist, Apollo Hospital Chennai, for helping me to interpret the

histopathological aspects of my Work.

l acknowledge Dr. Muraleedhar'an Nair (Senior Faculty) and Dr. Renjit, Retheesh and Ginsh Kumai; (Researchers) Dept of Chemical Oceanography for permitting and assisting me respectively, to use A tomic Abs oiption Spectrophotometer for my research.

l thank my uncle Dr. Ragha van (Consultant in Surgery; Palakkad Thankam Hospital ) and Dr. Nara yanan (Gastroenterologist, All\»¢5; Cochin) ﬂit their Well Wishes and support.

Sincere thanks to Dr: facoh Philip, Director, S YYC, Hat granting me permission to perform Gas Chromatography aspects of my research Work at S Tl C I am especially thankful

to technical staff,‘ S Tl C, Dr. Bifu and Sh yam forgiving me essential directions.

l am greatly obliged to Dr. T: V. Shankar, Principal Scientist, Biochemistry and Nutntion Di'vision, CIFYI for issuing consent to carryout GC-1%? analysis at CIFT laboratory. l also remain thankful to Dz R. Anandan, Scientist (Senior Scale ) Biochemistry and Nutntion Division, C1137, Cochin for helping me in carrying out GC-l\1§' analysis.

I am especially grateful to Dr. HK. Krishna lyer, Scientist (R etired), CYFY; Mn Steph y Thomas and Mr. fahir (Dept. of Statistics, CUS14 T) for their valuable help during statistical anal ysrs of data. l also thank Dr. Valsamma fosqoh, Lecturer (NCAAI-1) for her practical suggestions on statistical processing of data.

I express my gratitude to my dear fiiend Dr. Pradecp, Postdoc fellow (phaimacokinetics) South Korea, for providing me With latest of research articles during the preparation of manuscript;

Thanks a lot to my dear pals Rajani, Ahsaas Rasool (Research Scholar, CYFE, Mumhai), Deepika Harish, Sajini Chechi, Misha Mama Mathew and Elizabeth Xavier; for giving me so much of your love and care and encouraging me Whenever l Was badly in need

of them.

l thank my fiiends Meena unni, Zarin Shamsi, Rajesh Nair Dr. shamzila Natarajan (Postdoc fello W, UPElW\7 medical school, Phrla delphia), Dr. Arun Kumar (Postdoc fello W, Canada ) and Dr. Radhika Gopinath and Dr. Mujeeb (Postdoc felloWs, Dept. ofMa11ne Biology, lwicrohiology and Biochemisny, C USA Y) for motivating me a lot. ‘

(7)

l Will never fail to remember the timely helps l received from Anil Kumar; Ablzilash KR and Man0]' ( R esearch fello Ws, Dept. ofMai1'ne Biology, lilicrobiology and Biochemisn;/3

C USA T) throughout the period of research.

I am so thankful and lucky to get colleagues like Aniladevi Kunjamma KP, fisha _/ose, Remya Varadara/an and l-laizisankar. H.S for providing a highly innovative and genuine environment in the Workplace which Was very much constructive for research activities.

l express my sincere thanks to Dr. l\/andini Menon and Dr. Anupama Nair for their Well—timed helps With the correction ofmanuscnpt. Thanks ﬁr being With me all the Wa y through, helping me to cross all hurdles and giving me all sort of support Whenever l was down.

l express my gratitude to Dr. Lakshmi G. Nair and Dr. Venu G. Nair for their love

and care.

l share Warm and candid relationships With all research fellows of my department and 1 individually thank ea ch of them for their support and friendship.

l am grateful to Mrs. Sheela, Shenai NA, Principal, KWW College of Nursing Cherthala, forpeimitting me to use the library facilities.

l sincerely thank non -tea ching stafﬂ Department ofManne Biology; l\<G'crobiology and Bioch emistiy for their timely helps.

l thank Mi: Manuel, librarian, School of Marine Sciences Library for his constant

8HCOUl'2g€II7€HZ'.

Sincere thanks are due to the librarians of CMFRI, Cochin, CYFII Cochin, CYPE Mumbai and IISC Bangalore.

l record my thanks to Mt Shabeer (lndu Photos 6! Graphics ) and Mr. Anand

(CEDIX) for all the D Y? Works.

Special thanks to Venkateswaran Uncle and Laksmi Aunty for their love, support and encouragement.

Sincere thanks to foseph uncle (ARA O/1) for his great understanding and Well Wishes, who never interrupted when l was deeply involved in my manuscnpt preparation, despite of my duties as treasurer; I had to do at ARA OA.

I am so much indebted to my sister-in-law Mrs. Bindu Menon for her swirt and faultless qping skills that made data entering process so cool and easy for me.

l am short of right Words to describe how 'H>rtunate and blessed l feel m yself to ha ve such loving, caring and greatly understanding mother-in-law and father-in-law Who had been

(8)

the motivation in all endeavours and I feel lucky to have them with me, always. I/Vithour those strong pillars of support and encouragement, l wouldn ’t have finished this work successfully! I am deeply indebted to them. liﬁl special thanks.

I take this moment to reminisce all my family members, my cousins and well wishers for their blessings and prayers.

l am forever indebted to my beloved husband Mr. R. Raghu ( Senior Project Engineer;

Al Naboodah, Abudhabi) Whose vision made me reach the great world of research. He made strong efforts to make me believe in m yself: that “I can ” pursue the research and harvest the results.

l am very much grateful to my broth er Rahal Vija y for his support and encouragement through the tough times of my work. l am really fortunate to have a loving brother like him,

with whom i share a unique bond offiiendship.

l recall with gratitude, the Erin motivation my brother in law late. Mr. Uda y Menon used to give me splendidly.

l am so much thankful to my sweet, channing nephew Sidharth, for his innocent and lovely company that helped a lot to do away with pressure, at strenuous writing hours.’

I am really humbled, saying thanks to my lovely little daughter Archita who thinks l am a fish doctorf I will never forget the days 1 used to type my thesis, With my daughter sitting on my lap. l know Archi, my research took lots of the precious time you deserved And l am here to say my sweet 'tonnes ofsoriy and thanks’ to you for your little ways of understanding.

Not but the least, l extend my heartfelt gratitude for all those good people whom .l might have missed unknowingly but has helped me any time, any wa y during my thesis work.

l humbly of-fer with prayers, my respect and gratitude at the feet of my father Late Mr.

R V. Bhanu and my Grandmother Late. Mrs. Lakshmykutzy Amma whose heavenly blessings help me to achieve all the success in my life.

Finally but mostly, l take this opportunity to thank my dearly loved mother; the pillar of my strength, whose love and care made me reach this stage!

Above all I thank “GOD'§ the almighty without whose blessings this would never have been completed successﬁilly.

Smz1M"”'€MM

(9)

Preface

Several geopolitical factors such as negative environmental consequences of fossil fuels and concerns about petroleum supplies aggravated the worries concerned with global warming. This has fueled the search for and production of renewable sources of energy worldwide for the past few years. The increasing demand of alternative energy sources has created interest in the production of ethanol and ethanol blended fuels since the l97()’s. Amendments to the Clean Air Act in 1990 opened the door for increased ethanol use. Amendments to the Clean Air Act boosted growth in the ethanol industry, and now ban on Methyl Tertiary Butyl Ether could propel it even further. The use of alcohol as a fuel was first introduced in Brazil in the year 1970. But nowadays the world’s total ethanol production depends solely upon United States. Even though the usage of ethanol in vehicle as a fuel is increasing worldwide, the potential risks associated with it have not been examined.

Reports stated that unburned ethanol emissions results in a global-scale source of acetaldehyde larger than that ofdirect emissions. But the actual fate of ethanol in the environment and its effect on aquatic organisms apart from ﬁsh kill have not been studied in detail. Like oil spills, ethanol spills also bring about deleterious effects on aquatic environments. In this study, effect on aquatic environment brought about by ethanol is discussed in detail by using Orcoc/zromis mossambicus (Peters) as the animal model. This thesis studies the toxicity of ethanol using haematological.

biochemical and histopathologieal parameters.

The present study exhibited the behavioural changes brought about by the euryhaline teleost Oreochromis mosscunbicus (Peters) when subjected to lethal toxicity studies. These changes were mainly due to respiratory stress. GC/MS study exhibited the presence of ethanol in an effluent which seemed to be close to the sub lethal dosage value selected for the study. Presence of ethanol in blood which was

detected by GC seems to be dose and duration dependent. The decrease in

membrane bound enzymes suggests the damages in the gill architecture. Marked alterations were exhibited in the haematological parameters studied. RBC hemolysis was increased both in in vizfro and in vivo conditions. Trace elements levels such as copper, zinc and selenium were studied by atomic absorption spectrophotometry.

\

(10)

Decrease in lysosomal fraction of B glucuronidase and acid phosphatase activities were observed both in in vitro studies and in vivo studies. Increase in serum parameters such as AST, ALT, CK, cortisol, vitamin B12 and ferritin as well as decrease in serum protein, albumin was obtained. Biochemical parameters ol carbohydrate, protein and lipid metabolism exhibited marked alterations in serum, blood and tissues of Oreochromis mossambicus (Peters). Decrease in Cyt.c. oxidasc activity indicates the reduced availability of oxygen. The increase in serum uric acid

and creatinine values refer to the renal effects of ethanol. An increase in

lipoperoxidation products as well as decrease in enzymatic and non enzymatic antioxidants was observed in the tissues of Oreochromis mossambicus (Peters).

Histopathological analysis carried out in the tissues such as gills, liver, heart and kidney proved the deleterious impact brought by ethanol. All this points to the fact that the widespread usage of ethanol ultimately pollutes the aquatic environment thereby affecting the aquatic fauna mainly ﬁshes which have a major role in the economy of the country and serves as valuable dietary sources of protein. All the above mentioned tests are useful for evaluating the environmental hazard brought by ethanol.

\

(11)

eon/lie/wllo

(Page W0

Preface

General Introduction . .. ..

Review of Literature...

0 Review of the toxicity and metabolic effect of ethanol 0 Pathogenesis of ethanol toxicity

0 Review of the methods of determination of levels of ethanol 0 Review of the effect of ethanol on branchial ATPases

0 Review of the effect of ethanol on haematological parameters 0 Review of the effect of ethanol on serum trace elements Q Review of the effect of ethanol on serum parameters

~ Review of the effect of ethanol on erythrocyte membrane stability 0 Review of the effect of ethanol on metabolic profiles

Q Review of the effect of ethanol on lysosomal membrane stability

0 Review of the effect of ethanol on enzymatic and non enzymatic parameters Q Review of histopathological effects of ethanol

7 |\;|\,|\jf\J§|\J-5-5-A-\_¢-5

~16‘!-§l\J-»¢(D¢D&\JL'l1CD

1-7

-27

1.1 QUANTITATIVE ANALYSIS OF ETHANOL IN AN EFFLUENT ' u USING GAS CHROMATOGRAPHY/MASS

SPECTROMETRY... ...28-36

1.1 A Introduction

1.1B Material and Methods 1.10 Results

l.1D Discussion

1.2 DETERMINATION OF BLOOD ETHANOL IN

OREOCHROMIS MOSSAMBICUS (PETERS): srunnzs

28 31 32 36

USING GAS CHROMATOGRAPHY... ....37-47

1.2A Introduction

1.2B Material and Methods 1.2B.l

l.2B.2 l.2B.3 l.2B.4 l.2B.5 l.2B.6

Maintenance of Fish

Determination of [C50 of ethanol in 0. mossambicus {Peters}

Behavioural changes observed Bioassay Method

Experimental Design

Preparation of blood sample for gas chromatographic studies

-§J>CJQOJ¢OC4JOJOO

ZCJCDCD(.D%%\J

(12)

1.2B.7

1.2B.8

1.26 Results

Estimation of ethanol in blood using an analytical technique:

Gas Chromatography (BC)

Standard conditions employed for GB Analysis:

instrumentation and Chromatographic separation conditions.

1.2D Discussion

2 EFFECT OF ETHANOL ON BRANCHIAL ATPases... .. ....48-65

2.1A Introduction

2.28 Materials and Methods

2.28.1 Preparation of gill sample for experimental studies 2.2B.2 Extraction of the enzyme

2.2B.3 Estimation of experiments 2.36 Results

2.4D Discussion

C€%a»pt0o3

EFFECT OF ETHANOL ON HAEMATOLOGICAL PARAMETERS OF OREOCHROMIS MOSSAMBICUS (PETERS)...

3.1A introduction

3.28 Materials and Methods

3.2B.l Preparation of blood sample for haematological studies.

3.28.2 Methods used for the haematological studies

a.

b.

c.

d.

e.

f.

Q.

3.30 Results 3.3D Discussion

Determination oi RBC count Determination of WBC count

Determination of packed cell volume IPCV) Estimation of erythrocyte sedimentation rate (ESR) Estimation of fine packed red cells lFPR[I)

Determination of haemoglobin lHbl Determination of erythrocyte indices

1. Determination of mean corpuscular volume (Milli) 2. Determination of mean corpuscular haemoglobin (MCH)

3. Determination oi mean corpuscular haemoglobin concentration (MCHC) 40

41 42 46

d'>u'1Q'lU'1U‘IU1-D

|\;J>—~©C3QOO

66-93

66 68

‘\-l\J\.|\J\l\J\JUUU7Q')U)

CAJC»JC~JI\J|'\J—-'@CDOOCDCD

73 74 B9

(13)

3"."'-'- :1‘:-'-"I-' H -- " . '

EFFECT OF ETHANOL ON SERUM TRACE ELEMENTS OF OREOCHROMIS MOSSAMBICUS (PETERS): STUDIES BY

ATOMIC ABSORPTION SPECTROPHOTOMETRY ... "94-105

4.1A Introduction 94

4.28 Materials and Methods 95

4.28.1 Preparation of serum samples 95

4.28.2 Estimation of serum trace elements 96

4.30 Results 97

4.48 Discussions 103

‘€M;»wv 5

5.1 EFFECT OF ETHANOL ON SELECTED SERUM PARAMETERS OF OREOCHROMIS MOSSAMBICLIS (PETERS) ... ..106-130

5.1A Introduction 106

5.18 Materials and Methods 108

5.18.1 Preparation of serum samples 108

5.18.2 Method used for serum experiments 108

a. Estimation oi serum iron 109

b. Estimation of serum creatine kinase 110

c. Estimation of serum alanine transaminase 111 d. Estimation of serum aspartate transaminase 112 e. Estimation of serum alkaline phosphatase 113

f. Estimation serum total protein 114

5.10 Results 115

5.11] Discussion 126

5.2 EFFECT OF ETHANOL ON SELECTED SERUM PARAMETERS OF OREOCHROMIS MOSSAMBICUS (PETERS): STUDIES

USING AUTOANALYZER... ...131-148

5.24 Introduction 131

5.28 Materials and Methods 133

5.28.1 Estimation of serum parameters using immunoassay analyzer 133

a. Estimation of serum cortisol 133

(14)

b. Estimation of serum lolate 134

c. Estimation of serum vitamin B12 135

d. Estimation of serum ferritin 136

5.26 Results 138

5.2D Discussion 145

6

EFFECT OF ETHANOL ON RBC MEMBRANE STABIIJITYOQ149—164

6.1 Ill!-I/ITRO RBC Membrane stability studies 149

6.1 A Introduction 149

6.1 B Materials and Methods 150

6.18.1 /n-vifro studies 150

6.1 B.1.1 Isolation oi red blood cells 151

6.1B.2 Estimation of RBC membrane stability l/n-vitra condition) 151

6.1 C Results 152

6.1 D Discussion 154

6.2 Ill!-l/ll/0 RBC Membrane stability studies 157

6.2 A Introduction 157

6.2 B Materials and Methods 159

6.28.1 Estimaiton oi RBC membrane stability (/n-viva Condition) 159

6.2 C Results 160

6.2 D Discussion 161

7 EFFECT OF ETHANOL ON METABOLIC PROFILES ... ..165-279

7.1 EFFECT UF ETHANOL UN CARBOHYDRATE METABOLISM OF FRESH WATER

TELEOST, OREOCHROMIS MOSSAMBICUS (PETERS) 165

7.1 A Introduction 165

7.1 B Materials and Methods 166

7.18.1 Preparation of samples for experiments 167

7.18.2 Experimental procedures 167

a. Estimation of total carbohydrate 167

b. Estimation of blood glucose 168

c. Estimation of serum lactate dehydrogenase 169

7.16 Results 171]

7.1D Discussion 177

(15)

7.2 EFFECT OF ETHANOL ON MITOCHONDRIAL ELECTRON TRANSPORT CHAIN 182

7.2 A Introduction 182

7.2 8 Materials and Methods 183

7.28.1 Preparation of tisuue samples tor experiments 184

7.28.2 Method used for biochemical analysis 184

7.2 C Results 185

7.2 D Discussion 189

7.3 EFFECT OF ETHANOL ON PROTEIN METABOLISM 192

7.3A Introduction 192

7.38 Materials and methods 194

7.38.1 Preparation of serum samples for experimental studies 194

7.38.2 Experimental procedures 194

. Estimation of albumin 194

. Estimation of urea I95

. Estimation of uric acid 196 . Estimation of creatinine I97

. Estimation of serum ammonia I98

Estimation of total protein 199

. Estimation of tree amino acids (Ninhydrin positive substances) 199

Estimation of acid phosphatase 200

:'<::| _—--co £2.11 c-m

7.36 Results 282

7.31] Discussion 221

7.4 EFFECT OF ETHANOL ON LIPID METABOLISM 229

7.4A Introduction 229

7.48 Materials and Methods 231

7.48.1 Preparation of serum samples for experimental studies 231

7.48.2 Methods used for biochemical analysis 231

a. Estimation of 8-hydroxy-8-methylglutaryl-CoA reductase

IHMG CoA Reductasel activity 231

b. Extraction of lipids 233

c. Estimation of total lipid 234

d. Estimation of triacylglycerol (TAG) 235

e. Estimation of total cholesterol 236 i. Estimation of HOL cholesterol 237

g. Estimation of LDL+ VLDL cholesterol 238

h. Estimation of free fatty acids 239

i. Estimation ot phospholipids 240

j. Determination of serum lipase 241

(16)

7.46 Results 242

7.40 Discussion 271

8 EFFECT OF ETHANOL ON LYSOSOMAL MEMBRANE

STABILITY (In vitro and In vivo Conditions) ....280-303 8.20 Materials and Methods 282 8.1!-l Introduction 280

8.28.1 Preparation of tissue samples for experimental studies 282

ii) In viva Studies 282 liil In vitm Studies 282

8.2B.la Activity of Iysomal enzymes (B-glucuronidase and acid phosphatase in the various sub cellular fractions of liver tissue of Ureochromis

mossambicus (in virm and in viva conditions). 282

8.2B.1b Rate of release of [3 glucuronidase from the lysosomal-rich fraction

of liver (Lysosomal Enzyme Release Assay) 283 8.2B.1c Methods used for the biochemical analysis 283

8.30 Results 285

8.40 Discussion 301

‘€/more/09

EFFECT OF ETHANOL ON ANTIOXIDANT PARAMETERS (NON ENZYMATIC AND EN ZYMATIC ANTIOXIDANTS)

OF OREOCHROMIS MOSSAMBICUS (PETERS) ... "304-367

9.1A lntroduction 304

9.28 Materials and Methods 308

9.2B.1Methods used for the biochemical analysis 308

a. Estimation of lipid peroxidation 308

h. Estimation of superoxide dismutase 389

c. Estimation of catalase 310

d. Estimation of glutathione peroxidase 311 e. Estimation of glutathione-s-transferase 312

t. Estimation of glutathione reductase 313 g. Estimation of conjugated dienes 314 h. Estimation of hydroperoxides 314

i Estimation of total reduced glutathione 315

(17)

j. Estimation vitamin A 316

k. Estimation of ascorbic acid (vitamin C). 318 I. Estimation of vitamin E (ct-tocopheroi) 319 m. Estimation of total sulphhydryl group 320

n. Estimation of peroxidase 321

o. Estimation of glucose-B- phosphate dehydrogenase 322

9.3 C Results 323

9.4 D Discussion 357

10 ETHANOL INDUCED HISTOPATHOLOGICAL CHANGES IN THE DIFFERENT TISSUES OF OREOCHROMIS

MOSSAMBICLIS (PETERS)... .. ....3as-391

10.1A Introduction 363

10.2B Materials and Methods 370

10.2B.1 Preparation of tissue samples for histopathological studies. 370

i0.2B.2 Maior steps involved in histological procedures. 370

10.3C Results 374

10.40 Discussion 383

SUMMARY AND CONCLUSION ... "392-401 BlBl.l0GRAPHY ... ..402-462 APPENDICES ... ..4ﬁ3

>!<*>l<**&n€**>i<*>l=

(18)

LIST OF TABLES

arable lllo _f Title _{Page lllo}

Table 1.2.1

Table 2.3.1

Table 2.3.2

Table 3.1

Table 3.2

Table 4.1

Table 4.2

Table 5.1.1

Table 5.1.2

Table 5.2.1

Table 5.2.2

Table 6.1.1

Table 6.2.1

Table 7.1.1

Table 7.1.2 Table 7.2.1

Table 7.2.2

Table 7.3.1

Table 7.3.2

Effect of 7 and 21 days of exposure to different concentrations of ethanol in the blood of 0. mossambims.

Effect of exposure to different concentrations of ethanol for 7 days on the gill ATPase activities in 0.mossamb/bus.

Effect of exposure to different concentrations of ethanol for 21 days on the gill ATPase activities in Qmassambicus.

Effect of exposure to different concentrations of ethanol for 7 days on haematological parameters of 0. mossambicus.

Effect of exposure to different concentrations of ethanol for 21 days on haematological parameters of 0. mossambicus.

Effect of exposure to different concentrations of ethanol for 7 days on serum trace elements of 0. mossambicus.

Effect of exposure to different concentrations of ethanol for 21 days on serum trace elements of 0. mossambicus

Effect of exposure to different concentrations of ethanol for 7 days on serum parameters of 0. mossamlricus.

Effect of exposure to different concentrations of ethanol for 21 days on serum parameters of 0. mossambicus.

Percentage hemolysis in 0. mossambicus on exposure to different

concentrations of ethanol (In - wtro conditions}.

Percentage hemolysis in 0. mossamb/cus on exposure to different

concentrations of ethanol for 7 days and 21 days (in viva conditions).

Effect of exposure to different concentrations of ethanol for 7 days on carbohydrate metabolism of Ilmossambicus.

Effect of exposure to different concentrations of ethanol for 21 days on carbohydrate metabolism of 0. mossambicus.

Effect of exposure to different concentrations of ‘ethanol for 7 days on the

levels of cytochrome-c-oxidase activity in different tissues of

0. mossambicus.

Effect of exposure to different concentrations of ethanol for 21 days on the

levels of cytochrome-c-oxidase activity in different tissues of

0. massambicus.

44 55

55

74

75

97

98

115

116

138

153

160

170

171

185

186

r 202

203

(19)

Table 7.3.3

Table 7.3.4

Table 7.3.5

Table 7.3.6

Table 7.3.7

Table 7.3.8

Table 7.4.1

Table 7.4.2

Table 7.4.6

Table 7.4.7

Table 7.4.8

Table 7.4.9

Table 8.3.1

Table 8.3.4

Table 8.3.7

Table 8.3.10

Table 8.3.13

Table 9.3.1

Effect of exposure to different concentrations of ethanol for 7 days on total protein content present in different tissues of 0. mossambicus.

Effect of exposure to different concentrations of ethanol for 21 days on

total protein content present in different tissues of

0. mossambicus.

Effect of exposure to different concentrations of ethanol for 7 days on total

free amino acid content present in different tissues of

0. mossambicus.

Effect of exposure to different concentrations of ethanol for 21 days on

total free amino acid content present in different tissues of

0. mossambicus.

Effect of exposure to different concentrations of ethanol for 7 days on acid

phosphatase activity present in serum and different tissues of

0. mossambicus.

Effect of exposure to different concentrations of ethanol for 21 days on acid phosphatase activity present in serum and different tissues of 0. mossambicus.

levels of HMG CoA Reductase activity in different tissues of

0. mossambicus.

levels of HMG CoA Heductase activity in different tissues of

0. mossambicus.

Effect of exposure to different concentrations of ethanol for 7 days on lipid profile parameters of 0. massambicus.

Effect of exposure to different concentrations of ethanol for 7 days on lipid profile parameters of 0. mossambicus.

Effect of exposure to different concentrations of ethanol for 21 days on lipid profile parameters on 0. mossambicus.

Effect of different concentrations of ethanol on the sub cellular activity of acid phosphatase in 0. mossambicus (in virm).

Effect of different concentrations of ethanol on the sub cellular activity of B-glucuronidase in 0. mossambicus /in vitro).

Effect of different concentrations of ethanol on the sub cellular activity of [3 - glucuronidase in 0. mossambicus (in viva).

Effect of different concentrations of ethanol on the sub cellular activity of acid phosphatase in 0. mossambicus /in viva/.

Time dependent release of [3 Glucuronidase enzyme in 0. mossambicus exposed for 7 and 21 days.

Effect of sub lethal concentrations of ethanol in different tissues of 0. mossambicus exposed for 7 days and 21 days on malondialdehyde levels.

(20)

Table 9.3.3

Table 9.3.5

Table 9.3.7

Table 9.3.9

Table 9.3.11

Table 9.3.13

Table 9.3.15

Table 9.3.17

Table 9.3.19

Table 9.3.21

Table 9.3.23

Table 9.3.25

Table 9.3.27 Table 9.3.29

Effect of different sub lethal concentrations of ethanol in different tissues of 0. mossambicus exposed for 7 days and 21 days on the level of total reduced glutathione.

Effect of different sub lethal concentrations of ethanol in different tissues

of 0. mossambicus exposed for 7 and 21 days on the level of

conjugated Dienes (CD).

hydroperoxide.

Effect of different sub lethal concentrations of ethanol in different tissues of 0. mossambicus exposed for 7 and 21 days on the level of vitamin A content.

Effect of different sub lethal concentrations of ethanol in different tissues of 0. mossambicus exposed for 7 and 21 days on the level of vitamin C content.

Effect of different sub lethal concentrations of ethanol in different tissues of 0. mossambicus exposed for 7 and 21 days on the level of vitamin E content

Effect of different sub lethal concentrations of ethanol in different tissues of 0. mossambicus exposed for 7 and 21 days on the level of glutathione reductase.

glutathione S transferase.

Effect of different sub lethal concentrations of ethanol in different tissues of 0. mossambicus exposed for 7 and 21 days on the level of peroxidase.

superoxide dismutase.

Effect of different sub lethal concentrations of ethanol in different tissues of 0. mossambicus exposed for 7 and 21 days on the level of glucose-El phosphate dehvdrogenase

Effect of different sub lethal concentrations of ethanol in different tissues of 0. mossamlncus exposed for 7 and 21 days on the level of total sulphhydryl groups.

Effect of different sub lethal concentrations of ethanol in different tissues of 0. mossambicus exposed for 7 and 21 days on the level of catalase.

glutathione peroxidase.

(21)

LIST OF STATISTICAL TABLES

TableNo W Title N % 7 _ Page No

All the figures were corrected to three places of decimals Ill the ANUVA table Table 1.2.2a

Table 1.2.3 Table 2.3.3a Table 2.3.4a Table 2.3.5a Table 2.3.6a Table 2.3.7 Table 3.3a Table 3.4a Table 3.5a Table 3.6 a Table 3.7 a Table 3.8 a Table 3.9 a Table 3.10 a Table 3.11 a Table 3.12 Table 4.3a Table 4.4a Table 4.5a Table 4.6 Table 5.1.3a Table 5.1.4 a Table 5.1.5 a Table 5.1.6a Table 5.1.7 a Table 5.1.8a Table 5.1.9 Table 5.2.3a Table 5.2.4a Table 5.2.5a Table 5.2.6a

ANOVA Table for blood ethanol Multiple comparison test ANOVA table for total ATPase ANOVA table for l\la‘l K‘ ATPase ANUVA table for Ca“ ATPase ANOVA table for Mg” ATPase Multiple comparison test ANUVA table for Haemoglobin ANUVA table for BBC Count ANUVA table for WBC Count ANUVA table for PCV ANOVA table for ESR ANOVA table for FPRC ANOVA table for MCV ANOVA table for MCH ANOVA table for MCHC Multiple comparison test ANOVA table for copper ANOVA table for zinc ANUVA table for selenium Multiple comparison test ANUVA table for iron

ANUVA table for creatine kinase ANUVA table for alanine transaminase ANUVA table for aspartate transaminase ANUVA table for alkaline phosphatase ANOVA table for total protein Multiple comparison test ANUVA table for ferritin ANOVA table for cortisol ANUVA table for lolic acid ANDVA table for vitamin B12

(22)

Table 5.2.7 Table 6.1 .2a Table 6.1.3 Table 6.2.2 a Table 6.2.3 Table 7.1 .3a Table 7.1.4a Table 7.1.5a Table 7.1.6 Table 7.1.7 Table 7.2.3a Table 7.2.4 Table 7.2.5 Table 7.3.9a Table 7.3.l0a Table 7.3.l1a Table 7.3.12a Table 7.3.13a Table 7.3.14a Table 7.3.15a Table 7.3.16a Table 7.3.l7a Table 7.3.18 Table 7.3.19 Table 7.4.3a Table 7.4.4 Table 7.4.5 Table7.4.l0a Table 7.4.11a Table 7.4.12a Table 7.4.13a Table 7.4.14a Table 7.4.15a Table 7.4.16a Table 7.4.17a Table 7.4.l8a

Multiple comparison test ANOVA table for RBC (/n-virro) Multiple comparison test ANUVA table for RBC (In-viva) Multiple comparison test ANUVA table for blood glucose ANUVA table for lactate dehydrogenase ANOVA table for total carbohydrate Multiple comparison test(Concentration) Multiple comparison test (Tissue)

Three Factor ANUVA table for cytochrome-c-oxidase activity Multiple comparison test (Concentration)

Multiple comparison test (Tissue) ANUVA table for albumin ANOVA table for urea ANUVA table for uric acid ANUVA table forcreatinine ANOVA table for ammonia

ANUVA table for tissue total protein ANOVA table tor tree amino acids ANUVA table for acid phosphatase ANUVA table for acid phosphatase Multiple comparison test (Concentrations) Multiple comparison test (Tissues)

ANUVA table for HMG Coll reductase activity Multiple comparison test (Concentrations) Multiple comparison test (Tissues) ANUVA table for total lipids (Serum) ANOVA Table for Total Lipids(Tissues) ANUVA table for phospholipids (Serum) ANDVA table for phospholipids(Tissues) ANUVA table tor tree fatty acids (Serum) ANUVA table for tree fatty acids(Tissues) ANOVA table for lipase(Serum)

ANUVA table for triglycerides(Serum) ANUVA table for triglycerideslTissues)

(23)

Table 7.4.19a Table 7.4.2021 Table 7.4.21 a Table 7.4.22a Table 7.4.23 Table 7.4.24 Table 8.3.2a Table 8.3.2b Table 8.3.2c Table 8.3.3 Table 8.3.5a Table 8.3.5b Table 8.3.5c Table 8.3.6 Table 8.3.8a Table 8.3.8b Table 8.3.8c Table 8.3.9 Table 8.3.11a Table 8.3.11b Table 8.3.11c Table 8.3.12 Table 8.3.13a Table 8.3.14 Table 9.3.2a Table 9.3.4a Table 9.3.6a Table 9.3.8a Table 9.3.1[)a Table 9.3.12a Table 9.3.14a Table 8.3.16a Table 9.3.18a Table 9.3.2(Ja Table 9.3.22a Table 9.3.24a Table 9.3.26a

ANUVA table for HDL cholesterol (Serum) ANDVA table for total cholesterol (Serum) ANUVA table for total cholesterol(Tissues) ANOVA table for l.l]L + VLDL Cholesterol (Serum) Multiple comparison test (Serum)

Multiple comparison test (Tissues)

ANUVA table for nuclear acid phosphatase activity (in-w'rro) ANDVA table for soluble acid phosphatase activity (/'n-w'rr0/

ANUVA table for lysosomal acid phosphatase activity /in-virro) Multiple comparison test (Concentrations) /in-virrol

ANUVA table for nuclear 13- glucuronidase activity /in-virro) ANUVA table for soluble B ~ glucuronidase activity (in-virrol ANUVA table for lysosomal [3 - glucuronidase activity (r'n-w'tm/

Multiple comparison test (Concentration)

ANOVA table for nuclear )5» glucuronidase activitV //n-viva) ANOVA table for soluble B- glucuronidase activity /ln-w'v0/

ANOVA table for lysosomal 13- glucuronidase activity (/n-w'v0/

ANOVA table for nuclear acid phosphatase activity //n-wi/0) ANOVA table for soluble acid phosphatase activity //n-vii/0}

ANOVA table for lysosomal acid phosphatase activity {/n-vr'v0/

ANOVA Table for (3 Glucuronidase enzyme release Assay (/n-w'v0/

Multiple comparison test (Time)

Three — Factor ANOVA table tor malondialdehyde Three - Factor ANUVA table for total reduced glutathione Three — Factor ANUVA table for conjugated dienes Three - Factor ANOVA table for hydroperoxides Three-Factor ANOVA table for Vitamin A Three-Factor ANOVA table for Vitamin C Three-Factor ANOVA table for Vitamin E

Three- Factor ANUVA table tor glutathione reductase Three-Factor ANOVA table for glutathione S translerase Three-Factor ANOVA table for peroxidase

Three-Factor ANOVA table for superoxide dismutase

Three - Factor ANOVA table for glucose-6-phosphate dehydrogenase Three-Factor ANDVA table for total sulphhydryl groups

(24)

Table 9.3.28a Three-Factor ANOVA table for catalase

Table 9.3.30a Three Factor ANUVA table for glutathione peroxidase Table 9.3.31 Multiple comparison test (Concentration)

Table 9.3.32 Multiple comparison test {Tissue}

(25)

LIST OF FIGURES

Table No Title

Page No Figure 1A

Figure 1B Figure 1C Figure 1D Figure 1E Figure 1F Figure 16 Figure 2f-l Figure 2B Figure 2C Figure 2D Figure 2E Figure 2F Figure 2G Figure 1 Figure 2

Figure 3

Figure1.2.2

Figure 2.3.3

Figure 2.3.4

Figure 2.3.5

Figure 2.3.6

Figure 3.3

Figure 3.4

Figure 3.5

Standard chromatogram with solvent peak and ethanol peak.

Standard chromatogram with ethanol peak after masking solvent peak.

Standard chromatogram with ethanol peak.

Standard chromatogram with ethanol peak and area (for 5,ull1Uml).

Mass spectrum of pure ethanol obtained from the PE library.

Sample chromatogram with solvent peak and ethanol peak.

Sample chromatogram with ethanol peak after masking solvent peak.

Sample chromatogram with ethanol peak.

Sample chromatogram with ethanol peak and area.

Mass spectrum of sample obtained from PE library.

Mass spectrum of standard ethanol obtained from PE library.

Gas chromatograms showing standard ethanol peak.

Standard gas chromatograms showing ethanol peak in the blood of 0. mossambicus upon exposure to different sub lethal concentrations of ethanol for 7days.

Standard gas chromatograms showing ethanol peak in the blood of 0. mossambicus upon exposure to different sub lethal concentrations of ethanol for 21 days.

Levels of ethanol in the blood of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of Total ATPase activity in the gill of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of Na‘l K‘ ATPase activity in the gill of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of Caz‘ ATPase activity in the gill of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of Mgz‘ ATPase activity in the gill of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of haemoglobin in the blood of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of red blood cell count in the blood of 0. massambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of white blood cell count in the blood of 0. massambicus exposed for 7 days and 21 days to different concentrations of ethanol.

33 33 33 34 34 34 34 35 35 35 35 36 36 36 42 42

43

44 56 57

59

66

75

77

78

(26)

Figure 3.6

Figure 3.7

Figure 3.8

Figure 3.9

Figure 3.10

Figure 3.11

Figure 4.3

Figure 4.4

Figure 4.5

Figure 5.1.3

Figure 5.1.4

Figure 5.1.5

Figure 5.1.6

Figure 5.1.7

Figure 5.1.8

Figure 5.2.3

Figure 5.2.4

Figure 5.2.5

Figure 5.2.6

Figure 6.1.2

Figure 6.2.2

Levels of packed cell volume in the blood of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of erythrocyte sedimentation rate in the blood of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of fine packed red cells in the blood of 0. massambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of mean corpuscular volume in the blood of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of mean corpuscular haemoglobin in the blood of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of mean corpuscular haemoglobin concentration in the blood of

0. massambicus exposed for 7 days and 21 days to different

concentrations of ethanol.

Levels of copper in the serum of 0. mossamb/cus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of zinc in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of selenium in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of iron in the serum of 0. mossamhicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of creatine kinase in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of alanine transaminase in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of aspartate transaminase in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of alkaline phosphatase in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of protein in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of ferritin in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of cortisol in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of folate in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of vitamin B12 in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Percentage hemolysis in 0. mossambicus on exposure to different

concentrations of ethanol (In - vitro conditions).

Levels of RBC membrane stability (/n-Viva) in the blood of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

(27)

Figure 7.1.3

Figure 7.1.4

Figure 7.1.5

Figure 7.2.3

Figure 7.3.9

Figure 7.3.10

Figure 7.3.11

Figure 7.3.12

Figure 7.3.13

Figure 7.3.14

Figure 7.3.15

Figure 7.3.16

Figure 7.3.17

Figure 7.4.3

Figure 7.4.10

Figure 7.4.11

Figure 7.4.12

Figure 7.4.13

Levels of glucose in the blood of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of lactate dehydrogenase activity in the serum of 0. mossambicus exposed for 7 days and 21 days to different

Levels of total carbohydrate content in the different tissues of 0. mossamlticus exposed for 7 days and 21 days to different

Variations in cytochrome-c-oxidase activity in the different tissues of 0. mossambicus exposed for 7 days and 21 days to different

Levels of albumin in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of urea in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of uric acid in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of creatinine in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of ammonia in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of total protein content present in the different tissues of 0. mossambicus exposed for 7 days and 21 days to different

Levels of total free amino acids present in the different tissues of 0. mossambicus exposed for 7 days and 21 days to different

Levels of acid phosphatase activity in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of acid phosphatase activity in the different tissues of 0. massambicus exposed for 7 days and 21 days to different

Levels of HMG Coll Reductase activity in the different tissues of 0. mossambicus exposed for 7 days and 21 days to different

Levels of total lipids in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of total lipids in the tissues of 0. mossamlricus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of phospholipids in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of phospholipids in the tissues of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

(28)

Figure 7.4.14

Figure 7.4.15

Figure 7.4.16

Figure 7.4.17

Figure 7.4.18

Figure 7.4.19

Figure 7.4.20

Figure 7.4.21

Figure 7.4.22

Figure. 8.3.2

Figure 8.3.5

Figure 8.3.8

Figure 8.3.11

Figure 8.3.14

Figure 9.3.2

Figure 9.3.4

Figure 9.3.6

Figure 9.3.8

Levels of free fatty acids in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of free fatty acids in the tissues of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of lipase activity in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of triglycerides in the serum of 0. mossamb/cus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of triglycerides in the tissues of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of HDL cholesterol in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of total vholesterol in the serum of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of total cholesterol in the tissues of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of LDL+ VLDL cholesterol in the serum of 0. mossa/no/cos exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of sub cellular acid phosphatase activity in the hepatic tissue of 0./nossamlﬁcus.

Levels of sub cellular B - glucuronidase activity in the hepatic tissue of 0. massambicus //n vitro).

Levels of sub cellular B - glucuronidase activity in the hepatic tissue of

0. mossambicus exposed for 7 days and 21 days to different

concentrations of ethanol //n-viva/.

Levels of sub cellular acid phosphatase activity in the hepatic tissue of

0. mossambicus exposed for 7 days and 21 days to different

concentrations of ethanol //n-vii/o/.

Lysosomal enzyme release assay (B — Glucuronidase) in the hepatic tissue

of 0. massambicus exposed for 7 days and 21 days to different

concentrations of ethanol /In-viva/.

Levels of malondialdehyde in the different tissues of 0. mossambicus exposed for 7 days and 21 days to different sub lethal concentrations of ethanol.

Levels of total reduced glutathione in the different tissues of

0. mossambicus exposed for 7 and 21 days to different concentrations of ethanol.

Levels of conjugated dienes in the different tissues of

0. mossamhicus exposed for 7 and 21 days to different concentrations of ethanol.

Levels of hydroperoxides in the different tissues of 0. mossambicus exposed for 7 and 21 days to different concentrations of ethanol.

(29)

Figure 9.3.10

Figure 9.3.12

Figure 9.3.14

Figure 9.3.16

Figure 9.3.18

Figure 9.3.20

Figure 9.3.22

Figure 9.3.24

Figure 9.3.26

Figure 9.3.28

Figure 9.3.30

Levels of vitamin A content in the different tissues of 0. mossambicus exposed for 7 and 21 days to different concentrations of ethanol.

Levels of vitamin C content in the different tissues of 0. mossambicus exposed for 7 and 21 days to different concentrations of ethanol.

Levels of vitamin E content in the different tissues of 0. mossambicus exposed for 7 and 21 days to different concentrations of ethanol.

Levels of glutathione reductase in the different tissues of

0. massambicus exposed for 7 and 21 days to different concentrations of ethanol.

Levels of glutathione S transferase in the different tissues of

Levels of peroxidase in the different tissues of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of superoxide dismutase in the different tissues of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of glucose-6-phosphate dehydrogenase in the different tissues of 0. mossambicus exposed for 7 and 21 days to different concentrations of ethanol.

Levels of total sulphhydryl groups in the different tissues of

Levels of catalase in the different tissues of 0. mossambicus exposed for 7 days and 21 days to different concentrations of ethanol.

Levels of glutathione peroxidase in the different tissues of 0. mossambicus exposed for 7 days and 21 days to different

(30)

LIST OF PLATES

Plate No U

Title pg g L pg g I g

Plate10.1

Platel 0.1 a

Plate10.1 a

Plate10.1b

Plate10.1c

Plate10.1d

Plate 10.1 e

Plate 10.lf

Plate10.1g

Plate10.2

Platel 0.2a

Plate10.2b

PIate10.2c

Plate 10.2d

Plate10.2e

Histopathological changes observed in the gill tissues of 0. mossambicus exposed to different concentrations of ethanol for 7 days and 21 days.

Photomicrograph of the control gill of 0. mossambicus showing normal gill architecture with primary gill lamellae (PL) and secondary gill lamellae (SL).

W&EXlm

Photomicrograph of the control gill of 0. mossamlricus showing normal gill architecture with primary gill lamellae (PL) and secondary gill lamellae (SL). (H 81 E >< 40)

Photomicrograph of the gill of 0. mossamlzicus exposed for 7 days at 0.659)) ethanol showing hyperplasia of the epithelium (HP), dilation of the blood vessel (DBV) and oedema (0). (H & E >< 20)

Photomicrograph of the gill of 0. mossambicus exposed for 7 days at 1.3gll ethanol showing haemorrhages (H) and telangiectasis (T). (H & E >< 20)

Photomicrograph of the gill of 0. mossambicus exposed for 7 days at 2.6gll ethanol showing gill aneurysm (GA), sloughing towards the base of the primary gill filament (SL) as well as at the edges of the secondary gill lamellae.

(H 8: E >< 20)

Photomicrograph of the gill of 0. mossambicus exposed for 21 days at 0.659)) ethanol showing haemorrhages (H), hyperplasia (HP) and clubbing (CL).

(H &E >< 20)

Photomicrograph of the gill of 0. mossambicus exposed for 21 days at 1.3qll ethanol showing gill necrosis (GN) and haemorrhages (H). (H & E >< 20)

Photomicrograph of the gill of 0. mossambicus exposed for 21 days at 2.6gll ethanol showing complete epithelium desquamation (BED) as well as haemorrhages (H). (H &E >< 20)

Histopathological changes observed in the liver tissues of 0. mossambicus exposed to different concentrations of ethanol for 7 days and 21 days.

Photomicrograph of the control liver of 0. mossambiws showing normal liver structure with hepatocytes (H). (H & E >< 40)

Photomicrograph of the liver of 0. mossambicus exposed for 7 days at 0.6591) ethanol showing ceroid pigmentation (CP) as well as focal area of necrosis (FN).

(H &E >< 40)

Photomicrograph of the liver of 0. mossambicus exposed for 7 days at 0.65gll ethanol showing fatty changes (FC). (H & E >< 40)

Photomicrograph of the liver of 0. mossambicus exposed for 7 days at 1.3g(l ethanol showing elongated biliary proliferation (EBP). (H & E >< 40)

Photomicrograph of the liver of 0. mossambicus exposed for 7 days at 2.6gll ethanol showing hepatic cord disruption (H00). llllknotic nuclei (PN), extensive proliferation of the biliary epithelium (EPBE) and ceroid pigmentation (CP).

(H & E >< 40)

Biochemical effects of ethanol on oreochromis mossambicus (Peters)