TOXICANT INDUCED ANTIOXIDANT ACTIVITY IN OREOCHROMIS MOSSAMBICUS (Peters)

340 Pages·2010·11.41 MB·English

TOXICANT INDUCED ANTIOXIDANT ACTIVITY
IN OREOCHROMIS MOSSAMBICUS (Peters)

Thesis submitted to the
Cochin University of Science and Technology
In partial fulfilment of the requirements for the degree of
Doctor of Philosophy
in Marine Biology
Under the Faculty of Marine Science

By
KESAVAN K.

DEPARTMENT OF MARINE BIOLOGY, MICROBIOLOGY AND BIOCHEMISTRY
SCHOOL OF MARINE SCIENCES
COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
KOCHI – 682 016

MARCH 2010
This is to certify that the thesis entitled “TOXICANT INDUCED
ANTIOXIDANT ACTIVITY IN OREOCHROMIS MOSSAMBICUS
(Peters)” is an authentic record of the research work carried out by
Sri. Kesavan. K, under my scientific supervision and guidance in the
Department of Marine Biology, Microbiology and Biochemistry, School of
Marine Sciences, Cochin University of Science and Technology, in partial
fulfillment of the requirements for the degree of Doctor of Philosophy in
Marine Biology of Cochin University of Science and Technology and that no
part of the thesis has been presented before for the award of any other degree,
diploma or associateship in any University.

Prof. Dr. N. RAVINDRANATHA MENON
Hon. Director and Emeritus Professor
Centre for Integrated Management of Coastal Zones
School of Marine Sciences
Cochin University of Science and Technology
Kochi - 682016
March 2010.
Kochi – 682 016.
Declaration

I hereby declare that the thesis entitled “TOXICANT INDUCED
ANTIOXIDANT ACTIVITY IN OREOCHROMIS MOSSAMBICUS
(Peters)” is a genuine record of the research work done by me under the
scientific guidance and supervision of Prof. Dr. N. Ravindranatha
Menon, Hon. Director and Emeritus Professor, Centre for Integrated
Management of Coastal Zones, School of Marine Sciences, Cochin
University of Science and Technology, Kochi and that this has not
previously formed the basis for the award of any degree, diploma or
associateship in any University.

KESAVAN K.
Kochi – 16
March, 2010.
Acknowledgements

I wish to convey my ardent and humble sense of indebtedness to my Supervising
Guide Prof. Dr. N.R. Menon, Emeritus Professor and Hon. Director, C- IMCOZ,
School of Marine Sciences, Cochin University of Science and Technology for his untiring
guidance, invaluable advices, intellectual contributions, critical suggestions and
constant encouragement all through the tenure of my research work and preparation of
the manuscript. Besides, the consideration and affection he has showered upon me
during the course of my work is gratefully acknowledged; deep from my inner heart.
I record my unfeigned obligations to Dr. N. Chandramohanakumar, Professor and
Head, Department of Chemical Oceanography, School of Marine Sciences, CUSAT, for
his whole hearted support during the concluding phase of my research work.
I am thankful to Prof. Dr. K. Mohankumar, former Dean of Faculty of Marine
Sciences and Prof. Dr. H.S. Ram Mohan, Director and Dean, School of Marine Sciences
for their immense support during the tenure of my studies.
I am grateful to Prof. Dr. Aneykutty Joseph, Head, Department of Marine
Biology, Microbiology and Biochemistry, CUSAT, since she has contemplated a lot in
the successful completion of my research work.
I sincerely thank Dr. A.V. Saramma, former Head, Department of Marine
Biology, Microbiology and Biochemistry, CUSAT, for her kind considerations especially
when I was suffering from serious infirmity happened halfway between the progresses
of my laboratory works.
I would like to express my whole hearted gratitude to Prof. Dr. Babu Philip,
Department of Marine Biology, Microbiology and Biochemistry, CUSAT, for his timely
suggestions which helped me a lot for making modifications of laboratory experiments
wherever necessary. Thanks are due to Dr. Rosamma Philip, Department of Marine
Biology, Microbiology and Biochemistry, CUSAT for providing advanced
instrumentation facilities. Sincere thanks are also extended to Dr. C. K. Radhakrishnan,
retired Professor of the Department.
I realize the value of unlimited scientific suggestions proposed by Dr. K.C.
George, Senior Scientist, CMFRI, Kochi, in finalizing the histological studies. His
valuable suggestions are thankfully acknowledged.
The sisterly affection and backing imparted by Dr. N. Nandini Menon during the
preliminary strides of this assignment is specially remembered with great fervor. I wish
to sincerely thank Dr. K. Suresh for his invaluable recommendations when I was in the
formative phase of this work.
I am extremely glad to thankfully scribe and remember the precious help and back
up extended by Dr. A. Biju, Principal, MES Asmabi College, P. Vemballur, during the
finishing phases of the documentation.
Sincere help and cooperation provided by the administrative staff of Dept.
Marine Biology, Microbiology and Biochemistry is also acknowledged.
The unfailing help and cooperation offered by my friends; Mr. Harisankar, H.S
and Mr. K.B. Padmakumar are specially acknowledged. Besides, I wish to consign on
record my deepest thanks to my friends; Ms. Anupama Nair and Ms. Bindya Bhargavan
who have extended all possible support during the period of laboratory works. Sincere
supports afforded by my dear friend Mr. K.U. Abdul Jaleel and my colleague Mr. Shibu
A Nair are remembered with adoration. I would like to thank Mr. Anilkumar, P.R, Ms.
Smitha Banu, Ms. Remya Varadarajan and Ms. Soja Louis who have helped me in
some way or other during the tenure of this work.
I wish to express my deep sense of gratitude to University Grants Commission for
providing Junior Research Fellowship during the initial stages of the work and Teacher
Fellowship when it approached its completion. I extend my sincere thanks to the
Principal and Management of MES Asmabi College, P. Vemballur for sanctioning me to
continue the Ph.D programme under the Faculty Improvement Programme of University
Grants Commission.
My deepest thanks are due to my Mother and Father for their love and blessings
throughout the schedule of my research work. The fruitful conclusion of my studies
would not have been a reality without the understanding, endurance, love and sacrifices
from my beloved wife and my little daughter; Varsha K. Namboothiri.
Most of all, the heavenly blessings from the Supreme Power has guarded and
guided me all through the hardships when I was in pursuit of this assignment.
Kesavan. K
PREFACE

The presence of a xenobiotic compound in an aquatic ecosystem
does not, by itself, indicate injurious effects. Connections must be
established between external levels of exposure, internal levels of tissue
contamination and early adverse effects. Many of the hydrophobic organic
compounds and their metabolites, which contaminate aquatic ecosystem,
have yet to be identified and their impact on aquatic life has yet to be
determined. Therefore, the exposure, fate and effects of chemical
contaminants or pollutants in the aquatic ecosystem have been extensively
studied by environmental toxicologists.
Deleterious effects on populations are often difficult to detect in
feral organisms since many of these effects tend to manifest only after
prolonged cycles of ontogeny. Often the damages become conceivable
very late in the ecosystem, thus rendering remedial actions pointless. In an
environmental context, biomarkers offer promise as sensitive indicators
demonstrating that toxicants have entered organisms, have been
distributed in tissues, and are eliciting a toxic effect at critical targets. In
this respect, it is also interesting to study the development and application
of sensitive laboratory bioassays, based upon the responses of biomarkers.
Bioassays offer many advantages for comparing the relative toxicity of
specific chemicals or specific effluents.
Histopathological techniques are a rapid, sensitive, reliable and
comparatively inexpensive tool for the assessment of stress responses to
xenobiotics. Cytological and histopathological alterations provide a direct
record of trace effect. Evaluation of histopathological manifestations
provides insight into the degree of stress, susceptibility and adaptive
capability of the stressed organism. The route that the toxicant takes
during its metabolisation often dictates the choice of organs for examining
the effect of xenobiotics. In this context, gill, liver and the kidney of
teleost are the best suited organ system to analyse both the biochemical
and histopathological aberrations due to xenobiotic stress.
Fish are of special concern because of the properties of aquatic
environment and its relationship with native organisms. An aquatic
environment is characterized by marked spatial and temporal heterogeneity
of its physicochemical parameters and processes. Oreochromis mossambicus
(Peters), the cichlid species known by the common name tilapia, forms the
experimental animal in the present investigation because of its year round
availability, adaptability to varied situations and sensitivity to toxicant
exposures. Antioxidant defense studies on oxidative stress in fish open a
number of research lines aimed at providing greater knowledge of fish
physiology and toxicology. In addition, such studies would provide more
precise information concerning the response of antioxidant defenses in
different species under various circumstances as well as on the regulatory
mechanisms of this response. Such future studies will, no doubt, benefit
aspects related to fish farming and aquaculture practices.

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CONTENTS
Page No
List of Tables
List of Figures
Abbreviations & Symbols
Chapter -1
INTRODUCTION............................................................................01 - 06
Chapter -2
REVIEW OF LITERATURE.............................................................07 - 31
Chapter -3
LETHAL TOXICITY STUDIES ......................................................32 - 41
3.1 Introduction 32
3.2 Material and methods 33
3.2.1 Test animals 33
3.2.2 Laboratory conditioning of test animals 33
3.2.3 Toxicants 34
3.2.3.1 Copper 35
3.2.3.2 Metacid 50 35
3.2.3.3 Malachite green 35
3.2.4 Studies on Lethal Toxicity 36
3.2.4.1 Lethal Toxic Responses to Copper 36
3.2.4.2 Lethal Toxic responses to Metacid 50 37
3.2.4.3 Response to malachite green 37
3.3 Results 38
3.4 Discussion 40

Chapter -4
SUBLETHAL TOXICITY................................................................42 - 162
4.1 Introduction 42
4.1.1 Oxidative Stress and Antioxidant Defense Mechanisms 43
4.1.2 Target Organs 49
4.2 Material and Methods 50
4.2.1 General protocol adopted for Sublethal Toxicity Studies. 50
4.2.2 Assays of Lipid peroxidation, Antioxidant Enzymes
and Estimation of Protein. 52
4.2.2.1 Assay of Lipid peroxidation. 52
4.2.2.2 Assay of Antioxidant Enzymes 52
4.2.2.3 Estimation of Endogenous Antioxidants 55
4.2.3 Scheme Adopted for Histopathology 56
4.2.4 Statistical Analysis 58
4.3 Results 59
4.3.1 Effects of Exposure to Copper 59
4.3.2 Effects of Exposure to Malachite Green 88
4.3.3 Effects of Exposure to Metacid 50 114
4.4 Discussion 138

Chapter -5
EFFECT OF VITAMIN C ON TOXICITY.........................................162 - 269
5.1 Introduction 163
5.2 Material and Methods 164
5.3 Results 165
5.3.1 Effect of Vitamin C on Copper Toxicity. 165
5.3.2 Effect of Vitamin C on Malachite green toxicity. 193
5.3.3 Effect of Vitamin C on Toxicity of Metacid 50 226
5.4 Discussion 258

Chapter -6
SUMMARY AND CONCLUSION ..................................................270 - 274

BIBLIOGRAPHY ..........................................................i - xxvi
APPENDIX

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Abbreviations & Symbols used in the thesis

% percentage
& and
/ per
µg microgram
µl microlitre
µM micro molar
CAT catalase
CDNB 1-chloro-2,4-dinitrobenzene
cm centimeter
Cu copper
e.g example
EDTA ethylene diamine tetra acetic acid
Fig. Figure.
g gram
GSH glutathione (reduced)
HCl hydrochloric acid
hrs. hours
i.e that is
I.U International Units
kg kilogram
l litre
L-1 per litre

M molar
mal. green malachite green