STUDIES ON CYTOCHROME P450 IN SOME AIR BREATHING TELEOST FISH
A Thesis submitted to the University of North Bengal For the Award of Doctor of Philosophy in Zoology
BY Dawa Bhutia
SUPERVISOR Prof. Joydeb Pal, Ph. D.
Department of Zoology University of North Bengal
July, 2017
DECLARATION
I declare that the thesis entitled “Studies on Cytochrome P450 in some air breathing teleost fish” has been prepared by me under the guidance of Prof.
Joydeb Pal, Department of Zoology, University of North Bengal. No part of this thesis has formed the basis for the award of any degree or fellowship previously.
Dawa Bhutia
Department of Zoology
University of North Bengal
Raja Rammohanpur, P.O. N.B.U., Siliguri – 734013.
District – Darjeeling, West Bengal, India.
Date: 18th July, 2017
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CERTIFICATE
I certify that Dawa Bhutia has prepared the thesis entitled “Studies on
Cytochrome P450 in some air breathing teleost fish”, for the award of Ph. D. degree of the University of North Bengal, under my guidance. He has carried out the work at the Department of Zoology, University of North Bengal.
The content of the thesis has not been submitted elsewhere for any degree.
Dr. Joydeb Pal
Former Professor,
Department of Zoology
University of North Bengal
Raja Rammohanpur, P.O. N.B.U., Siliguri – 734013.
District – Darjeeling, West Bengal, India.
E-mail: [email protected]
Date: 18th July, 2017
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Dedicated to my Reverend Beloved Father Late Pasang Bhutia And Mother Late Kanchi Bhutia
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ACKNOWLEDGEMENTS A word of sincere thanks to the following personalities and institutions: . My mentor Prof. Joydeb Pal for his patient supervision, guidance, comments and insight throughout the course of this study. . Teachers of the Department of Zoology, Prof. Ananda Mukhopadhaya, Prof. Tapas Kumar Chaudhuri, Prof. Sudip Barat, Dr. Min Bahadur, Dr. Soumen Bhattacharjee, Dr. Dhiraj Saha and Mr. Tilak Saha, for providing me with encouragement, valuable suggestions and advice in many aspects of my research project. . My Research colleagues, Benoy Kishore Rai, Rujas Yonle, Tapan Sarkar, Ganesh Bahadur Thapa, Ritesh Biswa, Kumar Basnet, Gautam Debnath, Pokhraj Guha, Dr. Mahua Rudra, Dr. Manoj Lama, Dr. Priyankar Dey, Bappaditya Ghosh, Swati Singh, Ruksa Nur, Anjali Prasad, Sangita Khewa, Uttara Dey Bhowmick, Sutanaka Chattaraj and all the other scholars of the Department of Zoology for their cooperation and helping me to develop my idea which has guided me throughout this work and refine my ideas as well. . Department of Zoology, University of North Bengal for providing me with the laboratory facilities. . Mr. Ashis Sarkar and Mr. Simphu Sarkar for providing me with the fish samples along with the fisherman for helping me catch the fish samples from the field. . Non-teaching staffs of the Department of Zoology, library Staffs of the University of North Bengal, for their willing cooperation and inexhaustible patience in searching the chemicals, articles and making them available to me. . UGC (University Grants Commission). Without this funding I would have not had the opportunity to pursue my studies.
A personal word of appreciation and gratitude: . My beloved parents, Late Pasang Bhutia and Late Kanchi Bhutia for their support, encouragement and understanding. Without their sacrifice and support, I would not have been able to succeed in my academic career. . My friend’s and dada’s Anand Lama, Zigmee Khaling, Kuldeep Rai, Samten Tamang, Raju Subba, Anand Pariyar, Dhiraj Brahmin whose friendship and humour kept me sane throughout my studies. . And finally to my wife, Anamika Moktan, for her love, understanding and support when I needed it the most.
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ABSTRACT
Environmental contamination by pesticide is a worldwide problem and affects a number of non-target organisms other than the target organism. Pesticides are widely used throughout the world to protect the crops from the damage caused by the pests. The excessively used pesticides eventually get accumulated in the aquatic system through surface run off and affect the non-target organisms which are very much sensitive to the pesticide. Cytochrome P450 (CYP 450) is one of the most important phase I enzyme of biotransformation and is considered as an important biomarker for its sensitiveness towards a wide range of xenobiotics. CYP 450 is a very large and diverse superfamily of heme proteins found in all domains of life. Among the diverse family of CYP 450, family 1-3 and to some extent family 4 are of prime importance for the oxidation of xenobiotic compounds, including pesticides. Except for CYP 4501A (CYP1A), information on other CYP 450 families in fish is limited as compared to mammals.
The general aim of the present study was to examine CYP 450 of an economically important air breathing freshwater fish, Channa punctatus, Heteropneustes fossilis and Clarias batrachus. Microsomal isolation was performed based on conventional ultra-centrifugation at 1,00,000 g for 60 minutes. The carbon monoxide reduced CYP 450 showed the spectrum of absorption maximum at 450 nm in liver microsome. The CO-difference spectra of dithionite reduced liver microsomes of C. punctatus, H. fossilis and C. batrachus were studied at 1 minute interval over a period of 5 minutes. The maximum absorbance was noticed at 1 to 2 minutes and then a gradual decrease in absorbance was seen with the increase in time interval.
In the present study, CYP 450 content, EROD (ethoxyresorufin O-deethylase) activity, N, N- dimethylaniline demethylase (N,N-DMA) activity, aniline hydroxylase (AH) activity and erythromycin N-demethylase (ERND) activity have been reported from liver microsome of fish, H. fossilis to indicate the presence of multiple forms of CYP 450 in fish. Induction of specific enzyme activity with a significant difference upon treatment with specific inducers such as naphthalene (CYP1A), acetone (CYP2E1) and deflazacort (CYP3A4) was found except for phenobarbitone (CYP2B) which was found to be unresponsive. The results clearly showed that the specific inducers were responsible for the induction of specific CYP 450 isoforms and the presence of multiple CYP 450 isoforms in fish, H. fossilis. The results also indicated that CYP1A mediates EROD (ethoxyresorufin O-deethylase) activity, CYP2B mediates N, N-dimethylaniline demethylase (N,N-DMA) activity, CYP2E1 mediates aniline
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hydroxylase (AH) activity and CYP3A4 mediates erythromycin N-demethylase (ERND) activity.
β-naphthoflavone (β-NF), a proven inducer of CYP1A was also administered in all the 3 fish species, C. punctatus, H. fossilis and C. batrachus and CYP 450 in hepatic tissue was examined. β-naphthoflavone treatment significantly induced CYP 450 content and EROD activity in all the 3 fish species, C. punctatus, H. fossilis and C. batrachus along with induction of erythromycin N-demethylase activity.
The effects of 3 different classes of pesticide viz. pyrethroid (cypermethrin), organophosphate (ethion) and organochlorine (dicofol) on hepatic CYP 450 isoforms of an economically important freshwater air breathing fish, C. punctatus, H. fossilis and C. batrachus was also studied. The LC50 value of all the pesticide was determined and the fish treated with 1/3 sub-lethal concentration of calculated LC50 value for a period of 5, 10 and 15 days. The LC50 values in C. punctatus, H. fossilis and C. batrachus towards cypermethrin were calculated to be 19.9, 3.7 and 5.6 µg/L respectively, towards ethion 43.9, 54.8 and 48.7 µg/L respectively and towards dicofol 45.8, 36.1 and 51.8 µg/L respectively. Apart from the enzyme activities, liver somatic index (LSI), protein content and CYP 450 content were also examined which revealed a significantly higher value in the pesticide treated fish compared to their respective control group. The 3 fish species also revealed marked interspecies differences in all of the parameters studied when comparing their basal level activities.
Upon treatment with pesticide cypermethrin, CYP1A, CYP2B and CYP2E1 activity were significantly induced. These 3 isoforms were found to be the most important CYP 450, catalyzing cypermethrin metabolism, while CYP3A4 activity was significantly down regulated and did not catalyze the metabolism of cypermethrin in all the 3 fish species studied, C. punctatus, H. fossilis and C. batrachus when compared with their respective control group. However, C. punctatus displayed a negligible response in CYP2E1 activity. Upon treatment with ethion, CYP1A, CYP2E1 and CYP3A4 activity were induced and significantly catalyzed the metabolism of the pesticide in fish, C. punctatus. In general, CYP1A and CYP3A4 played a crucial and CYP2E1 a lesser role in the metabolism of ethion whereas CYP2B had no role in the metabolism of ethion. In H. fossilis, all the activities mediated by CYP1A, CYP2B, CYP2E1 and CYP3A4 were induced and significantly catalyzed the metabolism of ethion. However, CYP1A, CYP2B and CYP3A4 catalyzed ethion in C. batrachus while CYP2E1 had no role in the metabolism. Similarly, CYP1A and CYP3A4 activity were induced and catalyzed the metabolism of dicofol in all the 3 fish
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species with a significant difference when compared with their respective control group. In C. punctatus, CYP2B also played a role in the metabolism of dicofol while CYP2E1 was moderately inhibited. On the other hand, in H. fossilis all the activities mediated by CYP1A, CYP2B, CYP2E1 and CYP3A4 catalyze the metabolism of dicofol similar to that of ethion, whereas in C. batrachus, CYP2B and 2E1 played no role in the metabolism of dicofol. The results showed that in C. punctatus, H. fossilis and C. batrachus, all the CYP 450 isoforms viz. CYP1A, CYP2B, CYP2E1 and CYP3A4 play a role in biotransforming the pesticide and thereby ensure its survival.
In vitro kinetics of hepatic phase I biotransformation reactions in 3 air breathing teleost fish, C. punctatus, H. fossilis and C. batrachus were also examined. The fishes were treated with 1/3 sub-lethal concentration of the pesticide cypermethrin, ethion and dicofol for a period of 15 days and enzyme kinetics were studied against the control. Maximal velocity (Vmax), binding affinity (Km) and catalytic efficiency (Vmax/Km) were used as endpoints for comparison. All the 3 fish species displayed marked differences in maximal velocity (Vmax), binding affinity (Km) and catalytic efficiency (Vmax/Km). Intrinsic clearance expressed as Vmax/Km ratio for 7-ethoxyresorufin was seen to be highest followed by erythromycin, N, N-dimethylaniline and aniline in all the fish species studied. The results clearly demonstrated the vital role played by CYP1A and CYP3A4 isoforms and are subsequently considered to be important isoforms in metabolizing the xenobiotics. Marked interspecies differences in the expression and metabolism of pesticides by CYP 450 were also clearly seen.
In the present study, fishes, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus were collected from 9 different sites in Terai region of North Bengal though all the species were not present in every site. All the fish species displayed a variable range of CYP 450 content and CYP 450 mediated enzyme activities and were attributed to the exposure of fish to different physiological and environmental stress.
The microsomal fraction was also subjected to SDS-PAGE and heme staining by TMPD
(N,N,N’N’ -tetramethyl-p-phenylenediamine) which identified the presence of cytochrome P450 in all 3 fish species with a molecular weight ranging from 50-56 kDa. Microsomes from control and pesticide treated fish liver were separated and the bands were visualized in molecular mass ~54 kDa for C. punctatus, ~53 kDa for H. fossilis and ~51 kDa for C. batrachus with increased intensity in all the pesticide treated populations.
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The CYP1A genomic sequence for all 3 fish species, C. punctatus (2511 bp), H. fossilis (534 bp) and C. batrachus (509 bp) were obtained and successfully submitted in the GenBank. All the gene sequences were deposited in the GenBank/NCBI data bank except the gene of H. fossilis which was deposited in GenBank/EMBL with the accession numbers KP282054, LN736019 and KP336485 for C. punctatus, H. fossilis and C. batrachus, respectively.
The phylogenetic tree based on the amino acid sequences clearly showed C. punctatus and C. maculata CYP1A and H. fossilis and C. batrachus CYP1A to be more closely related to each other than CYP1A subfamily of other fish employed in the current study. H. fossilis and C. batrachus CYP1A was more closely related with Peltobagrus fulvidraco (yellow catfish) another member of the order Siluriformes. Comparing the orders, the order Siluriformes (Ancistrus sp., Peltobagrus fulvidraco, H. fossilis and C. batrachus) was closely related to order Cypriniformes (Catla catla and Cyprinus carpio). The order Perciformes (Scup, Nile tilapia, Channa maculate and Channa punctatus) was closely related to Salmoniformes (Rainbow trout).
The present study clearly established the efficacy of CYP 450 as an important biomarker for monitoring the aquatic pollution. Of all the CYP 450 isoforms (CYP1A, CYP2B, CYP2E1 and CYP3A4), CYP1A was the most responsive isoform towards pesticide metabolism and in accordance with various other studies proved itself to be one of the most reputable biomarker of CYP 450 family.
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PREFACE
Pesticides and agriculture are closely intertwined and immediate halt in the usage of the former would practically be impossible. The use of pesticides is necessary to combat the loss caused by the pests and the areas of Darjeeling, Terai and Dooars in North Bengal are frequently contaminated with pesticides due to vast stretches of tea plantations in the region. Besides the target organisms, a large number of non-target organisms are also affected and in turn, contaminates both the terrestrial and aquatic environment. The protection of aquatic ecosystems against adverse effects of anthropogenic chemicals is of great concern because of its high economic, genetic and recreational value.
In aquatic toxicology, most research on potential biochemical markers has been conducted on fish which have been suggested as indicators of chemical exposure and play a significant role in the monitoring of water pollution because of its high sensitivity to changes in the aquatic environment. Cytochrome P450 (CYP 450) with its broad substrate specificity is one of the most important phase I enzyme of biotransformation which has been widely studied throughout the world as a biomarker of environmental contamination and have been reported from different species of organisms including bacteria, fungi, plants, insects, molluscs, fishes and mammalian systems.
In India, very few research have been carried out regarding cytochrome P450 as a detoxifying enzyme or a biomarker to assess the contamination of the aquatic environment by domestic wastes, industrial by-products, pesticides, etc.
Therefore, in the present study, cytochrome P450 and its isoforms mainly of the family 1-3 (CYP1A, CYP2B, CYP2E1 and CYP3A4) responsible for the metabolism of xenobiotics in 3 fish species, Channa punctatus, Heteropneustes fossilis and Clarias batrachus have been examined. The water bodies of North Bengal region are contaminated through indiscriminate use of chemical/synthetic pesticides and their run-offs from the agricultural fields and tea gardens. Fishes are highly sensitive to such unwanted changes of the environment. Variations and induction of specific forms of cytochrome P450 can contribute to characterize specific genetic responses that not only may provide a mechanistic understanding of the fish to a chemical stress but may also be exploited as a potential biomarker for environmental hazards and provide evidence of the state of pollution in a comprehensive way.
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TABLE OF CONTENTS Page ACKNOWLEDGEMENTS IV ABSTRACT V PREFACE IX TABLE OF CONTENTS X LIST OF TABLES XIV LIST OF FIGURES XVIII ABBREVIATIONS XXI 1. INTRODUCTION ------1 2. OBJECTIVES ------4 3. LITERATURE REVIEW ------5 3.1. Pesticide in aquatic ecosystems ------5 3.2. Biological response of aquatic organism to environmental contaminants ------6 3.3. Xenobiotic biotransformation ------7 3.4. Biomarkers ------8 3.4.1. Bioindicators ------9 3.4.2. Biochemical markers ------9 3.5. Cytochrome P450 (CYP 450) ------10 3.5.1. Cytochrome P450 characterization ------10 3.5.2. Cytochrome P450 distribution and function------12 3.5.3. Nomenclature ------13 3.5.3.1. CYP1 family ------14 3.5.3.2. CYP2 family ------15 3.5.3.3. CYP3 family ------16 3.5.3.4. CYP4 Family ------17 3.6. Mechanisms of induction of cytochrome P450 in fish ------17 3.7. Role of cytochrome P450 in fish ------17 3.8. Cytochrome P450 in air breathing teleost fish ------23
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3.9. Cytochrome P450 and pesticide interaction ------24 4. MATERIALS AND METHODS ------28 4.1. Experimental animal ------28 4.2. Collection and maintenance of healthy fish in the laboratory ------29 4.3. Collection of healthy fish from the field ------29 4.3.1. Sample collection ------29 4.3.2. Sampling stations/sites ------29 4.4. Treatment of the fish with mammalian specific inducers of CYP 450 isoforms -- 32 4.5. Treatment of the fish with CYP1A specific inducer β-naphthoflavone ------32
4.6. Determination of LC50 value of pesticides------32 4.7. Treatment of the fish with 1/3 sub-lethal concentration of the pesticides, Cypermethrin 10% EC (RIPCORD), Ethion 50 % EC and Dicofol 18.5 % EC (COLONEL S) ------33 4.8. Surgery ------34 4.9. Liver Somatic Index (LSI) ------34 4.10. Preparation of microsomes ------34 4.11. Protein estimation ------36 4.12. Spectral analysis and quantification of cytochrome P-450 ------37 4.13. Enzyme aassay ------39 4.13.1. Determination of EROD (ethoxyresorufin-O-deethylase) activity ------39 4.13.2. Determination of N,N-dimethylaniline demethylase (N,N-DMA) activity-- 40 4.13.3. Determination of aniline hydroxylase (AH) activity ------41 4.13.4. Determination of erythromycin N-demethylase (ERND) activity ------42 4.14. Enzyme kinetics ------43 4.14.1. Specific experimental parameters for each substrate ------44 4.14.1.1. EROD ------44 4.14.1.2. N,N-dimethylaniline demethylation ------44 4.14.1.3. Aniline hydroxylation ------44 4.14.1.4. Erythromycin N-demethylation ------45
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4.15. Gel electrophoresis ------45 4.15.1. Heme staining ------46 4.16. Isolation of genomic DNA ------47 4.17. Amplification of CYP1A isoform from genomic DNA using PCR ------49 4.18. Phylogenetic analysis ------50 4.19. Statistical analysis ------51 5. RESULTS ------52 5.1. Spectral analysis of liver microsomes of C. punctatus, H. fossilis and C. batrachus ------52 5.2. Multiple forms of cytochrome P450 family in liver of Heteropneustes fossilis treated with mammalian specific inducers inducers of cytochrome P450 ------56 5.3. Treatment of the fish, C. punctatus, H. fossilis and C. batrachus with CYP1A specific inducer β-naphthoflavone ------59
5.4. Assessment of LC50 value in fish C. punctatus, H. fossilis and C. batrachus ------63 5.5. Assessment of cytochrome P450 in fish, C. punctatus, H. fossilis and C. batrachus administered with cypermethrin ------65 5.6. Assessment of cytochrome P450 in fish, C. punctatus, H. fossilis and C. batrachus administered with ethion ------72 5.7. Assessment of cytochrome P450 in fish, C. punctatus, H. fossilis and C. batrachus administered with dicofol ------79 5.8. Assessment of in vitro kinetics of hepatic phase I biotransformation reactions in 3 air breathing teleost fish, C. punctatus, H. fossilis and C. batrachus ------86 5.8.1. 7-Ethoxyresorufin O-deethylation (EROD) kinetics ------86 5.8.2. N,N-Dimethylaniline demethylation (N,N-DMA) kinetics ------89 5.8.3. Aniline hydroxylation (AH) kinetics ------92 5.8.4. Erythromycin N-demethylation (ERND) kinetics ------95
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5.9. Assessment of CYP450 from hepatic tissue in fish collected from local areas near the tea plantation in Chopra (Balarampur and Katagaon), Kalaghaj (Dangra Dangri and Ariagoan), Sonapur, Magurmari, Lotchka, Changa and Deomani ------99 5.10. SDS-PAGE and Heme staining ------108 5.11. Nucleotide sequence analysis of genomic CYP1A and phylogenetic analysis of 3 fish species, C. punctatus, H. fossilis and C. batrachus ------121 5.11.1. Comparison of amino acid sequences ------122 5.11.2. Phylogenetic analysis ------122 6. DISCUSSION ------127 7. BIBLIOGRAPHY ------156 8. INDEX ------188 9. APPENDICES ------191
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LIST OF TABLES Page
Table 4.1. Total CYP450 assay mixture for a final volume of 4mL. 38
Table 4.2. Reaction mixture and assay protocol for preparing 5% stacking gel and 10% resolving gel. 46 Table 4.3. Oligonucleotide primers used in PCR amplification of genomic CYP1A. 50 Table 5.1. CYP 450 content and absorption maxima of control fish, H. fossilis and fish administered with different inducers. 58 Table 5.2. EROD, N, N-dimethylaniline demethylase (N,N-DMA), aniline hydroxylase (AH) and erythromycin N-demethylase (ERND) activities in H. fossilis treated with different inducers. 58 Table. 5.3. Liver somatic index (LSI) and protein content of C. punctatus, H. fossilis and C. batrachus administered with β-naphthoflavone. 59 Table 5.4. CYP 450 content, EROD, N, N-dimethylaniline demethylase (N,N- DMA), aniline hydroxylase (AH) and erythromycin N-demethylase (ERND) activities of C. punctatus, H. fossilis and C. batrachus administered with β-naphthoflavone. 62
Table 5.5. Cumulative mortalities and LC50 values of the pesticide, cypermethrin for 3 different air breathing fish, C. punctatus, H. fossilis and C. batrachus. 64
Table 5.6. Cumulative mortalities and LC50 values of the pesticide, ethion for 3 different air breathing fish, C. punctatus, H. fossilis and C. batrachus. 64
Table 5.7. Cumulative mortalities and LC50 values of the pesticide, dicofol for 3 different air breathing fish, C. punctatus, H. fossilis and C. batrachus. 64 Table 5.8. Liver somatic index (LSI) of C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin. 68 Table 5.9. Microsomal protein content of C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin. 69 Table 5.10. CYP 450 content of C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin. 69
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Table 5.11. EROD (ethoxyresorufin O-deethylase) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin. 70 Table 5.12. N, N-dimethylaniline demethylase (N,N-DMA) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin. 70 Table 5.13. Aniline hydroxylase (AH) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin. 71 Table 5.14. Erythromycin N-demethylase (ERND) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin. 71 Table 5.15. Liver somatic index (LSI) of C. punctatus, H. fossilis and C. batrachus exposed to ethion. 75 Table 5.16. Microsomal protein content of C. punctatus, H. fossilis and C. batrachus exposed to ethion. 76 Table 5.17. CYP 450 content of C. punctatus, H. fossilis and C. batrachus exposed to ethion. 76 Table 5.18. EROD (ethoxyresorufin O-deethylase) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to ethion. 77 Table 5.19. N, N-dimethylaniline demethylase (N,N-DMA) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to ethion. 77 Table 5.20. Aniline hydroxylase (AH) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to ethion. 78 Table 5.21. Erythromycin N-demethylase (ERND) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to ethion. 78 Table 5.22. Liver somatic index (LSI) of C. punctatus, H. fossilis and C. batrachus exposed to dicofol. 82 Table 5.23. Microsomal protein content of C. punctatus, H. fossilis and C. batrachus exposed to dicofol. 83 Table 5.24. CYP 450 content of C. punctatus, H. fossilis and C. batrachus exposed to dicofol. 83
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Table 5.25. EROD (ethoxyresorufin O-deethylase) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to dicofol. 84 Table 5.26. N, N-dimethylaniline demethylase (N,N-DMA) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to dicofol. 84 Table 5.27. Aniline hydroxylase (AH) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to dicofol. 85 Table 5.28. Erythromycin N-demethylase (ERND) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to dicofol. 85 Table 5.29 Michaelis-Menten parameters for 7-ethoxyresorufin O-deethylase activity using 7-ethoxyresorufin as a substrate in hepatic microsomal suspensions prepared from control and treated fish. 88 Table 5.30. Comparison of microsomal intrinsic clearance values analyzed by
enzyme kinetic method: CIint = Vmax/Km for selected substrate 7- ethoxyresorufin between control and treated C. punctatus, H. fossilis and C. batrachus. 88 Table 5.31. Michaelis-Menten parameters for N,N-DMA demethylase activity using N, N- dimethylaniline as a substrate in hepatic microsomal suspensions prepared from control and treated fish. 91 Table 5.32. Comparison of microsomal intrinsic clearance values analyzed by
enzyme kinetic method: CIint = Vmax/Km for selected substrate N, N- dimethylaniline between control and treated C. punctatus, H. fossilis and C. batrachus. 91 Table 5.33. Michaelis-Menten parameters for aniline hydroxylase activity using aniline as a substrate in hepatic microsomal suspensions prepared from control and treated fish. 94 Table 5.34. Comparison of microsomal intrinsic clearance values analyzed by
enzyme kinetic method: CIint = Vmax/Km for selected substrate aniline between control and treated C. punctatus, H. fossilis and C. batrachus. 94
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Table 5.35. Michaelis-Menten parameters for erythromycin N-demethylase activity using erythromycin as substrate in hepatic microsomal suspensions 97 prepared from control and treated fish. Table 5.36. Comparison of microsomal intrinsic clearance values analyzed by
enzyme kinetic method: CIint = Vmax/Km for selected substrate erythromycin between control and treated C. punctatus, H. fossilis and C. batrachus. 97 Table 5.37. Liver somatic index (LSI) in air breathing teleost fish, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus brought from different sites. 105 Table 5.38. CYP 450 content in air breathing teleost fish, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus brought from different sites. 105 Table 5.39. EROD activities in air breathing teleost fish, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus brought from different sites. 106 Table 5.40. N, N-dimethylaniline demethylase (N,N-DMA) activities in air breathing teleost fish, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus brought from different sites. 106 Table 5.41. Aniline hydroxylase (AH) activities in air breathing teleost fish, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus brought from different sites. 107 Table 5.42. Erythromycin N-demethylase (ERND) activities in air breathing teleost fish, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus brought from different sites. 107 Table 5.43. Comparison of CYP1A exons and introns length (bp). 125 Table 5.44. Percent identities of deduced amino acid sequences of fish CYP1A gene subfamilies. 125
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LIST OF FIGURES Page Figure 4.1 Channa punctatus. 28
Figure 4.2. Heteropneustes fossilis. 28
Figure 4.3. Clarias batrachus. 29
Figure 4.4. Map and sampling stations in Terai region of North Bengal. 30 Figure 4.5. Sampling sites in the terai region of North Bengal. 31
Figure 4.6. Pesticide formulations: (a) Cypermethrin, (b) Ethion, and (c) Dicofol. 33
Figure 4.7. (a) Dissection of fish, and (b) Dissected liver. 35
Figure 4.8. Schematic representation for the preparation of microsomes. 36
Figure 4.9. A carbon monoxide preparing apparatus. 38 Figure 4.10. Chemical structures of substrates 7-ethoxyresorufin, aniline, N, N- dimethylaniline and erythromycin. 39 Figure 4.11. Diagram of reaction speed and Michaelis-Menten kinetics showing the increase in reaction velocity (V) with increase in substrate concentration ([S]). 43 Figure 4.12. Schematic representation for isolation of DNA from liver tissue. 48 Figure 5.1. Carbon monoxide difference spectra of dithionite reduced liver microsomes of (a) control, and (b) experimental C. punctatus at 1 min interval over a period of 5 min. 53 Figure 5.2. Carbon monoxide difference spectra of dithionite reduced liver microsomes of (a) control, and (b) experimental H. fossilis at 1 min interval over a period of 5 min. 54 Figure 5.3. Carbon monoxide difference spectra of dithionite reduced liver microsomes of (a) control, and (b) experimental C. batrachus at 1 min interval over a period of 5 min. 55 Figure 5.4. Carbon monoxide difference spectra of dithionite reduced liver microsomes of (a) control Heteropneustes fossilis and fish treated with different inducers (b) naphthalene, (c) phenobarbitone, (d) acetone, and (e) deflazacort. 57
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Figure 5.5. Michaelis-Menten plots for EROD, N,N- DMA, AH and ERND activities obtained from in vitro metabolism by CYP450 system: (a) C. punctatus, (b) H. fossilis, and (c) C.batrachus. 98 Figure. 5.6. Pesticide bottles and packets found near the pond sites from where fish were caught: (a) Balarampur, Chopra (b) Katagoan, Chopra and (c) Ariagoan, Kalaghaj. 99 Figure 5.7a. Differentially stained SDS-PAGE protein gels showing the analysis of CYP 450 enzymes from hepatic microsomes in C. punctatus. 109 Figure 5.7b. Differentially stained SDS-PAGE protein gels showing the analysis of CYP 450 enzymes from hepatic microsomes in H. fossilis. 110 Figure 5.7c. Differentially stained SDS-PAGE protein gels showing the analysis of CYP 450 enzymes from hepatic microsomes in C. batrachus. 111 Figure 5.8a. 10% SDS-PAGE of the microsomal fraction of liver of cypermethrin treated and control fish, C. punctatus stained with 0.025% CBB. 112 Figure 5.8b. 10% SDS-PAGE of the microsomal fraction of liver of ethion treated and control fish, C. punctatus stained with 0.025% CBB. 113 Figure 5.8c. 10% SDS-PAGE of the microsomal fraction of liver of dicofol treated and control fish, C. punctatus stained with 0.025% CBB. 114 Figure 5.9a. 10% SDS-PAGE of the microsomal fraction of liver of cypermethrin treated and control fish, H. fossilis stained with 0.025% CBB. 115 Figure 5.9b. 10% SDS-PAGE of the microsomal fraction of liver of ethion treated and control fish, H. fossilis stained with 0.025% CBB. 116 Figure 5.9c. 10% SDS-PAGE of the microsomal fraction of liver of dicofol treated and control fish, H. fossilis stained with 0.025% CBB. 117 Figure 5.10a. 10% SDS-PAGE of the microsomal fraction of liver of cypermethrin treated and control fish, C. batrachus stained with 0.025% CBB. 118 Figure 5.10b. 10% SDS-PAGE of the microsomal fraction of liver of ethion treated and control fish, C. batrachus stained with 0.025% CBB. 119
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Figure 5.10c. 10% SDS-PAGE of the microsomal fraction of liver of dicofol treated and control fish, C. batrachus stained with 0.025% CBB. 120 Figure 5.11. Nucleotide sequences (2511 bp) of C. punctatus genomic cytochrome CYP1A. 123 Figure 5.12. Nucleotide sequences (534 bp) of H. fossilis genomic cytochrome CYP1A. 124 Figure 5.13. Nucleotide sequences (509 bp) of C. batrachus genomic cytochrome CYP1A. 124 Figure 5.14. Phylogenetic tree of CYP1A genes constructed by the neighbour joining method using the amino acid sequences. 126
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ABBREVIATIONS
AH Aniline hydroxylase AhR Aryl hydrocarbon receptor APS Ammonium persulfate BaP Benzo-a-pyrene β-NF β- naphthoflavone BSA Bovine serum albumin CAR Constitutive androstane receptor CBB Coomassie brilliant blue CYP 450 Cytochrome P450 DDT Dichloro Diphenyl Trichloro Ethane EDTA Ethylenediaminetetra acetic acid 7-ER 7-ethoxyresorufin ERND Erythromycin N-demethylase EROD Ethoxyresorufin O-deethylase Fig. Figure FMO Flavin-containing monooxygenase gm Gram i.p. Intraperitoneal kDa Kilodalton LSI Liver somatic index 3-MC Methylcholantrene M Molar MFO Mixed function oxidase mg/L Milligram per liter µg/L Microgram per liter min Minute µl Microliter ml Milliliter
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µM Micromolar mM Milimolar N,N-DMA N,N-dimethylaniline demethylase NPAH Nitrated polyaromatic hydrocarbons nm Nanometer O.D Optical density p-aminophenol Para-aminophenol PAH Polycyclic aromatic hydrocarbons PCB Polychlorinated biphenyls PCDD Polychlorinated dibenzodioxins PCDF Polychlorinated dibenzofurans ppb Parts per billion PSMO Poly-substrate monooxygenase PXR Pregnane X receptor SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis TCA Tricarboxylic acid TEMED N,N,N′,N′-Tetramethylethane-1,2-diamine TMPD N,N,N’N’ -Tetramethyl-p-phenylenediamine
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1. INTRODUCTION
Pesticides and agriculture are closely intertwined and immediate halt in the usage of the former would practically be impossible. Regardless of the increasing public concern about the negative effects of pesticides on animal health, it continues to be used worldwide. As a result contamination of water resources such as ponds, lakes, rivers, lagoons and the oceans along with the organisms dwelling therein have been contaminated with persistent pesticides over the years (Tsuda et al., 1999; Ejobi et al., 2007; Adeyemi et al., 2008).
Pesticides have harmful effects on species biodiversity. These compounds have adverse effects either directly or indirectly on the normal life cycles of animals and their habitats. Different species of fish, amphibians, birds and aquatic mammals have been listed as endangered because of chronic exposure to pesticides and other related agrochemicals. Measures can be taken to lower the level of exposure to animals and their habitat. The flow of energy is disturbed when any level of the food chain/web is affected by compounds such as pesticides and other agrochemicals. This can lead to widespread adverse effects causing an imbalance in the ecosystem (Barlas, 1999).
Tea (Camellia sinensis) due to its tremendous export potential is one of the most important cash crops in India. Tea as a plantation crop was introduced in Darjeeling hills in 1852 but with time it has spread over vast stretches of the sub-Himalayan foothills and the adjoining regions of North Bengal covering an area of 1,07,479 hectares in the foothills and plains (Biswa and Mukhopadhyay, 2013). Pest, pathogens and weeds are severe constraints in the productivity and quality of tea. As a result, the tea planters use a broad range of pesticides to overcome these problems for high yield and economic returns. The principal control measures taken to keep the population of major tea pests down is by applying synthetic pesticides, e.g. chlorinated hydrocarbons, organophosphates, carbamates and synthetic pyrethroids. Indiscriminate use of pesticides not only in tea gardens but also in agricultural fields have resulted in serious problems concerning environmental pollution, both terrestrial and aquatic. Because of the known toxicity of pesticides in food products, there is increasing public concern of late regarding the pesticides residue in tea also. Information for pesticide residues in Indian tea ecosystem, specifically in the tea growing soils and adjacent water bodies is very scanty (Bishnu et al., 2009). Recently, Pal et al. (2011) and Singh et al. (2015) have reported the presence of pesticide residues in the water bodies, sediments and tissues of fish in and around the areas of tea garden in North Bengal.
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Natural and man-made foreign compounds (xenobiotics) enter the aquatic ecosystems by various routes, including direct discharge, land overflow, atmospheric deposition and food chain transfer (Livingstone et al., 1992). Natural xenobiotics comprise a broad range including plant products, animal toxins and natural hydrocarbons, whereas the fabrication of man-made xenobiotics (contaminants) increases on a daily basis in quantity and variety. Knowledge of the fate and effects of xenobiotics on biota is imperative in relation to evolutionary and ecological processes, whereas the same for contaminants is decisive for pollution monitoring, impact assessment and environmental management (Livingstone, 1993).
The complete chemical analysis for all possible environmental contaminants in sediments, water, air and from every species and sample of interest is not possible. Studies of entire ecosystems are expensive and do not provide specific information about the toxic levels of pollutants to individual species (Landis and Yu, 1995). Therefore, a true cascade of toxicity tests or bioassays must be performed which can be classified into three groups:
1) single species acute tests that give results in a short period of time
2) single species chronic tests that show results in weeks, months or years, and are used to analyse exposure effects on growth and reproduction, and
3) multispecies tests that allow analysis of the properties of ecological structures (Heliovaara and Rauno, 1993). These three kinds of tests are used to analyse effects of pesticides in different organisms.
Risk assessment needs reliable scientific information and one such root of information is understanding the metabolic fate and toxicokinetics of compounds. Toxicokinetic refers to the progression of a xenobiotic through the body which is carried out by processes including absorption, distribution, metabolism and excretion. Metabolism is one of the most significant factors that can influence the overall toxic profile of a pesticide. The biotransforming enzymes react with the xenobiotic leading to its metabolism by a two-phase process called phase I and Phase II reactions. In phase I reactions, adding or unmasking the functional groups by oxidation, reduction or hydrolysis give rise to a polar compound and the greatest importance is credited to oxidation enzymes involved in the metabolism of the majority of xenobiotics. This may result in reducing the efficacy of the metabolised xenobiotic, described as bioelimination or generates an active metabolite. The second-phase reaction involves the conjugation of metabolites produced in the first phase or seldom, the original substances also,
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with phase II conjugating enzymes which give rise to more polar compounds and facilitates in easy elimination from the body (Siroka and Drastichova, 2004).
In general, these enzymatic reactions are advantageous as they help in the elimination of foreign compounds but however, sometimes these enzymes may transform a harmless substance into a reactive form, a process known as metabolic activation (Guengerich, 2001). Biotransformation is the enzyme-catalyzed process of chemically modifying drugs and other xenobiotics to increase their water solubility in order to facilitate their elimination from the body. Actually, biotransformation is the process that alters the chemical properties of a xenobiotic away from lipophilicity to hydrophilicity that favours the elimination of xenobiotics in urine and faeces (Parkinson et al., 2010).
Among the phase I biotransforming enzymes, the cytochrome P450 (CYP 450) system lines first because of its high catalytic versatility and the sheer number of xenobiotic it detoxifies or activates to reactive intermediates (Guengerich, 2006; Wickramasinghe, 2017) and as such also regarded as one of the most important biomarker. In the fish, CYP 450 has primarily been examined as a biomarker signifying pollution of the aquatic environment by industrial or agricultural sewage. However, fish may differ from other species in response to xenobiotics (Siroka and Drastichova, 2004). The highest concentration of CYP 450 enzymes concerned with xenobiotic biotransformation is found in liver endoplasmic reticulum (microsomes), but CYP 450 enzymes are present in virtually all the tissues (Lewis, 2001).
The CYP 450 comprises a large multigene family of heme-thiolate proteins which play a significant role in the metabolism of xenobiotics and endobiotics and hepatic clearance is a principal way for elimination of xenobiotics (Pelkonen and Turpeinen, 2007). It is now possible to differentiate metabolic reactions and interactions and to assign which CYP 450 is involved in the metabolism of a certain xenobiotic by different in vitro approaches (Pelkonen and Raunio, 2005; Pelkonen et al., 2005; Hodgson and Rose, 2007).
North Bengal being one of the major tea producing areas in India, the use of pesticides is necessary to combat the pest, but the application of these pesticides may affect the non-target organisms apart from its designated pest. As in India, very few research have been carried out regarding CYP 450 as a detoxifying enzyme or a biomarker to assess the contamination of the aquatic environment. The rationale of the present work was to study the role of hepatic CYP 450 and its dependent enzyme activities in some air breathing fishes as a potential detoxifying enzyme of pesticides.
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2. OBJECTIVES
The overall goal of this study is to define potential metabolic and enzymatic pathways responsible for xenobiotic detoxification in Channa punctatus, Heteropneustes fossilis and Clarias batrachus. The main objectives are:
. To study the impact of insecticides on cytochrome P450 of the hepatic microsomal fraction of air breathing teleost fish in the laboratory.
. To characterize cytochrome P450 activity representing three fundamental generic groups of biotransformation reactions viz. aromatic hydroxylation, N-demethylation and O-deethylation using selected compounds (aniline, N, N-dimethylaniline, erythromycin and 7-ethoxyresorufin) as probes.
. To study the induction, or increased level of cytochrome P450 of treated fish by SDS polyacrylamide gel electrophoresis.
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3. LITERATURE REVIEW
3.1. Pesticide in aquatic ecosystems
The aquatic environment is the ultimate sink for many of those chemicals as the end stage collectors due to direct discharges of domestic and industrial wastes, urban and agricultural runoff, discharges from ships or hydrological and atmospheric process. The aquatic environment covers 70% of the earth, can neutralize some chemical wastes, however, many of the chemicals introduced into the environment during the last 50 years are highly persistent compounds such as polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), pesticides, alkyltin compounds and heavy metals (i.e. Pb, Hg, Cd, etc.). Because of their chemical stability and persistency, they tend to accumulate in the different compartment of the aquatic environment. The result is the exposure of the aquatic organisms to highly toxic chemicals (Havelkova et al., 2007).
Since pesticides are indispensable for agriculture to curtail the damage of crops, a wide range of hypotheses about the effects of pesticide on aquatic ecosystems should be considered, including impacts on ecosystem structure and function and its interactions with the abiotic environment. The main points regarding the ecological consequences of pesticide contamination are: 1) temporary and reversible reductions in plant productivity, 2) damage to the community structure of macrophytes and reduction of its ability to sustain populations of other organisms, 3) adverse effects on survival, growth, and/or reproduction of herbivores and carnivores, including fish (Solomon et al., 1996). In cases where pesticide seems not to be directly toxic to aquatic fauna, indirect effects through alterations of the algal food resource may provide the best explanation for observed faunal population and community responses in natural systems (Denoyelles et al., 1982).
In a developing nation like India where the agricultural sector is vast and occupies a pivotal role in the economy, the nation runs at a high risk of problems related to pesticide contamination. Positive detections of pesticides have been made in water samples of river Kaveri (Rajendran and Subramanian, 1997), sediment samples of lake Kolleru, Andhra Pradesh (Rao and Pillala, 2001), drinking water in Delhi (Mathur et al.,2003) and river water and fish samples of river Ganga around Kolkata (Aktar et al., 2009). Although the trend of pesticide accumulation differs between species and tissues depending on the fat content, yet residues tend to accumulate and its toxicity can be biomagnified (Kannan et al., 1995).
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In a study conducted by Malik et al. (2007), organochlorine pesticide residue, viz. DDT metabolites were detected in fish muscle samples of river Gomti in Uttar Pradesh showing contamination of the river with persistent organochlorines reflecting present or earlier usage of organochlorine pesticides. Kaur et al. (2008) have also reported organochlorine pesticide residue in Indian major carps and exotic carps in Punjab showing inland water contaminated by the use of pesticides in the agricultural fields.
North Bengal is one of the major tea growing areas of India and comprises of vast stretches of tea plantations particularly in the areas of Darjeeling, Terai and Dooars. Bishnu et al. (2009) in their study reported the presence of pesticide residues in tea soil and water bodies of the Dooars region, showing that these plantation areas serve as primary source of contamination of water bodies in this region. Pal et al. (2011) have also reported the presence of pesticide residues in the sediment and water of rivers flowing through the tea gardens from the Terai regions of Darjeeling district. Similarly, Singh et al. (2015) also reported the presence of chlorpyrifos, ethion and dicofol residues in the sediment and water of Deomani river flowing through the tea gardens of Terai region of West Bengal along with the presence of pesticide residues in the muscles of fish Puntius sp. collected from the river.
According to Hall et al. (2001), there are presently 900 pesticide products and 600 active ingredients in the market. Out of the millions of tons of pesticide that are applied annually, however, less than 5% are estimated to reach the target organism and the bulk gets deposited on the soil, water and non-target organisms (Pimental and Levitan, 1986).
3.2. Biological response of aquatic organism to environmental contaminants
Toxic organic contaminants such as PAHs, PCBs and dioxins found in the ecosystem, can be taken up by the organisms through the direct uptake from water by gills or skin (bioconcentration), through the uptake of suspended particles (ingestion) and through the consumption of contaminated food (biomagnification). Those chemicals are highly lipophilic in nature, and can easily enter the cell (Ejobi et al., 2007).
The first stage of the effects of a pollutant on the organisms is the interactions with endogenous molecules. These interactions can be divided into three main groups. In the first group, the contaminants can be sequestrated and then neutralized in the neutral lipid fraction. In the second group, contaminants can bind specifically to some cellular molecules such as receptors, for example, some xenoestrogenic chemicals like DDT, a kind of organochlorine
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pesticides largely used for agricultural purposes, bind to estrogen receptor and show estrogenic effects (Adeyemi et al., 2008). In the third group, contaminants can interact with the biotransformation enzymes. These enzymes either metabolize contaminants into nonreactive hydrophilic compounds which are easily excreted from the body or convert them into reactive metabolites which are more toxic than the parent compounds. All these interactions can result in accumulation of foreign chemicals in the body of organisms and lead to direct or indirect (via its metabolites) toxic effects or excretion of the parent compound and/or its metabolites.
Pollutants which are bioaccumulated in the organism, first cause changes at the molecular and cellular level. This may lead to the adverse effect on the organism which in turn may cause changes in the population and the community level in the years to come (Arinc et al., 2000). As stated above, for example, xenoestrogenic compounds first bind to the estrogen receptor at the cellular level and cause feminization of male species at the individual level and finally, this may threaten the existence of susceptible species at the population even community level (Gimeno et al., 1996). Growing number of toxic organic chemicals in the aquatic environment may cause deleterious effects on populations. For this reason, it is very important to detect the relationship between contaminant level (exposure) and their biological effects before any detrimental effect has been established at higher organization level (Van der Oost et al., 2003).
3.3. Xenobiotic biotransformation
Xenobiotic biotransformation is the process by which lipophilic foreign compounds are metabolized to hydrophilic metabolites that are eliminated directly or after conjugation with endogenous cofactors by renal or biliary excretion. These metabolic enzymes are divided into two groups, Phase I and Phase II enzymes (Oesch et al., 2000).
The Phase I involves reactions giving rise to more polar compounds. This may either result in reducing the effectiveness of the metabolized xenobiotic, also called as bioelimination or produce an active metabolite, also called as bioactivation in terms of both pharmacology and toxicology (Siroka and Drastichova, 2004). Phase I reactions, considered as functionalization reactions, add or uncover functional groups on xenobiotics with the purpose of increasing the polarity or nucleophilicity that create a suitable substrate for Phase II metabolism. Phase I reactions generally takes place by oxidation, reduction or hydrolytic reactions. These
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reactions are mediated primarily by the CYP 450 family of enzymes, but other enzymes (e.g. flavin monooxygenases, dehydrogenases, peroxidases, amine oxidases, xanthine oxidases) also catalyze the oxidation of certain functional groups. In addition to the oxidative reactions, there are different types of hydrolytic reactions catalyzed by enzymes like carboxylesterases and epoxide hydrolases (Hodgson and Goldstein, 2001; Parkinson, 2001).
Phase I products are not usually eliminated rapidly, but undergo a subsequent reaction in which an endogenous substrate such as acetic acid, glucuronic acid, sulfuric acid or an amino acid combines with the newly formed functional group to make a highly polar conjugate which can be easily excreted. Sulfation, glucuronidation and glutathione conjugation are the most prevalent classes of phase II metabolism, which may directly act on the parent compounds, or, as is usually the case, on functional groups added or exposed by phase I reactions (LeBlanc and Dauterman, 2001; Rose and Hodgson, 2004; Zamek-Gliszczynski et al., 2006).
3.4. Biomarkers
A biomarker is a measurable indicator of some biological state or condition. Most chemical analyses for environmental contaminants only establish their presence. Although chemical analyses are able to quantitatively measure a wide range of pollutants, these analyses are unable to reveal potential activity within an organism. Exposure to contaminants may produce increased activity of specific cytochrome P450 enzymes, which makes it a potential biomarker of contaminant exposure. The use of biochemical biomarkers fulfills this purpose (Guengerich, 2001).
Biomarker responses are of two kinds: those that measure only exposure to a pollutant (bio- indicators), and those that measure both exposure and toxic effect of environmental chemicals (biochemical biomarkers) (Peakall, 1992).
Van der Oost et al. (2003), mentioned that special attention ought to be given to interpret the data obtained through the use of a biomarker to differentiate between laboratory and field results, the biological and toxicological relevance of the induction of enzymatic activity, and possible confounding factors not related to the exposure, thereby producing the same effect as the causal factors (e.g., stress by excessive manipulation). Therefore, it is recommended to use several biomarkers and combine them with the quantification of accumulated xenobiotics to determine cause-effect relationships. In fish, physiological (e.g., gonadosomatic index,
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liver somatic index and morphometric parameters), reproductive (e.g., plasmatic vitellogenous level) and molecular (e.g., CYP1A1 activity and AChE inhibition) biomarkers have been frequently used (Orrego et al., 2006).
3.4.1. Bioindicators
A bioindicator is any biological species or group of species whose functions, population or status can reveal the qualitative status of the environment. A bioindicator response provides information on both the presence of a contaminant in the environment and that it has reached the affected tissue or organ in sufficient amounts for a period of time, long enough to cause the observed response (Perry et al., 1998). Freshwater biomonitoring uses organisms as indicators primarily of organic pollution in streams, lakes, ponds and rivers.
According to Manly (1995) and Beeby (2001), the model organisms, also called bioindicators, should be easily identifiable and abundant in the field representing a short life cycle, an accumulator of pollutants with capacity to reflect levels of pollutants and sources of emission, and with a well-known biology, ecology and distribution.
Fish are among the most important members of the aquatic ecosystems and represent about half of the currently known vertebrate species (Bucheli and Fent, 1995). The sudden death of fish may indicate heavy pollution and the effects can be measured in terms of physiological, biochemical or histological responses of the fish organism after exposure to sub-lethal levels of pollutants (Mondon et al., 2001). Furthermore, they provide a vital protein source and, hence, are of crucial importance for the nutrition of mankind. Therefore, the health and protection of these organisms are of pivotal importance. The fish plays an increasingly important role in the monitoring of water pollution as it responds with great sensitivity to changes in the aquatic environment (Saha and Kaviraj, 2003). Among fish, the main species for monitoring programs and toxicological studies are zebrafish, rainbow trout, channel catfish, Japanese medaka, mummichog, Atlantic salmon, scup and Nile tilapia (Arellano- Aguilar et al., 2009).
3.4.2. Biochemical markers
The biochemical marker is any biochemical compound like enzymes, antibody, antigen or hormone that is sufficiently altered and serves as an aid in predicting susceptibility.
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Biochemical markers are a response induced in the tissues by the presence of a specific group of contaminants that have the same mechanism of toxicity. These kinds of markers are measurable responses to the exposure of an organism to xenobiotics. Once a xenobiotic enters into an organism, it binds to specific receptors that may induce cellular processes that have toxic or other adverse effects at the cellular level (Siroka and Drastichova, 2004). Biochemical markers have been used in toxicology, ecotoxicology and pharmacology research, and involve both in vitro and in vivo assays.
One of the most intensively studied biomarkers, in both laboratory and field conditions, is cytochrome P450. Cytochrome P450’s represent an inducible enzyme system, and increased amounts of specific CYP 450 isozymes are observed after treatment of organisms with a variety of environmental contaminants (Scott, 1999). Its levels have been studied as a response of the organism to the presence of pollutants in an aquatic environment. The first knowledge of CYP 450 activity in aquatic organisms was reported in 1972 (Khan et al., 1972). Since then, cytochrome P450 enzymatic activities have been reported in aquatic arthropods, annelids, cnidarians, molluscs, poriferans, platyhelminths, echinoderms and insects (Livingstone, 1985; Chipman et al., 1991; Feyereisen, 1993; James and Boyle, 1998). Payne and Penrose (1975) were among the first to make use of this enzyme system as a biochemical biomarker, reporting elevated monooxygenase activity in fish from petroleum- contaminated sites in the aquatic environment. Payne (1976) suggested that level of monooxygenase activity (benzo-a-pyrene hydroxylase) is the indicator of environmental exposure of aquatic animals to pollutant mixtures. Cytochrome P450 has been used in fish cell lines as a biochemical biomarker of polychlorinated hydrocarbon contamination in sediments and landfill leachates (Fent, 2004) and also to detect contamination of toxic carcinogen pollutants, such as polychlorinated biphenyls (Arinc et al., 2000).
3.5. Cytochrome P450 (CYP 450)
3.5.1. Cytochrome P450 characterization
The name cytochrome P450 originated from its spectral properties before its catalytic function was known. Klingenberg (1958) and Garfinkel (1958) first reported that hepatic microsomes contain a pigment that binds carbon monoxide with unusual visible absorbance maxima of 450nm in its CO-reduced difference spectrum. Omura and Sato (1964) discovered that this pigment is a b-type cytochrome and called it Cytochrome P450. CYP 450 is believed
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to be more primitive than those considered and may even have emerged even before the advent of atmospheric oxygen probably very early in chemical evolution (Wickramasinghe and Villee, 1975).
CYP 450 belongs to one of the most intensively studied enzyme systems and represents a class of hemoproteins present in virtually all lineages of life (Wickramasinghe and Villee, 1976). The presence of a heme-thiolate bond in the enzyme active site is responsible for the various unique spectroscopic and catalytic properties of this class of enzymes (Estabrook and Werringloer, 1996). CYP 450 catalyzes a variety of reactions in structurally diverse compounds that serve as substrates for the enzyme. Endogenous substrates for the enzyme include cholesterol, steroid hormones and fatty acids and exogenous compounds (xenobiotics) such as drugs, food additives, polyaromatic hydrocarbons, pesticides and chemicals used for industry. CYP 450 is also involved in inactivation or activation of therapeutic agents; conversion of chemicals to highly reactive molecules that produces unwanted cellular damage, cell death or mutations; production of steroid hormones; metabolism of fatty acids, and enzyme inhibition or induction that results in drug-drug interactions and adverse effects (Okita and Masters, 1997).
Since the main function of CYP 450 involves the activation of molecular oxygen, the significance of the heme-thiolate linkage was suggested to be intimately involved in oxygen atom transfer (Wickramasinghe and Villee, 1976). CYP 450 is a one-electron acceptor, and the oxygen activation occurs in two steps (Peterson et al., 1977):
2+ 2+ 3+ - 1. O + P450-Fe → P450- Fe O ↔ P450- Fe O 2 2 2
3+ - - 2+ - 3+ = 2. P450- Fe O + e → P450- Fe O ↔ P450- Fe O 2 2 2
The catalytic cycle of CYP 450 mediated reactions involves the transfer of electrons from NADPH via. NADPH cytochrome P450-reductase, a flavoprotein that is present in much lower concentration than CYP 450. The CYP 450 cycle starts with the binding of the substrate. A second step is accomplished when the heme iron is reduced from the ferric form (Fe3+) to the ferrous (Fe2+) by the addition of an electron from cytochrome P450-reductase. Oxygen then binds to the CYP 450 in its ferrous state. The complex is converted to a Fe2+OOH state by the addition of a proton (H+) and a second electron derived from the + 2+ cytochrome P450-reductase or from cytochrome b5. A second H cleaves the Fe OOH and 3+ produces H20 and the (FeO) complex which transfers its oxygen atom to the substrate.
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When the oxidized substrate is released, the CYP 450 molecule is ready for a next cycle (Parkinson, 2001).
CYP 450 enzymes have the ability to react with a multitude of different chemical substrates. This mechanism of reaction includes oxygen activation, splitting and formation of very reactive oxygen. The catalytic power of cytochrome P450 is demonstrated in the following thermodynamic equation (Rein and Jung, 1993):
+ + RH + O + NADPH + H → ROH + H O + NADP 2 2
Where (R) is the substrate and (ROH) is the hydroxylated product.
The overall reaction is called monooxygenation due to the incorporation of only one of the two oxygen atoms into the substrate.
CYP 450 system contains a number of versatile isozymes in that they catalyze many types of reactions, including aliphatic and aromatic hydroxylations and epoxidations, N-oxidations, sulfoxidations, dealkylations, deaminations, dehalogenations, and others (Matsumura, 1985). These isozymes are responsible for the oxidation of different substrates or for different types of oxidative reactions of the same substrate.
CYP 450’s are typical monooxygenases and catalyze oxygenation of various organic substrates using NADPH and molecular oxygen as co-substrates. At the same time, these enzymes are known to be capable of utilizing organic hydroperoxides and hydrogen peroxide as co-substrates in hydroxylation reactions. CYP 450’s can also reduce certain compounds, acting as an electron carrier. CYP 450 dependent monooxygenations are accompanied by the release of superoxide radicals and hydrogen peroxide (Londono, 2005).
3.5.2. Cytochrome P450 distribution and function
CYP 450’s are membrane-bound monooxygenase proteins involved in the biotransformation of many endogenous and exogenous compounds. They have been characterized in both prokaryotic (Urlacher and Schmid, 2002; Lewis and Wiseman, 2005) and eukaryotic species such as plants (Bolwell et al., 1994); aquatic invertebrates (Snyder, 2000); annelids (Rewitz et al., 2004); crustacea (James and Boyle, 1998); fish (Andersson and Forlin, 1992); birds (Walker, 1998); and mammals (Seliskar and Rozman, 2007), with relatively few exceptions,
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like the enteric bacteria Escherichia coli and Salmonella typhimurium, are devoid of CYP 450 genes (Guengerich and Isin, 2008).
These enzymes are found predominantly in the endoplasmic reticulum and mitochondria of liver and adrenal glands, however, they are also distributed throughout the body in diverse areas such as the brain, heart, intestine, kidney, lung and skin (Sarasquete and Segner, 2000). In teleost fish, CYP 450’s are present in liver, intestine, gonads, kidneys, brains, gills, blood, spleen, skin (fins) and eyes (Arellano-Anguillar et al., 2009). Owing to the absence of mitochondrial P450’s in nematodes (C. elegans) and lower eukaryotes, it has been suggested that mitochondrial CYP 450’s did not originate independently but was picked up from the microsome, presumably by mistargeting of the latter gene (Nelson, 1998).
Functionally, CYP 450 enzymes may be broadly divided into those involved primarily in drug and other foreign xenobiotic metabolism, those functioning in steroidogenesis and those participating in other important endogenous functions. Despite a large number of CYP 450’s, the bulk of drug metabolism is catalysed by a relatively small number of CYP 450 enzymes found in families 1, 2, 3 and to a lesser extent by family 4. The substrates for cytochrome P450 include a host of xenobiotics, including substances that occur biologically although are foreign to animals, such as antibiotics and unusual compounds in plants, as well as synthetic organic chemicals, and a variety of steroids and other physiologically occurring lipids (Wickramasinghe, 1990; Ryan and Levin, 1990; Guengerich, 1991). CYP 450 enzymes comprise 70% - 80% of all Phase I xenobiotic metabolizing enzymes (Evans and Relling, 1999). The number of man-made environmental chemicals has been estimated as greater than 200000, most of which are thought to be potential substrates for CYP 450 and many may also serve as inducers or inhibitors of various isoforms (Murray et al., 2012). Since the synthetic modification of new and existing drugs and xenobiotics are possible, there is no upper limit on the number of compounds acted on by this enzyme family.
3.5.3. Nomenclature
Cytochrome P450 enzymes are known under a variety of names, including monooxygenases, mixed function oxidases (MFOs), poly-substrate monooxygenases (PSMOs), microsomal oxidases and heme-thiolate proteins (Feyereisen, 1999). The nomenclature of these enzyme designates all gene members of a cytochrome P450 superfamily with a CYP prefix, followed by Arabic numeral for the family, a letter for the subfamily and a numeral for the individual
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gene (Nebert and Gonzalez, 1987), for e.g. 1A1, 2B1, 3A4, etc. In the term CYP 450, the first two letters represent the cytochrome and the first letter in P450 is used as a preface to designate a gene or protein with a characteristic soret peak formed by absorbance of light at wavelengths near 450 nm when the heme iron is reduced (often with sodium dithionite) and complexed to carbon monoxide. Thus cytochrome P450 1A1 and P450 2B1 are designated CYP1A1 and CYP2B1 respectively in this nomenclature system. All members of a family share >40% identity at the amino acid sequence level, members of a subfamily share >55% identity and members that share more than 97% identity are considered to represent alleles unless there is evidence to the contrary (Nelson et al., 1996).
CYP 450’s are greatly conserved across vertebrate lineages. CYP 450 families and subfamilies, with the exception of subfamilies within CYP2’s, are nearly identical in vertebrate taxa and the total number of CYP 450 genes in vertebrate species is similar. For example, fish and mammalian lineages share all families except CYP39, which has arisen within mammals (Nelson, 2003). All other 17 CYP families have clear orthologs in both fish and mammals and the intron-exon boundaries are well conserved within CYP 450 families. CYP2’s are the least conserved family with extensive subfamily diversification (Nelson, 2003).
Among the many families of cytochrome P450, family 1–4 are the main contributors in the metabolism of xenobiotic compounds and are most widely studied throughout the world (Nelson et al., 1996). CYP 450’s are under the complex and distinct control of endogenous signals, and various xenobiotic compounds have been shown to influence their activity. As a result, individual CYP 450’s can show diverse expression patterns related to life stages, sex, tissues, strains or diet (Scott and Wen, 2001). Some forms are expressed at all life stages, such as CYP4D1 and CYP6A1, while others are adult specific, such as CYP6D1 or larval specific such as CYP6B2 (Scott et al., 1998). Similarly, CYP 450’s show great variation in response to inducers and some CYP 450’s have several known inducers, while others have none (Amichot et al., 1998).
3.5.3.1. CYP1 family
This family represents one of the most studied families in fish and have been reported in 64% of the total teleost fish studied (Arellano-Anguillar et al., 2009). It stands as the most searched for with respect to other isoforms due to its extensive use as a biomarker of
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xenobiotics exposure (Whyte et al., 2000; Siroka and Drastichova, 2004). Research on CYP 450’s in fish is more limited than that reported for mammals. The CYP1A family members, CYP1A1 and CYP1A2 have been well characterized as inducible genes and historically are linked to the aryl hydrocarbon receptor (AhR) (Dogra et al., 1998). In fish, the presence of only single CYP1A gene is reported and is considered ancestral to both mammalian CYP1A1 and CYP1A2 (Schlenk et al., 2008). Fragmented CYP1B1 research exists in fish with only a handful of reports of studies published to date. The presence of CYP1B like form in fish has been established through comparative studies with the mammalian isoform and by northern blot. CYP1B1 is constitutively expressed in most tissues but also inducible through the AhR pathway. CYP1B1 is involved in the metabolism of endogenous estrogens, as well as active in the biotransformation of heterocyclic amines found in charcoal broiled meats (Crofts et al., 1997). The CYP1C subfamily of cytochrome P450, which is present in fish but not in mammals, has only recently been discovered and so limited research on the subfamily is available. The vertebrate CYP1C subfamily was first described by Godard et al. (2005) when they identified a CYP1C1 and CYP1C2, the two CYP1C paralogs in fish, in scup and carp liver and head kidney.
3.5.3.2. CYP2 family
CYP2 is the largest CYP 450 family comprising 16 genes, 5 families and 13 subfamilies (Nebert and Russell, 2002). CYP2C and CYP2E1 account for more than 15% of liver CYP 450 enzymes, whereas the corresponding value for CYP2A6 is approximately 10% and for CYP2D6, CYP2C8, CYP2C19 less than 5% (Pelkonen et al., 2008). CYP2B6, CYP2C9, CYP2C19 and CYP2D6 presents the most important CYP 450 polymorphism clinically with ethnic differences in their distributions. Approximately 30% of all drugs are CYP2D6 substrates such as codeine and tramadol (Desmeules et al., 1991; Dayer et al., 1997). CYP2D6 can be inhibited by quinidine, fluoxetine or paroxetine (Otton et al., 1993; Heiskanen et al., 1998; Lemberg et al., 2010), but CYP2D6 seems not to be inducible by other drugs. In mammals, induction of CYP2B proceeds through activation of the constitutive androstane receptor (CAR) (Honkakoski and Negishi, 1997) but in fish, unlike their mammalian counterpart, the presence and expression of CYP2B are contradictory, in spite of the fact that this subfamily is important for the metabolism of foreign compounds such as pesticides and drugs (Arellano-aguillar et al., 2009). On the basis of metabolism of prototypical mammalian CYP2B substrates such as aldrin, benzphetamine, ethylmorphine,
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aminopyrine and alkoxyresorufins, the existence of piscine CYP2B-like enzymes have been supported (Stegemann, 1981; Eisele et al., 1984; Goksoyr et al., 1987; Buhler and Williams, 1989; Elskus and Stegemann, 1989; Kleinow et al., 1990; Haasch et al., 1994). Furthermore, protein purification followed by immune-cross-reactivity and sequence analysis with other fish species and rat CYP2B1 have extensively proven the occurrence of CYP2B form in fish such as scup (Stegemann and Hahn, 1994), rainbow trout (Miranda et al., 1990), tomtate, pinfish, Bermuda chub and sergeant major (Stegemann et al., 1997). Even though, CYP2B forms do exist in fish there seems to be an apparent lack of induction in response to the mammalian prototypical CYP2B inducer phenobarbital (Stegemann, 1981; Eisele et al., 1984; Goksoyr et al., 1987; Buhler and Williams, 1989; Haasch et al., 1994; Iwata et al., 2002).
3.5.3.3. CYP3 family
The CYP3A subfamily is the most important CYP 450 subfamily consisting of four isoenzymes CYP3A4, CYP3A5, CYP3A7 and CYP3A43 (Finta and Zaphiropoulos, 2000). CYP3A4 exhibits a dominant role among the CYP3A proteins. The CYP3 family accounts for the metabolism of 50% of the currently used pharmaceutical agents (Parkinson, 1996). In fish, they are regulated by the same receptors as in mammals, namely, through the pregnane X receptor (PXR) (Bresolin et al., 2005). Although several variant alleles of the CYP3A4 gene have been defined, most of them seem to be too rare to contribute significantly to the CYP3A4 variability (Eiselt et al., 2001). CYP3A4 is undetectable in fetal liver, but rapidly rises after the birth, and becomes the predominant CYP 450 in the adult liver (Schuetz et al., 1994). CYP3A4 accounts for approximately 30% of total CYP 450 proteins (Shimada et al., 1994). The CYP3A4 expression represents high inter-individual heterogeneity and its function has been estimated to vary over 11-fold in both liver microsomes and duodenal biopsies. The expression of CYP3A4 diminishes along the intestinal tract, thus the greatest CYP3A4 content is determined in the duodenum and lowest in the ileum (Paine et al., 1997). As in the liver, CYP3A4 is the most abundant cytochrome also in the intestine, representing approximately 50–70% of its total intestinal CYP 450 content (Paine et al., 2006).
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3.5.3.4. CYP4 family
The CYP4 family consists of 11 subfamilies (CYP4A- CYP4M) (Simpson, 1997), with the CYP4A subfamily perhaps being the most highly studied. The CYP4A subfamily is of interest in toxicology, due to its inducibility by multiple chemicals, including agents used as plasticizers, herbicides and solvents, e.g., di-(2-ethylhexyl) phthalate, 2,4- dichlorophenoxyacetic acid and trichloroethylene (Simpson, 1997). In the case of fish, the function of CYP4 family is unknown to date (Arellano-aguillar et al., 2009).
3.6. Mechanisms of induction of cytochrome P450 in fish
The induction of CYP 450’s is highly conserved and is found not only in humans but also in many other species. The inducible CYP 450’s (CYP1 - CYP3) make up a large percentage of CYP 450’s in the liver. In most cases, induction of CYP 450’s occurs by a process involving de novo RNA and protein synthesis through ligand activation of key receptor transcription factors including aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR) and others ultimately leading to increased transcription (Daujat et al., 1991). An alternative mechanism of CYP 450 induction involves compounds that stabilize translation or inhibit the protein degradation pathway (Woodcraft and Novak, 1998).
For many years, the mechanisms governing cytochrome P450 gene induction following xenobiotic exposure remained elusive. AhR–CYP1A interactions were well described, but the processes by which induction of CYP2, CYP3 and CYP4 families occurred had yet to be determined (Hahn et al., 2005). With the discovery of the nuclear receptors CAR and PXR, the mechanistic understanding of CYP 450 regulation was greatly enhanced. Both CAR and PXR are low-affinity (high substrate concentration) xenosensors in mammals, capable of regulating genes associated with the metabolism, transport and elimination of exogenous substrates.
3.7. Role of cytochrome P450 in fish
Initially, it was thought that fish lacked CYP 450 linked monooxygenases, but studies carried out in the late 1960s by Buhler and Rasmusson (1968) and Dewaide and Hendersson (1968) showed that these enzymes were present in the livers of rainbow trout and other fish. Subsequently, as observed in mammals, multiple CYP 450 forms were also discovered in
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fish, predominately in the liver, but also found in lower concentrations in other tissues, like kidney, gut, gall bladder, gonads, nervous tissue, endocrine cells, gills, etc. (Goksoyr and Forlin 1992; Sarasquete and Segner 2000). Since then a large number of CYP 450’s have been characterized in different species that have been studied (Nelson, 2009). Lester et al. (1993) and Lewis (2001) reported high levels of CYP 450 in fish liver, accounting for 1 to 2% mass of hepatocytes. Cytochrome P450 is not only of crucial importance for the transformation of xenobiotic or exogenous substances, but it also plays a major role in metabolizing various endogenous substances (Siroka and Drastichova, 2004). Even though there are differences between mammalian and fish monooxygenase systems (e.g., generally lower enzyme activity, different isoenzyme composition and lack of phenobarbital-type induction for the fish enzyme system), the overall enzyme system and characteristics seem to be very similar (Arellano-Aguilar et al., 2009).
It has been known for some time that various species of fish possess the enzymes involved in the biotransformation of xenobiotic chemicals, this area of research did not enjoy the intensity of effort in the past as did its mammalian counterpart. Recent interest in the aquatic environment and in aquatic toxicology has led to investigations with in vitro tissue preparations from several species of fish and it is now well documented that fish possess mixed-function oxidase enzymes and conjugating enzymes and that the former are inducible by many of the agents which induce mixed-function oxidases in mammals (Lech and Bend, 1980).
Schlenk et al. (1993) reported the presence of at least two constitutive forms of CYP 450 in channel catfish by performing western blots, utilizing polyclonal antibodies raised against five purified rainbow trout liver CYP 450 enzymes and revealed at least two protein bands that were approximately 50 kDa and 53 kDa. Schlenk (1994) reported the expression of CYP1A and CYP2K from liver and kidney in channel catfish (Ictalurus punctatus) exposed to 2-Methylisoborneol, which is a natural product produced by a mixture of microorganisms that inhabit ponds used for raising various fish species, using anti-trout CYP 450 polyclonal antibodies.
Although the full catalytic function of many fish CYP 450’s is still unknown, organ and cell- type distribution of major fish CYP 450’s have been investigated. Immunohistochemistry, PCR, Northern and Western blots are among the common tools used to determine mRNA and protein expression levels of CYP1A and 3A in fish tissues. CYP1A is found in the liver, heart, gill, kidney, and intestinal tract of fish (Husoy et al., 1994) and CYP3A is highly
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expressed in the liver and intestinal tract (Lee et al., 2001; Tseng et al., 2005). By genomic sequence analysis, the presence of CYP1B1 was reported in scup (Stenotomus chrysops), plaice (Pleuronectes platessa) and striped dolphin (Stenella coeruleoalba) (Godard et al., 2000). Hassanin et al. (2009) reported that Nile Tilapia CYP1A genomic sequence shows similarities of 50.8, 70.7, 71.8, 77.2, 72.4, 74.3, 77.6, 77.8, 79.1 and 80.5% with CYP1A of common carp, red sea bream, European eel, toad fish, Japanese eel, rainbow trout, European sea bass, butterfly fish, killifish, scup and European flounder respectively.
In fish, as in other animal species, there are great differences in cytochrome P450 catalytic activity among individuals of one population as well as among populations (Navas and Segner, 2001; Machala et al., 1997; Schlenk and Di Giulio, 2002). In addition, there are large inter-species differences and, in different species, the same substrate can be metabolized by different CYP 450 isoforms. Van der Oost et al. (2003) and Orrego et al. (2006) have considered CYP 450 as one of the most valuable and stable fish biomarkers for evaluating environmental risk assessment and environmental monitoring.
Studies in numerous fish species have revealed multiple cytochrome P450 proteins that have different physicochemical as well as catalytic properties. Procedures including protein purification, immunological reactivity and amino acid sequencing have been used to identify and classify different CYP 450 genes in fish. Stegemann et al. (1997) studied multiple CYP 450 expression in 10 tropical fishes from Bermuda, viz. Graysby bass, Grey snapper, Yellowtail snapper, Parrotfish, Slippery dick, Blue-striped grunt, Tomtate, Pinfish, Sergeant major and Bermuda chub. Bainy et al. (1999) have also reported multiple cytochrome P450 forms in Tilapia (Oreochromis niloticus) from Billings reservoir, Sao Paulo, Brazil. The induction of this enzyme system in fish is highly specific for certain classes of xenobiotics (e.g., PAHs and planar PCBs) and clearly attributes a biological reaction with exposure to those chemicals.
The more studied fish families are Cyprinidae, Ictaluridae, Salmonidae, Fundulidae, Cichlidae, Sparidae and Adrianichthyidae. These families include species with trade importance, easily maintained in captivity or ubiquitously distributed (Burr and Mayden, 1992). Zebrafish, channel catfish, rainbow trout and Japanese medaka are examples. As of 2009, at least 34 isoforms of CYP 450 have been described in fish, which comprise 19 subfamilies and 8 families, viz, CYP1, CYP2, CYP3, CYP4, CYP11, CYP17, CYP19 and CYP26 (Arellano-Aguilar et al., 2009). At least one CYP 450 enzyme has been described in more than 57 teleost fish species. The CYP1A isoform has been reported in over 64% of
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these, while CYP19A has been reported in 17.5%, CYP17C in 15.7% and CYP1B1, CYP1C, CYP17C and CYP2E1 between 14 - 15%. CYP2K and CYP2M isoforms have been reported in 10.5% and enzymes from CYP2N, CYP2P, CYP2X, CYP3A, CYP4T1, CYP11A1, and CYP26 subfamilies in smaller percentages. The high percentage of CYP1A compared to other isoforms is due to its broad application as a biomarker of exposure and thus making it the most studied one (Whyte et al., 2000; Siroka and Drastichova, 2004; Arellano-Aguilar et al., 2009).
Hepatic monooxygenase activities in rainbow trout (Oncorhynchus mykiss), flounder (Platichthys felsus) and dab (Limanda limanda) have been used as a monitoring tool for water bodies in northern Germany by industrial effluents (Pluta, 1993). CYP 450’s, principally the CYP1A1 isoform have been used to detect the presence of pollutants in environmental monitoring programs due to its high affinity to xenobiotics (Livingstone et al., 2000).
It has also been reported that enzymatic activity is higher in the field than in laboratory tests which are related to physiological and physical factors (Pluta, 1993; Schmitt et al., 2005; Hinck et al., 2006; Kammann et al., 2008). Hence, it has been suggested that monitoring programs should set up in situ basal levels of enzymatic activity in organisms from reference and polluted habitats. In other cases, CYP 450 enzymatic activity can also be compared between fish populations from reference clean sites to evaluate a possible risk of polluted sites. Another common practice in biomonitoring studies is the organism transplantation from unpolluted to polluted sites.
According to Whatley et al. (2009), channel catfish (Ictalurus punctatus) from field studies, Troy wastewater treatment plant (Alabama) induced transcription of mRNA for CYP1A enzyme production and increased EROD activity in channel catfish compared to catfish exposed to water from upstream of the wastewater treatment plant. Again, Whatley et al. (2010) suggested that in situ exposure to wastewater pollutants using caged experimental organisms provide a much more convenient local monitor of pollutant exposure and biological impact than ex situ toxicological studies. They also reported the induction and gene expression of CYP1 in channel catfish to be stronger in the field exposures over laboratory exposures on all sampling days and also reported the presence and induction of CYP1B when quantified by real-time reverse transcription polymerase chain reaction.
The CYP1 gene family represents one of the most studied families of CYP 450 in fish, primarily with regard to the role that CYP1A subfamily members play in the
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biotransformation of environmentally persistent aromatic hydrocarbons (e.g., PCBs, PAHs) and their relationship with disease processes resulting from exposure to compounds of this nature (Stegmann and Hahn, 1994). CYP1A induction is mediated by Ah receptor (AhR), a xenobiotic-binding protein present in the cytosol (Lewis, 2001). Pacheco and Santos (2002) reported that naphthalene, a class of PAH, have been found to be specific for the induction of CYP1A in European eel. Induction of CYP1A in fish has been widely used as a sensitive and convenient early warning signal of exposure to aryl hydrocarbon agonists such as polychlorinated biphenyls, polyaromatic hydrocarbons, dioxins, furans and organochlorine pesticides (Jonsson, 2003). In fish, CYP1A activities are often used as a marker to determine the quantities of persistent organic pollutants (Havelkova et al., 2007).
Willett et al. (2006) reported the induction of CYP 1B in channel catfish in both the laboratory treated with benzo-a-pyrene (BaP) and from the three field site, Lake Roebuck in Itta Bena, Bee Lake in Thornton and Sunflower River in Indianola. The CYP1B transcript was higher in gills compared to other tissues in both laboratory and wild catfish when quantified with real-time reverse transcriptase PCR. 3-methylcholantrene (3-MC), a specific inducer of CYP 1A in mammals is also reported to induce CYP 1A in catfish, Ancistrus multispinis (Klemz et al., 2010).
As in mammals, induction of the cytochrome P450 2B family in fish by phenobarbital is unclear, however, over the last decades, fish liver microsomes have been shown to metabolize prototypical mammalian CYP2B substrates, like aldrin, benzphetamine, ethylmorphine, aminopyrine and alkoxyresorufins (Stegemann, 1981; Eisele et al., 1984; Goksoyr et al., 1987; Elskus and Stegemann, 1989; Buhler and Williams, 1989; Kleinow et al., 1990; Haasch et al., 1994; Sadars et al., 1996; Iwata et al., 2002). A CAR immunoreactive protein was also detected in scup liver cytosol and nucleus using antibodies against human CAR. Studies on scup (Klotz et al., 1986), rainbow trout (Miranda et al., 1990) and some tropical fishes from the Bermuda Archipelago (Stegemann et al., 1997) have also been shown to metabolize prototypical mammalian CYP2B substrates. CYP2B1 and CYP2B2 are the primary members expressed in rats. In rodents, phenobarbital and other barbiturates usually induce enzymes from this subfamily and are inhibited by metyrapone (Mimura et al., 1993). A piscine CYP2B gene ortholog has so far not been reported, however, the existence of piscine CYP2B-like enzymes was further supported by protein purification and immuno-cross-reactivity studies. N-terminal analysis of scup CYP 450B showed 50% sequence identity with rat CYP2B1 and CYP2B2, furthermore, proteins from
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different taxa, including several fish species, show cross-reactivity with antibodies against both scup CYP 450B and rat CYP2B1 (Stegemann and Hahn, 1994; Stegemann et al., 1997).
The possible existence of CYP2E1 form in fish (Poeciliopsis monacha-lucida) was proposed based on hybridization with rat CYP2E1 49-base oligonucleotide, antibodies to rat CYP2E1, as well as responsiveness to ethanol treatment. This CYP 450 form was suggested to be involved in the CYP 450 mediated dealkylation of the fish carcinogen diethylnitrosamine (Kaplan et al., 1991). Furthermore, metabolism of the mammalian CYP2E substrate chlorzoxazone by hepatic microsomes in winter flounder (Pleuronectes americanus) and in viviparous Poeciliopsis monacha and Poeciliopsis viriosa supported the presence of CYP2E- like enzymes in fish (Wall and Crivello, 1998; Kaplan et al., 2001). The CYP2E1 isoform is particularly induced by ethanol, acetone, isoniazid and by starvation in animal models (Lieber, 1997). CYP2E1 is responsible for the metabolism of a large number many endogenous or exogenous small molecules and also potential bioactivation of many low-molecular-weight pharmaceuticals and other xenobiotic compounds, like acetaminophen, glycerol, halogenated anaesthetics, carbon tetrachloride, nitrosamines and benzene (Lee et al., 1996; Gonzalez, 2005). Chlorzoxazone and p-nitrophenol are routinely used as substrate probes to measure CYP2E1 activity, although chlorzoxazone can also be metabolized by CYP1A2.
CYP3A-like proteins were initially purified from several teleost species including scup, rainbow trout, and Atlantic cod (Klotz et al., 1986; Celander et al., 1989; Miranda et al., 1989). CYP3A are the major constitutive CYP 450 forms expressed in the liver and intestine of fish and is considered to play a vital role as a biochemical defence to prevent bioaccumulation of xenobiotics (Celander et al., 1989). CYP3A enzymes are among the most versatile forms of CYP 450’s as they have unusually broad substrate specificities for both endogenous and exogenous substrates, including steroids, bile acids, eicosanoids, retinoids, xenobiotics such as pharmaceuticals and procarcinogens (Aoyama et al., 1990; Gillam et al., 1993; Li et al., 1995; Waxman et al., 1998). James et al. (2005) also reported the presence and expression of CYP3A family isozymes in the intestine of channel catfish (Ictalurus punctatus).
3.8. Cytochrome P450 in air breathing teleost fish
Studies on cytochrome P450 in teleost fish have been rapidly progressing after it was first reported about 4 decades ago. In fish, cytochrome P450 induction has been widely used as a
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biomarker in analyzing exposures and responses of aquatic organisms to contaminants. Of all the teleost fish species, however, studies on cytochrome P450 in air breathing fish have been reported only in a few species.
The best studied example of CYP 450 is found in the CYP1A subfamily, which has been shown to be highly inducible in numerous aquatic species (Bucheli and Fent, 1995). Al-Arabi and Goksoyr (2002) examined the phase I cytochrome P450 monooxygenase activity and response in two tropical fish species, riverine catfish (Rita rita) and marine mudfish (Apocryptes bato) for the first time. Ethoxyresorufin O-deethylase (EROD) activity mediated by CYP1A was shown to be significantly induced in two tropical fish species after they were given a single intraperitoneal injection of two selected inducing compounds, β- naphthoflavone (β-NF) and a polychlorinated biphenyl mixture (Clophen A50) for 3 and 10 days. But the fishes when treated with the heavy metal compound cadmium chloride, showed significant inhibition of CYP1A activity in both the treated fish.
Yu (2000) reported dose-effect induction of gut and hepatic cytochrome P450 content and CYP1A dependent EROD activity in Channa striatus when exposed to deltamethrin, malathion and endosulfan for 48 hours. Mdegela et al. ( 2006 ) also reported the induction of CYP 1A in liver and gills of African sharptooth catfish (Clarias gariepinus) exposed to waterborne benzo-a-pyrene for 24 hours at different concentrations of 1.60, 3.44, and 18.21 gm/L. Liver genes that regulate detoxification are important in the metabolism of xenobiotics in various aquatic organisms. Similarly, Hassanain et al. (2007) also reported the induction of EROD activity in the liver of African sharptooth catfish, Clarias gariepinus exposed to benzo-a-pyrene while inhibition of the same was seen with cadmium. Qun et al. (2008) obtained the CYP1A cDNA of Taiwan snakehead (Channa maculata) by RT-PCR method. They reported that the cloned cDNA fragments were 908, 902 and 684 bp in length encoding 302, 300 and 228 amino acids.
In India, very little work has been done on cytochrome P450. Sadat et al. (2009) reported the increased level of hepatic and renal microsomal protein content in Heteropneustes fossilis which was correlated with increased N, N-dimethylaniline demethylase and aniline hydroxylase activity when treated with sub-lethal concentration of cypermethrin. The cytochrome P450 band intensity in SDS-PAGE was also higher in treated fish in contrast to the control fish. Radhakrishnan (2010) reported the induction of CYP 4501A in gills of Heteropneustes fossilis after exposure to benzo-a-pyrene for 45 days. Similarly, Pal et al. (2011) reported the induction of multiple forms of CYP 450 (viz. CYP1A, CYP2B, CYP2E1,
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CYP3A4) in Channa punctatus exposed to sub-lethal concentrations of dicofol and chlorpyrifos for a period of 15 days. According to Ghosh and Ray (2013), in Heteropneustes fossilis, carbofuran augments total phospholipid in the liver and accelerates CYP1A activity by changing its structural conformation and thus controls the detoxification of xenobiotics.
3.9. Cytochrome P450 and pesticide interaction
There are many pathways by which pesticides leave their sites of application and distribute throughout the environment and enter the aquatic ecosystem. The major route of pesticides to water ecosystems is through rainfall runoff and atmospheric deposition and another source of water contamination by pesticides are from domestic and industrial discharge. Most pesticides eventually find their route into rivers, lakes and ponds and have been found to be highly toxic to non-target organisms that inhabit natural aquatic environments apart from their target organisms (Vryzas et al., 2009; Werimo et al., 2009; Arjmandi et al., 2010; Maqbool and Ahmed, 2013). The contamination of water bodies by pesticides is known to have deleterious effects on the reproduction, survival and growth of aquatic animals. In the past few years, the problems caused by pesticides runoff coupled with intense agricultural practices has drawn scholars attention due to the increase in death among the fish in various ponds, lakes and streams of around the world because of their high sensitivity to the environmental contamination of water. Different concentrations of pesticides are present in many types of wastewater and numerous studies have found them to be toxic to aquatic organisms, especially fish species (Uner et al., 2006; Banaee et al., 2008). Certain physiological and biochemical processes may be significantly damaged when pollutants such as pesticides enter into the organs of fishes (John, 2007; Banaee et al., 2011).
After exposure to different concentrations of pesticides in water, the fish absorbs them in its gills, skin or gastrointestinal tract. In other words, due to their lipophilicity, most pesticides easily permeate the biological membranes and it increases the sensitivity of fish to aqueous pesticides. Then, these compounds are rapidly metabolized and may be bioconcentrated in various tissues of fish. Bioaccumulation occurs if the pesticides are slowly metabolized or excreted from the body. As the amount of pesticide increases, it becomes more harmful to the consumer or animal. Accumulated pesticides can cause death or long term damage. The low biodegradation and the high lipid solubility of some pesticides such as organochlorine pesticides have led to problems with the bioconcentrations of these compounds in different
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tissues of fish. In addition, since some fish are lower on the food chain, bioaccumulation of pesticides may increase in tissues of their predators and consumers, such as humans and thus affect their health and survival. So, the bioaccumulation of these contaminants in fish and the potential biomagnification in humans are perceived as threats (Favari et al., 2002). Ballesteros et al. (2011) showed that pesticides may be transformed in various tissues of fish during the initial 24 hours of exposure. However, some difference in the distribution of the original compounds and their metabolites may exist between tissues relating to differences in metabolism rates.
Bioaccumulation rate of pesticides in fish depends on the species, life stages, the amount of fat reserve in different tissues and diet of fish, chemical and physical properties of pesticides and of water. For the elimination and detoxification of toxic compounds, fishes have developed detoxification mechanisms involving the liberation of biotransforming enzymes collectively termed xenobiotic metabolizing enzymes. Enzymatic biotransformation of pesticides can potentially alter their activity and toxicity. Enzymes participating in the biotransformation of pesticides are classified into phase I and phase II enzymes. The phase I enzymes, particularly cytochrome P450 involving CYP1A and CYP3A, are commonly involved in the biotransformation of both exogenous and endogenous compounds, thereby creating a more polar hydrophilic compound. The common pathways of biotransformation of different kinds of pesticides include three cytochrome P450 mediated reactions: O- dealkylation, hydroxylation and epoxidation of pesticides (Kitamura et al., 2000; Straus et al., 2000; Behrens and Segner, 2001; Nebbia, 2001).
Cytochrome P450 isoenzymes or isoforms are of vital significance in the metabolism of many xenobiotics and endogenous compounds (Wickramasinghe, 2017), and levels of total cytochrome P450 have been employed to detect the presence of pollutants in aquatic environment owing to its high sensitivity to xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), and have been so far proved to be the most responsive indicator (Schlenk and Di Giulio, 2002). EROD activity have been seen to be induced in liver microsomes of rainbow trout exposed to dieldrin and endrin (Janz et al., 1992), Ancistrus multisipinis exposed to deltamethrin (Assis et al., 2009), roundnose grenadier in response to DDT exposure (Lemaire et al., 2010) and Indian major carp, L. rohita exposed to sub-lethal concentrations of chlorpyrifos (Rai et al., 2010). Pal et al. (2011) also reported the induction of CYP1A, CYP2B, CYP2E1 and CYP3A4 in Channa punctatus exposed to chlorpyrifos and dicofol.
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In general, the metabolic transformation of pesticides in living organisms goes through various metabolic reactions. Phase I reactions primarily carried out by microsomal mixed function oxidase is responsible for converting xenobiotics into more soluble products. Although these products are generally less toxic than the parent compound, more toxic metabolites may also occur, as in the cases of parathion (Chambers et al., 1991) and malathion (Buratti et al., 2005) and endosulfan (Lee et al., 2006). Such in vitro studies with phase I enzyme provide explicit details of the chemical nature of metabolites and intermediates, the pattern of their formation and the metabolic pathways of xenobiotics (Kim et al., 2005).
Perkins et al. (1999) reported an induction of several cytochrome P450 isoforms with low specificity in Ictalurus punctatus upon exposure to aldicarb, a carbamate pesticide and the fungicide propiconazole has also been reported to induce the expression of hepatic cytochrome P4501A in brown trout (Salmo trutta) (Egaas et al., 1999). Strauss et al. (2000) showed the induction of CYP1A activity in fingerling channel catfish when exposed to chlorpyrifos and parathion and also reported that aroclor 1254 did not induce the CYP 450’s responsible for the metabolism of the phosphorothionate insecticides. According to Stuchal et al. (2006), CYP1 and CYP3 family isozymes in channel catfish demethylated methoxychlor in liver and intestine. Their results suggested that the formation of estrogenic metabolites from methoxychlor would be more rapid in catfish co-exposed to CYP1A inducer, 3- methylcholanthrene. Some of the pesticides are extremely poisonous to fish species and may affect the fish population largely due to the inhibition of cytochrome P450 enzyme which reduces the capability of fish to face chemical stress. One such pesticide carbofuran has recently been reported to inhibit EROD activity in sea bass (Dicentrarchus labrax) (Moreno et al. 2011). Manar et al. (2011) have reported induction and inhibition of cytochrome P450, mainly of the family 1-3 by 18 pesticides belonging to organophosphorous, pyrethroids and benzoyl urea pesticides.
CYP2E1 activity shows induction as well as inhibition in humans and other experimental animals, based on the type of xenobiotic exposure. Studies have reported an induction of CYP2E1 activity upon exposure to pesticides such as carbaryl (Tang et al., 2002), imidacloprid (Schultz-Jander and Casida, 2002) and parathion (Mutch et al., 2003) while inhibition of the same upon exposure to pesticide chlorpyrifos have also been reported in humans (Tang et al., 2002). A similar result has been published in Atlantic cod (Gadus
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morhua) exposed to ketokonazole, ethynylestradiol and nonylphenol (Hasslberg et al., 2005). Rai et al. (2010) also reported inhibition of CYP2E1 by chlorpyrifos in fish, Labeo rohita.
Liver being the main organ of detoxification, effects on fish liver are of increasing importance to environmental toxicologists and have been the subjects of many recent reports. Scientists from throughout the world have reported the effects of pesticides on hepatic cytochrome P450 experimenting with different fish species. The diversity of these reports is illustrated by the following examples: Barnhill et al. (2003) reported that dieldrin stimulated the expression of CYP 450 in Rainbow Trout, Oncorhyncus mykiss when fed with 0.3 mg dieldrin/kg/day for 9 weeks and then an intraperitoneal (ip) dose of benzo-a-pyrene (10 µmol/kg). Female Nile Tilapia, Oreochromis niloticus, showed higher induction of EROD activity than their male counterpart when exposed to herbicide, paraquat (Figueiredo- Fernandes et al., 2006). Mortensen and Arukwe, (2006) reported CYP3A and PXR mRNA induction in the liver of Atlantic salmon, Salmo salar exposed to p, p’ DDE (Dichlorodiphenyldichloroethylene). Barberm et al. (2007) reported the induction of CYP2 and CYP 3 families in largemouth bass Micropterus salmoides after exposure to p, p’-DDE. Similarly, Blum et al. (2008) also reported an induction of CYP3A in largemouth bass Micropterus salmoides exposed to methoxychlor. Gasterosteus aculeatus, a three-spined stickleback showed moderate induction of EROD activity in a transient manner after treatment with different doses of prochloraz fungicide for different periods of time suggesting an extensive metabolization (Sanchez et al., 2008).
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4. MATERIALS AND METHODS
4.1. Experimental animal
Channa punctatus, Heteropneustes fossilis and Clarias batrachus were choosen as the experimental animal for this research work (Fig. 4.1, 4.2 and 4.3). These 3 fish species are native to South and South-east Asian countries (Froese and Pauly, 2011; Debnath, 2011; Khan et al., 2012). They are freshwater air breathing teleost fish with accessory respiratory organ and one of the most important fish species of flood plains. They are abundantly found in ponds, swamps, beels and canals generally in the plains. They prefer stagnant waters in muddy streams and rivers. They have a great demand in the market because of its high nutritional value.
Figure 4.1. Channa punctatus. Common name: Spotted snakehead Local name: Lata,taki,okol
Figure 4.2. Heteropneustes fossilis. Common name: Stinging catfish Local name: Singhi
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Figure 4.3. Clarias batrachus. Common name: Walking catfish Local name: Magur
4.2. Collection and maintenance of healthy fish in the laboratory
Healthy fish, Channa punctatus, Heteropneustes fossilis and Clarias batrachus weighing
30±5 gm (body length 8-12 cm) were collected and after a bath in 0.1% KMnO4 solution for 1 min to avoid any infection, the fishes were kept in glass aquaria measuring 90x35x35 cm and the depth of water were maintained at 20 cm. The supplier provided the fish caught from areas with no record of pesticide contamination. The fishes were acclimatized for a period of two weeks under laboratory conditions before the start of the experiment. They were fed regularly with small pieces of chopped fish at fixed rate during the course of the experiment.
4.3. Collection of healthy fish from the field
4.3.1. Sample collection A survey was done to identify the resident fishes in the rivers and ponds flowing through the tea plantation areas of Terai region of North Bengal. Fishes were collected during November, 2012 to June, 2013 using cast net and brought to the laboratory in live condition and immediately sacrificed for microsome preparation.
4.3.2. Sampling stations/sites
Nine sampling sites (Site 1-Site 9) were selected based on the criterion of easy accessibility, proximity to conventional tea gardens and abundance of local fish species (Fig. 4.4 and 4.5). The sampling was done between 2012 and 2013 during pre- and post-monsoon season.
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Site 1: Pond in Balarampur, Chopra (26.351855N, 88.365612E)
Site 2: Pond in Katagaon, Chopra (26.354489N, 88.384723E)
Site 3: Pond in Dangra Dangri, Kalagach (26.439045N, 88.338341E)
Site 4: Pond in Ariagoan, Kalagach (26.437831N, 88.359505E)
Site 5: Pond in Jaikhori, Sonapur (26.424007N, 88.269311E)
Site 6: Magurmari river, NBU (26.712514N, 88.355991E)
Site 7: Lotchka river, NBU (26.713385N, 88.343561E)
Site 8: Changa river (26.69131N, 88.25442E)
Site 9: Deomani river (26.69231N, 88.26339E)
Figure 4.4. Map showing sampling stations in Terai region of North Bengal.
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Figure 4.5. Sampling sites in the Terai region of North Bengal.
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4.4. Treatment of the fish with mammalian specific inducers of CYP 450 isoforms
Fish, Heteropneustes fossilis were given a single intraperitoneal (i.p.) injection of 20 mg/kg bodyweight of naphthalene, phenobarbitone and deflazacort and 2 ml/kg body weight of acetone. The inducers were dissolved in sesame oil and the control group was administered with only sesame oil. The fish were anaesthetized by dipping the fish in eugenol (0.03%) water. Once the animal was in a state of deep anaesthesia, the required dose of i.p. injection was administered. After an exposure of 72 hours, the enzyme activities were studied with respect to control groups.
4.5. Treatment of the fish with CYP1A specific inducer β-naphthoflavone
Fish, Channa punctatus, Heteropneustes fossilis and Clarias batrachus were treated by i.p. injections of soyabean oil containing 50 mg/kg body weight doses of β- naphthoflavone (β- NF). Control fish received soyabean oil alone. The fishes were anaesthetized by dipping the fish in eugenol (0.03%) water. Once the animal was in a state of deep anaesthesia, the required dose of i.p. injection was administered.
4.6. Determination of LC50 value of pesticides
Reagents/Pesticides
. Cypermethrin (Ripcord) 20 µg/ml Stock Solution: 0.1 ml cypermethrin was dissolved in 49.9 ml distilled water. . Ethion 100 µg/ml Stock Solution: 0.1 ml ethion was dissolved in 49.9 ml distilled water. . Dicofol (Colonel-S) 44 µg/ml Stock Solution: 0.1 ml dicofol was dissolved in 49.9 ml distilled water.
Probit analysis (Finney, 1971) was adopted to calculate the LC50 value. Initially, all the 3 fish species, C. punctatus, H. fossilis and C. batrachus were kept in a large glass aquarium separately for acclimatization. Thereafter, the fish were separated in a group of 10 fish per aquarium. A total of 50 fish were taken for conducting a single experiment. The fish were administered with 5 different concentrations of the pesticide, cypermethrin (Ripcord 10% EC), ethion (ethion 50 % EC) and dicofol (COLONEL S 18.5 % EC) while the control fish
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received no pesticide application (Fig. 4.6). The range of the pesticide concentration varied in all the experimental fish. The mortality was recorded for 24, 48, 72 and 96 hours. The fishes were not provided with food during the course of an experiment. The lethal concentration –
50% end point (LC50) was calculated from the relationship between the probits of percentage mortalities and the logs of concentration of the pesticide application.
Figure 4.6. Pesticide formulations: (a) Cypermethrin, (b) Ethion, and (c) Dicofol.
4.7. Treatment of the fish with 1/3 sub-lethal concentration of the pesticides, Cypermethrin 10% EC (RIPCORD), Ethion 50 % EC and Dicofol 18.5 % EC (COLONEL S)
10 fishes were randomly taken in a group each for control and treated. A 1/3 sub-lethal concentration of the 96 hour LC50 value of the pesticides, cypermethrin, ethion and dicofol based on acute toxicity data generated from the laboratory experiments was used to treat the experimental fish. The water was renewed every 48 hours with a fresh pesticide for the treated groups and only water for the control group. Homogeneity was maintained in all the groups by providing similar experimental conditions.
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4.8. Surgery
The fish were killed by a knock on the head and a midline incision was made along the abdomen. The gastrointestinal tract was retracted and the liver was excised using blunt-ended scissors, taking care not to break the bile duct (Fig. 4.7. a and b).
4.9. Liver Somatic Index (LSI)
Livers were excised, weighed and the liver somatic index was calculated as the percentage ratio of liver weight to body weight. The LSI was calculated by using the formula:
LSI = (liver weight/total live weight) X 100.
Since the liver samples were too small to be processed independently for enzyme activity, they were pooled before homogenization.
4.10. Preparation of microsomes
Reagents
. Perfusion buffer (1.15% KCl, 1mM EDTA, pH 7.4): 5.75 gm KCl and 0.186 gm EDTA was dissolved in 450 ml distilled water. The pH was adjusted to 7.4 using HCl and the final volume was maintained at 500 ml. . Homogenization buffer (1.15% KCl, 1mM EDTA and 0.05M Tris, pH 7.4): 1.15gm KCl, 0.037 gm EDTA and 0.605 gm Tris were dissolved in 80 ml distilled water. The pH was adjusted to 7.4 using HCl and the final volume was maintained at 100 ml. . Resuspension buffer (0.05M Tris, 1mM EDTA and 20% Glycerol, pH 7.4): 0.605 gm Tris, 0.037 gm EDTA and 20 ml glycerol were dissolved in 80 ml distilled water. The pH was adjusted to 7.4 using HCl and the final volume was maintained at 100 ml.
The procedure for preparation of microsomes was based on the method reported by Chang and Waxman (1998) with minor modifications (Fig. 4.8). Livers were perfused with a large volume of ice cold perfusion buffer (1.15% KCl, 1mM EDTA, pH 7.4) to get rid of unwanted tissues, fat bodies and blood and homogenized (1.15% KCl, 1mM EDTA and 0.05M Tris, pH 7.4) using a glass-teflon homogenizer at 4°C (4 ml per gram of tissue). The homogenized
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tissues were centrifuged in a cooling centrifuge (Sigma 3K30) at 12,000 g for 20 minutes at a temperature of 4°C. The supernatant (post-mitochondrial supernatant, PMS) were carefully harvested, without disturbing the pellet and further centrifuged in a super speed vacuum centrifuge (VS35SMTi) at 1,00,000 g for 60 minutes at 4⁰C. The supernatant was discarded (cytosol fraction) and the microsomal pellet was resuspended in a resuspension buffer containing 0.05M Tris, 1mM EDTA and 20% Glycerol, pH 7.4 (2 ml per gm of original tissue weight). The fraction was maintained on ice for immediate analysis or stored at -20°C for future use.
Figure 4.7. (a) Dissection of fish (b) Dissected out liver.
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Figure 4.8. Schematic representation for the preparation of microsomes.
4.11. Protein estimation
Reagents
. 0.1 M Sodium hydroxide (NaOH): 0.4 gm NaOH was dissolved in 100 ml distilled water.
. Solution A: 2% Na2CO3 was dissolved in 0.1M NaOH (2g of Na2CO3 in 100ml of 0.1M NaOH)
. Solution B: 1% CuSO4 and 2% sodium potassium tartrate was dissolved in 100 ml distilled water. . 1N Folin - Ciocalteu reagent: Folin - Ciocalteu was diluted [1:1] with distilled water from a 2N commercial preparation and made fresh daily. . Bovine Serum Albumin (BSA): 1 mg BSA was dissolved in 1 ml distilled water.
Protein in the microsomal fraction was estimated as described by Lowry et al. (1951). 2 gm sodium carbonate (Na2CO3) was dissolved in 0.1 M sodium hydroxide (NaOH)- (Solution A). 1% copper sulphate and 2% sodium potassium tartarate were prepared by mixing together in 100 ml distilled water (Solution B). A fresh alkaline solution was then prepared by mixing Solution A and Solution B in a ratio of 50:1 (A:B). In a test tube, 20 µL microsomal protein was diluted with 80 µL distilled water to make the volume 1 ml and to it 5 ml alkaline solution and 1 N 0.5 ml folin-ciocalteau reagent was added. The mixture was then incubated
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at room temperature for 30 minutes and the resulting blue colour intensity was measured at 660 nm. The standard curve of BSA from 20 to 100 μg was plotted and used for determination of protein concentration of samples.
4.12. Spectral analysis and quantification of cytochrome P450
Reagents
. 0.2 M Potassium Phosphate Buffer (pH 7.4): 2.721 gm KH2PO4 and 3.483 gm
K2HPO4 was dissolved independently in 100 ml distilled water. Then 19 ml of
KH2PO4 and 81 ml of K2HPO4 mixed together to get a pH 7.4. . Sulfuric acid and Formic acid for the preparation of carbon monoxide gas. . Potassium hydroxide solution . Sodium dithionite
Spectral analysis of cytochrome P450 in the hepatic microsomal fraction was done using the method described by Omura and Sato (1964) with minor modification. The microsomal preparation was diluted in 0.1M phosphate buffer and saturated with carbon monoxide for 40- 60 seconds. Carbon monoxide was prepared in a glass bottle by mixing sulfuric acid and formic acid and then passed to the cuvette through another bottle having a solution of potassium hydroxide (Fig. 4.9).
The sample was equally divided between two cuvettes. The baseline (400nm to 500nm) was recorded following which only one of the cuvette was mixed with a few mg of sodium dithionite in order to reduce CYP 450 (Table 4.1) and the absorbance was measured in a spectrophotometer (RAY LEIGH, UV-2601). The data was used to construct a graph using Microsoft office excel 2007.
The CYP 450 content was determined by using the extinction coefficient (ΔE450-490) of 91 -1 -1 mM cm .
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Figure 4.9. A carbon monoxide preparing apparatus.
Table 4.1. Total CYP 450 assay mixture for a final volume of 4mL.
Potassium Phosphate Buffer (pH 7.4) 3.5 mL
Microsomes 0.5 mL
Sodium Dithionite Few mg
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Figure 4.10. Chemical structures of substrates 7-ethoxyresorufin, aniline, N, N- dimethylaniline and erythromycin.
Chemical structures of substrates selected for the study of major detoxification reactions including O-deethylation using 7- ethoxyresorufin as probe substrate, aromatic hydroxylation using aniline as probe substrates and N-demethylation using N, N-dimethylaniline and erythromycin as probe substrates are shown in Fig. 4.10.
4.13. Enzyme assay
The methods CYP1A mediated EROD, CYP2B mediated N, N-dimethylaniline demethylation (N,N-DMA), CYP2E1 mediated aniline hydroxylation (AH) and CYP3A4 mediated erythromycin N-demethylation (ERND) assay were standardized to determine the activities of microsomal mixed function oxidase (CYP 450).
4.13.1. Determination of EROD (ethoxyresorufin-O-deethylase) activity
Reagents
. 0.1 M Tris buffer: 1.2114 gm Tris-HCl was dissolved in 80 ml distilled water and the pH was adjusted to 7.8. The final volume was maintained at 100 ml. . 0.1 M NaCl: 0.588 gm NaCl was dissolved in 100 ml distilled water. . 7-Ethoxyresorufin (2 µM): 25 µl of 124 µM stock solution of 7-Ethoxyresorufin was dissolved in 1.5 ml of the reaction mixture to get a solution of 2µM.
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. 0.01 M NADPH: 8 mg NADPH was dissolved in 1 ml of 1% NaHCO3 (1 gm in 100 ml). . 20% TCA: 20 gm TCA was dissolved in 100 ml distilled water.
CYP1A dependent EROD (7-Ethoxyresorufin O-deethylase) activity of fish liver microsomes was determined by the spectrophotometric method of Klotz et al. (1984) with minor modifications. The reaction mixture of 1.5 ml consisted of 1.35 ml (0.1 M) tris buffer-pH 7.8, 25 µL (0.1 M) NaCl, 25 µL (2 µM) 7-ethoxresorufin, and 50 µL of microsomes. After pre- incubation at 32°C for 5 minutes, the reaction was initiated by the addition of 50 µL (0.01 M) NADPH and the reaction was stopped following 10 minutes of incubation at the same temperature with 0.5 ml of ice-cold trichloroacetic acid (TCA) and centrifuged at 20,000 g for 10 min. The resulting brown colour of resorufin formed in the supernatant was measured at 572 nm.
Resorufin solution was used as a standard. 4 different standard concentrations of resorufin (50, 100, 150 and 200 pmoles) and other incubation constituents were run under the same conditions as for reaction mixture for constructing a standard calibration curve and used for calculation of enzyme activities.
4.13.2. Determination of N, N-dimethylaniline demethylase (N,N-DMA) activity
Reagents
. 0.1 M Na-K Buffer (pH 7.4): 1.56 gm NaH2PO4 and 3.483 gm K2HPO4 was
dissolved separately in 100 ml distilled water. Then 19 ml of NaH2PO4 and 81ml of
K2HPO4 was mixed together to get a pH 7.4.
. 0.15 M MgCl2: 0.305 gm MgCl2 was dissolved in 10 ml distilled water. . 0.01 M N,N-DMA: 0.012 ml N, N-Dimethylaniline was dissolved in 10 ml acetone. . 0.1 M Semicarbazide: 0.111 gm Semicarbazide was dissolved in 10 ml distilled water.
. 0.01 M NADPH: 8 mg NADPH was dissolved in 1 ml of 1% NaHCO3 (1 gm in 100 ml).
. 25% ZnSO4: 25 gm ZnSO4 was dissolved in 100 ml distilled water.
. Saturated Ba(OH)2: Ba(OH)2 was dissolved in 100 ml distilled water and allowed to settle down. Then the clear solution was filtered through Watman paper.
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. Nash Reagent: 15 gm ammonium acetate, 0.3 ml acetic acid and 0.2 ml acetylacetone was mixed together in a final volume of 50 ml.
CYP2B dependent N,N-DMA assay was determined by the method of Schenkman et al. (1967) with minor modifications. The reaction mixture of 1 ml consisted of 300 µL (0.1 M)
Na-K buffer-pH 7.4, 70 µL (0.15 M) MgCl2, 30 µL (0.1 M) semicarbazide, 50 µL (0.01 M) N, N-dimethylaniline and 500 µL microsomes. The mixture was incubated for 5 minutes at 32°C and the reaction started by adding 50 µL (0.01 M) NADPH instead of NADPH- generating system (NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase and
MgCl2). Following aerobic incubation for another 30 minutes, the reaction was terminated by adding 0.5 ml each of 25% zinc sulfate and saturated barium hydroxide. After centrifugation at 10,000 g for 10 min, 1 ml of the supernatant was mixed with 2 ml of double strength Nash reagent and incubated at 60°C for 30 min. The yellow colour of formaldehyde formed as the end product of N,N-DMA activity was measured by the method of Nash (1953) at 412 nm.
A freshly prepared formaldehyde solution was used as a standard. The tubes contained 5 standard concentrations (20, 40, 60, 80 and 100 nmoles) as well as other incubation constituents mixed by Nash reagent and incubated at 60°C for 30 minutes to give the same colour reaction. A standard formaldehyde calibration curve was constructed and used for calculation of enzyme activities.
4.13.3. Determination of aniline hydroxylase (AH) activity
Reagents
. 0.12 M Tris buffer: 1.453 gm Tris-HCl was dissolved in 80 ml distilled water and the pH was adjusted to 7.4. The final volume was maintained at 100 ml.
. 0.1 M MgCl2: 0.203 gm MgCl2 was dissolved in 10 ml distilled water. . 0.1 M Aniline: 0.091 ml aniline was dissolved in 10 ml distilled water.
. 0.01 M NADPH: 8 mg NADPH was dissolved in 1 ml of 1% NaHCO3 (1 gm in 100
ml). . 20% TCA: 20 gm TCA was dissolved in 100 ml distilled water.
. 1 M Sodium carbonate: 10.6 gm Na2CO3 was dissolved in 100 ml distilled water.
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. Phenol reagent (2% phenol in 0.2 N NaOH): 0.8 gm NaOH was dissolved in 100 ml distilled water to get 0.2 N NaOH. Then 2 ml saturated phenol was dissolved in 98 ml of 0.2 N NaOH to get a solution of phenol reagent.
CYP2E1 dependent aniline hydroxylase assay was carried out by the procedure of Schenkman et al. (1967) with minor modifications. The reaction mixture of 1 ml consisted of
250 µL (0.12 M) tris-pH 7.4, 100 µL (0.1 M) MgCl2, 100 µL (0.1 M) aniline and 500 µL microsomes. The temperature of thereaction mixture was raised to 32°C by incubation in a water bath for 5 minutes and the reaction was started by adding 50 µL (0.01 M) NADPH. The reaction was terminated after 30 minutes by adding 0.5 ml of ice-cold 20% trichloroacetic acid. The precipitate was removed by centrifugation at 10,000 g for 10 min and 1 ml of supernatant was added to 1 ml of a solution containing 2% phenol in (0.2 M) NaOH and 1 ml of (1 M) Na2CO3. After 30 min of incubation at 32°C, the blue colour of p-aminophenol formed as the end product of aniline hydroxylase activity was measured at 630 nm.
The p-aminophenol solution was used as a standard. 6 different standard concentrations of p- aminophenol (5, 10, 15, 20, 25 and 30 nmoles) and other incubation constituents were run under the same conditions as for reaction mixture. A standard calibration curve was constructed and used for calculation of enzyme activities.
4.13.4. Determination of erythromycin N-demethylase (ERND) activity
Reagents
. 0.05 M Potassium Phosphate Buffer (pH 7.4): 0.680 gm KH2PO4 and 0.870 gm
K2HPO4 was dissolved independently in 100 ml distilled water. Then 28 ml of
KH2PO4 and 72 ml of K2HPO4 was mixed together to get a pH 7.2.
. 0.15 M MgCl2: 0.305 gm MgCl2 was dissolved in 10 ml distilled water.
. 0.01 M Erythromycin: 0.073 gm erythromycin was dissolved in 10 ml acetone.
. 0.01 M NADPH: 8 mg NADPH was dissolved in 1 ml of 1% NaHCO3 (1 gm in 100
ml). . 20% TCA: 20 gm TCA was dissolved in 100 ml distilled water. . Nash Reagent: 15 gm ammonium acetate, 0.3 ml acetic acid and 0.2 ml acetylacetone was mixed together in a final volume of 50 ml.
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CYP3A4 dependent erythromycin N-demethylase assay was carried out following the method of Werringloer (1978). The reaction mixture of 1 ml consisted of 250 µL (0.05 M) phosphate buffer-pH 7.25, 100 µL (0.15 M) MgCl2, 100 µL (0.01 M) erythromycin and 500 µL microsomes. Reactions were initiated by the addition of 50 µL (0.01 M) NADPH and the mixture were incubated for 10 min at 32°C. The reaction was terminated by adding 0.5 ml of 20% trichloroacetic acid. Precipitated proteins were removed by centrifugation at 10,000 g for 10 min. 1 ml of supernatant were added to 1 ml of Nash reagent and incubated at 60°C for 30 minutes. The yellow colour of formaldehyde formed as the end product was measured at 412 nm.
The procedure used for measuring the erythromycin N-demethylase activity was identical with that used for N, N-dimethylaniline demethylase activity as same standard curve was used. All the experiment was run in duplicates and repeated. The average values were considered.
4.14. Enzyme kinetics
Michaelis-Menten constant (Km) and maximal velocity (Vmax) were used to define the enzymatic activity. Km and Vmax values were determined by use of nonlinear regression analysis (rate of metabolite formation against substrate concentration) using solver module in Microsoft excel 2007 (Fig. 4.11).
Figure 4.11. Diagram of reaction speed and Michaelis-Menten kinetics showing the increase in reaction velocity (V) with the increase in substrate concentration ([S]).
As the substrate concentration increases, saturation of substrate binding to the enzyme active site eventually occurs and a maximal reaction velocity (Vmax) is reached. The substrate concentration at a reaction velocity which is half Vmax is called the Km and is a measure of
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the affinity of the enzyme for the substrate. The reaction velocity (V) at any particular substrate concentration ([S]) is given by one site binding model in our study (Fig. 4.11):
In vitro kinetics of hepatic phase I biotransformation reactions in 3 air breathing teleost fish, C. punctatus, H. fossilis and C. batrachus were carried out. The fishes were treated with a sub-lethal concentration of the pesticide cypermethrin, ethion and dicofol for a period of 15 days and enzyme kinetics studied against the control. Maximal velocity (Vmax), binding affinity (Km) and catalytic efficiency (Vmax/Km) were used as endpoints for comparison.
4.14.1. Specific experimental parameters for each substrate
4.14.1.1. EROD
7-ethoxyresorufin concentrations ranged from 0.125 μM to 3.0 μM. Microsomal protein concentration was approximately 150 μg/ml and the reaction mixture was incubated for 10 min. The metabolite standard for 7-ethoxyresorufin metabolism was resorufin. The assay protocol is the same as mentioned in section 4.13.1.
4.14.1.2. N, N-dimethylaniline demethylation
The N, N-dimethylaniline concentration ranged from 0.025 mM to 1.2 mM. The microsomal protein concentration was approximately 1 mg/ml. Incubation times were 30 min. The formation of formaldehyde at the end of the reaction was measured for N, N-dimethylaniline metabolism. The assay protocol is the same as mentioned in section 4.13.2.
4.14.1.3. Aniline hydroxylation
Aniline concentrations ranged from 0.25 mM to 12 mM. The microsomal protein concentration was approximately 1 mg/ml and the incubation time of the reaction mixture was 30 min. Metabolite standard for aniline metabolism was p-aminophenol. The assay protocol is the same as mentioned in section 4.13.3.
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4.14.1.4. Erythromycin N-demethylation
Erythromycin concentration ranged from 0.025 mM to 1.2 mM. The microsomal protein concentration was approximately 1mg/ml and the incubation time was 10 min. The formation of formaldehyde at the end of the reaction was measured for erythromycin metabolism. The assay protocol is the same as mentioned in section 4.13.4.
4.15. Gel electrophoresis Reagents
. 30% Stock Acrylamide: 29.2 gm acrylamide and 0.8 gm bisacrylamide were dissolved in 100 ml distilled water. . 1.5 M Tris buffer: 18.171 gm Tris was dissolved in 80 ml distilled water. The pH was adjusted to 8.8 using HCl and the final volume was maintained at 100 ml. . 0.5 M Tris buffer: 6.057 gm Tris was dissolved in 80 ml of distilled water. The pH was adjusted to 6.8 using HCl and the final volume was maintained at 100 ml. . 20% SDS (Sodium dodecyl sulfate): 2 gm SDS was dissolved in 10 ml distilled water. . 10% APS (Ammonium persulfate): 0.1 gm APS was dissolved in 1 ml distilled water. . Sample buffer for SDS-PAGE (reducing- 5 ml): 0.625 ml Tris pH-6.8, 0.5 ml β- mercaptoethanol, 1 ml glycerol and 1 ml 10% SDS was dissolved in 1.875 ml distilled water. . Electrode buffer pH 8.3: 1.5 gm Tris, 7.15 gm glycine and 1 gm SDS was dissolved in 500 ml distilled water. . Bromophenol blue (0.05%): 0.05 gm bromophenol blue was dissolved in 100 ml distilled water. . 0.25% Coomassie Blue staining solution: 0.25 gm Coomassie Blue was dissolved in a solution containing 50 ml methanol, 10 ml acetic acid and 40 ml distilled water. . Destaining solution: 50 ml methanol, 10 ml Acetic acid and 40 ml distilled water was mixed together to make a solution of 100 ml. . TEMED . β-Mercaptoethanol
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The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of microsomes prepared from the liver of different fish was done by the method of Laemmli (1970). SDS- PAGE was performed in a vertical electrophoresis unit using a 10% separating gel and 5% stacking gel with a discontinuous tris-glycine buffer system for differentiating the CYP 450 bands. Bromophenol blue was used as tracking dye. The gel was stained by 0.25% coomassie blue stain for overnight and destained the following day to visualize the protein band. The methods for preparing 10% separating gel and 5% stacking gel is presented in table 4.2.
Table 4.2. Reaction mixture and assay protocol for preparing 5% stacking gel and 10% resolving gel.
Reagents 5% stacking gel 10% resolving gel
30% Stock Acrylamide 1.7 ml 10.0 ml
Tris 1.8 ml (pH 6.8) 11.2 ml (pH 8.8)
SDS (Sodium dodecylsulfate) 0.10 ml 0.15 ml
Distilled water 6.5 ml 8.65 ml
TEMED 0.05 ml 0.1 ml
APS (Ammonium persulfate) 0.05 ml 0.1 ml
4.15.1. Heme staining
Reagents
. 0.05 M Tris-HCl - pH 7.0: 0.605 gm Tris was dissolved in 80 ml distilled water. The pH was adjusted to 7.0 using HCl and the final volume was maintained at 100 ml. . TMPD (N,N,N’N’ -tetramethyl-p-phenylenediamine) . 30% hydrogen peroxide
The electrophoresis procedure was employed to visualize the heme-containing proteins chiefly CYP 450. PAGE gels were prepared as described earlier except that SDS was omitted from the gels (Thomas et al., 1976). Prior to loading, the gels were prepared and pre-run for
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20 min at 180 V to remove excess ammonium persulfate and to allow SDS from the running buffer to enter the gels before protein was loaded. Microsomal protein was diluted with sample buffer and incubated for 5 min at 70°C before being loaded into individual wells. As β-mercaptoethanol inhibits the peroxidase staining reaction, it was omitted from the sample buffer (Welton and Aust, 1974). Gels were run at a constant 120 V till the tracking dye reached the end of the gel. Gels were then placed into 50 ml of Tris–HCl (0.05 M, pH 7.0) containing 15 mg TMPD (N,N,N’N’ -tetramethyl-p-phenylenediamine) and 75 μl of 30% hydrogen peroxide to visualize hemeprotein bands.
4.16. Isolation of genomic DNA
Reagents
. Proteinase K (10 mg/ml Stock solution): 10 mg proteinase K was dissolved in 1 ml of distilled water. . SET buffer pH-8: 0.3 M Sucrose (10.26 gm in 100 ml), 0.5 M EDTA (18.612 gm EDTA in 100 ml) and 1 M Tris pH-8 (12.114 gm Tris in 100 ml) were mixed in equal volume. . Lysis buffer (TEN): 1 M Tris pH-8 (12.114 gm Tris in 100 ml), 0.5 M EDTA (18.612 gm EDTA in 100 ml) and 5 M NaCl (29.22 gm NaCl in 100 ml) were mixed in equal volume. . Tris/EDTA buffer (TE) pH-8: 1 M Tris pH-8 (12.114 gm Tris in 100 ml) and 0.5 M EDTA (18.612 gm EDTA in 100 ml) were mixed in equal volume. . Chloroform/ isoamyl alcohol (24:1): 24 ml chloroform and 1 ml isoamyl alcohol was mixed together. . 5 M NaCl: 29.22 gm NaCl was dissolved in 100 ml distilled water.
Genomic DNA was isolated from 1 gm of the liver sample by phenol chloroform isoamyl method (Sambrook and Russel, 2001). The schematic DNA extraction method is presented in Figure 4.12. DNA concentration and purity were determined spectrophotometrically by measuring the absorbance at 260 nm and 280 nm. The relative purity was evaluated by computing the ratio of A260 to A280 where a ratio of 1.8-2.0 was considered highly pure. An O.D value of 1 corresponds to 50 μg of double stranded DNA. The DNA was electrophoresed on 1.0% agarose gel and visualized after staining with 0.2% ethidium bromide.
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DNA extraction protocol 1. The tissue was rinsed and homogenized in cold SET buffer followed by proteinase K (0.1 mg/ml) digestion at 4°C for 15 minutes. The solution was then centrifuged at 5000 rpm X 5 minutes. 2. An aliquot of the pellet (200-500 µl) was taken. The volume was made up to 900 µl with TEN buffer and 100 µl of 10% SDS was added and incubated at 50°C for 10 minutes. 3. The solution was then cooled to room temperature and 250 µl 5M NaCl was added and kept on ice for 30 minutes after gentle vortexing. The solution was then centrifuged at 10000 rpm X 15 minutes. 4. The clear volume was transferred to a fresh centrifuge tube. An equal volume of saturated phenol was added and centrifuged at 8000 rpm X 10 minutes at 4°C. 5. The clear volume was transferred to a fresh centrifuge tube. An equal volume comprising of saturated phenol + chloroform: isoamyl alcohol (24:1) was added and centrifuged at 8000 rpm X 10 minutes at 4°C. 6. The clear volume was transferred to a clean centrifuge tube. An equal volume of chloroform/isoamyl alcohol (24:1) solution was added and centrifuged in 8000 rpm X 10 minutes at 4°C. 7. The clear volume was transferred into a clean centrifuge tube and double volume of chilled absolute ethanol was added and stored at -20°C for 30 minutes. The solution was centrifuged at 13000 rpm X 5 minutes at 4°C. 8. The ethanol was flushed from the tubes and a good volume of 70% ethanol (cold) was added and centrifuged at 13000 rpm X 5 minutes at 4°C. 9. The ethanol was flushed and the tube was dried at 50°C until no trace of ethanol or moisture was detected. 10. The extract was then dissolved in 50 µl TE Buffer and stored at 4°C overnight to assure dissolving to be homogenous. 11. Finally, the extract was transferred to -20°C for storage and future use.
Figure 4.12. Schematic representation for isolation of DNA from liver tissue.
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4.17. Amplification of CYP1A isoform from genomic DNA using PCR
Reagents
. 25 µl Master Mix for PCR amplification: Reaction buffer - 2.5 µl dNTP - 0.5 µl primer - 2 µl Double distilled water - 17.75 µl DNA polymerase - 0.25 µl Template - 2 µl
The CYP1A isoform from all the three fish species, Channa punctatus, Heteropneustes fossilis and Clarias batrachus was amplified from isolated genomic DNA by PCR using the primers presented in table 4.3 and sent to SciGenome Lab for sequencing.
The PCR program or cycle conditions for Channa punctatus:
Initial denaturation for 5 min at 94°C, 35 cycles Denaturation for 45 sec at 94°C, Annealing for 1min at 65°C, Extension for 1 min 30 sec at 72°C. Final extension 8 min at 72°C.
The PCR program or cycle conditions for Heteropneustes fossilis and Clarias batrachus :
Initial denaturation for 5 min at 94°C, 35 cycles Denaturation for 25 sec at 98°C, Annealing for 45 sec at 58°C, Extension for 1 min 30 sec at 72°C. Final extension 8 min at 72°C
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Table 4.3. Oligonucleotide primers used in PCR amplification of genomic CYP1A
Channa punctatus-
Sense (F)1 5’- ACA ACG GTG TGT CTG GTC TAC -3’
Antisense (R) 1 5’- GAC AAT GCA GTG GTG ACA GTG -3’
Sense (F) 2 5’- CTG CCG ACT TCA TCC CCA TT -3’
Antisense (R) 2 5’- TGA ACC TTA CAG TTC CTG AGT GA -3’
Sense (F) 3 5’- AGA TGT TGC GGC ACT CTT CA -3’
Antisense (R) 3 5’- TCT GCT CCG TAC AGA AAC CTC -3’
Heteropneustes fossilis and Clarias batrachus-
Sense (F) 5’- CGA GGG TGA GAG TTC TGA GT -3’
Antisense (R) 5’- CAG CTT CCT GTC CTC ACA GT -3’
4.18. Phylogenetic analysis
DNA sequences with the following Genbank accession numbers were retrieved from the database and used in the phylogenetic analysis along with the genomic DNA sequences of Channa punctatus, Heteropneustes fossilis and Clarias batrachus which has been submitted by us in the Genbank : JX480500.2 (Catla catla CYP1A), AB048939.1 (Cyprinus carpio CYP1A), AF015660.1 (rainbow trout CYP1A), EU683728.1 (Channa maculate CYP1A), U14162.1 (scup CYP1A), FJ389918.2 (Nile tilapia CYP1A), EF584508.1 (Peltobagrus fulvidraco CYP1A), HM043796.1 (Ancistrus sp. CYP1A).
In order to determine homology among CYP1A family cDNAs or deduced amino acid sequences from various species, sequence alignment was performed by the CLUSTAL W method using Mega6 (Ver. 6.06).
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4.19. Statistical analysis
All the data are presented as mean ± standard deviation (SD). Statistical analysis was carried out using t-test and one-way analysis of variance (ANOVA). The means were compared using Dunnett’s test and Tukey HSD test. Km and Vmax values were determined by using nonlinear regression analysis (rate of metabolite formation against substrate concentration) using solver module in Microsoft excel 2007. The statistical significance was tested at 0.1, 1 and 5 % levels. All the analysis were performed using SPSS version 16.0. The documentation of SDS-PAGE and TMPD stained gels was done using Spectronics ImageAide software, version 3.06.04 and phylogenetic analysis with sequence alignment was performed by the CLUSTAL W method using Mega6 (Ver. 6.06).
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5. RESULTS
5.1. Spectral analysis of liver microsomes of C. punctatus, H. fossilis and C. batrachus
The carbon monoxide difference spectra of dithionite reduced liver microsomes of C. punctatus, H. fossilis and C. batrachus studied at 1 minute interval over a period of 5 minutes showed the maximum absorbance at 1 to 2 minutes and then a gradual decrease in absorbance was seen with the increase in time interval (Fig. 5.1, 5.2 and 5.3). Two soret peaks were seen in the dithionite reduced spectra of liver microsomes in all the 3 control and experimental fish, C. punctatus, H. fossilis and C. batrachus. These peaks consisted of the characteristic absorbance at around 450 nm for the reduced CYP 450-CO complex and the other of lesser magnitude at around 420-425 nm. Both the control and experimental groups displayed similar patterns of soret peak.
(a)
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(b)
Figure 5.1. Carbon monoxide difference spectra of dithionite reduced liver microsomes of (a) control, and (b) experimental C. punctatus at 1 min interval over a period of 5 min.
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(a)
(b)
Figure 5.2. Carbon monoxide difference spectra of dithionite reduced liver microsomes of (a) control, and (b) experimental H. fossilis at 1 min interval over a period of 5 min.
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(a)
(b)
Figure 5.3. Carbon monoxide difference spectra of dithionite reduced liver microsomes of (a) control, and (b) experimental C. batrachus at 1 min interval over a period of 5 min.
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5.2. Multiple forms of cytochrome P450 family in liver of Heteropneustes fossilis treated with mammalian specific inducers of cytochrome P450
Spectral analysis: Carbon monoxide difference spectra upon dithionite reduction followed by bubbling of CO, showed the presence of cytochrome P450 in hepatic microsomal fractions of control and fish treated with naphthalene, phenobarbitone, deflazacort and acetone (Fig. 5.4). The peak absorbance of the control fish showed a maximum peak at 450 nm and the fish treated with naphthalene, phenobarbitone, acetone and deflazacort showed peaks at 451 nm, 452 nm, 450 nm and 449 nm respectively (Fig. 5.4 and Table 5.1).
CYP 450 content: In the control fish, cytochrome P450 (CYP 450) content was 0.214±0.102 nmole/mg protein and in experimental fish treated with naphthalene, phenobarbitone, acetone and deflazacort, the CYP 450 content were 0.330±0.089, 0.378±0.083, 0.428±0.011 and 0.440±0.096 nmole/mg protein respectively (Table 5.1). All the experimental fish displayed a significant difference [naphthalene and phenobarbitone (p<0.05); acetone and deflazacort (p< 0.01)] when compared to the control fish.
EROD activity: EROD (ethoxyresorufin O-deethylase) activity in control fish was 17.989±12.034 pmole resorufin formed/mg protein/min while in the experimental fish treated with naphthalene, phenobarbitone, acetone and deflazacort was 66.274±23.059, 41.053±9.506, 58.591±23.376 and 38.982±7.446 pmole resorufin formed/mg protein/min respectively though only naphthalene treated fish displayed a significant induction (p<0.05) in the activity (Table 5.2).
N,N-DMA activity: No significant difference were observed in N,N-dimethylaniline (N, N- DMA) demethylase activity between the control and the experimental groups. The N,N-DMA activity in control fish was 0.054±0.045 nmole formaldehyde formed/mg protein/min and in experimental fish treated with naphthalene, phenobarbitone, acetone and deflazacort, the N,N-DMA activity was 0.052±0.048, 0.060±0.013, 0.036±0.005 and 0.059±0.004 nmole formaldehyde formed/mg protein/min respectively (Table 5.2).
AH activity: In the control fish, aniline hydroxylase (AH) activity was 0.037±0.028 nmole p- aminophenol formed/mg protein/min and in the fish treated with naphthalene, phenobarbitone, acetone and deflazacort were 0.020±0.005, 0.015±0.002, 0.277±0.008 and 0.053±0.012 nmole p-aminophenol formed/mg protein/min respectively. Only acetone treated fish showed significant induction (p<0.01) as compared to control fish in AH activity. The
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fish treated with naphthalene and phenobarbitone showed down regulation in AH activity as compared to control fish but was not significant (Table 5.2).
ERND activity: Erythromycin N-demethylase (ERND) activity in control fish was 0.898±0.062 nmole formaldehyde formed/mg protein/min whereas in naphthalene, phenobarbitone, acetone and deflazacort were 0.815±0.714, 0.506±0.103, 0.127±0.022 and 3.129±0.545 nmole formaldehyde formed/mg protein/min respectively. Only deflazacort (synthetic corticosteroid) treated fish showed significant elevation (p<0.01) in the activity while the other treated groups showed down regulation in the activity than that of the control group with significant difference seen in phenobarbitone (p<0.05) and acetone (p<0.01) treated groups (Table 5.2).
Figure 5.4. Carbon monoxide difference spectra of dithionite reduced liver microsomes of (a) control Heteropneustes fossilis and fish treated with different inducers (b) naphthalene, (c) phenobarbitone, (d) acetone, and (e) deflazacort.
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Table 5.1. CYP 450 content and absorption maxima of control fish, H. fossilis and fish administered with different inducers.
INDUCER/dose CYP 450 CONTENT Absorption maxima (nmole/mg protein) CONTROL VEHICLE 0.214 ±0.102 (n=3) 450 nm (Water/Sesame oil) NAPTHELENE 0.330 ±0.089 (n=4)* 451 nm (20 mg/kg body weight) PHENOBARBITAL 0.378±0.083 (n=3)* 452 nm (20 mg/kg body weight) ACETONE 0.428 ±0.011 (n=3)** 450 nm (2 ml/kg body weight) DEFLAZACORT 0.440 ±0.096 (n=3)** 449 nm (20 mg/kg body weight)
Values are the mean of (n) number of experiments ± SD. Significantly different from control * (p ≤ 0.05), ** (p ≤ 0.01)
Table 5.2. EROD, N, N-dimethylaniline demethylase (N,N-DMA), aniline hydroxylase (AH) and erythromycin N-demethylase (ERND) activities in H. fossilis treated with different inducers.
Enzyme Activity EROD activity N,N-DMA activity AH activity ERND activity
(pmole resorufin (nmole (nmole (nmole formed/mg formaldehyde p-aminophenol formaldehyde INDUCER/dose protein/mi n) formed/mg formed/mg formed /mg protein/min) protein/min) protein/min) CONTROL VEHICLE 17.989±12.034 0.054±0.045 0.037±0.028 0.898±0.062 (Water/Sesame oil) (n=3) (n=3) (n=3) (n=3)
NAPTHELENE 66.274±23.059* 0.052±0.048 0.020±0.005 0.815±0.714 (20 mg/kg body weight) (n=4) (n=4) (n=4) (n=4)
PHENOBARBITONE 41.053±9.506 0.060±0.013 0.015±0.002 0.506±0.103* (20 mg/kg body weight) (n=3) (n=3) (n=3) (n=3)
ACETONE 58.591±23.376 0.036±0.005 0.277±0.008** 0.127±0.022** (2 ml/kg body weight) (n=3) (n=3) (n=3) (n=3)
DEFLAZACORT 38.982±7.446 0.059±0.004 0.053±0.012 3.129±0.545** (20 mg/kg body weight) (n=3) (n=3) (n=3) (n=3)
Values are the mean of (n) number of experiments ± SD. Significantly different from control * (p ≤ 0.05), ** (p ≤ 0.01)
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5.3. Treatment of the fish, C. punctatus, H. fossilis and C. batrachus with CYP1A specific inducer β-naphthoflavone
Liver somatic index (LSI) and Microsomal protein content: Table 5.3 presents data for LSI and liver microsomal protein content of control and experimental fish, C. punctatus, H. fossilis and C. batrachus treated with β- naphthoflavone. C. punctatus (1.066±0.098) showed the highest basal value for LSI followed by H. fossilis (0.988±0.140) and C. batrachus (0.912±0.155). Though LSI values were higher in all the 3 treated fish, only C. batrachus showed significantly (p<0.05) higher LSI value (1.219±0.076) while C. punctatus (1.095±0.024) and H. fossilis (1.192±0.296) showed no significant differences in the LSI value in comparison to their respective control group.
The microsomal protein content in control fish was highest in H. fossilis (3.324±0.577 mg/gm liver) followed by C. batrachus (3.284±0.719 mg/gm liver) and C. punctatus (2.875±0.612 mg/gm liver). Exposed C. punctatus showed higher liver microsomal protein content (3.353±3.355 mg/gm liver) than the control group but was not significant. In contrast, H. fossilis with 4.639±1.282 mg/gm liver (p<0.05) and C. batrachus with 5.073±0.450 mg/gm liver (p<0.01) showed a significant difference when compared with their respective control group.
Table 5.3. Liver somatic index (LSI) and protein content of C. punctatus, H. fossilis and C. batrachus administered with β-naphthoflavone (n= 4).
Fish Control Treated
Liver somatic index [LSI %]
C. punctatus 1.066±0.098 1.095±0.024 H. fossilis 0.988±0.140 1.192±0.296 C. batrachus 0.912±0.155 1.219±0.076*
Microsomal protein content (mg/gm)
C. punctatus 2.875±0.612 3.353±1.402 H. fossilis 3.324±0.577 4.639±1.282* C. batrachus 3.284±0.719 5.073±0.450**
Values are the means ± sd. Significantly different from control * (p ≤ 0.05), **(p ≤ 0.01)
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CYP 450 content, EROD, N,N-DMA, AH and ERND activities: CYP 450 content, EROD, N, N-dimethylaniline demethylase (N,N-DMA), aniline hydroxylase (AH) and erythromycin N-demethylase (ERND) activities of control and β-naphthoflavone administered fish are illustrated in Table 5.4. The CYP 450 content of control fish was higher in H. fossilis (0.313±0.061 nmole/mg protein) followed by C. batrachus (0.296±0.071 nmole/mg protein) and C. punctatus (0.280±0.054 nmole/mg protein). The CYP 450 content in all the treated fish increased significantly in comparison to that of their respective control group [C. punctatus, 0.432±0.097 (p<0.05); H. fossilis, 0.509±0.069 (p<0.001); C. batrachus, 0.466±0.118 nmole/mg protein (p<0.05)].
EROD activity was positively detected in all the fish species studied and was significantly (p<0.001) elevated in all the treated fish in comparison to their respective control group. The basal EROD activity in C. punctatus, H. fossilis and C. batrachus were 50.498±7.197, 44.685±3.340 and 55.631±1.275 pmole resorufin formed/mg protein/min respectively. The highest elevation was observed in H. fossilis with 5.7 fold increase (255.415±9.615 pmole resorufin formed/mg protein/min) followed by C. punctatus with 3.8 fold increase (194.811±7.961 pmole resorufin formed/mg protein/min) and C. batrachus with 3.3 fold increase (185.252±22.159 pmole resorufin formed/mg protein/min).
N,N-DMA activities in control fish, C. punctatus, H. fossilis and C. batrachus were 0.100±0.007, 0.129±0.050 and 0.207±0.042 nmole formaldehyde formed/mg protein/min respectively. The treatment of fish to β-naphthoflavone had no effect on N,N-DMA activities in all the 3 fish although H. fossilis showed a slight increase (0.169±0.033 nmole formaldehyde formed/mg protein/min) and C. punctatus a slight decrease (0.077±0.140 nmole formaldehyde formed/mg protein/min) in the activity but was not significant. The N,N-DMA activity in treated C. batrachus (0.201±0.035 nmole formaldehyde formed/mg protein/min) was homogeneous with respect to the control.
The AH activities in β-naphthoflavone treated fish were homogeneous with respect to the control. AH activities in control fish, C. punctatus, H. fossilis and C. batrachus were 0.109±0.003, 0.169±0.061 and 0.215±0.054 nmole p-aminophenol formed/mg protein/min and in β-naphthoflavone treated fish were 0.103±0.022, 0.166±0.010 and 0.221±0.035 nmole p-aminophenol formed/mg protein/min respectively.
ERND activity was positively detected in all the fish species and control fishes were homogeneous with respect to the activity. ERND activities in control fish, C. punctatus, H.
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fossilis and C. batrachus were 0.812±0.005, 0.743±0.168 and 0.782±0.272 nmole formaldehyde formed/mg protein/min respectively. ERND activity in experimental fish was significantly elevated (p<0.05) only in H. fossilis (1.026±0.078 nmole formaldehyde formed/mg protein/min) while C. punctatus (1.156±0.321 nmole formaldehyde formed/mg protein/min) and C. batrachus (1.117±0.265 nmole formaldehyde formed/mg protein/min) though revealed an increase in the enzyme activity, no significant difference was seen in comparison to their respective control group.
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Table 5.4. CYP 450 content, EROD, N, N-dimethylaniline demethylase (N,N-DMA), aniline hydroxylase (AH) and erythromycin N-demethylase (ERND) activities of C. punctatus, H. fossilis and C. batrachus administered with β-naphthoflavone (n= 4).
Fish Control Treated
CYP 450 content (nmole/mg protein) C. punctatus 0.280±0.054 0.432±0.097* H. fossilis 0.313±0.061 0.509±0.069*** C. batrachus 0.296±0.071 0.466±0.118* EROD activity (pmole resorufin formed/mg protein/min) C. punctatus 50.498±7.197 194.811±7.961*** H. fossilis 44.685±3.340 255.415±9.651*** C. batrachus 55.631±1.275 185.252±22.159*** N,N-DMA activity (nmole formaldehyde formed/mg protein/min) C. punctatus 0.100±0.007 0.077±0.140 H. fossilis 0.129±0.050 0.169±0.033 C. batrachus 0.207±0.042 0.201±0.035 AH activity (nmole p-aminophenol formed/mg protein/min) C. punctatus 0.109±0.003 0.103±0.022 H. fossilis 0.169±0.061 0.166±0.010 C. batrachus 0.215±0.054 0.221±0.035 ERND activity (nmole formaldehyde formed/mg protein/min) C. punctatus 0.812±0.005 1.156±0.321 H. fossilis 0.743±0.168 1.026±0.078* C. batrachus 0.782±0.272 1.117±0.265
Values are the mean of (n) number of experiments ± SD. Significantly different from control * (p ≤ 0.05), *** (p ≤ 0.001)
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5.4. Assessment of LC50 value in fish, C. punctatus, H. fossilis and C. batrachus
The details of the pesticide concentration, fish mortality and LC50 values are presented in Tables 5.5, 5.6 and 5.7. The tested concentration of the pesticide, cypermethrin (Ripcord 10% EC), ethion (ethion 50 % EC) and dicofol (COLONEL S 18.5 % EC) varied between the fish, C. punctatus, H. fossilis and C. batrachus. All the applied pesticide belonged to a different class. Cypermethrin belonged to pyrethroid, ethion belonged to organophosphate and dicofol belonged to organochlorine class of pesticide.
The calculated LC50 values of the pesticide, cypermethrin in C. punctatus, H. fossilis and C. batrachus were 19.9 µg/L, 3.7 µg/L and 5.6 µg/L respectively. H. fossilis was observed to be highly susceptible towards cypermethrin followed by C. batrachus and C. punctatus (Table 5.5). The fish C. punctatus, H. fossilis and C. batrachus were administered with 6.6,1.2 and
1.9 µg/L of cypermethrin (1/3 of LC50 value) for a period of 5, 10 and 15 days.
The calculated LC50 values of the pesticide, ethion in C. punctatus, H. fossilis and C. batrachus were 43.9 µg/L, 54.8 µg/L and 48.7 µg/L respectively. C. punctatus was observed to be highly susceptible towards cypermethrin followed by C. batrachus and H. fossilis (Table 5.6). The fish C. punctatus, H. fossilis and C. batrachus were administered with 14.5,
18.1 and 16.1 µg/L of ethion (1/3 of LC50 value) for a period of 5, 10 and 15 days.
The calculated LC50 values of the pesticide, dicofol in C. punctatus, H. fossilis and C. batrachus were 45.8 µg/L, 36.1 µg/L and 51.8 µg/L respectively. H. fossilis was observed to be highly susceptible towards dicofol followed by C. punctatus and C. batrachus (Table 5.7). The fish C. punctatus, H. fossilis and C. batrachus were administered with 15.2, 12.1 and
17.2 µg/L of dicofol (1/3 of LC50 value) for a period of 5, 10 and 15 days.
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Table 5.5. Cumulative mortalities and LC50 values of the pesticide, cypermethrin for 3 different air breathing fish, C. punctatus, H. fossilis and C. batrachus. Dose C. punctatus Dose H. fossilis Dose C. batrachus (µg/L) (N/10) (µg/L) (N/10) (µg/L) (N/10) 18 1/10 2 1/10 4 2/10 19 3/10 3 3/10 5 3/10 20 6/10 4 5/10 6 6/10 21 8/10 5 7/10 7 8/10 22 9/10 6 9/10 8 9/10
LC50 19.9 µg/L LC50 3.7 µg/L LC50 5.6 µg/L
N/10 = number of dead fish/ total number of fish LC50 = calculated concentration (Dose) required to kill 50% of the fish (Calculated from relationship between probits of percentage mortalities and the logs of pesticide concentration applied)
Table 5.6. Cumulative mortalities and LC50 values of the pesticide, ethion for 3 different air breathing fish, C. punctatus, H. fossilis and C. batrachus. Dose C. punctatus Dose H. fossilis Dose C. batrachus (µg/L) (N/10) (µg/L) (N/10) (µg/L) (N/10) 42 2/10 53 2/10 46 1/10 43 4/10 54 3/10 47 3/10 44 5/10 55 6/10 48 4/10 45 6/10 56 7/10 49 7/10 46 8/10 57 9/10 50 9/10
LC50 43.9 µg/L LC50 54.8 µg/L LC50 48.7 µg/L
N/10 = number of dead fish/ total number of fish LC50 = calculated concentration (Dose) required to kill 50% of the fish (Calculated from relationship between probits of percentage mortalities and the logs of pesticide concentration applied)
Table 5.7. Cumulative mortalities and LC50 values of the pesticide, dicofol for 3 different air breathing fish, C. punctatus, H. fossilis and C. batrachus. Dose C. punctatus Dose H. fossilis Dose C. batrachus (µg/L) (N/10) (µg/L) (N/10) (µg/L) (N/10) 44 2/10 34 1/10 50 2/10 45 4/10 35 3/10 51 4/10 46 5/10 36 5/10 52 6/10 47 7/10 37 7/10 53 8/10 48 9/10 38 8/10 54 9/10
LC50 45.8 µg/L LC50 36.1 µg/L LC50 51.8 µg/L
N/10 = number of dead fish/ total number of fish LC50 = calculated concentration (Dose) required to kill 50% of the fish (Calculated from relationship between probits of percentage mortalities and the logs of pesticide concentration applied)
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5.5. Assessment of cytochrome P450 in fish, C. punctatus, H. fossilis and C. batrachus administered with cypermethrin
Liver somatic index (LSI): Table 5.8 presents the LSI values for all the control and cypermethrin treated fish, C. punctatus, H. fossilis and C. batrachus. The LSI values of all the 3 control groups of fish displayed a homogeneous range. The LSI values in control fish, C. punctatus, H. fossilis and C. batrachus were 0.914±0.113, 0.997±0.173 and 0.976±0.162 respectively. All the treated fish displayed an elevated LSI values. The LSI values in C. punctatus for 5, 10 and 15 days treated groups were 0.985±0.096, 1.009±0.120 and 1.042±0.109 respectively. The LSI values in H. fossilis for 5, 10 and 15 days treated groups were 1.181±0.337, 1.214±0.280 and 1.245±0.144, and in C. batrachus were 1.090±0.070, 1.126±0.088 and 1.167±0.222 respectively. The significant difference (p<0.05) was shown by only 15 days treated group of C. punctatus and H. fossilis while C. batrachus showed no significant difference in any treated groups in comparison to their respective control group.
Microsomal protein content: Table 5.9 illustrates the microsomal protein content in control and cypermethrin treated fish. The microsomal protein content in control fish, C. punctatus was 2.796±0.397 mg/gm liver and in 5, 10 and 15 days treated groups were 3.622±0.725, 3.765±0.707 and 4.125±0.669 mg/gm liver respectively. The values were significantly different [5 and 10 days (p<0.05); 15 days (p<0.01)] from the control. Among H. fossilis, the microsomal protein content for control fish was 3.147±0.657 mg/gm liver and in 5, 10 and 15 days treated groups were 3.803±0.729, 4.566±0.709 and 4.981±0.633 mg/gm liver respectively. Although 5 days treated group showed an elevation in microsomal protein content, no significant difference was seen but 10 days and 15 days treated groups displayed a significant difference (p<0.01) in comparison to the control. In C. batrachus, the microsomal protein content for control, 5, 10 and 15 days treated groups were 3.068±0.537, 4.112±0.281, 4.262±0.148 and 4.994±0.385 mg/gm liver respectively. All the treated groups displayed a significant difference [5 and 10 days (p<0.05); 15 days (p<0.01)] in comparison to the control group. The 15 days treated group was observed to have higher protein content than 5 days and 10 days treated group.
CYP 450 content: Table 5.10 depicts the CYP 450 content in all 3 fish species for control and cypermethrin treated groups. In C. punctatus, the CYP 450 content in control group was
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0.276±0.089 nmole/mg protein and in 5, 10 and 15 days treated groups were 0.359±0.020, 0.382±0.042 and 0.425±0.053 nmole/mg protein respectively. The CYP 450 content in control group of H. fossilis was 0.325±0.129 nmole/mg protein and in 5, 10 and 15 days treated groups were 0.404±0.083, 0.495±0.082 and 0.523±0.234 nmole/mg protein respectively. In C. batrachus, the CYP 450 content in control group was 0.296±0.082 nmole/mg protein and in 5, 10 and 15 days treated groups were 0.382±0.034, 0.461±0.091 and 0.484±0.131 nmole/mg protein respectively. The 10 days (p<0.05) and 15 days (p<0.01) treated groups displayed significant differences in all the fish studied compared to their respective control group. The 5 days treated group showed only a marginal increase in the CYP 450 content (Table 5.10).
EROD activity: Table 5.11 illustrates EROD activity from liver microsomes in control and cypermethrin treated fish. EROD activities in control fish, C. punctatus was 35.424±15.553 pmole resorufin formed/mg protein/min and in 5, 10 and 15 days treated groups were 71.119±17.685, 81.570±26.395 and 99.341±13.451 pmole resorufin formed/mg protein/min respectively. In H. fossilis, EROD activities in control, 5, 10 and 15 days treated groups were 42.935±11.097, 79.397±9.555, 94.886±16.453 and 97.516±15.619 pmole resorufin formed/mg protein/min respectively. In C. batrachus, EROD activities in control group was 37.891±14.398 pmole resorufin formed/mg protein/min and in 5, 10 and 15 days treated groups were 75.249±17.273, 99.284±14.351 and 123.934±29.316 pmole resorufin formed/mg protein/min respectively. Of the different treated groups, the 15 days treated groups revealed the highest elevation in EROD activity than 10 days and 5 days treated groups in all the 3 fish studied. Metabolism of 7-ER was higher in all the cypermethrin treated groups of 3 fish, C. punctatus, H. fossilis and C. batrachus displaying a significant difference (p<0.001) compared with their respective control group (Table 5.11).
N,N-DMA activity: Table 5.12 represents N, N-dimethylaniline demethylase (N,N-DMA) activity in hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin. In C. punctatus, 5 days (0.108±0.042 nmole formaldehyde formed/mg protein/min) and 10 days (0.116±0.052 nmole formaldehyde formed/mg protein/min) treated group showed no significant increase in N,N-DMA activity. The activity was significantly induced only in 15 days treated group (0.186±0.082 nmole formaldehyde formed/mg
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protein/min) when compared to the control (0.091±0.044 nmole formaldehyde formed/mg protein/min). H. fossilis, on the other hand, revealed a significant increase in all the treated groups. The mean value increased from 0.135±0.056 nmole formaldehyde formed/mg protein/min in control to 0.229±0.084 nmole formaldehyde formed/mg protein/min after 5 days (p<0.05), 0.285±0.111 nmole formaldehyde formed/mg protein/min after 10 days (p<0.001) and 0.217±0.051 nmole formaldehyde formed/mg protein/min after 15 days (p<0.05). C. batrachus displayed a similar trend of response to that of C. punctatus with only 15 days treated group (0.338±0.147 nmole formaldehyde formed/mg protein/min) showing a significant difference (p<0.05) in activity. No significant difference was observed in 5 days (0.330±0.085 nmole formaldehyde formed/mg protein/min) and 10 days (0.331±0.138 nmole formaldehyde formed/mg protein/min) treated groups in comparison to that of the control group, 0.210±0.072 nmole formaldehyde formed/mg protein/min (Table 5.12).
AH activity: Table 5.13 illustrates aniline hydroxylase (AH) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin. C. punctatus showed no significant variation in the treated groups in AH activity. The AH activity in control, 5, 10 and 15 days treated groups were 0.119±0.046, 0.114±0.043, 0.110±0.056 and 0.170±0.061 nmole p-aminophenol formed/mg protein/min respectively. H. fossilis, on the other hand, displayed a significant difference (p<0.05) only in 15 days treated group (0.318±0.152 nmole p-aminophenol formed/mg protein/min). No elevation was observed in 5 days treated group (0.183±0.075 nmole p-aminophenol formed/mg protein/min) while a slight increase in 10 days treated group (0.274±0.094 nmole p-aminophenol formed/mg protein/min) was observed in comparison to the control (0.188±0.066 nmole p-aminophenol formed/mg protein/min). C. batrachus displayed elevated activity with a significant difference (p<0.01) in 10 days (0.395±0.059 nmole p-aminophenol formed/mg protein/min) and 15 days (0.387±0.143 nmole p-aminophenol formed/mg protein/min) treated groups in comparison to the control group (0.224±0.055 nmole p-aminophenol formed/mg protein/min). The 5 days treated group (0.248±0.093 nmole p-aminophenol formed/mg protein/min) displayed no change in the activity.
ERND activity: Table 5.14 illustrates erythromycin N-demethylase (ERND) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin.
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Control C. punctatus (0.847±0.220 nmole formaldehyde formed/mg protein/min), H. fossilis (0.836±0.277 nmole formaldehyde formed/mg protein/min) and C. batrachus (0.886±0.223 nmole formaldehyde formed/mg protein/min) scored a value which was almost homogeneous with respect to the activity. All the fish exposed to cypermethrin showed down regulation in the enzyme activity. In C. punctatus, the ERND activities for 5, 10 and 15 days were 0.674±0.163, 0.561±0.139 and 0.611±0.139 nmole formaldehyde formed/mg protein/min respectively. In H. fossilis, the activities for 5, 10 and 15 days were 0.578±0.107, 0.564±0.201 and 0.504±0.142 nmole formaldehyde formed/mg protein/min respectively and in C. batrachus the activities for 5, 10 and 15 days were 0.637±0.149, 0.606±0.108 and 0.676±0.136 nmole formaldehyde formed/mg protein/min respectively. In C. punctatus and H. fossilis only 10 and 15 days treated groups resulted in significant difference (p<0.05) whereas C. batrachus revealed a significant difference in all the treated groups (5, 10 and 15 days) with respect to their control group (Table 5.14).
Table 5.8. Liver somatic index (LSI) of C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin (n= 6).
Liver Somatic Index [LSI] C. punctatus H. fossilis C. batrachus Control 0.914±0.113 0.997±0.173 0.976±0.162 5 days 0.985±0.096 1.181±0.337 1.090±0.070 10 days 1.009±0.120 1.214±0.280 1.126±0.088 15 days 1.042±0.109* 1.245±0.144* 1.167±0.222 F value 1.924 2.637 1.849 P value 0.155 0.070 0.177
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05)
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Table 5.9. Microsomal protein content of C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin (n= 6).
Microsomal protein content (mg/gm liver) C. punctatus H. fossilis C. batrachus Control 2.796±0.397 3.147±0.657 3.068±0.537 5 days 3.622±0.725* 3.803±0.729 4.112±0.281* 10 days 3.765±0.707* 4.566±0.709** 4.262±0.148* 15 days 4.125±0.669** 4.981±0.633** 4.994±0.385** F value 3.329 7.759 16.600 P value 0.039 0.022 0.008
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different *(p<0.05), ** (p<0.01)
Table 5.10. CYP 450 content of C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin (n= 6).
CYP 450 content (nmole/mg protein) C. punctatus H. fossilis C. batrachus Control 0.276±0.089 0.325±0.129 0.296±0.082 5 days 0.359±0.020 0.404±0.083 0.382±0.034 10 days 0.382±0.042* 0.495±0.082* 0.461±0.091** 15 days 0.425±0.053** 0.523±0.234** 0.484±0.131** F value 6.190 3.491 5.730 P value 0.003 0.029 0.007
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), ** (p<0.01)
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Table 5.11. EROD (ethoxyresorufin O-deethylase) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin (n= 6).
EROD activity (pmole resorufin formed/mg protein/min) C. punctatus H. fossilis C. batrachus Control 35.424±15.553 42.935±11.097 37.891±14.398 5 days 71.119±17.685** 79.397±9.555*** 75.249±17.273** 10 days 81.570±26.395*** 94.886±16.453*** 99.284±14.351*** 15 days 99.341±13.451*** 97.516±15.619*** 123.934±29.316*** F value 17.032 27.455 29.461 P value 0.000 0.000 0.000
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different ** (p<0.01), *** (p<0.001)
Table 5.12. N, N-dimethylaniline demethylase (N,N-DMA) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin (n= 6).
N,N-DMA demethylase activity (nmole formaldehyde formed/mg protein/min) C. punctatus H. fossilis C. batrachus Control 0.091±0.044 0.135±0.056 0.210±0.072 5 days 0.108±0.042 0.229±0.084* 0.330±0.085 10 days 0.116±0.052 0.285±0.111*** 0.331±0.138 15 days 0.186±0.082* 0.217±0.051* 0.338±0.147* F value 3.301 6.586 2.438 P value 0.039 0.002 0.098
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), *** (p<0.001)
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Table 5.13. Aniline hydroxylase (AH) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin (n= 6).
Aniline hydroxylase activity (nmole p-aminophenol formed/mg protein/min) C. punctatus H. fossilis C. batrachus Control 0.119±0.046 0.188±0.066 0.224±0.055 5 days 0.114±0.043 0.183±0.075 0.248±0.093 10 days 0.110±0.056 0.274±0.094 0.395±0.059** 15 days 0.170±0.061 0.318±0.152* 0.387±0.143** F value 1.326 3.454 6.279 P value 0.291 0.031 0.004
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), ** (p<0.01)
Table 5.14. Erythromycin N-demethylase (ERND) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin (n= 6).
Erythromycin N-demethylase activity (nmole formaldehyde formed/mg protein/min) C. punctatus H. fossilis C. batrachus Control 0.847±0.220 0.836±0.277 0.886±0.223 5 days 0.674±0.163 0.578±0.107 0.637±0.149* 10 days 0.561±0.139** 0.564±0.201* 0.606±0.108** 15 days 0.611±0.139* 0.504±0.142** 0.676±0.136* F value 3.632 3.507 3.870 P value 0.030 0.035 0.025
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), ** (p<0.01)
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5.6. Assessment of cytochrome P450 in fish, C. punctatus, H. fossilis and C. batrachus administered with ethion
Liver somatic index (LSI): Table 5.15 presents the LSI values of all the 3 fish species exposed to ethion. All of the treated fish displayed elevated LSI values. In C. punctatus, the significant difference (p<0.01) was shown by 10 days (1.075±0.085) and 15 days (1.155±0.122) treated groups in comparison to the control group (0.927±0.312). No significant difference was observed in 5 days exposure (1.051±0.044). H. fossilis and C. batrachus showed no significant elevation in LSI values in any of the treated groups. LSI values in H. fossilis for control, 5, 10 and 15 days exposed groups were 0.968±0.283, 1.026±0.119, 1.057±0.156 and 1.073±0.120 and in C. batrachus were 0.992±0.237, 1.033±0.053, 1.045±0.024 and 1.068±0.040 respectively. 15 days treated group showed the maximum increase in LSI value compared to 5 days and 10 days treated group in all the fish studied.
Microsomal protein content: Table 5.16 illustrates the microsomal protein content of C. punctatus, H. fossilis and C. batrachus exposed to ethion. All the ethion treated fish species displayed an increase in the microsomal protein content but only 15 days treated group showed a significant difference when compared with their respective control group. The microsomal protein content for control, 5, 10 and 15 days exposed groups in C. punctatus were 2.988±0.361, 3.272±0.763, 3.393±0.853 and 4.055±0.236 (p<0.05) mg/gm liver respectively. Among H. fossilis, the microsomal protein content in control, 5, 10 and 15 days exposed groups were 3.081±0.792, 3.435±0.192, 3.605±0.441 and 4.136±0.403 (p<0.01) mg/gm liver and in C. batrachus, the microsomal protein content for control, 5, 10 and 15 days exposed groups were 3.239±0.891, 3.447±0.062, 3.571±0.111 and 4.110±0.563 (p<0.05) mg/gm liver respectively.
CYP 450 content: Table 5.17 represents the CYP 450 content of all 3 fish species for control and ethion treated group. All the treated groups (5, 10 and 15 days) displayed an elevated level in CYP 450 content when compared with their respective control group. In C. punctatus, the mean value increased from 0.291±0.065 in control to 0.319±0.021 after 5 days, 0.417±0.187 after 10 days (p<0.05) and 0.550±0.120 nmole/mg protein after 15 days (p<0.001) of exposure. In H. fossilis, the mean value increased from 0.352±0.112 in control
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to 0.438±0.074 after 5 days, 0.487±0.179 after 10 days and 0.599±0.159 nmole/mg protein after 15 days (p<0.001) of exposure. In C. batrachus, the mean value increased from 0.316±0.098 in control to 0.377±0.065 after 5 days, 0.424±0.133 after 10 days (p<0.05) and 0.542±0.084 nmole/mg protein after 15 days (p<0.001) of exposure.
EROD activity: Table 5.18 represents EROD activities from liver microsomes by using 7- ethoxyresorufin as a substrate in control and ethion treated fish. In the case of C. punctatus, EROD activity for the control group was 37.812±12.293 pmole resorufin formed/mg protein/min. The 5 days treated group displayed the highest induction (99.867±19.907 pmole resorufin formed/mg protein/min) while in 10 days and 15 days, the values were 88.593±11.527 and 92.195±34.060 pmole resorufin formed/mg protein/min respectively. The EROD activity in H. fossilis for control, 5, 10 and 15 days exposed groups were 40.153±13.381, 58.915±14.793, 67.624±21.636 and 86.944±21.720 pmole resorufin formed/mg protein/min respectively. In C. batrachus, the mean value increased from 42.101±16.173 in control to 74.925±16.197 after 5 days, 111.933±20.631 after 10 days and 104.447±24.162 pmole resorufin formed/mg protein/min after 15 days of exposure. All the treated groups of 3 fish species displayed a significant difference (p<0.001) when compared with their respective control group.
N,N-DMA activity: Table 5.19 displays the N, N-dimethylaniline demethylase (N,N-DMA) activity from hepatic microsomes of control and ethion treated fish, C. punctatus, H. fossilis and C. batrachus. N,N-DMA activities in C. punctatus for control, 5, 10 and 15 days treated groups were 0.094±0.059, 0.092±0.026, 0.098±0.010 and 0.120±0.020 nmole formaldehyde formed/mg protein/min respectively. C. punctatus hardly revealed any variation in N,N-DMA activity and only showed a marginal increase in 15 days treated group. H. fossilis revealed significant increase in 5 days (0.212±0.070 nmole formaldehyde formed/mg protein/min; p<0.05) and 10 days (0.249±0.077 nmole formaldehyde formed/mg protein/min; p<0.001) treated groups when compared to the control (0.148±0.086 nmole/mg protein/min), while the 15 days treated group (0.189±0.054 nmole formaldehyde formed/mg protein/min) displayed only a marginal increase in the activity. In C. batrachus, the N,N-DMA activity was significantly (p<0.05) induced only in the 5 days treated group (0.312±0.064 nmole formaldehyde formed/mg protein/min) in comparison to that of control group (0.208±0.095
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nmole formaldehyde formed/mg protein/min) while 10 days (0.209±0.041 nmole formaldehyde formed/mg protein/min) and 15 days (0.273±0.075 nmole formaldehyde formed/mg protein/min) treated groups displayed a negligible response.
AH activity: Table 5.20 displays aniline hydroxylase (AH) activity of control and ethion treated fish. C. punctatus showed a significant increase (p<0.05) only in 10 days treated group (0.177±0.045 nmole p-aminophenol formed/mg protein/min). 5 days (0.086±0.046 nmole p-aminophenol formed/mg protein/min) and 15 days (0.121±0.050 nmole p- aminophenol formed/mg protein/min) treated groups displayed a negligible response in AH activity compared to the control group (0.109±0.052 nmole p-aminophenol formed/mg protein/min). H. fossilis, on the other hand, displayed a significant difference [10 days (p<0.001); 5 and 15 days (p<0.001)] in all the treated groups with respect to its control group. The AH activity in control, 5, 10 and 15 days treated groups were 0.197±0.086, 0.380±0.133, 0.316±0.076 and 0.455±0.078 nmole p-aminophenol formed/mg protein/min respectively. C. batrachus showed a negligible response in AH activity in all the treated groups. The AH activity in control, 5, 10 and 15 days treated groups were 0.214±0.075, 0.248±0.108, 0.172±0.032 and 0.219±0.064 nmole p-aminophenol formed/mg protein/min respectively. The 5 days treated group revealed a slight increase and 10 days a slight decrease, whereas 15 days treated group showed negligible response when compared to its control group.
ERND activity: Table 5.21 displays erythromycin N-demethylase activity (ERND) of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to ethion. The ERND activity in control C. punctatus was 0.827±0.420 nmole formaldehyde formed/mg protein/min. The 5 days treated group (1.642±0.612 nmole formaldehyde formed/mg protein/min; p<0.01) reflected the highest induction. The significant increase in ERND activity were also noticed in 10 days (1.297±0.053 nmole/mg protein/min; p<0.05) and 15 days (1.585±0.309 nmole formaldehyde formed/mg protein/min; p<0.01) treated groups. In H. fossilis, the ERND activity varied significantly. The ERND activity in control was 0.878±0.327 and in 5, 10 and 15 days treated group were 1.743± 0.570 (p<0.05), 3.978±1.067 (p<0.001) and 3.630±1.058 (p<0.001) nmole formaldehyde formed/mg protein/min respectively. In C. batrachus, the ERND activity in control, 5, 10 and 15 days treated group were 0.836±0.291, 1.073±0.334, 1.314±0.210 and 1.579±0.520 nmole
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formaldehyde formed/mg protein/min respectively. The activity differed significantly (p<0.05) only after 15 days of exposure in comparison to its control group.
Table 5.15. Liver somatic index (LSI) of C. punctatus, H. fossilis and C. batrachus exposed to ethion (n= 6).
Liver Somatic Index [LSI] C. punctatus H. fossilis C. batrachus Control 0.927±0.312 0.968±0.283 0.992±0.237 5 days 1.051±0.044 1.026±0.119 1.033±0.053 10 days 1.075±0.085** 1.057±0.156 1.045±0.024 15 days 1.155±0.122** 1.073±0.120 1.068±0.040 F value 6.173 0.401 0.672 P value 0.004 0.754 .580
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different ** (p<0.01)
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Table 5.16. Microsomal protein content of C. punctatus, H. fossilis and C. batrachus exposed to ethion (n= 6).
Microsomal protein content (mg/gm liver) C. punctatus H. fossilis C. batrachus Control 2.988±0.361 3.081±0.792 3.239±0.891 5 days 3.272±0.763 3.435±0.192 3.447±0.062 10 days 3.393±0.853 3.605±0.441 3.571±0.111 15 days 4.055±0.236* 4.136±0.403** 4.110±0.563* F value 2.501 4.396 2.943 P value 0.108 0.021 0.070
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), ** (p<0.01)
Table 5.17. CYP 450 content of C. punctatus, H. fossilis and C. batrachus exposed to ethion (n= 6).
CYP 450 content (nmole/mg protein) C. punctatus H. fossilis C. batrachus Control 0.291±0.065 0.352±0.112 0.316±0.098 5 days 0.319±0.021 0.438±0.074 0.377±0.065 10 days 0.417±0.187* 0.487±0.179 0.424±0.133* 15 days 0.550±0.120*** 0.599±0.159*** 0.542±0.084*** F value 5.416 5.858 7.182 P value 0.007 0.004 0.002
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), *** (p<0.001).
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Table 5.18. EROD (ethoxyresorufin O-deethylase) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to ethion (n= 6).
EROD activity (pmole resorufin formed/mg protein/min) C. punctatus H. fossilis C. batrachus Control 37.812±12.293 40.153±13.381 42.101±16.173 5 days 99.867±19.907*** 58.915±14.793 74.925±16.197** 10 days 88.593±11.527*** 67.624±21.636** 111.933±20.631*** 15 days 92.195±34.060*** 86.944±21.720*** 104.447±24.162*** F value 15.322 8.938 29.610 P value 0.000 0.000 0.000
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different ** (p<0.01), *** (p<0.001)
Table 5.19. N, N-dimethylaniline demethylase (N,N-DMA) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to ethion (n= 6).
N,N-DMA demethylase activity (nmole formaldehyde formed/mg protein/min) C. punctatus H. fossilis C. batrachus Control 0.094±0.059 0.148±0.086 0.208±0.095 5 days 0.092±0.026 0.212±0.070* 0.312±0.064* 10 days 0.098±0.010 0.249±0.077*** 0.209±0.041 15 days 0.120±0.020 0.189±0.054 0.273±0.075 F value 0.471 5.102 2.315 P value 0.706 0.006 0.115
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), *** (p<0.001)
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Table 5.20. Aniline hydroxylase (AH) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to ethion (n= 6).
Aniline hydroxylase activity (nmole p-aminophenol formed/mg protein/min) C. punctatus H. fossilis C. batrachus Control 0.109±0.052 0.197±0.086 0.214±0.075 5 days 0.086±0.046 0.380±0.133*** 0.248±0.108 10 days 0.177±0.045* 0.316±0.076** 0.172±0.032 15 days 0.121±0.050 0.455±0.078*** 0.219±0.064 F value 2.768 17.656 1.146 P value 0.067 0.000 0.358
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), ** (p<0.01), *** (p<0.001)
Table 5.21. Erythromycin N-demethylase (ERND) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to ethion (n= 6).
Erythromycin N-demethylase activity (nmole formaldehyde formed/mg protein/min) C. punctatus H. fossilis C. batrachus Control 0.827±0.420 0.878±0.327 0.836±0.291 5 days 1.642±0.612** 1.743±0.570* 1.073±0.334 10 days 1.297±0.053* 3.978±1.067*** 1.314±0.210 15 days 1.585±0.309** 3.630±1.058*** 1.579±0.520* F value 7.474 25.462 4.382 P value 0.003 0.000 0.020
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), ** (p<0.01), *** (p<0.001).
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5.7. Assessment of cytochrome P450 in fish, C. punctatus, H. fossilis and C. batrachus administered with dicofol
Liver somatic index (LSI): Table 5.22 shows the LSI values of control and dicofol treated fish, C. punctatus, H. fossilis and C. batrachus. In C. punctatus, the mean value of LSI increased significantly from 0.936±0.349 in control to 1.079±0.186 (p<0.01) after 5 days, 1.161±0.126 (p<0.05) after 10 days and 1.177±0.140 (p<0.01) after 15 days of exposure. In H. fossilis, the 10 days (1.171±0.091; p<0.05) and 15 days (1.202±0.085; p<0.01) treated group revealed a significant increase in LSI value. The 5 days treated group (1.110±0.194) only showed a marginal increase in comparison to the control (0.964±0.316). In C. batrachus, the mean value increased significantly (p<0.01) from 0.982±0.296 in control to 1.531±0.488 after 5 days of exposure. Thereafter, the LSI value decreased to 1.410±0.284 (p<0.01) after 10 days and further to 1.263±0.049 (p<0.05) after 15 days of exposure.
Microsomal protein content: Table 5.23 illustrates the microsomal protein content in control and dicofol treated fish, C. punctatus, H. fossilis and C. batrachus. In C. punctatus, the microsomal protein content in control, 5, 10 and 15 days were 3.096±0.417, 3.352±0.961, 3.948±0.479 (p<0.05) and 4.264±0.656 (p<0.01) mg/gm liver respectively. In H. fossilis, the microsomal protein content in control, 5, 10 and 15 days were 3.281±0.693, 3.774±0.898, 3.986±0.422 (p<0.05) and 4.205±0.450 (p<0.01) mg/gm liver respectively. C. batrachus revealed a significant difference (p<0.05) only in 15 days treated group (4.380±1.128 mg/gm liver) with respect to its control group (2.996±0.732 mg/gm liver). The microsomal protein content in 5 and 10 days treated groups were 3.533±1.083 and 3.885±0.380 mg/gm liver respectively.
CYP 450 content: Table 5.24 illustrates the CYP 450 content in control and dicofol treated fish, C. punctatus, H. fossilis and C. batrachus. In C. punctatus, no significant difference was seen between control (0.288±0.091 nmole/mg protein) and 5 days treated group (0.318±0.085 nmole/mg protein) while the 10 days (0.401±0.107 nmole/mg protein; p<0.05) and 15 days (0.524±0.031 nmole/mg protein; p<0.001) treated groups displayed a significant difference. H. fossilis revealed a significant difference (p<0.001) only in 15 days treated group (0.713±0.397 nmole/mg protein) in comparison to its control group (0.338±0.157 nmole/mg protein). The CYP 450 content in 5 days and 10 days treated groups were 0.433±0.072 and
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0.547±0.306 nmole/mg protein respectively. In C. batrachus, the CYP 450 content varied significantly (p<0.001) in all the treated groups compared to the control group (0.306±0.098 nmole/mg protein). The CYP 450 content in 5, 10 and 15 days treated groups were 0.534±0.111, 0.613±0.105 and 0.705±0.069 nmole/mg protein respectively.
EROD activity: Table 5.25 represents the EROD activities from liver microsomes in both the control and dicofol treated groups of C. punctatus, H. fossilis and C. batrachus. The entire dicofol treated groups of 3 fish species varied significantly (p<0.001) compared to their respective control group. In C. punctatus, the mean value increased from 39.142±14.964 pmole resorufin formed/mg protein/min in control to 128.861±29.076 pmole resorufin formed/mg protein/min after 5 days, then a drastic increase was seen at 10 days (173.690±30.021 pmole resorufin formed/mg protein/min), thereafter, the activity decreased slightly after 15 days of exposure (135.096±26.195 pmole resorufin formed/mg protein/min). On the other hand, the 15 days treated groups of C. batrachus and H. fossilis revealed the highest elevation in EROD activity. In H. fossilis, EROD activity in control, 5, 10 and 15 days treated groups were 41.621±13.147, 99.236±8.225, 115.037±11.420 and 153.767±18.840 pmole resorufin formed/mg protein/min respectively. In C. batrachus, EROD activity in control, 5, 10 and 15 days treated groups were 44.573±16.246, 130.736±5.375, 140.843±14.073 and 156.055±8.652 pmole resorufin formed/mg protein/min respectively.
N,N-DMA activity: Table 5.26 displays the N, N-dimethylaniline demethylase (N,N-DMA) activity from hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to dicofol. In C. punctatus and H. fossilis, only the 15 days treated group displayed a significant induction (p<0.001) in comparison to the control. The N,N-DMA activities in C. punctatus for control, 5, 10 and 15 days treated group were 0.102±0.052, 0.105±0.013, 0.119±0.011 and 0.291±0.091 nmole formaldehyde formed/mg protein/min respectively. In H. fossilis, the N,N-DMA activities in control, 5, 10 and 15 days treated group were 0.141±0.074, 0.150±0.016, 0.175±0.028 and 0.267±0.063 nmole formaldehyde formed/mg protein/min respectively. C. batrachus displayed a slight decrease in the activity with no significant difference at 5 days (0.142±0.055 nmole formaldehyde formed/mg protein/min) and 10 days (0.156±0.067 nmole formaldehyde formed/mg protein/min) treated groups compared to its
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control group (0.221±0.095 nmole formaldehyde formed/mg protein/min). The activity recovered and increased to 0.250±0.053 nmole formaldehyde formed/mg protein/min after 15 days of exposure but was not significant.
AH activity: Table 5.27 displays aniline hydroxylase (AH) activities of control and dicofol treated fish species from hepatic microsomes. In C. punctatus, the activity decreased from 0.116±0.051 nmole p-aminophenol formed/mg protein/min in control to 0.098±0.022 after 5 days, 0.079±0.019 after 10 days and 0.081±0.034 nmole p-aminophenol formed/mg protein/min after 15 days of exposure but was not significant. H. fossilis, on the other hand, displayed a significant increase (p<0.001) in all the treated groups. The AH activities in control, 5, 10 and 15 days treated groups were 0.197±0.082, 0.583±0.172, 0.491±0.087 and 0.726±0.230 nmole p-aminophenol formed/mg protein/min respectively. C. batrachus displayed a decrease in AH activity in 5 days treated group (0.169±0.031 nmole p- aminophenol formed/mg protein/min), then recovered after 10 days (0.241±0.118 nmole p- aminophenol formed/mg protein/min) and 15 days (0.212±0.064 nmole p-aminophenol formed/mg protein/min) when compared to the control group (0.219±0.079 nmole p- aminophenol formed/mg protein/min). No significant difference was observed in any of the treated groups.
ERND activity: Table 5.28 displays erythromycin N-demethylase (ERND) activity of hepatic microsomes in control and dicofol treated C. punctatus, H. fossilis and C. batrachus. The ERND activity of C. punctatus in control, 5, 10 and 15 days treated groups were 0.858±0.412, 2.183±0.213 (p<0.05), 5.889±0.523 (p<0.001) and 9.566±1.807 (p<0.001) nmole formaldehyde formed/mg protein/min respectively. In H. fossilis, only 5 days treated group (3.156±1.410 nmole formaldehyde formed/mg protein/min) showed a significant difference (p<0.001) in comparison to its control group (0.844±0.327 nmole formaldehyde formed/mg protein/min). The ERND activities in 10 days and 15 days treated groups were 1.234±0.423 and 1.278±0.260 nmole formaldehyde formed/mg protein/min respectively. C. batrachus, on the other hand, revealed highest induction (2.168±0.392 nmole formaldehyde formed/mg protein/min) and a significant difference (p<0.001) only in 15 days treated group with respect to its control group (0.878±0.298 nmole formaldehyde formed/mg protein/min). The 5 days treated group showed a marginal increase (1.171±0.121 nmole formaldehyde
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formed/mg protein/min) in the activity, thereafter, it decreased in 10 days (0.913±0.142 nmole formaldehyde formed/mg protein/min) of exposure.
Table 5.22. Liver somatic index (LSI) of C. punctatus, H. fossilis and C. batrachus exposed to dicofol (n= 6).
Liver somatic index [LSI %] C. punctatus H. fossilis C. batrachus Control 0.936±0.349 0.964±0.316 0.982±0.296 5 days 1.079±0.186* 1.110±0.194 1.531±0.488** 10 days 1.161±0.126** 1.171±0.091* 1.410±0.284** 15 days 1.177±0.140** 1.202±0.085** 1.263±0.049* F value 6.714 3.447 6.555 P value 0.002 0.030 0.003
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), ** (p<0.01)
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Table 5.23. Microsomal protein content of C. punctatus, H. fossilis and C. batrachus exposed to dicofol (n= 6).
Microsomal protein content (mg/gm liver) C. punctatus H. fossilis C. batrachus Control 3.096±0.417 3.281±0.693 2.996±0.732 5 days 3.352±0.961 3.774±0.898 3.533±1.083 10 days 3.948±0.479* 3.986±0.422* 3.885±0.380 15 days 4.264±0.656** 4.205±0.450* 4.380±1.128* F value 4.708 2.951 1.746 P value 0.014 0.059 0.203
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), ** (p<0.01)
Table 5.24. CYP 450 content of C. punctatus, H. fossilis and C. batrachus exposed to dicofol (n= 6).
CYP 450 content (nmole/mg protein) C. punctatus H. fossilis C. batrachus Control 0.288±0.091 0.338±0.157 0.306±0.098 5 days 0.318±0.085 0.433±0.072 0.534±0.111*** 10 days 0.401±0.107* 0.547±0.306 0.613±0.105*** 15 days 0.524±0.031*** 0.713±0.397*** 0.705±0.069*** F value 12.992 4.551 29.980 P value 0.000 0.010 0.000
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), *** (p<0.001)
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Table 5.25. EROD (ethoxyresorufin O-deethylase) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to dicofol (n= 6).
EROD activity (pmole resorufin formed/mg protein/min) C. punctatus H. fossilis C. batrachus Control 39.142±14.964 41.621±13.147 44.573±16.246 5 days 128.861±29.076*** 99.236±8.225*** 130.736±5.375*** 10 days 173.690±30.021*** 115.037±11.420*** 140.843±14.073*** 15 days 135.096±26.195*** 153.767±18.840*** 156.055±8.652*** F value 48.939 88.325 195.437 P value 0.000 0.000 0.000
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different *** (p<0.001).
Table 5.26. N, N-dimethylaniline demethylase (N,N-DMA) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to dicofol (n= 6).
N,N-DMA demethylase activity (nmole formaldehyde formed/mg protein/min) C. punctatus H. fossilis C. batrachus Control 0.102±0.052 0.141±0.074 0.221±0.095 5 days 0.105±0.013 0.150±0.016 0.142±0.055 10 days 0.119±0.011 0.175±0.028 0.156±0.067 15 days 0.291±0.091*** 0.267±0.063*** 0.250±0.053 F value 17.732 7.160 2.603 P value 0.004 0.008 0.082
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different *** (p<0.001)
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Table 5.27. Aniline hydroxylase (AH) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to dicofol (n= 6).
Aniline hydroxylase activity (nmole p-aminophenol formed/mg protein/min) C. punctatus H. fossilis C. batrachus Control 0.116±0.051 0.197±0.082 0.219±0.079 5 days 0.098±0.022 0.583±0.172*** 0.169±0.031 10 days 0.079±0.019 0.491±0.087*** 0.241±0.118 15 days 0.081±0.034 0.726±0.230*** 0.212±0.064 F value 2.009 28.615 0.831 P value 0.138 0.000 0.493
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different *** (p<0.001)
Table 5.28. Erythromycin N-demethylase (ERND) activity of hepatic microsomes in C. punctatus, H. fossilis and C. batrachus exposed to dicofol (n= 6).
Erythromycin N-demethylase activity (nmole formaldehyde formed/mg protein/min) C. punctatus H. fossilis C. batrachus Control 0.858±0.412 0.844±0.327 0.878±0.298 5 days 2.183±0.213* 3.156±1.410*** 1.171±0.121 10 days 5.889±0.523*** 1.234±0.423 0.913±0.142 15 days 9.566±1.807*** 1.278±0.260 2.168±0.392*** F value 103.195 11.735 27.492 P value 0.000 0.000 0.000
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), *** (p<0.001)
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5.8. Assessment of in vitro kinetics of hepatic phase I biotransformation reactions in 3 air breathing teleost fish, C. punctatus, H. fossilis and C. batrachus
Results for Vmax and Km values of EROD, N,N-DMA, AH and ERND kinetics are presented in Tables 5.29, 5.31, 5.33 and 5.35 respectively. Vmax/Km ratios are depicted in Tables 5.30, 5.32, 5.34 and 5.36. Representative plots of enzyme kinetic data obtained from in vitro metabolism of various compounds by CYP 450 system and fitted in the Michaelis- Menten kinetics model is presented in Figure 5.5.
5.8.1. 7-Ethoxyresorufin O-deethylation (EROD) kinetics
Vmax and Km values of EROD reaction in 3 fish, C. punctatus, H. fossilis and C. batrachus are illustrated in Table 5.29. In C. punctatus, EROD Vmax values in control, cypermethrin, ethion and dicofol exposed groups were 42.785±4.560, 114.517±13.222, 109.952±8.493 and 161.665±12.252 pmole resorufin formed/mg protein/min respectively. In H. fossilis, EROD Vmax values in control, cypermethrin, ethion and dicofol exposed groups were 49.685±3.835, 111.190±10.412, 98.632±7.103 and 160.332±11.976 pmole resorufin formed/mg protein/min respectively. Similarly, in C.batrachus, EROD Vmax value in control, cypermethrin, ethion and dicofol exposed groups were 40.490±6.359, 147.740±13.296, 122.107±10.756 and 180.770±12.185 pmole resorufin formed/mg protein/min respectively. The entire experimental groups of all the fish studied displayed a significant difference (p<0.001) in Vmax value when compared to their respective control with the highest induction seen in dicofol exposure.
In C. punctatus, Km values in control, cypermethrin, ethion and dicofol exposed groups were 0.421±0.035, 0.479±0.020, 0.715±0.073 and 0.494±0.080 µM respectively. Only ethion exposed group showed a statistically significant difference (p<0.001) from the control. H. fossilis displayed lower Km value with a significant difference in all the treated groups when compared with the control (0.731±0.021 µM). The Km values in cypermethrin, ethion and dicofol treated groups were 0.507±0.017 (p<0.001), 0.477±0.013 (p<0.001) and 0.633±0.017 (p<0.05) µM respectively. In C. batrachus, all the pesticide treated groups showed a significant difference (p<0.001) from the control (0.427±0.019 µM). The Km values in cypermethrin, ethion and dicofol treated groups were 0.629±0.043, 0.669±0.023 and 0.630±0.017 µM respectively. The highest Km value was recorded for C. punctatus treated with ethion (0.715 µM) and lowest for ethion treated H. fossilis (0.477 µM) (Table 5.29).
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Intrinsic clearance expressed as Vmax/Km ratio for 3 fish, C. punctatus, H. fossilis and C. batrachus are shown in Table 5.30. In case of EROD activity in C. punctatus, Vmax/Km ratio varied significantly when compared to the control. Vmax/Km ratio in control, cypermethrin, ethion and dicofol were 102.416±24.325, 238.687±52.658 (p<0.01), 154.074±35.321 and 337.881±62.318 (p<0.001) respectively. In H. fossilis, Vmax/Km ratio in control, cypermethrin, ethion and dicofol were 68.056±12.534, 218.709±48.153, 206.580±39.452 and 252.870±42.158 respectively.The values were significantly different (p<0.001) from the control. In C.batrachus, Vmax/Km ratio in control was 94.469±18.348 and in cypermethrin, ethion and dicofol exposed groups were 236.519±34.572 (p<0.001), 183.097±31.257 (p<0.05) and 287.371±52.876 (p<0.001) respectively. In all the 3 fish species, the highest Vmax/Km ratio was seen in dicofol exposure followed by cypermethrin and ethion (Table 5.30).
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Table 5.29. Michaelis-Menten parameters for 7-ethoxyresorufin O-deethylase activity using 7-ethoxyresorufin as a substrate in hepatic microsomal suspensions prepared from control and treated fish (n= 4).
Subject of Study Vmax Km (pmole resorufin (µM) formed/mg protein/min) Control 42.785±4.560 0.421±0.035 Cypermethrin 114.517±13.222*** 0.479±0.020 C. punctatus Ethion 109.952±8.493*** 0.715±0.073*** Dicofol 161.665±12.252*** 0.494±0.080
Control 49.685±3.835 0.731±0.021 Cypermethrin 111.190±10.412*** 0.507±0.017*** H. fossilis Ethion 98.632±7.103*** 0.477±0.013*** Dicofol 160.332±11.976*** 0.633±0.017*
Control 40.490±6.359 0.427±0.019 Cypermethrin 147.740±13.296*** 0.629±0.043*** C. batrachus Ethion 122.107±10.756*** 0.669±0.023*** Dicofol 180.770±12.185*** 0.630±0.017***
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), *** (p<0.001)
Table 5.30. Comparison of microsomal intrinsic clearance values analyzed by enzyme kinetic method: CIint = Vmax/Km for selected substrate 7-ethoxyresorufin between control and treated C. punctatus, H. fossilis and C. batrachus (n=4).
Intrinsic clearance
C. punctatus H. fossilis C. batrachus
Control 102.416±24.325 68.056±12.534 94.469±18.348
Cypermethrin 238.687±52.658** 218.709±48.153*** 236.519±34.572***
Ethion 154.074±35.321 206.580±39.452*** 183.097±31.257*
Dicofol 337.881±62.318*** 252.870±42.158*** 287.371±52.876***
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), ** (p<0.01), *** (p<0.001)
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5.8.2. N, N-Dimethylaniline demethylation (N,N-DMA) kinetics
N, N-Dimethylaniline demethylase kinetic parameters for 3 fish examined are presented in Table 5.31. In C. punctatus, the Vmax values varied significantly in all the experimental groups when compared to the control group. Vmax values in control, cypermethrin, ethion and dicofol exposed groups were 0.099±0.012, 0.198±0.016 (p<0.01), 0.144±0.018 (p<0.05) and 0.305±0.019 (p<0.001) nmole formaldehyde formed/mg protein/min respectively. In H. fossilis, the entire experimental groups showed a significant difference (p<0.001) when compared to the control (0.138±0.019 nmole formaldehyde formed/mg protein/min). Vmax values in cypermethrin, ethion and dicofol exposed groups were 0.289±0.020, 0.224±0.024 and 0.278±0.011 nmole formaldehyde formed/mg protein/min respectively. In C.batrachus, the Vmax value with a significant difference was seen in cypermethrin (0.357±0.021 nmole formaldehyde formed/mg protein/min; p<0.001) and ethion (0.279±0.019 nmole formaldehyde formed/mg protein/min; p<0.01) exposure with respect to its control group (0.222±0.015 nmole formaldehyde formed/mg protein/min). The Vmax value in dicofol exposed group was 0.255±0.018 nmole formaldehyde formed/mg protein/min.
All the tested fish species displayed a mixed response for the Km value. C. punctatus displayed the highest basal Km value (0.285±0.021 mM) followed by C. batrachus (0.244±0.018 mM) and H. fossilis (0.182±0.013 mM). The entire experimental groups in C. punctatus displayed a lower Km value with a significant difference compared to its control group. The Km value in C. punctatus for cypermethrin, ethion and dicofol exposure was 0.212±0.018 (p<0.05), 0.165±0.033 (p<0.01) and 0.203±0.016 (p<0.05) mM respectively. In H. fossilis a significant difference was observed in cypermethrin (0.156±0.014 mM; p<0.05) and dicofol exposure (0.294±0.020 mM; p<0.001). The Km value was homogeneous in ethion (0.186±0.012 mM) exposed group with respect to its control. Dicofol exposed group displayed a higher Km value while cypermethrin and ethion exposed groups displayed a lower Km value. In C. batrachus, the Km values were almost homogeneous in all the experimental groups compared to the control (0.244±0.023 mM). Km values for cypermethrin, ethion and dicofol exposure were 0.225±0.018, 0.216±0.015 and 0.236±0.020 mM respectively (Table 5.31).
Intrinsic clearance expressed as Vmax/Km ratios in case of N,N-DMA for 3 fish, C. punctatus, H. fossilis and C. batrachus are presented in Table 5.32. In C. punctatus all the experimental groups displayed a higher Vmax/Km ratio with a significant difference (p<0.001) compared to its control group (0.350±0.059). The Vmax/Km ratios in
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cypermethrin, ethion and dicofol exposure were 0.936 ±0.218, 0.884±0.246 and 1.511±0.464 respectively. Both H. fossilis and C. batrachus displayed a significant difference (p<0.001) in cypermethrin and ethion exposed groups. The Vmax/Km ratio in control, cypermethrin, ethion and dicofol exposure in H. fossilis were 0.758±0.229, 1.861±0.418, 1.207±0.425 and 0.946±0.324 respectively and in C. batrachus, the Vmax/Km ratio were 0.911±0.314, 1.592±0.498, 1.299±0.512 and 1.083±0.435 respectively (Table 5.32).
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Table 5.31. Michaelis-Menten parameters for N,N-DMA demethylase activity using N, N- dimethylaniline as a substrate in hepatic microsomal suspensions prepared from control and treated fish (n= 4).
Subject of Study Vmax Km (nmole formaldehyde (mM) formed/mg protein/min) Control 0.099±0.012 0.285±0.021 Cypermethrin 0.198±0.016** 0.212±0.018* C. punctatus Ethion 0.144±0.018* 0.165±0.033** Dicofol 0.305±0.019*** 0.203±0.016*
Control 0.138±0.019 0.182±0.013 Cypermethrin 0.289±0.020*** 0.156±0.014* H. fossilis Ethion 0.224±0.024*** 0.186±0.012 Dicofol 0.278±0.011*** 0.294±0.020***
Control 0.222±0.015 0.244±0.023 Cypermethrin 0.357±0.021*** 0.225±0.018 C. batrachus Ethion 0.279±0.019** 0.216±0.015 Dicofol 0.255±0.018 0.236±0.020
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), ** (p<0.01), *** (p<0.001)
Table 5.32. Comparison of microsomal intrinsic clearance values analyzed by enzyme kinetic method: CIint = Vmax/Km for selected substrate N, N-dimethylaniline between control and treated C. punctatus, H. fossilis and C. batrachus (n=4).
Intrinsic clearance
C. punctatus H. fossilis C. batrachus
Control 0.350±0.059 0.758±0.229 0.911±0.314
Cypermethrin 0.936 ±0.218*** 1.861±0.418*** 1.592±0.498***
Ethion 0.884±0.246*** 1.207±0.425*** 1.299±0.512***
Dicofol 1.511±0.464*** 0.946±0.324 1.083±0.435
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different *** (p<0.001)
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5.8.3. Aniline hydroxylation (AH) kinetics
Table 5.33 illustrates Vmax and Km values of AH reaction in 3 fish, C. punctatus, H. fossilis and C. batrachus. In C. punctatus, cypermethrin and ethion exposed groups displayed a significant difference in Vmax value compared to the control. Vmax value for AH activity in control was 0.122±0.015 nmole p-aminophenol formed/mg protein/min, and in cypermethrin, ethion and dicofol were 0.181±0.010 (p<0.001), 0.149±0.015 (p<0.05) and 0.107±0.011 nmole p-aminophenol formed/mg protein/min respectively. H. fossilis showed a significant difference (p<0.001) in all the exposed groups compared to control (0.189±0.019 nmole p- aminophenol formed/mg protein/min). The Vmax values in cypermethrin, ethion and dicofol were 0.315±0.021, 0.446±0.033 and 0.720±0.027 nmole p-aminophenol formed/mg protein/min respectively. In C. batrachus, only cypermethrin (0.345±0.029 nmole p- aminophenol formed/mg protein/min) exposed group displayed a significant increase (p<0.001) in Vmax value when compared to the control group (0.229±0.014 nmole p- aminophenol formed/mg protein/min) while ethion (0.215±0.019 nmole p-aminophenol formed/mg protein/min) and dicofol (0.210±0.017 nmole p-aminophenol formed/mg protein/min) exposed groups showed reduced activity with no significant difference.
In C. punctatus, the Km values in control, cypermethrin, ethion and dicofol exposure were 1.056±0.159, 1.068±0.090, 1.120±0.102 and 1.102±0.125 mM respectively. In H. fossilis, ethion (1.133±0.033 mM; p<0.05) and dicofol (0.842±0.045 mM; p<0.01) exposed groups displayed a lower Km value with a significant difference in comparison to the control (1.834±0.120 mM). The cypermethrin exposed group (1.781±0.164 mM) did not show any difference with the control. In C. batrachus, only cypermethrin (1.588±0.087 mM) exposed group with a lower Km value displayed a significant difference (p<0.01) with respect to its control group (2.170±0.130 mM). The Km values in ethion and dicofol exposed groups were 2.286±0.179 mM and 2.346±0.150 mM respectively (Table 5.33).
Intrinsic clearance expressed as Vmax/Km ratios for 3 fish, C. punctatus, H. fossilis and C. batrachus in relation to aniline hydroxylation are presented in Table 5.34. In C. punctatus, Vmax/Km ratio revealed a significant difference only in cypermethrin exposure (0.170±0.053), showing it to be catalytically efficient (p<0.01) in comparison to the control (0.116±0.023). The Vmax/Km ratio in ethion and dicofol exposed groups were 0.133±0.025 and 0.098±0.015 respectively. In H. fossilis, Vmax/Km ratio in control was 0.103±0.014 and in cypermethrin, ethion and dicofol exposure were 0.179±0.046, 0.394±0.086 and 0.857±0.123 respectively. The dicofol exposed group (p<0.001) was seen to be the most
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catalytically efficient of all followed by ethion (p<0.001) while cypermethrin exposed group showed a negligible variation with respect to the control. In C. batrachus, only cypermethrin exposed group (0.217±0.065) was seen to be catalytically efficient (p<0.001) in comparison to the control (0.106±0.017) while the ethion (0.094±0.014) and dicofol (0.090±0.012) exposed groups showed lower Vmax/Km ratio with negligible variation (Table 5.34).
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Table 5.33. Michaelis-Menten parameters for aniline hydroxylase activity using aniline as a substrate in hepatic microsomal suspensions prepared from control and treated fish (n= 4).
Subject of Study Vmax Km (nmole p-aminophenol (mM) formed/mg protein/min) Control 0.122±0.015 1.056±0.159 Cypermethrin 0.181±0.010* 1.068±0.090 C. punctatus Ethion 0.149±0.015* 1.120±0.102 Dicofol 0.107±0.011 1.102±0.125
Control 0.189±0.019 1.834±0.120 Cypermethrin 0.315±0.021*** 1.781±0.164 H. fossilis Ethion 0.446±0.033*** 1.133±0.033* Dicofol 0.720±0.027*** 0.842±0.045**
Control 0.229±0.014 2.170±0.130 Cypermethrin 0.345±0.029*** 1.588±0.087** C. batrachus Ethion 0.215±0.019 2.286±0.179 Dicofol 0.210±0.017 2.346±0.150
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), ** (p<0.01), *** (p<0.001)
Table 5.34. Comparison of microsomal intrinsic clearance values analyzed by enzyme kinetic method: CIint = Vmax/Km for selected substrate aniline between control and treated C. punctatus, H. fossilis and C. batrachus (n=4).
Intrinsic clearance
C. punctatus H. fossilis C. batrachus
Control 0.116±0.023 0.103±0.014 0.106±0.017
Cypermethrin 0.170±0.053*** 0.179±0.046 0.217±0.065***
Ethion 0.133±0.025 0.394±0.086*** 0.094±0.014
Dicofol 0.098±0.015 0.857±0.123*** 0.090±0.012
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different *** (p<0.001)
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5.8.4. Erythromycin N-demethylation (ERND) kinetics
Erythromycin N-demethylase kinetics parameters are shown in Table 5.35 for 3 fish, C. punctatus, H. fossilis and C. batrachus examined in this study. All the control groups showed a homogeneous Vmax value. In C. punctatus, the Vmax value for control was 0.896±0.071 nmole formaldehyde formed/mg protein/min and in cypermethrin, ethion and dicofol exposed groups, the Vmax values were 0.817±0.027, 1.578±0.096 and 5.633±0.203 nmole formaldehyde formed/mg protein/min respectively. Dicofol (p<0.001) and ethion (p<0.01) exposed group displayed a significant difference while cypermethrin displayed a lower value with a negligible difference in comparison to the control. In both H. fossilis and C. batrachus, the Vmax values varied significantly with a significant increase seen in ethion and dicofol exposure and a significant reduction in cypermethrin exposed group. In H. fossilis, the Vmax values for control, cypermethrin, ethion and dicofol exposed groups were 0.872±0.033, 0.615±0.021 (p<0.05), 3.817±0.118 (p<0.001) and 1.648±0.066 (p<0.01) nmole formaldehyde formed/mg protein/min respectively. In C. batrachus, the Vmax values for control, cypermethrin, ethion and dicofol exposed groups were 0.842±0.023, 0.717±0.018 (p<0.05), 1.713±0.060 (p<0.01) and 2.201±0.033 (p<0.001) nmole formaldehyde formed/mg protein/min respectively.
C. punctatus exposed to cypermethrin (0.233±0.016 mM) displayed a significant elevation (p<0.001) while ethion exposure (0.041±0.015 mM) displayed a significant reduction (p<0.001) in Km values when compared to control (0.105±0.014 mM). The dicofol exposure (0.084±0.012 mM) displayed a lower Km value with negligible variation. In H. fossilis only cypermethrin exposure (0.210±0.016 mM) displayed a significant difference (p<0.01) in Km value compared to its respective control group (0.167±0.019 mM). The ethion exposure (0.184±0.018 mM) displayed a slightly higher and dicofol exposure (0.146±0.013 mM) a slightly lower Km values. On the other hand, C. batrachus showed a homogeneous value for Km in all the experimental groups along with the control. The Km values in control, cypermethrin, ethion and dicofol were 0.137±0.015, 0.146±0.010, 0.145±0.018 and 0.142±0.017 mM respectively. The basal Km value was highest in H. fossilis followed by C. batrachus and C. punctatus (Table 5.35).
Intrinsic clearance (Vmax/Km) in case of erythromycin N-demethylation for 3 fishes, C. punctatus, H. fossilis and C. batrachus are presented in Table 5.36. The Vmax/Km ratio in C. punctatus for control, cypermethrin, ethion and dicofol were 8.533±1.258, 3.523±0.823, 42.968±7.238 and 67.979±11.687 respectively. The Vmax/Km ratio in H. fossilis for control,
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cypermethrin, ethion and dicofol were 5.278±1.671, 2.947±0.762, 20.913±4.687 and 11.283±1.897 respectively, and in C. batrachus, the Vmax/Km ratio were 6.205±1.982, 4.953±1.245, 11.942±2.634 and 15.636±5.179 respectively. Vmax/Km ratio indicated that the dicofol and ethion exposed groups were observed to be catalytically efficient (p<0.001) while the cypermethrin exposed group showed a lower Vmax/Km ratio with no significant difference.
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Table 5.35. Michaelis-Menten parameters for erythromycin N-demethylase activity using erythromycin as a substrate in hepatic microsomal suspensions prepared from control and treated fish (n= 4).
Subject of Study Vmax Km (nmole formaldehyde (mM) formed/mg protein/min) Control 0.896±0.071 0.105±0.014 Cypermethrin 0.817±0.027 0.233±0.016*** C. punctatus Ethion 1.578±0.096** 0.041±0.015*** Dicofol 5.633±0.203*** 0.084±0.012
Control 0.872±0.033 0.167±0.019 Cypermethrin 0.615±0.021* 0.210±0.016** H. fossilis Ethion 3.817±0.118*** 0.184±0.018 Dicofol 1.648±0.066** 0.146±0.013
Control 0.842±0.023 0.137±0.015 Cypermethrin 0.717±0.018* 0.146±0.010 C. batrachus Ethion 1.713±0.060** 0.145±0.018 Dicofol 2.201±0.033*** 0.142±0.017
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different * (p<0.05), ** (p<0.01), *** (p<0.001)
Table 5.36. Comparison of microsomal intrinsic clearance values analyzed by enzyme kinetic method: CIint = Vmax/Km for selected substrate erythromycin between control and treated C. punctatus, H. fossilis and C. batrachus (n=4).
Intrinsic clearance
C. punctatus H. fossilis C. batrachus
Control 8.533±1.258 5.278±1.671 6.205±1.982
Cypermethrin 3.523±0.823 2.947±0.762 4.953±1.245
Ethion 42.968±7.238*** 20.913±4.687*** 11.942±2.634***
Dicofol 67.979±11.687*** 11.283±1.897*** 15.636±5.179***
Values are the means ± SD. Means were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Significantly different *** (p<0.001)
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Figure 5.5. Michaelis-Menten plots for EROD, N,N-DMA, AH and ERND activities obtained from in vitro metabolism by CYP 450 system: (a) C. punctatus, (b) H. fossilis, and (c) C.batrachus.
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5.9. Assessment of CYP450 from hepatic tissue in fish collected from local areas near the tea plantation in Chopra (Balarampur and Katagaon), Kalaghaj (Dangra Dangri and Ariagoan), Sonapur, Magurmari, Lotchka, Changa and Deomani
The fish, Channa punctatus, Channa striatus, Channa gachua, Heteropneustes fossilis and Clarias batrachus were collected from different sites, though all the species were not found in every sites. The fish, C. punctatus and H. fossilis were collected from all the sites except Changa and Deomani, while C. batrachus were not found in Balarampur (Chopra), Dangra Dangri and Ariagoan in Kalaghaj, Changa and Deomani. C. striatus was found only in Katagaon (Chopra) and C. gachua was found only at sites, Changa and Deomani. N,N-DMA, AH and ERND activities for C. gachua could not be performed due to lack of enzyme quantity. Fishes were collected during November, 2012 to June, 2013. During the survey and collection of fishes, pesticide bottles and packets were also found near the pond sites in Balarampur, Katagoan, and Ariagoan (Fig. 5.6).
Figure 5.6. Pesticide bottles and packets found near the pond sites from where fish were caught: (a) Balarampur, Chopra (b) Katagoan, Chopra and (c) Ariagoan, Kalaghaj.
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Liver somatic index (LSI): Table 5.37 shows LSI values of fishes collected from different sites. Among C. punctatus, the fish collected from Magurmari (1.16±0.39) displayed a significant difference (p<0.05) in LSI value in comparison to the fish collected from Dangra Dangri (0.92±0.31). The LSI values in C. punctatus collected from Balarampur, Katagaon, Ariagoan, Sonapur and Lotchka were 0.98±0.34, 0.972, 0.95±0.46, 1.09±0.40 and 1.08±0.32 respectively. The LSI value of C. striatus collected from Katagaon was 0.970 and the LSI values of C. gachua collected from Changa and Deomani were 1.02±0.41 and 1.04±0.53 respectively (Table 5.37).
The LSI values in H. fossilis collected from all the sites displayed a homogeneous range. The LSI values in H. fossilis collected from Balarampur, Katagaon, Dangra Dangri, Ariagoan, Sonapur, Magurmari and Lotchka were 1.26±0.42, 1.285, 1.07±0.41, 1.114, 1.23±0.52 1.20±0.40 and 1.18±0.43 respectively (Table 5.37).
In the case of C. batrachus, the fish collected from Lotchka (1.42±0.42) displayed a significant difference (p<0.05) in LSI value compared to fish collected from Magurmari (1.18±0.41).The LSI values in fish caught from Katagaon and Sonapur were 1.360 and 1.202 respectively (Table 5.37).
CYP 450 content: CYP 450 content in hepatic microsomes of fishes collected from different sites has been tabulated in Table 5.38. Among C. punctatus, fish brought from Balarampur (0.56±0.21 nmole/mg protein), Magurmari (0.48±0.22 nmole/mg protein) and Lotchka (0.58±0.28 nmole/mg protein) displayed a significant difference (p<0.05) in comparison to fish collected from Dangra Dangri (0.34±0.12 nmole/mg protein) and Ariagoan (0.32±0.09 nmole/mg protein). The CYP 450 content in fish collected from Katagoan and Sonapur were 0.711 and 0.42±0.18 nmole/mg protein respectively. Overall, C. punctatus collected from Katagaon showed the highest and the fish from Ariagoan showed the lowest CYP 450 content. Among C. gachua, the fish caught from Changa showed higher CYP 450 content (0.28±0.08 nmole/mg protein) than that caught from Deomani (0.19±0.07 nmole/mg protein). The CYP 450 content of C. striatus caught only in Katagaon was 0.400 nmole/mg protein (Table 5.38).
The CYP 450 content in H. fossilis collected from Balarampur (0.66±0.24 nmole/mg protein), Magurmari (0.84±0.31 nmole/mg protein) and Lotchka (0.74±0.33 nmole/mg protein) displayed a significant difference (p<0.05) compared to fish caught from Dangra Dangri
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(0.25±0.08 nmole/mg protein) and Sonapur (0.42±0.12 nmole/mg protein). The CYP 450 content in fish collected from Katagoan and Ariagoan were 0.710 and 0.325 nmole/mg protein respectively. H. fossilis collected from Magurmari showed the highest and the fish from Dangra Dangri showed the lowest CYP 450 content (Table 5.38).
No statistical significance could be found among C. batrachus. CYP 450 content in C. batrachus brought from Katagaon, Sonapur, Magurmari and Lotchka were 1.066, 0.557, 0.57±0.28 and 0.54±0.22 nmole/mg protein respectively. Overall C. batrachus brought from Katagaon showed the highest CYP 450 content and the fish from Lotchka showed the lowest (Table 5.38).
EROD activity: Table 5.39 shows the EROD activity in all the species of fish collected from different sites. All the fishes displayed a variable range of EROD activity. Among C. punctatus, the fish brought from Sonapur (163±42 pmole resorufin formed/mg protein/min) and Magurmari (160±45 pmole resorufin formed/mg protein/min) displayed a significant difference (p<0.05) compared to fish brought from Dangra Dangri (107±34 pmole resorufin formed/mg protein/min) and Ariagoan (100±24 pmole resorufin formed/mg protein/min). The EROD activity in C. punctatus brought from Balarampur, Katagoan and Lotchka were 154±32, 153 and 147±28 pmole resorufin formed/mg protein/min respectively. C. striatus collected from Katagoan showed an EROD activity of 155 pmole resorufin formed/mg protein/min. Among C. gachua, the fish collected from Changa (83±28 pmole resorufin formed/mg protein/min) displayed a significant difference (p<0.05) from the fish collected from Deomani (22±12 pmole resorufin formed/mg protein/min) (Table 5.39).
Among H. fossilis, the fish collected from Sonapur (157±46 pmole resorufin formed/mg protein/min) and Magurmari (167±64 pmole resorufin formed/mg protein/min) displayed a significant difference (p<0.05) from the fish collected from Dangra Dangri (106±59 pmole resorufin formed/mg protein/min). The EROD activity in H. fossilis collected from Balarampur, Katagoan, Ariagoan and Lotchka were 132±48, 127, 95 and 146±51 pmole resorufin formed/mg protein/min respectively. Overall the fish collected from Magurmari showed the highest EROD activity and the fish collected from Ariagoan the lowest (Table 5.39).
No statistical significance was seen between C. batrachus in EROD activity. The fish brought from Magurmari (187±52 pmole resorufin formed/mg protein/min) displayed the highest
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activity followed by Sonapur (167 pmole resorufin formed/mg protein/min), Katagoan (153 pmole resorufin formed/mg protein/min) and Lotchka (150±56 pmole resorufin formed/mg protein/min) (Table 5.39).
N,N-DMA activity: N, N-dimethylaniline demethylase (N,N-DMA) activity in air breathing teleost fish, C. punctatus, C. striatus, H. fossilis and C. batrachus brought from different sites have been presented in Table 5.40. The N,N-DMA activity in fish, C. punctatus brought from Dangra Dangri (0.64±0.31 nmole formaldehyde formed/mg protein/min) and Ariagoan (0.73±0.49 nmole formaldehyde formed/mg protein/min) showed a significant difference (p<0.05) than that collected from other sites. The N,N-DMA activity in fish, C. punctatus collected from Balarampur, Katagoan, Sonapur, Magurmari and Lotchka were 0.21±0.11, 0.282, 0.17±0.12, 0.27±0.13 and 0.21±0.10 nmole formaldehyde formed/mg protein/min respectively. C. striatus collected from Katagoan showed 0.137 nmole formaldehyde formed/mg protein/min of N,N-DMA activity (Table 5.40).
Among H. fossilis, the fish collected from Dangra Dangri (0.89±0.43 nmole formaldehyde formed/mg protein/min) and Magurmari (0.36±0.16 nmole formaldehyde formed/mg protein/min) displayed a significant difference (p<0.05) in N,N-DMA activity compared to fish collected from all other sites. A significant difference (p<0.05) in N,N-DMA activity was also seen between the fish collected from Dangra Dangri and Magurmari. The N,N-DMA activity in fish, H. fossilis collected from Balarampur, Katagoan, Ariagoan, Sonapur and Lotchka were 0.20±0.11, 0.272, 0.756, 0.16±0.09 and 0.31±0.17 nmole formaldehyde formed/mg protein/min respectively (Table 5.40).
No statistical significance was observed among C. batrachus caught from various site. The highest N,N-DMA activity was observed in fish collected from Lotchka (0.32±0.16 nmole formaldehyde formed/mg protein/min) followed by Magurmari (0.31±0.14 nmole formaldehyde formed/mg protein/min), Sonapur (0.295 nmole formaldehyde formed/mg protein/min) and Katagoan (0.198 nmole formaldehyde formed/mg protein/min) (Table 5.40).
AH activity: Aniline hydroxylase (AH) activity in air breathing teleost fish, C. punctatus, C. striatus, H. fossilis and C. batrachus collected from different sites have been presented in Table 5.41. Among C. punctatus, the fish collected from Sonapur (0.19±0.09 nmole p- aminophenol formed/mg protein/min) and Magurmari (0.18±0.10 nmole p-aminophenol
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formed/mg protein/min) displayed a significant difference (p<0.05) in AH activity compared to fish collected from other sites. The AH activity in C. punctatus collected from Balarampur, Katagoan, Dangra Dangri, Ariagoan and Lotchka were 0.12±0.05, 0.137, 0.09±0.02, 0.08±0.03 and 0.12±0.06 nmole p-aminophenol formed/mg protein/min respectively. Overall, C. punctatus collected from Sonapur showed the highest and Ariagoan showed the lowest AH activity. The AH activity in C. striatus collected from Katagoan was 0.119 nmole p- aminophenol formed/mg protein/min (Table 5.41).
Among H. fossilis, the fish collected from Balarampur (0.34±0.12 nmole p-aminophenol formed/mg protein/min) displayed a significant difference (p<0.05) in AH activity than the fish collected from Lotchka (0.20±0.09 nmole p-aminophenol formed/mg protein/min). The AH activity in fish collected from Katagoan, Dangra Dangri, Ariagoan, Sonapur and Magurmari were 0.409, 0.24±0.09, 0.236, 0.25±0.11 and 0.21±0.14 nmole p-aminophenol formed/mg protein/min respectively (Table 5.41).
On the other hand, C. batrachus displayed homogeneous activity from all the sampling sites with no significant difference. The AH activity in fish collected from Katagoan, Sonapur, Magurmari and Lotchka were 0.341, 0.327, 0.30±0.12, 0.33±0.18 nmole p-aminophenol formed/mg protein/min respectively (Table 5.41).
ERND activity: Erythromycin N-demethylase (ERND) activity in air breathing teleost fish, C. punctatus, C. striatus, H. fossilis and C. batrachus collected from different sites have been presented in Table 5.42. Among C. punctatus, the fish brought from Dangra Dangri (2.58±0.91 nmole formaldehyde formed/mg protein/min), Ariagoan (2.18±0.98 nmole formaldehyde formed/mg protein/min), Sonapur (3.03±1.25 nmole formaldehyde formed/mg protein/min) and Magurmari (2.09±1.21 nmole formaldehyde formed/mg protein/min) displayed a significant difference (p<0.05) in ERND activity when compared to fish collected from Balarampur (0.98±0.42 nmole formaldehyde formed/mg protein/min). The fish collected from Lotchka and Katagoan showed 1.69±0.99 and 0.866 nmole formaldehyde formed/mg protein/min of ERND activity. The ERND activity of C. striatus collected from Katagoan was 1.097 nmole formaldehyde formed/mg protein/min (Table 5.42).
Among H. fossilis, the fish collected from Dangra Dangri (3.55±1.58 nmole formaldehyde formed/mg protein/min), Magurmari (4.13±1.52 nmole formaldehyde formed/mg protein/min) and Lotchka (3.14±1.32 nmole formaldehyde formed/mg protein/min) displayed
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a significant difference (p<0.05) from Balarampur (1.37±0.85 nmole formaldehyde formed/mg protein/min) and Sonapur (1.69±0.86 nmole formaldehyde formed/mg protein/min). The ERND acivity of H. fossilis collected from Katagoan and Ariagoan were 1.402 and 4.080 nmole formaldehyde formed/mg protein/min respectively (Table 5.42).
No significant difference was observed in C. batrachus caught from different sites. The highest ERND activity was observed in fish caught from Magurmari (3.21±1.43 nmole formaldehyde formed/mg protein/min) followed by Sonapur (2.917 nmole formaldehyde formed/mg protein/min), Lotchka (2.34±1.02 nmole formaldehyde formed/mg protein/min) and Katagoan (0.944 nmole formaldehyde formed/mg protein/min) (Table 5.42).
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Table 5.37. Liver somatic index (LSI) in air breathing teleost fish, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus brought from different sites. Values are the mean of (n) number of experiments.
Liver Somatic Index [LSI%] Sampling site Channa Channa Channa Heteropneustes Clarias punctatus striatus gachua fossilis batrachus Balarampur 0.98±0.34 ab (2) - - 1.26±0.42a (2) - Katagaon 0.972 (1) 0.970 (1) - 1.285 (1) 1.360 (1) Dangra Dangri 0.92±0.31a (3) - - 1.07±0.41a (2) - Ariagoan 0.95±0.46 ab (2) - - 1.114 (1) - Sonapur 1.09±0.40 ab (2) - - 1.23±0.52 a (2) 1.202 (1) Magurmari 1.16±0.39b (4) - - 1.20±0.40 a (3) 1.18±0.41a (3) Lotchka 1.08±0.32ab (2) - - 1.18±0.43 a (2) 1.42±0.42b (2) Changa - - 1.02±0.41a (2) - - Deomani - - 1.04±0.53a (2) - - Values are the means ± sd. Means were compared using Tukey HSD. Values with different superscripts in a column are significantly different at p<0.05
Table 5.38. CYP 450 content in air breathing teleost fish, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus brought from different sites. Values are the mean of (n) number of experiments.
CYP 450 content (nmole/mg protein) Sampling site Channa Channa Channa Heteropneust Clarias punctatus striatus gachua es fossilis batrachus Balarampur 0.56±0.21 b (2) - - 0.66±0.24 b (2) - Katagaon 0.711 (1) 0.400 (1) - 0.710 (1) 1.066 (1) Dangra Dangri 0.34±0.12 a (3) - - 0.25±0.08 a (2) - Ariagoan, 0.32±0.09 a (2) - - 0.325 (1) - Sonapur 0.42±0.18 ab (2) - - 0.42±0.12 a (2) 0.557(1) Magurmari 0.48±0.22 b (4) - - 0.84±0.31 b (3) 0.57±0.28 a (3) Lotchka 0.58±0.28 b (2) - - 0.74±0.33 b (2) 0.54±0.22 a (2) Changa - - 0.28±0.08 a (2) - - Deomani - - 0.19±0.07 a (2) - - Values are the means ± sd. Means were compared using Tukey HSD. Values with different superscripts in a column are significantly different at p<0.05
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Table 5.39. EROD activities in air breathing teleost fish, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus brought from different sites. Values are the mean of (n) number of experiments.
EROD activity (pmole resorufin formed/mg protein/min) Sampling site Channa Channa Channa Heteropneustes Clarias punctatus striatus gachua fossilis batrachus Balarampur 154±32 ab (2) - - 132±48 a (2) - Katagaon 153 (1) 155 (1) - 127 (1) 153(1) Dangra Dangri 107±34 a (3) - - 106±59a (2) - Ariagoan, 100±24 a (2) - - 95 (1) - Sonapur 163±42 b (2) - - 157±46 b (2) 167 (1) Magurmari 160±45 b (4) - - 167±64 b (3) 187±52 a (3) Lotchka 147±28 ab (2) - - 146±51ab (2) 150±56 a (2) Changa - - 83±28 b (2) - - Deomani - - 22±12 a (2) - - Values are the means ± sd. Means were compared using Tukey HSD. Values with different superscripts in a column are significantly different at p<0.05
Table 5.40. N, N-dimethylaniline demethylase (N,N-DMA) activities fish, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus brought from different sites. Values are the mean of (n) number of experiments.
N,N-DMA demethylase activity (nmole formaldehyde formed/mg protein/min) Sampling site Channa Channa Channa Heteropneustes Clarias punctatus striatus gachua fossilis batrachus Balarampur 0.21±0.11a (2) - - 0.20±0.11 a (2) - Katagaon 0.282 (1) 0.137(1) - 0.272 (1) 0.198 (1) Dangra Dangri 0.64±0.31b (3) - - 0.89±0.43 c (2) - Ariagoan, 0.73±0.49 b (2) - - 0.756 (1) - Sonapur 0.17±0.12 a (2) - - 0.16±0.09 a (2) 0.295 (1) Magurmari 0.27±0.13 a (4) - - 0.36±0.16 b (3) 0.31±0.14 a (3) Lotchka 0.21±0.10 a (2) - - 0.31±0.19 a (2) 0.32±0.16 a (2) Changa - - - - - Deomani - - - - - Values are the means ± sd. Means were compared using Tukey HSD. Values with different superscripts in a column are significantly different at p<0.05
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Table 5.41. Aniline hydroxylase (AH) activities in air breathing teleost fish, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus brought from different sites. Values are the mean of (n) number of experiments.
Aniline hydroxylase activity (nmole p-aminophenol formed/mg protein/min) Sampling site Channa Channa Channa Heteropneustes Clarias punctatus striatus gachua fossilis batrachus Balarampur 0.12±0.05 a (2) - - 0.34±0.12 b (2) - Katagaon 0.137 (1) 0.119 (1) - 0.409 (1) 0.341 (1) Dangra Dangri 0.09±0.02 a (3) - - 0.24±0.10 ab (2) - Ariagoan, 0.08±0.03 a (2) - - 0.236 (1) - Sonapur 0.19±0.09 b (2) - - 0.25±0.11 ab (2) 0.327 (1) Magurmari 0.18±0.10 b (4) - - 0.21±0.14 ab (3) 0.30±0.12a (3) Lotchka 0.12±0.06 a (2) - - 0.20±0.09 a (2) 0.33±0.18a (2) Changa - - - - - Deomani - - - - - Values are the means ± sd. Means were compared using Tukey HSD. Values with different superscripts in a column are significantly different at p<0.05
Table 5.42. Erythromycin N-demethylase (ERND) activities in air breathing teleost fish, C. punctatus, C. striatus, C. gachua, H. fossilis and C. batrachus brought from different sites. Values are the mean of (n) number of experiments.
Erythromycin N-demethylase activity (nmole formaldehyde formed/mg protein/min) Sampling site Channa Channa Channa Heteropneustes Clarias punctatus striatus gachua fossilis batrachus Balarampur 0.98±0.42 a (2) - - 1.37±0.85 a (2) - Katagaon 0.866 (1) 1.097 (1) - 1.402 (1) 0.944 (1) Dangra Dangri 2.58±0.91 b (3) - - 3.55±1.58 b (2) - Ariagoan, 2.18±0.98 b (2) - - 4.080 (1) - Sonapur 3.03±1.25 b (2) - - 1.69±0.86 a (2) 2.917 (1) Magurmari 2.09±1.21 b (4) - - 4.13±1.52 b (3) 3.21±1.43a (3) Lotchka 1.69±0.99 ab (2) - - 3.14±1.32 b (2) 2.34±1.02a (2) Changa - - - - - Deomani - - - - - Values are the means ± sd. Means were compared using Tukey HSD. Values with different superscripts in a column are significantly different at p<0.05
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5.10. SDS-PAGE and Heme staining
Electrophoresis of hepatic microsomal preparations from 3 fish species, C. punctatus, H. fossilis and C. batrachus were stained for heme-related peroxidase activity to estimate the relative molecular mass of the CYP 450 enzyme. Following heme staining by N,N,N,N- tetramethyl-p-phenylenediamine (TMPD), proteins in the vicinity of molecular mass 50-56 kDa were stained in each of the 3 fish species along with other bands at around 20-29 kDa as shown by densitometric curve (Fig. 5.7 a, b and c; left panel). Similar enhancement was observed in SDS-PAGE gels as evidenced by coomassie brilliant blue (CBB) staining (Fig. 5.7 a, b and c; left panel).
The SDS electrophoretic profile of microsomal proteins from control and pesticide treated fish species, C. punctatus, H. fossilis and C. batrachus are shown in figure 5.8, 5.9 and 5.10 a, b and c. In C. punctatus, the molecular mass 53-54 kDa protein increased in intensity when treated with all 3 pesticides, cypermethrin, ethion and dicofol but was most pronounced in the dicofol treated (53.4 kDa) followed by the ethion (54 kDa) and cypermethrin (53.5 kDa) treated groups (Fig. 5.8 a, b and c) as evidenced by densitometric curve.
In H. fossilis, a protein band of approximately 52-53 kDa exhibited higher intensity in the pesticide treated fish in comparison to that of control fish. The highest intensity was seen in cypermethrin (52.6 kDa) followed by ethion (52.4 kDa) and dicofol (52.7 kDa) treated fish population (Fig. 5.9 a, b and c).
In microsomal preparation from control and pesticide treated C. batrachus, bands were visualized in molecular mass 50-52 kDa with increased intensity in all pesticide treated groups. The highest intensity in band was seen in dicofol (50.6 kDa) followed by ethion (50.4 kDa) and cypermethrin (51.5 kDa) treated groups (Fig. 5.10 a, b and c).
Many other bands in the different molecular mass range were also seen to have higher intensities in microsomes of treated fish species but were not verified as they were not relevant to the present study.
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MWM LS*
Figure 5.7a. Differentially stained SDS-PAGE protein gels showing the analysis of CYP 450 enzymes from hepatic microsomes in C. punctatus. Left panel: Protein gel developed with TMPD/hydrogen peroxide stain specific for heme. Right panel: Protein gel stained with Coomassie dye. A faint band corresponding to free heme at around ~53.55 kDa can be seen as indicated by the arrow.
MWM= Molecular weight marker; LS = liver sample
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MWM LS*
Figure 5.7b. Differentially stained SDS-PAGE protein gels showing the analysis of CYP 450 enzymes from hepatic microsomes in H. fossilis. Left panel: Protein gel developed with TMPD/hydrogen peroxide stain specific for heme. Right panel: Protein gel stained with Coomassie dye. A faint band corresponding to free heme at around ~52.9 kDa can be seen as indicated by the arrow.
MWM= Molecular weight marker; LS = liver sample
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MWM LS*
Figure 5.7c. Differentially stained SDS-PAGE protein gels showing the analysis of CYP 450 enzymes from hepatic microsomes in C. batrachus. Left panel: Protein gel developed with TMPD/hydrogen peroxide stain specific for heme. Right panel: Protein gel stained with Coomassie dye. A faint band corresponding to free heme can be seen at around ~50.1 kDa as indicated by the arrow.
MWM= Molecular weight marker; LS = liver sample
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MWM LSC* LST*
Figure 5.8a. 10% SDS-PAGE of the microsomal fraction of liver of cypermethrin treated and control fish, C. punctatus stained with 0.025% CBB. The documentation was done using Spectronics ImageAide software, version 3.06.04. When the gel was analysed using gel documentation software, the protein bands in the region of ~53.5 kDa was visualized.
MWM= Molecular weight marker; LSC= liver sample control; LST = liver sample treated
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MWM LSC* LST*
Figure 5.8b. 10% SDS-PAGE of the microsomal fraction of liver of ethion treated and control fish, C. punctatus stained with 0.025% CBB. The documentation was done using Spectronics ImageAide software, version 3.06.04. When the gel was analysed using gel documentation software, the protein bands in the region of ~54 kDa was visualized.
MWM= Molecular weight marker; LSC= liver sample control; LST = liver sample treated
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MWM LSC* LST*
Figure 5.8c. 10% SDS-PAGE of the microsomal fraction of liver of dicofol treated and control fish, C. punctatus stained with 0.025% CBB. The documentation was done using Spectronics ImageAide software, version 3.06.04. When the gel was analysed using gel documentation software, the protein bands in the region of ~53.4 kDa was visualized.
MWM= Molecular weight marker; LSC= liver sample control; LST = liver sample treated
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MWM LSC* LST*
Figure 5.9a. 10% SDS-PAGE of the microsomal fraction of liver of cypermethrin treated and control fish, H. fossilis stained with 0.025% CBB. The documentation was done using Spectronics ImageAide software, version 3.06.04. When the gel was analysed using gel documentation software, the protein bands in the region of ~52.6 kDa was visualized.
MWM= Molecular weight marker; LSC= liver sample control; LST = liver sample treated
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MWM LSC* LST*
Figure 5.9b. 10% SDS-PAGE of the microsomal fraction of liver of ethion treated and control fish, H. fossilis stained with 0.025% CBB. The documentation was done using Spectronics ImageAide software, version 3.06.04. When the gel was analysed using gel documentation software, the protein bands in the region of ~52.4 kDa were visualized.
MWM= Molecular weight marker; LSC= liver sample control; LST = liver sample treated
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MWM LSC* LST*
Figure 5.9c. 10% SDS-PAGE of the microsomal fraction of liver of dicofol treated and control fish, H. fossilis stained with 0.025% CBB. The documentation was done using Spectronics ImageAide software, version 3.06.04. When the gel was analysed using gel documentation software, the protein bands in the region of ~52.7 kDa were visualized.
MWM= Molecular weight marker; LSC= liver sample control; LST = liver sample treated
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MWM LSC* LST*
Figure 5.10a. 10% SDS-PAGE of the microsomal fraction of liver of cypermethrin treated and control fish, C. batrachus stained with 0.025% CBB. The documentation was done using Spectronics ImageAide software, version 3.06.04. When the gel was analysed using gel documentation software, the protein bands in the region of ~51.5 kDa were visualized.
MWM= Molecular weight marker; LSC= liver sample control; LST = liver sample treated
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MWM LSC* LST*
Figure 5.10b. 10% SDS-PAGE of the microsomal fraction of liver of ethion treated and control fish, C. batrachus stained with 0.025% CBB. The documentation was done using Spectronics ImageAide software, version 3.06.04. When the gel was analysed using gel documentation software, the protein bands in the region of ~50.4 kDa were visualized.
MWM= Molecular weight marker; LSC= liver sample control; LST = liver sample treated
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MWM LSC* LST*
Figure 5.10c. 10% SDS-PAGE of the microsomal fraction of liver of dicofol treated and control fish, C. batrachus stained with 0.025% CBB. The documentation was done using Spectronics ImageAide software, version 3.06.04. When the gel was analysed using gel documentation software, the protein bands in the region of ~ 50.6 kDa were visualized.
MWM= Molecular weight marker; LSC= liver sample control; LST = liver sample treated
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5.11. Nucleotide sequence analysis of genomic CYP1A and phylogenetic analysis of 3 fish species, C. punctatus, H. fossilis and C. batrachus
The CYP1A genes of 3 fish species, C. punctatus, H. fossilis and C. batrachus were successfully amplified by using the primers presented in Table 4.3 in materials and methods.
In C. punctatus, the 3 sequences of CYP1A genomic DNA were amplified with 3 different primers and were submitted to the GenBank. The received accession number of the sequences were KP203843 (1041 bp), KP231221 (1498 bp) and KP271996 (1108 bp). These 3 sequences of genomic DNA were joined together to make it a single genomic DNA. Sequence analysis showed that the obtained sequence contained CYP1A structural gene of approximately 2511 bp in length for C. punctatus (Fig.5.11). In both H. fossilis and C. batrachus, a single primer was used to amplify CYP1A genomic DNA. Sequence analysis showed 534 bp for H. fossilis (Fig. 5.12) and 509 bp for C. batrachus (Fig. 5.13). All the gene sequences were successfully deposited in the GenBank/NCBI data bank except the gene of H. fossilis which was deposited in GenBank/EMBL. The accession numbers of CYP1A genes received from GenBank were KP282054 for C. punctatus, LN736019 for H. fossilis and KP336485 for C. batrachus.
Comparison of genomic DNA of C. punctatus with CYP1A cDNA sequence of C. punctatus obtained from NCBI (EU930319) identified 6 exons and 5 introns. The nucleotide size of 6 exons were 720, 127, 90, 124, 87 and 298 bp respectively with 389 bp of untranslated sequence in 3’ region while the nucleotide size of 5 introns were 124, 102, 106, 123 and 221 bp respectively (Table 5.43). cDNA of the CYP1A gene obtained from NCBI had 1566 bp of the coding region but in the genomic DNA of the present study initial 120 bp were not amplified and only 1446 bp sequence were obtained coding 481 amino acid.
The amplified sequences of H. fossilis (534 bp) and C. batrachus (509 bp) showed highest similarity/homogeneity with cDNA sequences of Peltobagrus fulvidraco (yellow catfish). When comparing their genomic DNA with CYP1A cDNA sequence of P. fulvidraco obtained from NCBI, 2 exons were identified for each fish species with a nucleotide size of 381 and 57 bp for H. fossilis coding 146 amino acids, and 366 and 46 bp for C. batrachus coding 137 amino acids. Both the species had 1 intron with H. fossilis having nucleotide size of 94 bp and C. batrachus having nucleotide size of 97 bp respectively (Table 5.43). All the introns begin with the sequence GT and end with AG.
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5.11.1. Comparison of amino acid sequences
Table 5.44 shows the percent identities of deduced amino acid sequences of C. punctatus, H. fossilis and C. batrachus CYP1A between and with the other fish CYP1A genes employed in the present study. The highest identity was 93% between C. punctatus and C. maculata and between H. fossilis and C. batrachus. Closely followed was the 86% and 85% identity of H. fossilis and C. batrachus with P. fulvidraco.
5.11.2. Phylogenetic analysis
The phylogenetic tree based on the amino acid sequences were used to assess the relationship of CYP1A of C. punctatus, H. fossilis and C. batrachus with those of other fish species belonging to order Cypriniformes, Salmoniformes, Perciformes and Siluriformes. Figure 5.14 clearly shows C. punctatus and C. maculata CYP1A and H. fossilis and C. batrachus CYP1A to be more closely related to each other than to other fish CYP1A. H. fossilis and C. batrachus CYP1A was more closely related with P. fulvidraco (yellow catfish). Comparing the orders, the order Siluriformes (Ancistrus sp., Peltobagrus fulvidraco, H. fossilis and C. batrachus) was closely related to order Cypriniformes (Catla catla and Cyprinus carpio). The order Perciformes (Stenotomus chrysops, Oreochromis niloticus, Channa maculate and C. punctatus) was closely related to Salmoniformes (Oncorhynchus mykiss).
>Channa (2511 bp)
>gi|794541524|gb|KP282054.1| Channa punctata isolate CHANNA cytochrome p450 CYP1A (cyp1A) gene, partial cds
1 GGGCTTTGTC GTCTCCCTGG CCCAAAGCCC TTGCCTCTCA TCGGGAATGT GCTGGAGCTT 61 CGCCACAAAC CTTATCAGAG TCTCACTGCT ATGAGCAAGC GTTATGGTCA TGTCTTCCAG 121 ATCCACATTG GCACACGTCC TGTGGTTGTG TTGAGTGGCA GTGAGACGGT TCGTCAGGCT 181 CTCATCAAGC AAGGGGAAGA GTTCGCAGGC AGACCTGACT TGTACAGCTT TCAATTCATC 241 AATGACGGAA AAAGTCTGGC TTTCAGTACA GATCAGTCTG GTGTCTGGCG TGCTCGCAGA 301 AAGCTGGCTT ACAGTGCCCT GCGCTCCTTT TCCAACCTGG AGAGCAAGAA CTCAGAGTAC 361 TCCTGTGTTC TAGAAGAACA CGTCAGTAAA GAGGCAGAGT ATCTAATCAA ACGACTCTGC 421 ACTGTCATGA AGGCAGACGG CAGCTTCGAC CCCGTCCGTC ACATTGTCGT CTCTGTGGCA 481 AATGTCATCT GCGGAATTTG CTTTGGCCGA CGCTACAGCC ACGATGACCA GGAGCTGCTC 541 AGCTTAGTGA CCCTTGCTGA TGACTTTAAC CAGGTGGCGG GAAGTGGGAA CCCTGCTGAC
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601 TTCATCCCCA TTCTCCAGTA TCTGCCTAGC AGAAACATGA AGAATTTTAT GGACCTCAAT 661 GCTCGCTTCA ACAGCTTTGT GCAAAAAATA GTCAGAGAAC ACTATGCCAC CTACGACAAG 721 gtacaccgca aactaaatca ctcatgaaat gagttcacgt tttctcaaag ccataatttc 781 cttattattt ttgtgtgtat ttgtatgtac gtatgtgatt gtgattgttc ttaacttttt 841 tcagGACAAC ATCCGTGATA TCACAGACTC CCTCATTGAT CACTGCGAGG ACAGGAAGCT 901 GGATGAGAAC TTCAATGTTC AGGTGTCAGA TGAGAAGATT GTGGGAATTG TCAATGACCT 961 ATTTGGGGCT Ggtacgctca cttcctatgt actgaataaa cctattgttg atatggtgat 1021 gtctgcctaa agaaaacaaa agtgccctca cataattaac atctcccttt cagGTTTTGA 1081 CACTGTCACC ACTGCATTGT CATGGTCAGT GATGTACATG GTGGCTTACC CAGAGATACA 1141 AGAGAGGCTT TATGATGAGC TGAgtaagta cactgatttt ggattttaca gttctattgc 1201 aaaaccactg aaggaatgtg gactaaaatc caaaaaaacg catactgagc atggatgact 1261 ttttttcagA GGTCAATGTG GGTCTGGAGC GGAGCCCGCG TCTCTCTGAT AAACCCAATT 1321 TACCATTTCT GGAGGCCTTC ATCCTGGAGA TGTTGCGGCA CTCTTCATTC CTGCCCTTTA 1381 CTATCCCACA CTGgtaaggt tcaactcaaa aagggtgaaa atagagctgt taagtgcata 1441 cttagtttta caggaccaca caaaaacaga tgcatgggat cgtgaggaga ctaagccttt 1501 gtttttcttt tctcagCACC ACAAAAGACA CGTCTCTGAA TGGCTACTTC ATTCCAAAAG 1561 ATACCTGTGT CTTCATCAAT CAGTGGCAGA TTAACCATGA TCCgtaagtt ctttttgtta 1621 cattttaaaa actttaaaac aaagcatgag gtgtaccaac ctaacagtta cacagtaaaa 1681 aaactgaagc agaaaactac ttacataaaa ccattaaagg aataagtaca gaaataagtt 1741 tgtcctaatg gaatttggag ctaaagccaa agatttattg tgcttttgta tatgacttac 1801 atttcctttc tctacccctc tcagTGAGCT GTGGAAAGAT CCATTTTCCT TCAACCCAGA 1861 CCGCTTCTTG AGCGCTGATA GCACTGAGGT CAACAAGGTG GAAGGGGAGA AGGTAGTGGC 1921 TTTCGGCCTA GGAAAGCGGC GCTGCATCGG CGAGGTCATT GCACGAAATG AAGTCTACCT 1981 CTTCTTGGCA ATTCTTATCC AGAAGCTAGA GTTCCACCAA ATGCCTGGGG TACCACTGGA 2041 CATGACGCCA CAATATGGTC TCACAATGAA ACACAAACCC TGCCACCTGA GAGCCACAAT 2101 GCGAGCAATC AATGAGCAGT GAAACTATTT ATATATTTAC TGTATTCAGA ATGAATTACT 2161 CAACAGTTGA TAATTGTTCA CTCAAGAACT GTAAGGTTCA GTGAAACAAG CATCTTTCTC 2221 AGAATGTACG GCAATGGTTG CCAAATCTGA TACATAGAGC AAATGGATTT GAAGCAAATA 2281 GGTGAAATTT GCTTGCTTGC TTGCAGACTG TCAGATATTT CTGGGTTTGT AAGATGAGGG 2341 GTTCCTCTAT ATGATCGATG CGTCTTGAGG AAGAATAAGC AGATACTTGG TTTTCCTGCT 2401 GTGTTTTTTT GCTGTGCTGG AAACGTAACT GTTCTTATGT AGAAGTTGTA TAGCACACAA 2461 ACTATGTTGC TTCAATCAAC TTTGGGACAC ATTATGTTTT ACTGGATATG C
Figure 5.11. Nucleotide sequences (2511 bp) of C. punctatus genomic cytochrome CYP1A. The nucleotides are represented in uppercase letters for the exons and in lowercase letters for the introns. The nucleotide marked by yellow colour represents 389 bp of untranslated sequence in 3’ region.
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H. fossilis (534 bp)
>gi|748762338|emb|LN736019.1| Heteropneustes fossilis partial cyp4501a gene for Cytochrome P450 1A
1 TCGAGGGTGA GAGTTCTGAG TATTCCTGTG CCCTGGAGGA ACACATCAGC AAAGAGGGCC 61 TGTACCTGAT CGAGAGGCTG CACAGTGTTA TGAAGGCCAA TGGTGGATTC GACCCGTTCC 121 GTCACATTGT GGTGTCTGTG ACAAACGTGA TCTGTGGCAT GTGCTTTGGC CGACGCTACA 181 GCCACGACGA CCACGAGCTG TTAAGCCTGG TGAACTTAAG CGAAGAGTTC AACCAAGTGG 241 TGGGCAGCGG AAACCCGGCC GACTTCATTC CCTTCCTGCG CCTCCTGCCC AGCACGAGCA 301 TGAATAAATT CCTGGCCATC AACCAGCGGT TTAACGTGTT CATGCAGAAG CTGGTCAGAG 361 AGCATTACGA GACATTCAAT AAGgttcgtg cacagtgtat acaaatttca tgacgagatt 421 ctgtcaaaat tcaggatgct cacactgtct tgtatcttct gattttttgt tttttagGAC 481 AACATCCGTG ATATCACTGA CTCTCTCATC GATCACTGTG AGGACAGGAA GCTG
Figure 5.12. Nucleotide sequences (534 bp) of H. fossilis genomic cytochrome CYP1A. The nucleotides are represented in uppercase letters for the exons and in lowercase letters for the introns.
C. batrachus (509 bp) >gi|794541545|gb|KP336485.1| Clarias batrachus isolate HM514 cytochrome p450 CYP1A (cyp1A) gene, partial cds
1 GAGTACTCCT GCGCCCTGGA GGAACACATC AGCAAGGAAG GCCTGTACCT GATTGAGAGG 61 CTGCACAGTG TTATGAAGGC CAGCGGCGGA TTCGACCCGT TCAGTCACAT TGTTGTGACT 121 GTCACGAACG TGATCTGCGG CATGTGCTTT GGTCGCCGCT ACAGCCACGA TGACCGGGAA 181 CTGTTAAGCC TGGTGAACTT AAGCGAAGAG TTCAACCAAG TGGTGGGCAG CGGAAACCCG 241 GCTGACTTCA TTCCCTTCCT GCGCCTCCTG CCCAGCACGA GCATGAAGAA ATTCCTGGCC 301 ATCAACGAGC GCTTTAACGT GTTCATGCAG AGGCTGGTCA AAGAGCATTA CGAGACATAC 361 AATAAGgttc gtgcacactt tgatcaagtg tatccgaata gcgtgatgag attccatcaa 421 aaattagcat gttcacgctt tgtgtttttt tctttccctc cagGACAACA TTCGTGATAT 481 CACAGACTCT CTCATCGATC ACTGTGAGG
Figure 5.13. Nucleotide sequences (509 bp) of C. batrachus genomic cytochrome CYP1A. The nucleotides are represented in uppercase letters for the exons and in lowercase letters for the introns.
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Table 5.43. Comparison of CYP1A exons and introns length (bp).
C. punctatus H. fossilis C. batrachus Exons Introns Exons Introns Exons Introns 720 bp 124 bp 381 bp 94 bp 366 bp 97 bp 127 bp 102 bp 57 bp 46 bp 90 bp 106 bp 124 bp 123 bp 87 bp 221 bp 298 bp
Table 5.44. Percent identities of deduced amino acid sequences of fish CYP1A gene subfamilies.
Cyprinus Rainbow Channa Scup Nile P. Ancistrus Channa H. Clarias carpio trout maculate tilapia fulvidraco sp. punctatus fossilis batrachus Catla catla 92 77 76 76 73 78 76 75 82 80 Cyprinus 80 77 79 74 79 78 76 81 78 carpio Rainbow 81 82 74 75 76 78 75 75 trout Channa 83 77 76 76 93 75 74 maculate Scup 79 77 76 81 76 74 Nile tilapia 71 70 78 73 72 Peltobagrus 80 76 86 85 fulvidraco Ancistrus sp. 74 76 74 Channa 74 73 punctatus Heteropneustes 93 fossilis
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Figure 5.14. Phylogenetic tree of CYP1A genes constructed by the neighbour joining method using the amino acid sequences. A number at each branch and the length of the stem indicate bootstrap value. Evolutionary analyses were conducted in MEGA6.
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6. DISCUSSION
Spectral analysis
The principal method for spectral analysis of CYP 450 involves what is termed the ferrous- CO difference spectrum. In our study, two soret peaks were seen in the dithionite reduced spectra of liver microsomes in control and experimental groups of all 3 fish studied, C. punctatus, H. fossilis and C. batrachus. These peaks consisted of the characteristic absorbance at around 450 nm for the reduced CYP 450-CO complex and the other of lesser magnitude at around 420-425 nm (Fig. 5.1, 5.2 and 5.3 a and b). The maximum absorbance was noticed at 1–2 min and then a gradual decrease in absorbance was seen with the increase in time interval. This time interval corresponds to the duration of incubation necessary for maximum reduction of the enzyme by sodium dithionite.
The CYP 450 is converted to a stable denatured state CYP 420 under long periods of incubation thus adding to the absorbance at 420-425 nm or may be possibly due to the absorbance of contaminating haemoglobin or cytochrome b5 (Klingenberg, 1958; Garfinkle, 1958; Omura and Sato, 1962; Arinc and Cakir, 1999). Not only CYP 450 binds CO in its reduced (ferrous) form, haemoglobin and other hemoproteins also bind CO in reduced form (Schenkman and Jansson, 2006; Guengerich et al., 2009).
The spectral absorbance at 420-425 nm was also described in a study with Neotropical freshwater fish species and adopted tissue perfusion method to decrease the influence of haemoglobin contamination of microsomes (Leitao et al., 2000). Similar unusual spectra were obtained when fish, Hypostomus punctatus and Ancistrus multispinis were examined for spectral analysis (Klemz et al., 2010). The peak at 420-425 nm in the current study may be due to the haemoglobin contamination since tissues could not be perfused adequately. Overall, the spectral analysis with the peak absorbance at around 450 nm also indicated the presence of active CYP 450 enzyme in hepatic microsomes.
Multiple forms of cytochrome P450
The spectral analysis of H. fossilis treated with naphthalene, phenobarbitone, deflazacort and acetone revealed dissimilar absorption peaks in comparison to that of control fish. The control fish showed a maximum peak at 450 nm, naphthalene at 451 nm, phenobarbitone at 452 nm, acetone at 450 nm and that of deflazacort treated at 449 nm respectively (Fig. 5.4
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and Table 5.1). The hepatic CYP 450 system consists of a family of inducible isoenzymes that possess different spectral and immunological characteristics, substrate preferences and primary protein structures (Wickramasinghe et al., 1980; Guengerich et al., 1982). Purification and separation procedures for CYP 450 often take advantage of the fact that different prominent forms are induced by particular xenobiotics and one to five forms has been accomplished in rat liver microsomes treated with polychlorinated biphenyls, phenobarbital, 3-methylcholanthrene and β-naphthoflavone, which displayed a spectral characteristics of CYP 450 peaks ranging from 446 to 452 nm (Kotake and Funae, 1980; Lau and Strobel, 1982; Seidel and Shires, 1986).
In the present study, H. fossilis treated with naphthalene (66.274±23.059 pmole resorufin formed/mg protein/minute) displayed a significant induction (p<0.05) in EROD activity when compared with the control (17.989±12.034 pmole resorufin formed/mg protein/minute) and phenobarbitone, acetone and deflazacort showed marginal increase in EROD activity (Table 5.2). So the fish, H. fossilis treated with naphthalene indicated its specificity to induce CYP1A isoform. Pacheco and Santos (2002) have reported that naphthalene also acts as a specific inducer of CYP1A in European eel. Although several isoforms have been identified in fish, CYP1A has received the most attention as the major hydrocarbon-inducible CYP 450.
CYP1A induction is mediated by the Ah receptor (AhR), a xenobiotic-binding protein present in the cytosol (Lewis, 2001). Well-established inducers of CYP1A and EROD are organic contaminants belonging to polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), nitrated polyaromatic hydrocarbons (NPAHs), dioxins, some pesticides (Machala et al., 1997; Jung et al., 2001) and have been so far proved to be the most sensitive indicator (Schlenk and Di Giulio, 2002).
There were no significant difference in N, N-dimethylaniline demethylase (N,N-DMA) activity between the control (0.054±0.045 nmole formaldehyde formed/mg protein/minute) and the phenobarbitone treated group (0.060±0.013 nmole formaldehyde formed/mg protein/minute) of fish, H. fossilis as well as the fish treated with naphthalene, acetone and deflazacort. The acetone treated group (0.036±0.005 nmole formaldehyde formed/mg protein/minute) showed a slightly lower activity but was not significant (Table 5.2). Thus the inducers phenobarbitone, naphthalene, acetone and deflazacort have no effect on CYP2B isoform responsible for mediating N,N-DMA activity. Goksoyr and Forlin (1992) concluded that subfamily CYP2B is totally absent in fish, however, over the last decades, fish liver microsomes have been shown to metabolize prototypical mammalian CYP2B substrates
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(Elskus and Stegemann, 1989; Haasch et al., 1994). In mammals, phenobarbital is an in vivo inducer of the CYP2B family, whereas in teleosts CYP 450 induction by phenobarbital is unclear (Sadars et al., 1996). In contrast, Stegemann et al. (1997) reported the presence of CYP2B like proteins in four tropical fish species (Perca fluviatilis, Cephalopholis cruentata, Haemulon aurolineatum and Abudefdud saxatilis) using immunoblot assay with rat CYP2B1 and CYP2B-like scup P450B antibodies.
Acetone treatment in fish, H. fossilis showed significant induction (p<0.01) in aniline hydroxylase (AH) activity (0.277±0.008 nmole p-aminophenol formed/mg protein/minute) as compared to control (0.037±0.028 nmole p-aminophenol formed/mg protein/minute). The fish treated with naphthalene and phenobarbitone showed a marginal decrease in aniline hydroxylase activity while deflazacort showed a marginal increase in the activity (Table 5.2). CYP2E1 metabolizes a number of low-molecular-weight pharmaceutical and other xenobiotic compounds, like acetaminophen, ethanol, isoniazid, halogenated anaesthetics, acetone and benzene (Tu et al., 1983; Lee et al., 1996; Gonzalez, 2005). The present study showed that in H. fossilis acetone induces AH activity indicating the presence of CYP2E1. CYP2E1 is also reported to be induced by starvation in animal models (Lieber, 1997). CYP 450 dependent aniline hydroxylation enzyme system is known to be a possible way for metabolism in the liver of fish species and aniline hydroxylase is known to be a member of mixed-function oxidases catalyzing the transformation of toxic aniline to nontoxic p-aminophenol (Zen and Korkmaz, 2009).
Deflazacort (synthetic corticosteroid) treated H. fossilis showed significant (p<0.01) induction of erythromycin N-demethylase (ERND) activity (3.129±0.545 nmole formaldehyde formed/mg protein/minute) whereas the naphthalene, phenobarbitone and acetone treated groups showed lower activity than that of the control (0.898±0.062 nmole formaldehyde formed/mg protein/minute) with significant difference seen in phenobarbitone (0.506±0.103 nmole formaldehyde formed/mg protein/minute; p<0.05) and acetone (0.127±0.22nmole formaldehyde formed/mg protein/minute; p<0.01) treated group (Table 5.2). CYP3A plays an important role in the metabolism of endogenous substances and xenobiotics including pharmaceuticals and is inducible by steroidal chemicals and a variety of naturally occurring compounds and synthetic glucocorticoids and macrolide antibiotics (Wickramasinghe, 1990; Quattrochi and Guzelian, 2001). Induction of CYP3A expression by dexamethasone has been shown to occur in fish (Tseng et al., 2005). As there is increase in ERND activity after treatment of the fish with deflazacort, a synthetic corticosteroid, it can be
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concluded that CYP3A4 is present and induced in H. fossilis. Strong similarities in the structure and catalytic function of CYP3A4 between fish and mammals have been reported through the use of polyclonal antibodies (Popovic et al., 2013).
The inducers, naphthalene, phenobarbitone, acetone and deflazacort administered to fish, H. fossilis did not have a profound effect on other CYP 450 isoform apart from its chosen specific isoform. The CYP 450 family- CYP1A, CYP2E1 and CYP3A4 show similar biotransformation reaction as that reported for various mammalian species (Chan et al., 2009) but CYP2B did not follow the trend though still a lot of studies have to be conducted.
Studies with various mammalian species have clearly confirmed the presence of multiple forms of hepatic (Lu and Levin, 1974) and extrahepatic (Wolf et al., 1978) CYP 450 and have shown that various CYP 450 isoforms have divergent substrate specificities and are under different regulatory control. However, almost every isoform has its specific substrate that can be used for its identification (Anzenbacherova and Anzenbacher, 2001; Lewis, 2001). In contrast, bacterial and mitochondrial CYP 450 isoforms are highly substrate- specific (Lewis 2001). Baron et al. (2001) reported the presence of multiple CYP 450 isoforms in humans, especially CYP1A1, 1B1, 2B6, 2E1, and 3A by carefully studying on RNA, protein, morphologic and catalytic level by reverse transcriptase-PCR, immunoblot, immunohistochemistry and catalytic assays.
Induction of microsomal mixed-function oxidase system can influence the metabolism and toxicity of xenobiotics in several ways. Due to the similarities between xenobiotic metabolism in fish and mammalian species, it is not surprising that similar toxicological responses are observed with some chemicals in fish and mammals (Lech and Bend, 1980). However, some standard compounds that act in mammals as CYP 450 inducers are not acting as inducers in fish, such as phenobarbital or methoxychlor do not induce CYP2B or CYP3A, respectively (Ruus et al., 2002; Stuchal et al., 2006).
β-naphthoflavone (β-NF) is reported as a specific inducer of CYP1A in all of the vertebrate taxa (Prettia et al., 2001). All the 3 fish, C. punctatus (194.811±7.961 pmole resorufin formed/mg protein/minute), H. fossilis (255.415±9.651 pmol resorufin formed/mg protein/minute) and C. batrachus (185.252±22.159 pmole resorufin formed/mg protein/minute) treated with β-naphthoflavone displayed a significant increase (p<0.001) in EROD activity suggesting its specificity to induce CYP1A isoform (Table 5.4). Significant differences were also observed between control and all the 3 β-naphthoflavone treated fish
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species in CYP 450 content. Prettia et al. (2001) have reported an increase in CYP 450 content and EROD activity in gilthead seabream after treatment with β-NF.
The fish, C. punctatus, H. fossilis and C. batrachus showed a negligible response towards β- naphthoflavone treatment in N, N-dimethylaniline demethylase and aniline hydroxylase activity compared to their respective control group indicating no effect on CYP2B and CYP2E1 isoform (Table 5.4). However, all the fish, C. punctatus, H. fossilis and C. batrachus administered with β-NF displayed an elevated level in erythromycin N- demethylase activity catalysed by CYP3A4 isoform though only H. fossilis (1.026±0.078 nmole formaldehyde formed/mg protein/minute) showed a significant difference (p<0.05) when compared to their respective control (Table 5.4). Husoy et al. (1994) have showed the occurrence of CYP3A-like isozymes in many organs of the body in control and β–NF treated cod by immunohistochemistry analysis.
All the 3 fish species, C. Punctatus, H. fossilis and C. batrachus administered with β-NF displayed an induction in CYP1A activity but had no major influence in CYP2B, CYP2E1 and CYP3A4 activities. The results from the current study suggest that fish CYP 450 responds differentially to different xenobiotics and indicated the presence of multiple forms of CYP 450 in fish (CYP1A, CYP2B, CYP2E1 and CYP3A4).
Acute toxicity (LC50)
The LC50 value represents the mortality of 50% of the tested organisms exposed to particular toxicants. Determination of LC50 value for the pesticide to a particular fish is important as they provide information about the safe level of pesticide use in the agricultural or household or any other application. In the present study, we also calculated the LC50 value of the pesticides cypermethrin (Ripcord 10% EC, pyrethroid), ethion (ethion 50 % EC, organophosphate) and dicofol (COLONEL S 18.5 % EC, organochlorine) in C. punctatus, H. fossilis and C. batrachus. The LC50 values of cypermethrin in C. punctatus, H. fossilis and C. batrachus was calculated to be 19.9 µg/L, 3.7 µg/L and 5.6 µg/L respectively (Table 5.5).
However, different authors have reported different LC50 values in their study. Saha and
Kaviraj (2003), Kumar et al. (2007) and Begum (2007) reported the LC50 value of cypermethrin as 0.67 μg/L, 0.4 mg/L, and 0.07 mg/L in H. fossilis, C. punctatus and C. batrachus respectively.
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The LC50 values of ethion in C. punctatus, H. fossilis and C. batrachus was calculated to be 43.9 µg/L, 54.8 µg/L and 48.7 µg/L respectively (Table 5.6). However, Bhatnagar et al.
(2003) reported the LC50 value of 0.43 mg/L for common carp, Cyprinus carpio when exposed to pesticide Match (Ethion-50% EC) which is higher than the value found in our study. The LC50 value of chlorpyrifos, the other class of organophosphate pesticide in fish C. punctatus, C. batrachus and H. fossilis was cited as 0.81 mg/L (Ali et al., 2008), 16.5 mg/L (Narra et al., 2011) and 2.84 mg/L (Khatun and Mahanta, 2014) respectively which is much higher than ethion reported in our study. The LC50 value of ethion in C. punctatus, H. fossilis and C. batrachus was lacking in literature.
The LC50 values of dicofol in C. punctatus, H. fossilis and C. batrachus was calculated to be
45.8 µg/L, 36.1 µg/L and 51.8 µg/L respectively (Table 5.7). The LC50 value, however, is lower than the 0.72 mg/L reported by Veeraiah et al. (2013) for C. punctatus exposed to dicofol 18.5% EC. The LC50 value of dicofol for H. fossilis and C. batrachus was lacking in literature though the LC50 value of endosulfan, another class of organochlorine pesticide was reported to be 0.75 mg/L (Singh and Srivastava, 1981), 0.6 mg/L (Tripathi and Verma, 2004) and 22.15 μg/L (Haloi et al., 2014) for H. fossilis, C. batrachus and C. punctatus respectively.
Of the 3 pesticides tested (cypermethrin, ethion and dicofol), cypermethrin was found to be the most sensitive to all the fish studied. It is in accordance with the previous study reported by Bradbury and Coates (1989), that fish have poor ability to metabolize and eliminate pyrethroid pesticide as compared to those of higher vertebrates. It is difficult to compare the toxicity of an individual pesticide to different species of fish as it is found to be influenced by several factors like physicochemical parameters of water (temperature, hardness, pH and dissolved oxygen content, etc), age, size and formulations of chemicals (Pandey et al., 2011).
Higher LC50 values are generally considered to be less toxic because greater concentrations are required to produce 50% mortality in animals (Hedayati et al., 2010). The acute toxicity test is used to determine the susceptibility and survival potential of animals to a toxic agent that produces a deleterious effect on a group of experimental organisms during a short-term exposure under controlled conditions, and to measure the effect of toxic chemicals in the biology of the tested species (Rani et al., 2011). Though acute toxicity test is not an accurate method of getting the exact value of LC50, however, it is somewhat the best method till now to get an idea of the threshold level of pesticide toxicity to a particular fish or any aquatic organism (Haloi et al., 2014).
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All the experimental fish, C. punctatus, H. fossilis and C. batrachus were observed to visit the surface of water much more frequently to gulf the air after the application of tested pesticide. Further dose-dependent abnormal behavioural changes in fish were noticed indicating the stages of stress response. The present study showed that the pesticide, cypermethrin, ethion and dicofol are highly toxic to fish, C. punctatus, H. fossilis and C. batrachus even if exposed to a minute concentration.
Liver somatic index (LSI), Microsomal protein content and CYP 450 content
The liver contains the bulk of the detoxification potential and it can be inferred that the addition of liver mass by the animals could be an effective metabolic investment directed towards increased overall functional capacity of the organ to allow for more efficient detoxification. In this study, all the experimental fish reflected an increase in their LSI values compared to that of the control group (Table 5.3, 5.8, 5.15 and 5.22). The LSI values were higher in all the 3 β-naphthoflavone treated fish, C. punctatus, H. fossilis and C. batrachus but the significant difference (p<0.05) was revealed only by C. batrachus (1.219±0.076) compared to their respective control group (Table 5.3).
All the cypermethrin treated fish displayed elevated LSI values but the significant difference (p<0.05) was shown by only 15 days treated group of C. punctatus (1.042±0.109) with 1.1 fold increase and H. fossilis (1.245±0.144) with 1.2 fold increase while C. batrachus showed no significant increase in any treated groups compared to their respective control group (Table 5.8). In case of ethion treatment, the significant difference (p<0.01) in LSI values were shown by 10 days (1.075±0.085) and 15 days (1.155±0.122) treated groups of C. punctatus, while H. fossilis and C. batrachus showed no significant increase in any of the treated groups (5, 10 and 15 days) compared to their respective control group. Highest elevation with 1.2 fold increase in LSI value was displayed by 15 days treated C. punctatus (Table 5.15). All of the dicofol treated fish displayed significant difference in LSI values when compared with their respective control group. Highest elevation with 1.5 fold increase was shown by 5 days (1.531±0.488) treated C. batrachus (Table 5.22).
The increase in LSI is commonly seen in fish exposed for longer periods of time to contaminants in both the laboratory and field (Whatley et al., 2010). This increase is not unexpected since the liver is regarded as the main detoxification organ and functions in storing and metabolising toxicants. LSI has frequently been used as a biomarker for
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examining fish exposed to environmental contaminants and also to determine the physiological status that can establish the welfare and condition of fish (Goede and Barton, 1990; Sole et al., 2009). Although, identification of the cause for LSI changes was not within the scope of this study, the higher LSI value in experimental fish compared to the control fish suggests that it may be due to the effect of pesticides. LSI values are generally elevated in vertebrates experiencing stimulation of hepatic microsomal CYP 450 for the detoxification of organic compounds and the elevation in LSI may be due to the altered allowance of energy reserves for detoxification of organic compounds (Miller et al., 2009).
The increase in LSI may be associated with the increased capacity to metabolize xenobiotics and may be considered as adaption to the presence of pollution rather than a dysfunction (Heath, 1995). This increase could also be linked to an increase in cell numbers, which is the response of the organism to the toxins leading to an increase in liver size coupled with pollutant exposure (Goede and Barton, 1990; Face et al., 2005). However, a histological assessment was not carried out on the liver, therefore, this increase in liver size could either be linked to an increase in the number (hyperplasia) or an increase in the size (hypertrophy) of the hepatocytes. Further investigation is needed.
The amount of microsomal protein per gram of liver is a critical scaling factor used in toxicology study models to extrapolate in vitro rates of metabolism to xenobiotic clearance in vivo. The liver is the main site of metabolic clearance and an accumulation of liver protein could result from an increased rate of synthesis or a decreased rate of breakdown. In this study, we found that the microsomal protein content was significantly higher in all the experimental groups of fish, C. punctatus, H. fossilis and C. batrachus when compared to their respective control group. The microsomal protein content increased with increasing days of exposure (5, 10 and 15 days) to the pesticide tested (Table 5.9, 5.16 and 5.23).
In cypermethrin exposed group, the highest elevation in microsomal protein content was shown by C. batrachus (4.994±0.385 mg/gm liver; p<0.001) with 1.6 fold increase followed by H. fossilis (4.981±0.633 mg/gm liver; p<0.001) with 1.5 fold increase and C. punctatus (4.125±0.669 mg/gm liver; p<0.001) with 1.4 fold increase when compared to their respective control after 15 days of exposure (Table 5.9). In ethion exposed group, the highest increase in microsomal protein content was shown by C. punctatus (4.055±0.236 mg/gm liver; p<0.05) with 1.35 fold increase followed by H. fossilis (4.136±0.403 mg/gm liver; p<0.01) with 1.34 fold increase and C. batrachus (4.110±0.563 mg/gm liver; p<0.05) with 1.26 fold increase after 15 days of exposure (Table 5.16). In dicofol exposed group, the
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highest increase in microsomal protein content was shown by 15 days C. batrachus (4.380±1.128 mg/gm liver; p<0.05) with 1.4 fold increase followed by C. punctatus (4.205±0.450 mg/gm liver; p<0.01) with 1.3 fold increase and H. fossilis (4.264±0.656 mg/gm liver; p<0.05) with 1.2 fold increase after 15 days of exposure (Table 5.23).
A considerable increase in liver weight, microsomal protein and many microsomal enzyme activities can be produced by treating animals for several days with toxins (Orrenius et al., 1965; Shuster and Jick, 1966; Kato et al., 1968). The increase in protein content in the fish exposed to pesticides suggests that any possible protein loss is compensated by increasing the tissue protein synthesis to meet increased demand to detoxify the pesticide and necessitate enhanced synthesis of detoxification enzyme proteins (Gill et al., 1990). Muley et al. (1996) also reported an increased level of protein content in gill, muscles and kidney of Tilapia mosambica exposed to 0.016 ppb of endosulfan for 168 hours. The increase in microsomal protein content in the present study may be the result of an elevation in tissues metabolic activity induced by pesticides.
H. fossilis treated with deflazacort (0.440±0.096 nmole/mg protein) recorded the highest content of CYP 450 compared to 0.214±0.102 nmole/mg protein in control group (Table 5.1). The CYP 450 content in β-naphthoflavone (β-NF) treated fish, C. punctatus (0.432±0.097 nmole/mg protein), H. fossilis (0.509±0.069 nmole/mg protein) and C. batrachus (0.466±0.118 nmole/mg protein) were also significantly higher (p<0.001) compared to their respective control (Table 5.4). The induction of CYP 450 was positively correlated with an elevation in LSI and microsomal protein content after the treatment with β-NF (Table 5.3).
The activities of CYP 450 enzymes was slightly higher in 15 days pesticide treated group compared to 5 days and 10 days treated groups in most of the cases studied in the present work. This could be explained by the increase in protein and CYP P450 content in fish exposed for longer duration (Table 5.9, 5.10, 5.16, 5.17, 5.23 and 5.24). The CYP 450 content after 15 days of exposure to cypermethrin in C. punctatus, H. fossilis and C. batrachus were 0.425±0.053, 0.523±0.234 and 0.484±0.131 nmole/mg protein respectively. These values reflected 1.4, 1.5 and 1.6 fold increase compared to their respective control group (Table 5.10). Of the ethion treated groups, CYP 450 content in C. punctatus, H. fossilis and C. batrachus were 0.550±0.120, 0.599±0.159 and 0.542±0.084 nmole/mg protein respectively and reflected 1.9, 1.84 and 1.83 fold increase compared to their respective control group after 15 days of exposure (Table 5.17). In dicofol treatment, C. batrachus (0.705±0.069 nmole/mg protein) showed the highest induction with 2.3 fold increase
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followed by. H. fossilis (0.713±0.397 nmole/mg protein) with 2.1 fold and C. punctatus (0.524±0.031 nmole/mg protein) with 1.8 fold increase when compared with their respective control group after 15 days of exposure (Table 5.24). It has been reported that the enzyme activity is higher if the experimental organism is exposed for a longer duration to the xenobiotics as it brings about the physiological differences within the organism (Arellano- Aguilar et al., 2009).
CYP 450 family of catalytic enzymes are inducible when animals are exposed to toxins and this explains why CYP 450 content was higher in experimental fish exposed to pesticides in comparison to the control. However, CYP 450 proteins only constitute a limited percentage of total microsomal protein content. Another microsomal enzyme system involved in pesticide metabolism is flavin-containing monooxygenase (FMO) (Hodgson et al., 1995). FMO enzyme is also inducible despite the fact that this enzyme is not under the same regulatory control as CYP 450 enzymes (Parkinson, 1996). Thus, the difference of microsomal protein content between control and experimental fish may be the result of induction of both CYP 450 system and FMO system.
The capability of an animal to survive adverse effects of ingested toxins must be considered from the perspective of the potency of the detoxification system. A useful index of the potency of the detoxification system is the amount of enzyme involved in the detoxification (Ling, 2005). Notably, all important activities of the hepatic microsomal detoxification system were considerably higher in experimental fish.
In the present investigation, it was evident that fish have the capacity to increase their detoxifying power when exposed to pesticides. CYP 450 plays a vital role in protecting tissue from oxidative stress and the increase in this enzyme activity in liver indicates the development of a defensive mechanism to counter the effect of pesticides and may reflect the organisms ability to provide more efficient protection against pesticide toxicity. Our studies suggest that pesticide exposed fish may improve their detoxification capacity by enhancing the amount of CYP 450 enzyme system.
Metabolism of pesticides by hepatic cytochrome P450
In this study, all the fish treated with 3 class of pesticides, cypermethrin, ethion and dicofol displayed an elevated level of EROD activity in 5, 10 and 15 days with a significant difference (p<0.001), probably due to EROD`s higher detection sensitivity (Table 5.11, 5.18
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and 5.25). In cypermethrin treatment, the 15 days treated group showed higher induction than 5 and 10 days treated group compared to the control. The EROD activity after 15 days of exposure to cypermethrin in C. punctatus, H. fossilis and C. batrachus were 99.341±13.451, 97.516±15.615 and 123.934±29.316 pmole resorufin formed/mg protein/min respectively (Table 5.11). Of the ethion treated groups, 5 days exposed C. punctatus (99.867±19.907 pmole resorufin formed/mg protein/min) showed the highest induction, while among H. fossilis, 15 days (86.944±21.720 pmole resorufin formed/mg protein/min) and among C. batrachus, 10 days (111.933±20.613 pmole resorufin formed/mg protein/min) exposed group showed the highest induction (Table 5.18). In dicofol treatment, the 10 days exposed C. punctatus (173.690±30.021 pmole resorufin formed/mg protein/min) showed the highest induction with 4.9 fold increase while among H. fossilis (153.767±18.840 pmole resorufin formed/mg protein/min) and C. batrachus (156.055±8.625 pmole resorufin formed/mg protein/min), the 15 days exposed group showed the highest induction when compared with their respective control group (Table 5.25). Though EROD activity was significantly induced in all the fish species examined, the induction level was of lesser magnitude with ethion exposure compared to cypermethrin and dicofol exposure. This increase in EROD activity proves the important role played by CYP1A isoform.
The main reaction in the metabolism of deltamethrin, cypermethrin or cyhalothrin in rats/mice is the ester cleavage by carboxylesterase (Demoute, 1989). Fish primarily metabolize pyrethroids by oxidative degradation, probably involving hepatic CYP 450- dependent monooxygenase with ester hydrolysis being a secondary reaction (Deer et al., 1996). Cypermethrin is metabolized and eliminated significantly more slowly by fish than by mammals or birds (Srivastava et al., 2008) compared to ethion and dicofol which may explain this compound’s high toxicity in fish compared to other organisms. Although, the evaluation of CYP1A stimulation by EROD activity is a common tool for quantifying environmental exposure to aryl hydrocarbon receptor (AhR) ligands, a number of studies have established CYP1A stimulation in response to pesticides (Haluzova et al., 2011). Assis et al. (2009) have reported induced level of EROD activity in liver microsomes of Ancistrus multispinis in response to deltamethrin exposure. Similar results have also been confirmed in fishes exposed to DDT (Lemaire et al., 2010), chlorpyrifos (Rai et al., 2010) and dicofol (Pal et al., 2011). Somnuek et al. (2012) reported chlorpyrifos and carbaryl at high concentrations resulted in significant elevation of CYP1A gene expression. CYP1A subfamily is the most studied CYP 450 isoform and is responsible for a wide range of xenobiotic biotransformation
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whose catalytic activity is expressed as the activity of EROD. EROD activity is best viewed as an indicator of contaminant exposure rather than of effect and this biomarker may also serve as a predictive tool for contaminant risk assessment (Popovic et al., 2013).
H. fossilis exposed to cypermethrin displayed a significant difference in N, N-dimethylaniline demethylase (N,N-DMA) activity in all the treated groups (5, 10 and 15 days) with the highest elevation seen in 10 days (0.285±0.111 nmole formaldehyde formed/mg protein/min; p<0.05) with 2.2 fold increase, while only 15 days treated group displayed a significant difference (p<0.05) in C. punctatus (0.186±0.082 nmole formaldehyde formed/mg protein/min) with 2.1 fold increase and C. batrachus (0.338±0.147 nmole formaldehyde formed/mg protein/min) with 1.6 fold increase in comparison to their respective control (Table 5.12). In ethion treatment, C. punctatus revealed negligible variation throughout the 15 days of exposure in N,N-DMA activity and only showed a marginal increase in 15 days treated group when compared to the control group. On the other hand, 5 days (0.212±0.070 nmole formaldehyde formed/mg protein/min; p<0.05) and 10 days (0.249±0.077 nmole formaldehyde formed/mg protein/min; p<0.001) treated H. fossilis and 5 days treated C. batrachus (0.312±0.064 nmole formaldehyde formed/mg protein/min; p<0.05) displayed a significant difference in comparison to their respective control group (Table 5.19). In case of dicofol treatment, there was a general trend of increase in N,N-DMA activity in all the fish species studied but only 15 days treated C. punctatus (0.291±0.091 nmole formaldehyde formed/mg protein/min) and H. fossilis (0.267±0.063 nmole formaldehyde formed/mg protein/min) displayed a significant difference (p<0.001), whereas, no difference was seen in C. batrachus (Table 5.26). The present work demonstrated that the increase in N,N-DMA activity is due to the presence of CYP2B isoform in all the fish species studied.
Earlier studies have reported that phenobarbital, a specific inducer of CYP2B in mammals have not been found to induce CYP2B in fish, however, CYP2B like activities in fish has been reported and suggested that some nutritional or environmental factors might control the expression of these genes in fish (Stegemann et al., 1997). It is likely that some CYP 450 differ in their functions among mammals and fish and follow different mechanisms of induction (Price et al., 2008) and it may be possible that pesticides do induce CYP2B like activities in fish. The metabolism of organophosphates to their oxon metabolites are likely mediated by CYP1A2, 2B6, 2B1/2, 2D6 and CYP3A4 (Sams et al., 2000; Tang et al., 2001). CYP2B6 has the highest desulfuration activity, whereas dearylation activity is highest for 2C19. CYP3A4 has high activity for both dearylation and desulfuration (Tang et al., 2001).
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Rai et al. (2010) reported the induction of CYP2B like activities in fish, Laboe rohita after exposure to the pesticide, chlorpyrifos. Pal et al. (2011) have also reported a significant induction in CYP2B mediated N,N-DMA activity in C. punctatus when exposed to 0.2 mg/kg body weight of chlorpyrifos and dicofol. In consequence, whenever CYP 450 enzymes are used as biomarkers in monitoring programs, one should be cautious with metabolic differences, even among fish species. Though specific CYP 450’s are necessary to metabolize xenobiotics, pesticides have the ability to induce one or more forms of CYP 450 simultaneously.
C. punctatus treated with cypermethrin displayed no response in aniline hydroxylase (AH) activity, whereas, a significant elevation was seen in 15 days treated H. fossilis (0.318±0.152 nmole p-aminophenol formed/mg protein/min; p<0.05), and 10 (0.395±0.059 nmole p- aminophenol formed/mg protein/min; p<0.01) and 15 days (0.387±0.143 nmole p- aminophenol formed/mg protein/min; p<0.01) treated C. batrachus when compared with their respective control group. Of the 3 fish species, 10 days treated C. batrachus revealed the highest induction with 1.7 fold increase in the activity (Table 5.13). In ethion treatment, only 10 days treated C. punctatus (0.177±0.045 nmole p-aminophenol formed/mg protein/min) displayed a significant induction (p< 0.05) in the activity compared to their control group, while all the treated groups (5, 10 and 15 days) of H. fossilis varied significantly in AH activity with 2.3 fold increase seen after 15 days of exposure (0.455±0.078 nmole p- aminophenol formed/mg protein/min). C. batrachus showed no response in AH activity towards ethion treatment when compared to its control, 0.214±0.075 nmole p-aminophenol formed/mg protein/min (Table 5.20). C. punctatus showed a marginal decrease in AH activity towards dicofol exposure in all the treated groups (5, 10 and 15 days) compared to the control (0.116±0.051 nmole p-aminophenol formed/mg protein/min) but the value was not significant while in H. fossilis, AH activity varied significantly (p<0.001). The values reflected a 3.1, 2.6 and 3.8 fold elevation after 5, 10 and 15 days of exposure when compared to its control (0.197±0.082 nmole p-aminophenol formed/mg protein/min). C. batrachus showed no response in aniline hydroxylase activity towards dicofol treatment when compared to its control, 0.219±0.079 nmole p-aminophenol formed/mg protein/min (Table 5.27). The present study indicated that the increase in AH activity is due to the presence of CYP2E1 isoform.
CYP2E1 dependent aniline hydroxylase activity has been shown to be a possible way for metabolism in the liver of fish species as in mammals. Although CYP2E1 is responsible for the metabolism of a number of low-molecular-weight xenobiotic compounds, studies have
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reported a stimulation of CYP2E1 activity upon exposure to pesticides carbaryl (Tang et al., 2002) and parathion (Mutch et al., 2003). A study by Sadat et al. (2009) reported its role in the metabolism of pesticide with long-term exposure. However, Labeo rohita exposed to chlorpyrifos displayed a significant inhibition of CYP2E1 activity (Rai et al., 2010). In contrast, Pal et al. (2011) reported a significant induction in aniline hydroxylase activity in C. punctatus when exposed to pesticides chlorpyrifos and dicofol.
All the treated groups (5, 10 and 15 days) of fish, C. punctatus, H. fossilis and C. batrachus showed a significant inhibition in erythromycin N-demethylase (ERND) activity after exposure to cypermethrin (Table 5.14). The lowest inhibition in C. punctatus (0.561±0.139 nmole formaldehyde formed/mg protein/min; p<0.01) and C. batrachus (0.606±0.108 nmole formaldehyde formed/mg protein/min; p<0.01) were shown by 10 days, and in H. fossilis by 15 days (0.504±0.142 nmole formaldehyde formed/mg protein/min; p<0.01) when compared to their respective control group. The result was in sharp contrast with that of ethion and dicofol treatment where ERND activity was significantly induced (Table 5.21 and 5.28). In ethion treatment, the highest induction was shown by 5 days (1.642±0.612 nmole formaldehyde formed/mg protein/min) exposed group in C. punctatus with 1.9 fold increase, by 10 days (3.978±1.067 nmole formaldehyde formed/mg protein/min) exposed group in H. fossilis with 4.7 fold increase and by 15 days (1.579±0.520 nmole formaldehyde formed/mg protein/min) exposed group in C. batrachus with 1.8 fold increase (Table 5.21). In dicofol treatment, the highest induction in C. punctatus (9.566±1.807 nmole formaldehyde formed/mg protein/min) and C. batrachus (2.168±0.392 nmole formaldehyde formed/mg protein/min) with 11.2 and 2.4 fold increase was shown by 15 days exposed group, while in H. fossilis, 5 days (3.156±1.410 nmole formaldehyde formed/mg protein/min) exposed group with 3.7 fold increase showed the highest induction in ERND activity compared to their respective control group (Table 5.28). The induction and inhibition in ERND activity in the 3 fishes studied proves the vital role played by CYP3A4 isoform.
CYP3A is one of the most abundant CYP 450 isoforms in fish liver possessing a broad range of substrate specificity and accounts for the metabolism of almost 50% of the currently used pharmaceutical agents (Parkinson, 1996; Hegelund and Celander, 2003). Methoxychlor, a structural analogue of the DDT pesticide, is known to induce rat hepatic CYP2B and 3A mRNAs and the corresponding proteins (Blizard et al., 2001). It is seen that CYP3A4 is the predominant isoform responsible for metabolizing pyrethroid pesticides in mammals (Scollon et al., 2009; Yang et al., 2009), thus, a probable explanation for high pyrethroid toxicity in
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fish may be due to the inhibition of CYP3A4 isoform in the liver and other tissues. CYP1A, CYP2B and CYP3A4 were also reported to be induced in channel catfish (Stuchal et al., 2006) and Labeo rohita (Rai et al., 2010) after exposure to methoxychlor and chlorpyrifos. However, Pal et al. (2011) have reported a marginal decrease in CYP3A4 mediated ERND activity in C. punctatus when exposed to chlorpyrifos and dicofol.
The biochemical responses assessed in the present study are the earliest indicators of exposure to stressors that can be detected in an organism. Many biochemical and physiological changes in aquatic organisms are caused by pesticides which influence the activities of several enzymes (Khan and Law, 2005) and pose a long-term risk to mammals, birds, amphibians and fish (Ali et al., 2011). Hepatic CYP 450 expression of fish can be induced by exposure to xenobiotics because of the presence of high percentage of CYP 450 in the liver as opposed to other organs.
A great diversity of cytochrome P450 enzymes in fish has been recognized and each isoform of the CYP 450 participates in the metabolism of many different compounds, but one substrate can also be metabolized by several different isoforms (Stegemann and Hahn, 1994; Siroka and Drastichova, 2004). Pesticides are primarily metabolized by the phase I enzymes that include the CYP 450 and flavin monooxygenases, and these enzymes can be either induced or inhibited by the pesticides or their reactive metabolites (Furnes and Schlenk, 2005). Recent studies have identified the presence of CYP1A, CYP2B, CYP2E1, CYP2K1 and CYP3A in the liver of some freshwater fish (Nabb et al., 2006) which play a vital role in the detoxification of carbamate and organophosphate insecticides (Ferrari et al., 2007).
Most lipophilic chemicals including drugs, carcinogens, pesticides, environmental pollutants and naturally occurring compounds undergo enzyme-mediated oxidative, hydrolytic or conjugative biotransformations in liver and in extrahepatic tissues yielding more polar compounds which can be easily excreted and are useful as early warning indices of environmental alteration, so that remedial measures may be taken by resource protection agency in time to prevent permanent decline in fish populations (Thomas, 1990). The great majority of them are almost devoid of any activity but in certain instances, metabolites are produced that may retain or augment the effects of the parent compounds or even acquire different pharmacological or toxicological properties. Thus, metabolism plays a critical role in determining both the efficacy and the residence time of xenobiotics in the body as well as in modulating the response to toxic chemicals (Akdogan and Sen, 2010).
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Thus, the present study demonstrates evidence of CYP 450 related xenobiotic metabolism in the liver of C. punctatus, H. fossilis and C. batrachus exposed to cypermethrin, ethion and dicofol. This study also established the presence of CYP2B activity which was thought to be absent in fish. The induction of CYP 450 mediated catalytic activity may also serve as a very efficient adaptive strategy of the fish species to increase tolerance to the pesticides and guarantee its survival.
Kinetics of cytochrome P450 mediated metabolism
Vmax and Km are the two parameters that define the kinetic behaviour of an enzyme as a function of substrate concentration. Km is an approximate measure of the affinity of the substrate for the enzyme and Vmax is the maximum rate of an enzyme catalyzed reaction (i.e. when the enzyme is saturated by the substrate) while Vmax/Km ratio acts as an indicator of enzymatic efficiency.
Considering 7-ethoxyresorufin as a substrate for EROD activity, in C. punctatus only ethion treated group (0.715±0.073 µM) showed a significantly (p<0.001) higher Km value compared to its control (0.421±0.035 µM). H. fossilis showed a significantly lower Km value in the entire treated group compared to control (0.731±0.021 µM) with the lowest value seen in ethion treated group (0.477±0.013 µM; (p<0.001), while C. batrachus showed a significantly (p<0.001) higher Km value in the entire treated group when compared to their control group, 0.427±0.019 µM (Table 5.29). This result shows that ethion treatment in C. punctatus and all the pesticide, cypermethrin, ethion and dicofol treatment in C. batrachus require high substrate concentration to carry out the reaction suggesting a lower binding affinity (high Km value), while in H. fossilis all the pesticide, cypermethrin, ethion and dicofol treatment require low substrate concentration to carry out the reaction suggesting a higher binding affinity (low Km value).
Considering N, N- dimethylaniline as a substrate for N,N-DMA activity in C. punctatus, all the pesticide treated group showed a significantly lower Km value compared to control (0.285±0.021 mM) with the lowest value seen in ethion treated group (0.165±0.033 mM; p<0.01). In H. fossilis, the dicofol treated group (0.294±0.020 mM; p<0.001) showed higher and cypermethrin treated group (0.156±0.014 mM; p<0.05) showed lower Km values compared to its control (0.182±0.013 mM) while in C. batrachus, the Km values were homogeneous in all the treated groups with respect to control, 0.244±0.023 mM (Table 5.31).
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This result shows that all the pesticide treatment in C. punctatus have a higher binding affinity for the substrate while in H. fossilis, the dicofol treatment have a lower binding affinity and cypermethrin treatment a higher binding affinity to the substrate.
Considering aniline as a substrate for AH activity in C. punctatus, the Km values were almost homogeneous in all the treated groups with respect to control (1.056±0.159 mM). In H. fossilis, both the ethion (1.133±0.033 mM; p<0.05) and dicofol (0.842±0.045 mM; p<0.01) treated group showed a significantly lower Km values when compared to the control (1.834±0.120 mM). In C. batrachus, only cypermethrin treated group (1.588±0.087 mM) showed a significantly (p<0.01) lower Km value in comparison to its control group, 2.170±0.130 mM (Table 5.33). This result shows that ethion and dicofol treatment in H. fossilis and cypermethrin treatment in C. batrachus have a higher binding affinity to the substrate.
Considering erythromycin as a substrate for ERND activity in C. punctatus, cypermethrin treated group (0.233±0.016 mM) showed a significantly (p<0.001) higher Km value and ethion treated group (0.041±0.015 mM) showed a significantly (p<0.001) lower Km value in comparison to the control (0.105±0.014 mM). In H. fossilis, only cypermethrin treated group (0.210±0.016 mM) showed a significantly (p<0.01) higher Km value with respect to its control (0.167±0.019 mM). In C. batrachus, the Km values were almost homogeneous in all the pesticide treated groups with respect to its control group, 0.137±0.015 mM (Table 5.35). This result shows that ethion treatment in C. punctatus have a higher binding affinity for the substrate, while cypermethrin treatment in C. punctatus and H. fossilis have a lower binding affinity to the substrate.
In the situation where Km value is high, the substrate would be expected to be poorly metabolized until it reached a certain concentration. However, if the inherent toxicity of such a substrate is high, systemic toxicity can take place before the compound is metabolized at a toxicologically significant rate. Thus, in fishes, compounds that undergo detoxification via the reaction having high Km would be expected to have higher ability to produce a toxic effect than compounds that are detoxified via the pathways having low Km (Ling, 2005).
Examination of the Vmax values for EROD activity revealed a significant difference (p<0.001) in all the pesticide treated groups with respect to the control. All the fish, C. punctatus (42.785±4.560 pmole resorufin formed/mg protein/min), H. fossilis (49.685±3.835 pmole resorufin formed/mg protein/min) and C. batrachus (40.490±6.359 pmole resorufin
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formed/mg protein/min) displayed a homogeneous range in the control group for EROD activity with the highest Vmax value shown by dicofol treated group followed by cypermethrin and ethion treated group (Table 5.29). In the present study, EROD Vmax values were quite similar among the species and despite statistically significant difference, all values were below 200 pmole resorufin formed/ mg protein/ min/. EROD activity is mainly studied to determine its suitability as a biomarker of pollution. Gonzalez et al. (2009) reported similar EROD Vmax value when studying kinetic phase I reaction in eight finfish species.
The Vmax for N,N-DMA activity had a variable range in values for both control and treated groups. All the pesticide treated fish, C. punctatus, H. fossilis and C. batrachus displayed a significant difference in N,N-DMA activity compared to their respective control group. The highest Vmax value in C. punctatus was shown by dicofol treated group (0.305±0.019 nmole formaldehyde formed/mg protein/min; p<0.001), while in H. fossilis (0.289±0.020 nmole formaldehyde formed/mg protein/min; p<0.001) and C. batrachus (0.357±0.021 nmole formaldehyde formed/mg protein/min; p<0.001), the cypermethrin treated group showed the highest Vmax value (Table 5.31).
The Vmax value for aniline hydroxylase activity in C. punctatus displayed a significant difference in cypermethrin (0.181±0.010 nmole p-aminophenol formed/mg protein/min; p<0.001) and ethion (0.149±0.015 nmole p-aminophenol formed/mg protein/min; p<0.05) treated group compared to the control. In H. fossilis, the Vmax value was significantly higher (p<0.001) in the entire pesticide treated groups compared to the control (0.189±0.019 nmole p-aminophenol formed/mg protein/min) with the highest value shown by dicofol treated group (0.720±0.027 nmole p-aminophenol formed/mg protein/min). In C. batrachus, only cypermethrin treated group (0.345±0.029 nmole p-aminophenol formed/mg protein/min) showed a significant difference (p<0.001) in comparison to the control, 0.229±0.014 nmole p-aminophenol formed/mg protein/min (Table 5.33).
The Vmax value for ERND activity in all the control groups of fish, C. punctatus (0.896±0.071 nmole formaldehyde formed/mg protein/min), H. fossilis (0.872±0.033 nmole formaldehyde formed/mg protein/min) and C. batrachus (0.842±0.023 nmole formaldehyde formed/mg protein/min) displayed a homogeneous range. The cypermethrin treated group in all the fish, C. punctatus, H. fossilis and C. batrachus displayed a significantly lower (p<0.05) Vmax value, while ethion and dicofol treated groups displayed a significantly higher (p<0.01) Vmax value in comparison to their respective control group (Table 5.35).
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A large variability in catalytic activities of hepatic CYP 450 enzymes (CYP1A, CYP2B, CYP2E1 and CYP3A4) was confirmed in our study. In particular, high variation between and within control, and pesticide treated fish species was noted for all the substrate tested. This can be explained by the fact that the activities of various metabolic pathways examined in our study were comprised of the sum of activities of different CYP 450 isoforms. The differences found in Km or Vmax values for given compounds can eventually lead to different toxic outcomes. Variability of catalytic activity of CYP 450 enzymes is a common characteristic of this enzyme system, and this is mainly associated with a large range of variability in the expression of enzyme protein (Lin and Lu, 2001).
All the fish species treated with cypermethrin was found to be a poor metabolizer in ERND activity (Table 5.35) when compared with other CYP 450 enzyme activities. Since the metabolic capacity of CYP 450 enzyme system was not equal in all the fish species examined, the species tested for this research were divided into two subgroups as poor metabolizers, representing those having lower Vmax values and strong metabolizers, having high Vmax values with respect to the control. As a result of variation in metabolism, the conversion and excretion rate of toxins would be expected to vary among individuals. For the compounds with a high intrinsic clearance, increased enzyme activity will have little effect on hepatic clearance (Labaune, 1989). This is normal for the xenobiotic metabolism catalyzed by cytochrome P450 system (McKinnon and Evans 2000).
In the present study, intrinsic clearance expressed as Vmax/Km ratio for 7-ethoxyresorufin revealed that in C. punctatus, the dicofol (337.881±62.318) and cypermethrin (238.687±52.658) treated groups were efficient in intrinsic clearance while in H. fossilis and C. batrachus, all the pesticide treated groups were efficient in intrinsic clearance when compared to their respective control group (Table 5.30). Vmax/Km ratio for N, N- dimethylaniline revealed that in C. punctatus, all the pesticide treated groups were efficient in intrinsic clearance with the highest clearance observed in dicofol treated group (1.511±0.464), while in H. fossilis and C. batrachus, cypermethrin and ethion treated groups were observed to be efficient in intrinsic clearance compared to their respective control (Table 5.32). Vmax/Km ratio for aniline revealed that in C. punctatus (0.170±0.053) and C. batrachus (0.217±0.065), only cypermethrin treated group was efficient in intrinsic clearance while in H. fossilis, ethion (0.394±0.086) and dicofol (0.857±0.123) treated groups were observed to be efficient in intrinsic clearance compared to their respective control group (Table 5.34). Vmax/Km ratio for erythromycin in all the fish, C. punctatus, H. fossilis and C.
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batrachus revealed that ethion and dicofol treated groups were efficient in intrinsic clearance. The Vmax/Km ratio was lower in all cypermethrin treated fish but was not significant when compared to their respective control group (Table 5.36).
Intrinsic clearance expressed as Vmax/Km ratio for 7-ethoxyresorufin was seen to be highest followed by erythromycin, N, N- dimethylaniline and aniline in all the fish species studied. This result clearly demonstrates the vital role played by CYP1A and CYP3A4 isoforms as they are considered to be important isoforms in metabolising the xenobiotics.
Vmax/Km is a pure measure of enzyme activity and is not influenced by other physiological factors of liver clearance. The Vmax/Km ratio is an important parameter that will determine the toxicity of a given compound in systemic clearance (Houston, 1994; Cotreau et al., 2005). To extrapolate the in vitro kinetic data to metabolic activity in vivo, the concept of intrinsic clearance (Vmax/Km ratio) is very useful and important and therefore, liver microsomal intrinsic clearance values can be scaled and used to predict hepatic clearance (Lin, 1998). For compounds with low intrinsic clearance values, the elimination rate of the compounds depends on enzymatic activity. This means enhancement in hepatic enzyme activity, resulting from the addition of an inducer, will have a more profound effect on clearance of such compounds (Stuchal et al., 2006). Conversely, enzyme inhibitors will have the opposite effect (Vaccaro, 2003; Zamaratskaia and Zlabek, 2011). Species that displayed low biotransformation capabilities (low Vmax) may have similar catalytic efficiencies than the ones with high maximum velocities due to a higher binding affinity (low Km) of their enzymatic systems (Gonzalez et al., 2009).
The study identified a noteworthy interspecies variation existing in the CYP450-mediated metabolism of substrates including resorufin, N, N-dimethylaniline, aniline and erythromycin and there were also differences in the intrinsic clearance between the control and pesticide exposed fish species for all selected substrates. Therefore, in essence, our data support the principle that animals exposed to toxins increase CYP 450 enzyme and thus acquire resistance.
Cytochrome P450 as environmental monitoring tool
In the present study, we caught fish species from 9 water sources present in and around the tea plantation areas in the Terai region of North Bengal where pesticides are routinely used to control the pest population (Fig. 5.6). Altogether, 5 species of fish was caught with the help
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of local fisherman. Major CYP 450 dependent mixed function oxidase activities such as CYP1A mediated EROD activity, CYP2B mediated N,N-DMA activity, CYP2E1 mediated AH activity and CYP3A4 mediated ERND activity were characterized in Channa punctatus, Channa striatus, Channa gachua, Heteropneustes fossilis and Clarias batrachus for the first time. All the fish species collected from the field displayed variable range of activity than those of the fish acclimatized in the laboratory. The fish collected from different sites also displayed a variable range of activity.
Among all the fishes collected from different sampling sites, C. batrachus collected from Lotchka showed the highest LSI value (1.42±0.42) and C. punctatus collected from Dangra Dangri showed the lowest LSI value (0.92±0.31). In C. punctatus, the fish collected from Magurmari (1.16±0.39) displayed a significant difference (p<0.05) in comparison to the fish collected from Dangra Dangri (0.92±0.31), and in C. batrachus, the fish collected from Lotchka (1.42±0.42) displayed a significant difference (p<0.05) compared to fish collected from Magurmari (1.18±0.41). The LSI values were almost homogeneous in all the groups of H. fossilis (Table 5.37). The CYP 450 content was higher in fish collected from Katagaon than that collected from other sites. Overall, C. batrachus collected from Katagaon (1.066 nmole/mg protein) showed the highest content of CYP 450 than that for C. punctatus, C. striatus, C. gachua and H. fossilis (Table 5.38).
Of all the fishes, EROD activity was highest in C. batrachus (187±52 pmole resorufin formed/mg protein/min) brought from Magurmari than that collected from other sites and lowest activity was found in C. gachua (22±12 pmole resorufin formed/mg protein/min) collected from Deomani (Table 5.39). N,N-DMA activity was found to be highest in H. fossilis (0.89±0.43 nmole formaldehyde formed/mg protein/min) collected from Dangra Dangri and lowest in C. striatus (0.137 nmole formaldehyde formed/mg protein/min) collected from Katagoan. The fish collected from Dangra Dangri and Ariagoan showed the highest activity than that collected from other sites (Table 5.40). Overall, AH activity was highest in H. fossilis (0.409 nmole p-aminophenol formed/mg protein/min) collected from Katagoan while C. punctatus (0.08±0.03 nmole p-aminophenol formed/mg protein/min) collected from Ariagoan showed the lowest activity than that collected from other sites (Table 5.41). Of all the fish species, ERND activity was highest in H. fossilis (4.13±1.52 nmole formaldehyde formed/mg protein/min) collected from Magurmari and the lowest in C. punctatus (0.866 nmole formaldehyde formed/mg protein/min) collected from Katagoan (Table 5.42). These differences in enzyme activities could be ascribed to duration,
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physiological status and environmental stress faced by the fish living in different water bodies in different locations. The water bodies of Terai region of North Bengal receive a considerable influx of anthropogenic pollutants especially from agricultural run-off, domestic sewage and tea gardens (Pal et al., 2011; Singh et al., 2015).
Some of the earliest changes to occur in an organism following exposure to an environmental pollutant occur at the cellular level including induction or inhibition of certain enzyme activity and alterations in genetic material or gene expression as a result of xenobiotic and molecular interactions (El-Shehawi et al., 2007; Maier et al., 2009; Whatley et al., 2010).
CYP1A response measured as EROD activity has already been incorporated into some major monitoring programs such as the National Status and Trends Program in the United States (Collier et al., 1992) and North Sea Task Force Monitoring Master Plan of the North Sea Nations in Europe (Goksoyr and Forlin, 1992). In fish, EROD activity with its high sensitivity, specificity, feasibility and simplicity of its measurement have been of advantage in using it as a biomarker (Bucheli and Fent, 1995).
The indiscriminate use of pesticides is considered one of the central factors in changing the environment by causing imbalances in the ecosystem, especially in the aquatic system (Sancho et al., 1998). Thus the growth and survival of fish are greatly disturbed by the use of pesticides in or near a body of water (Ewing, 1999). Several studies have shown that most fish sampled from the agricultural areas contained detectable levels of pesticides (Sayeed et al., 2003; Singh et al., 2015). In Canada, losses among 62 imperilled species were significantly more related to rates of pesticide use and species loss was highest in areas with intensive agriculture (Gibbs et al., 2009). Over the last decade, CYP 450 isozymes have been employed as a useful tool for measuring the environmental pollution by pesticides. Although CYP1A was classically recognized as being specifically induced by PAHs and pesticide exposure, it is now known that it is not particularly responsive to one unique class of pollutants. It can be used as a biomarker to monitor the ecological risk of various pollutants in the environment (Whatley et al., 2010).
Apart from CYP1A activity, CYP2B, 2E1 and CYP3A4 activity has also been reported to be a useful monitoring tool for assessing the environmental pollution. Boyunegmez (2004) studied 3 different fish, leaping mullet, common sole and annular seabream along the Izmer Bay, Turkey and reported that the area was heavily contaminated with PAH, PCB and other organochlorine type persistent organic precarcinogen/carcinogen chemicals by studying
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major CYP 450 dependent mixed function oxidase activities such as ethoxyresorufin O- demethylation and methoxyresorufin O-demethylation (CYP1A), benzphetamine N- demethylation (CYP2B), ethylmorphine N-demethylation (CYP3A) and aniline 4- hydroxylation (CYP2E1). Similarly, the CYP 450 monooxygenase activity (CYP1A and CYP2B) of different populations of Nile tilapia has been used to alert possible human health hazards by organochlorate pollution in the Guanadu River of Brazil, a source of drinking water for Rio de Janeiro (Parente et al., 2004). Zhu et al. (2006) evaluated CYP 450 activity (CYP1 and CYP2 families) in stripey seaperch that were collected from unpolluted sites and placed in cages along a region where oil was discharged in Western Australia. These authors concluded that CYP 450 isoforms act as potential indicators of environmental pollution by hydrocarbon in fish populations.
Field and laboratory investigations have proved that pesticides are highly toxic to a number of non-target organisms even at very low concentrations (Oudou et al., 2004). However, it has been reported that variability of enzymatic activity in the field is higher than in laboratory tests due to factors such as physiological differences in species, migration, gender, period of exposure and concentrations of accumulated pollutants during the life of each organism (Schmitt et al., 2005; Hinck et al., 2006; Kammann et al., 2008). Our study revealed a variable range of CYP 450 activities in all the fish species studied. This may be due to the fact that the factors like gender, age, exposure period and concentration of pollutants were not taken into consideration.
Vindimian et al. (1993) reported the induction of EROD activity in fish exposed to a mixture of pesticide runoff from nearby vineyards. These findings indicate the multifarious nature of the fundamental mechanisms by which organisms bear exposure to mixtures of pollutants in their environment. Therefore, it is possible that the toxic nature of pesticide mixture is more complicated than a simple concentration or a response addition model typically used for predicting the probable impact of mixtures in aquatic systems (Broderius et al., 1995). Carbamate, pyrethroid, organophosphate and organochlorine pesticides are commonly used for controlling a number of agriculturally important pests, as a result, there is a strong possibility that mixtures of these pesticides co-occur in the environment, and therefore, the combined effects of these pesticides should be considered. Herbicide-insecticide mixtures have also been reported to influence organisms in different ways. Some interactions result in augmenting toxicity while others reduce toxicity (Miota, 2000). Singh et al. (2015) have also
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reported residues of chlorpyrifos, ethion and dicofol in water, sediment and fish tissue from river Deomoni flowing through the tea gardens of Terai region of West Bengal, India.
The results of the present study suggest that CYP 450 isoforms can be used as a valuable monitoring tool for assessing the pollution of the water bodies contaminated with pesticides of different class that can disrupt the development, reproduction and survival of aquatic organisms.
Interspecies variability in in vitro metabolism by cytochrome P450
In spite of observed induction of cytochrome P450 (CYP 450) in 3 fish species in response to pesticide contamination, there were important variations between species concerning the level of enzyme activities. The CYP 450 content and enzyme activities in all the 3 fish studied, C. punctatus, H. fossilis and C. batrachus showed a variable range in β- naphthoflavone treatment (Table 5.4). The response to the pesticide cypermethrin, ethion and dicofol also varied between the fish studied. Among the pesticides in terms of LC50 value, C. punctatus and C. batrachus were observed to be highly sensitive towards cypermethrin followed by ethion and dicofol, while H. fossilis was highly sensitive to cypermethrin followed by dicofol and ethion (Table 5.5, 5.6 and 5.7).
The results of the present study demonstrated that the CYP 450 enzyme activities varied differently between and within each pesticide treated fish species. EROD, N,N-DMA and AH activities were involved in cypermethrin metabolism while ERND activity was inhibited and did not catalyze the metabolism of cypermethrin in all the 3 fish species studied (Table 5.11, 5.12, 5.13 and 5.14). In C. punctatus, the pesticide, ethion increased EROD, AH and ERND activities while no response was observed in N,N-DMA activity. In H. fossilis all the activities viz. EROD, N,N-DMA, AH and ERND were increased by ethion treatment. However, in C. batrachus, EROD, N,N-DMA and ERND were induced indicating their role in the metabolism of ethion whereas no response was noted in AH activity (Table 5.18, 5.19, 5.20 and 5.21). EROD and ERND activities, in particular, were induced by the pesticide, dicofol in all the 3 fish species. In C. punctatus, N,N-DMA activity was also induced after dicofol treatment while AH activity was moderately inhibited. In H. fossilis, similar to ethion treatment, all the activities viz. EROD, N,N-DMA, AH and ERND were induced after dicofol treatment. On the other hand, in C. batrachus, N,N-DMA and AH activities were not induced by dicofol treatment (Table 5.25, 5.26, 5.27 and 5.28).
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All the 3 fish species displayed a marked difference in maximal velocity (Vmax), binding affinity (Km) and catalytic efficiency (Vmax/Km) between and within species population (Table 5.29, 5.30, 5.31, 5.32, 5.33, 5.34, 5.35 and 5.36).
The fish, Channa punctatus, Channa striatus, Channa gachua, Heteropneustes fossilis and Clarias batrachus collected from different sites in Terai region of North Bengal displayed a variable range of CYP 450 activities (Table 5.38, 5.39, 5.40, 5.41, and 5.42). All these results establish the interspecies differences in metabolizing the pesticides and environmental xenobiotics by CYP 450 enzymes.
Large interspecies differences are seen in organisms and in different species, the same substrate can be metabolised by different CYP 450 isoforms. There are generally marked differences between the species in CYP 450 enzyme content, activity, and susceptibility. It could explain that the organisms have different physiology, habitats, behaviour and feeding habitats and the ability to accumulate, distribute and metabolize contaminants (Bucheli and Fent, 1995). Basal activities may also vary considerably both among and within species (Whyte et al., 2000).
In fish, as in other animal species, there are great differences in CYP 450 catalytic activity among individuals of one population as well as among populations which is related to factors such as species, sex (Navas and Segner 2001), diet or season (Machala et al., 1997; Aas et al., 2001; Schlenk and Di Giulio 2002; Ruus et al., 2002; Jorgensen et al., 2002).
In the present study, the intra-individual difference among the population was not carried out as the gender was not taken into consideration. Although, the difference in species was noted when comparing their basal CYP 450 mediated monooxygenase activities as well as their Vmax/Km ratio. One of the most important aspects of CYP 450 enzyme system is that the metabolic capacity of CYP 450 enzyme system is not equal to all members of a population, and as a result, the metabolic conversion and excretion rate of toxins varies among individuals ranging from extremely slow to ultra fast (McKinnon and Evans, 2000) caused by genetic polymorphisms or by inhibition or induction of toxin metabolism (Ling, 2005). Individual variability has been noted in populations of Fundulus heteroclitus, with evolutionarily adaptive changes in gene expression thought to account for much of this variation (Oleksiak et al., 2005; Crawford and Oleksiak, 2007).
Numerous authors observed differences in species, from which no generalized picture can be obtained. EROD activity was generally higher (3 to 10 fold) in salmonids than in cyprinids
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(Monod et al., 1988). Lindstrom-Seppa and Oikari (1990) found that bream (Abramis bramd) responded well as an indicator, whereas perch (Perca fluviatilis) and roach (Rutilus rutilus) showed lower inducibility. Herbivorous fish may show enhanced basal enzyme activities due to natural inducers (Vindimian et al., 1991), such as those present in plants or coral reefs (Vrolijk et al., 1993). Conversely, barbel (Barbus barbus), a benthic carnivore, was more sensitive to pollution than the herbivorous species nase (Chondrostoma nasus) (Vindimian et al., 1991). Another carnivorous fish, chub (Leuciscus cephalus) has been found to have considerably high enzyme activity (Vindimian et al., 1993).
Various mechanisms can be associated with differences between species in response to a xenobiotic, which includes genetic defect in a particular metabolic pathway, differences in the Km and Vmax of specific enzyme, the existence of different isozymes, differences in the ratios of important specific isozymes and differences in the ratio of activities of separate enzyme systems that act together to metabolize a specific xenobiotic (deBethizy and Hayes, 1989; Ling, 2005). Thus, it is possible to trace species differences in xenobiotic metabolism to variations in CYP 450 levels, their inducibilities and the existence of different CYP 450 isoforms of the same protein family or subfamily in various species (Lewis et al., 1998; Ling, 2005). Generally, the basic reactions and major metabolites of a xenobiotic are similar among species. However, small differences in metabolism can result in major differences in susceptibility to toxicity.
Electrophoresis
Microsomes are the suspension of membrane protein preparations and are thought to possess CYP 450 and other drug metabolising proteins. A careful analysis of gels in hepatic microsomes from C. punctatus, H. fossilis and C. batrachus led to the identification of protein bands at around 50-56 kDa along with other bands at around 20-29 kDa when stained with N,N,N’N’ -Tetramethyl-p-phenylenediamine (TMPD) for visualizing peroxidase activity (Fig. 5.7 a, b and c). The bands at around 50-56 kDa are expected to be that of CYP 450 and the other band could be that of haemoglobin and other heme proteins. A coomassie brilliant blue (CBB) stained gel of the proteins (right panel) demonstrated that the lack of β- mercaptoethanol in the sample buffer is responsible for smearing of the protein bands. The gels were stained for peroxidase activity and then stained for protein. The ability to stain the same gel for peroxidase activity and protein allows one to superimpose the gel scans or
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photographs of the two stains to verify which protein staining bands contain heme. Such a comparison is much less precise if made by staining two different SDS gels.
Comparisons of SDS-PAGE of microsomal proteins from control and pesticide treated fish, C. punctatus, H. fossilis and C. batrachus identified a protein band of increased intensity with a molecular weight between 50-56 kDa (Fig. 5.8, 5.9 and 5.10 a, b and c). Similar enhancement was observed in a heme-containing protein as evidenced by heme staining (peroxidase activity) of PAGE gels by TMPD (Fig. 5.7 a, b and c).
The hemeproteins CYP 450 have been previously reported to have H2O2-peroxidase activity on SDS polyacrylamide gels using TMPD (Scharf et al., 2000; Miota, 2000). One of the larger families of CYP 450-dependent microsomal monooxygenases are the heme-thiolate membrane-associated proteins with a molecular weight between 45-60 kDa (Miota, 2000). Gel electrophoresis separates proteins based on independent physical characteristics and is a powerful tool for resolving complex mixtures of proteins (Palzkill, 2002). All CYP 450 isoforms possess a common heme cofactor (iron protoporphyrin IX) that is required for binding oxygen and creation of the active oxidant species (Henne et al., 2001).
SDS–PAGE results are in agreement with CYP 450 mediated monooxygenase assays viz. CYP1A, CYP2B, CYP2E1 and CYP3A4 presented in the results section (5.5, 5.6 and 5.7), although some of the activities displayed a negligible response in experimental groups (Table 5.10, 5.11, 5.12, 5.13, 5.14, 5.17, 5.18, 5.19, 5.20, 5.21, 5.24, 5.25, 5.26, 5.27 and 5.28). The band intensities, however, appear to directly correspond with CYP 450 monooxygenase activity results. Together, these findings suggest that potential CYP 450 isoforms are constitutively overexpressed in pesticide resistant populations, while induction of various forms is greater for more susceptible populations. Multiple CYP 450 forms are clearly present, possibly several in a given heme-stained band, the isolation of distinct electrophoretic forms will be necessary to assist future studies of CYP 450 in this species.
Gene sequencing and Phylogenetic analysis
The CYP1A genomic sequence for H. fossilis with 534 bp coding 146 amino acid and C. batrachus with 509 bp coding 137 amino acid submitted by us is the first report of a partial genomic sequence of any CYP 450 gene of H. fossilis and C. batrachus (Fig. 5.12 and 5.13), while the cDNA sequence for C. punctatus have been previously submitted in the Genbank (Raisuddin and Lee, 2008; EU930319). The studies of xenobiotic metabolizing genes in
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Siluriformes fish have attracted lesser attention as compared to fish from other orders (Kim et al., 2008). Since C. punctatus, H. fossilis and C. batrachus is commercially and ecologically important fish in various parts of Asia, its genomics may provide impulsion for its sustainable commercial exploitation and preservation and its relationship with other species.
C. punctatus CYP1A structural gene (2511 bp) contained 6 exons and 5 introns while H. fossilis (534 bp) and C. batrachus (509 bp) contained 2 exons and 1 intron (Table 5.43). In C. punctatus, no differences were found between the CYP1A cDNA and exons of the CYP1A genomic DNA obtained in the present study. The large structural gene of C. punctatus (2511 bp) is due to the fact that 3 different sequences of genomic DNA amplified by using 3 different primers were attached together to make it a single structural genomic DNA (Fig. 5.11). All the introns begin with the sequence GT and end with AG, consistent with the GT/AG rule of exon-intron junction sequences (Hassanin et al., 2009). The phylogenetic tree based on the amino acid sequences clearly shows C. punctatus and C. maculata CYP1A and H. fossilis and C. batrachus CYP1A to be more closely related to each other than to other CYP1A subfamilies with 93% identity. H. fossilis CYP1A with 86% identity and C. batrachus CYP1A with 85% identity were more closely related to Peltobagrus fulvidraco, another member of the order Siluriformes (Table 5.44 and Fig. 5.14).
The CYP1A isoform is only considered for gene sequencing in the present study due to its high sensitivity towards a vast majority of xenobiotics compared to other isoforms. The position of the ancestral CYP1A locus remains a question and opposing orientations of CYP1A in pufferfish and frog genes pose a dilemma in ascertaining whether CYP1A1 or CYP1A2 resides in the ancestral CYP1A gene locus (Goldstone and Stegemann, 2006). It remains confusing and somewhat arbitrary and accordingly, in fish, CYP1A is not being provided with a number following the subfamily.
The CYP1A gene subfamily comprises a single ancestral representative of most fish species and two representatives in higher vertebrates (Kimura et al., 1984; Gilday et al., 1996; Morrison et al., 1998). Fish CYP1A is thought to be a hybrid protein coded by a gene ancestral to both mammalian CYP1A1 and CYP1A2 forms, therefore, the name CYP1A rather than CYP1A1 has been suggested (Stegemann et al., 1997). Although CYP1A seems to be well conserved across vertebrate taxa (Goldstone et al., 2007), levels and inducibility of CYP1A protein and catalytic activity exhibit a rather large variability between fish species and populations (Parente et al., 2009).
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Liver CYP1A induction in fish by certain classes of chemicals has been widely applied as a biomarker in field studies. The cloning and sequencing of CYP1A gene have been conducted from many fish species for use in assessing the contamination of the aquatic environment (Meyer et al., 2002; Fent, 2003; Moore et al., 2003; Kim et al., 2008). Pesticides like chlorpyrifos and carbaryl were also found to increase CYP1A both on protein and gene levels and the induction was characterized by a dose-dependent increase of EROD activity, which was correlated well with CYP1A mRNA levels in liver (Somnuek et al., 2012). Members of subfamily CYP1A are involved in the metabolism of environmental pollutants leading to its detoxification or bioactivation. Understanding the functional evolution of these genes is indispensable for predicting and interpreting species differences in sensitivity to toxicity by environmental pollutants.
From the above discussion and results of the experiments, it can be concluded that CYP 450 isoforms (CYP1A, CYP2B, CYP2E1 and CYP3A4) fulfill the criteria to be used as biomarkers in environmental monitoring. It can also be inferred that the induction of CYP 450 isoforms may be a defensive mechanism, whereby the cell can detoxify potentially lethal xenobiotic compounds that might otherwise accumulate. This knowledge of the mechanisms of induction may provide insights into the strategies that organisms use to adapt to a changing environment and provide a better understanding of the biochemical pathways which control, respond and recognize the chemical stimuli occurring within the cell.
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8. INDEX
A absorbance ...... 10, 14, 37, 47, 52, 56, 127 acetone ...... 22, 32, 40, 42, 56, 57, 127, 128, 129, 130 amphibians ...... 1, 141 Ancistrus ...... 21, 25, 50, 122, 125, 127, 137 aniline ...... 4, 23, 39, 41, 42, 44, 56, 58, 60, 62, 67, 74, 81, 92, 94, 129, 131, 139, 143, 144, 145, 146, 149 annelids ...... 10, 12 aquatic invertebrates ...... 12 Ariagoan ...... 30, 99, 100, 101, 102, 103, 104, 105, 106, 107, 147 aromatic hydroxylations...... 12 aryl hydrocarbon receptor ...... 15, 17, 137
B β-naphthoflavone ...... 23, 32, 59, 60, 62, 128, 130, 131, 133, 135, 150 Balarampur...... 30, 99, 100, 101, 102, 103, 104, 105, 106, 107 basal ...... 20, 59, 60, 89, 95, 151, 152 benzo-a-pyrene ...... 10, 21, 23, 24, 27 bioindicator ...... 9 biomarkers ...... 8, 10, 19, 139, 155 birds ...... 1, 12, 137, 141
C Camellia sinensis ...... 1 carbamates ...... 1 Catla catla...... 50, 122, 125 centrifuge ...... 35, 48 Changa ...... 30, 99, 100, 101, 105, 106, 107 Channa gachua ...... 99, 147, 151 Channa maculate ...... 50, 122, 125 Channa punctatus ...... 4, 24, 26, 28, 29, 32, 49, 50, 99, 105, 106, 107, 125, 147, 151 Channa striatus ...... 23, 99, 105, 106, 107, 147, 151 Chopra ...... 30, 99 Clarias batrachus ...... 4, 28, 29, 32, 49, 50, 99, 105, 106, 107, 124, 125, 147, 151 constitutive androstane receptor ...... 15, 17 crustacea ...... 12 cytochrome P450 (CYP 450). .... 3, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 26, 27, 32, 37, 38, 39, 46, 52, 56, 58, 60, 62, 65, 69, 72, 76, 79, 83, 86, 98, 100, 101, 105, 108, 109, 110, 111, 127, 128, 129, 130, 131, 133, 134, 135, 136, 137, 138, 140, 141, 142, 145, 146, 147, 148, 149, 150, 151, 152, 153, 155 CYP1A ..... 15, 17, 18, 19, 20, 23, 24, 25, 26, 32, 39, 40, 49, 50, 59, 121, 122, 123, 124, 125, 126, 128, 130, 131, 137, 141, 145, 146, 147, 148, 153, 154, 155 CYP2B ...... 15, 21, 24, 26, 39, 41, 128, 130, 131, 138, 140, 141, 142, 145, 147, 148, 153, 155 CYP2E1 ...... 15, 20, 22, 24, 26, 39, 42, 129, 130, 131, 139, 141, 145, 147, 149, 153, 155 CYP3A4...... 16, 24, 26, 39, 43, 130, 131, 138, 140, 145, 146, 147, 148, 153, 155 cypermethrin .... 23, 32, 33, 44, 63, 64, 65, 66, 67, 68, 69, 70, 71, 86, 87, 89, 90, 92, 95, 108, 112, 115, 118, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 142, 143, 144, 145, 150 Cyprinus carpio ...... 50, 122, 125, 132
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D Dangra Dangri ...... 30, 99, 100, 101, 102, 103, 105, 106, 107, 147 Darjeeling ...... 1, 6 deflazacort ...... 32, 56, 57, 127, 128, 129, 130, 135 Deomani...... 6, 30, 99, 100, 101, 105, 106, 107, 147 dicofol ..... 6, 24, 26, 32, 33, 44, 63, 64, 79, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 92, 95, 108, 114, 117, 120, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 150 Dooars...... 6
E environment ...... 3, 5, 7, 9, 10, 18, 24, 25, 148, 149, 155 enzyme...... 3, 10, 11, 13, 18, 19, 20, 26, 32, 34, 40, 41, 42, 43, 44, 61, 68, 86, 88, 91, 94, 97, 99, 108, 127, 129, 135, 136, 141, 142, 145, 146, 147, 148, 150, 151, 152 EROD ... 20, 23, 25, 26, 27, 39, 40, 44, 56, 58, 60, 62, 66, 70, 73, 77, 80, 84, 86, 87, 98, 101, 106, 128, 130, 136, 137, 142, 143, 147, 148, 149, 150, 151, 155 erythromycin ...... 4, 39, 42, 43, 45, 58, 60, 62, 67, 74, 81, 95, 97, 129, 131, 140, 143, 145, 146 Erythromycin N-demethylase...... 57, 71, 78, 85, 95, 103, 107 ethion… ...... 6, 32, 33, 44, 63, 64, 72, 73, 74, 75, 76, 77, 78, 86, 87, 89, 90, 92, 95, 108, 113, 116, 119, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 142, 143, 144, 145, 150 eugenol ...... 32 exons ...... 121, 123, 124, 125, 154
G Genbank ...... 50, 153 genomic DNA ...... 47, 49, 50, 121, 154
H heme-thiolate ...... 3, 11, 13, 153 Heteropneustes fossilis ...... 4, 23, 28, 29, 32, 49, 50, 56, 57, 99, 105, 106, 107, 124, 125, 147, 151 hydroxylase ...... 10, 23, 41, 42, 56, 58, 60, 62, 67, 71, 74, 78, 81, 85, 94, 102, 107, 129, 131, 139, 144
I inducers ...... 13, 14, 32, 56, 57, 58, 128, 130, 152 inhibitors ...... 13, 146 intrinsic clearance ...... 87, 88, 89, 91, 92, 94, 95, 97, 146 introns ...... 121, 123, 124, 125, 154 isoform ...... 15, 19, 20, 22, 49, 128, 130, 131, 137, 138, 139, 140, 141, 154
K Kalagach ...... 30 Katagaon ...... 30, 99, 100, 101, 105, 106, 107, 147 Km ...... 43, 44, 51, 86, 87, 88, 89, 91, 92, 94, 95, 97, 142, 143, 145, 146, 151, 152
L liver...... 3, 9, 13, 15, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27, 34, 35, 40, 46, 47, 48, 52, 53, 54, 55, 56, 57, 59, 65, 66, 69, 72, 73, 76, 79, 80, 83, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 127, 128, 129, 133, 134, 135, 136, 137, 139, 140, 141, 142, 146, 147, 155 Lotchka ...... 30, 99, 100, 101, 102, 103, 104, 105, 106, 107, 147
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M Magurmari ...... 30, 99, 100, 101, 102, 103, 104, 105, 106, 107, 147 monooxygenase ...... 10, 12, 18, 20, 23, 136, 137, 149, 151, 153
N N, N-dimethylaniline ...... 4, 23, 39, 40, 41, 43, 44, 58, 60, 62, 66, 70, 73, 77, 80, 84, 91, 102, 106, 128, 131, 138, 146 NADP ...... 12, 41 NADPH ...... 11, 12, 40, 41, 42, 43 naphthalene ...... 21, 32, 56, 57, 127, 128, 129, 130 Nile Tilapia ...... 19, 27 Nomenclature ...... 13 North Bengal ...... 1, 3, 6, 29, 30, 31, 146, 148, 151 Nucleotide sequences...... 123, 124
O oligonucleotide ...... 22 organochlorines ...... 6
P Peltobagrus fulvidraco ...... 50, 121, 122, 125, 154 pesticides...... 1, 2, 3, 5, 6, 7, 11, 15, 21, 24, 25, 26, 27, 32, 33, 108, 128, 131, 132, 134, 135, 136, 137, 138, 140, 141, 142, 146, 148, 149, 150, 151 phenobarbitone ...... 32, 56, 57, 127, 128, 129, 130 phylogenetic tree ...... 122, 154 pregnane X receptor...... 16, 17 primers ...... 49, 50, 121, 154
R rainbow trout ...... 9, 16, 17, 18, 19, 20, 21, 22, 25, 50
S scup...... 9, 15, 16, 19, 21, 22, 50, 129 Sesame oil ...... 58 Sonapur ...... 30, 99, 100, 101, 102, 103, 104, 105, 106, 107 Spectral ...... 37, 52, 56, 127 spectrophotometer ...... 37 substrates...... 11, 12, 13, 15, 17, 21, 22, 39, 128, 146 supernatant ...... 35, 40, 41, 42, 43 synthetic pyrethroids...... 1
T temperature ...... 35, 37, 40, 42, 48, 132 Terai ...... 6, 29, 30, 31, 146, 148, 150, 151
V Vmax ...... 43, 44, 51, 86, 87, 88, 89, 91, 92, 94, 95, 97, 142, 143, 144, 145, 146, 151, 152
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9. APPENDICES Appendix A List of published research papers in Journals:
. Dawa Bhutia, Benoy K. Rai, Joydeb Pal. 2015. Hepatic Cytochrome P450 as Biomarkers of Cypermethrin Toxicity in Freshwater Teleost, Channa punctatus (Bloch). Brazilian Archives of Biology and Technology.58: 130-135. (ISSN 1516- 8913).
. Dawa Bhutia, Benoy K. Rai, Joydeb Pal. 2013. Detection of Multiple Cytochrome P450 in Hepatic Tissue of Heteropneustes fossilis (Bloch) Exposed to Cypermethrin. Proceedings of the Zoological Society. 66: 14–19. (ISSN 0373-5893).
. Dawa Bhutia, Benoy K. Rai and J. Pal. 2010. Multiple forms of cytochrome P450 family in liver of fresh water teleost fish, Heteropneustes fossilis (Bloch). N.B.U. Journal of Animal Sciences. 4: 28-35. (ISSN 0975-1424).
List of submitted genomic DNA sequences (CYP1A) in the GenBank:
Channa punctata isolate CHANNA cytochrome p450 CYP1A (cyp1A) gene, partial cds GenBank: KP282054.1---- Dawa Bhutia, Benoy K. Rai and Joydeb Pal. 2015 Channa punctata isolate NBUC1 cytochrome p450 CYP1A (cyp1A) gene, partial cds GenBank: KP203843.1---- Dawa Bhutia, Benoy K. Rai and Joydeb Pal. 2015 Channa punctata isolate NBUC2 cytochrome p450 CYP1A (cyp1A) gene, partial cds GenBank: KP231221.1---- Dawa Bhutia, Benoy K. Rai and Joydeb Pal. 2015 Channa punctata isolate NBUC3 cytochrome p450 CYP1A (cyp1A) gene, partial cds GenBank: KP271996.1---- Dawa Bhutia, Benoy K. Rai and Joydeb Pal. 2015 Heteropneustes fossilis partial cyp4501a gene for Cytochrome P450 1A GenBank: LN736019.1---- Dawa Bhutia, Benoy K. Rai and Joydeb Pal. 2015 Clarias batrachus isolate HM514 cytochrome p450 CYP1A (cyp1A) gene, partial cds GenBank: KP336485.1---- Dawa Bhutia, Benoy K. Rai and Joydeb Pal. 2015
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Appendix B List of abstracts published in proceedings of Seminars/ Conferences/ Workshops 1. Dawa Bhutia, Benoy K. Rai, Joydeb Pal. 2010. Cytochrome P450 induction in liver microsomes of air breathing teleost fish, Channa punctatus, Heteropneustes fossilis and Clarias batrachus after exposure to pyrethroid insecticide, Cypermethrin. National Symposium on “Pesticide stress on Target, Non-Target Organisms and Human Health”. Department of Zoology, University of North Bengal (oral presentation). 2. Dawa Bhutia, Benoy K. Rai, Joydeb Pal. 2010. “Studies on some Cytochrome P450 family in freshwater teleost fish, Heteropneustes fossilis and Labeo rohita”. National Conference on “Evaluation of Biodiversity of Eastern Himalaya and Adjoining Plains”. Department of Zoology, University of North Bengal in collaboration with Centre for Environment and Development, Kolkata (oral presentation). 3. Participated in the “UGC Sponsored Research Scholars’ Training Programme”, from 30th June 2011 to 1st July 2011 at UGC Academic Staff College, University of North Bengal. 4. Dawa Bhutia, Benoy K. Rai, Joydeb Pal. 2012. “Expression of hepatic Cytochrome P450 in Heteropneustes fossilis (Bloch) upon exposure to Cypermethrin”. National Conference on “Research in Animal Science: Development and Evaluation”. Department of Zoology, University of North Bengal (oral presentation). 5. Dawa Bhutia, Benoy K. Rai, Joydeb Pal. 2013. Expression of multiple forms of cytochrome P450 family in liver of freshwater teleost fish, Heteropneustes fossilis (bloch). National Conference on “Man, Animal and Environment interaction in the perspective of modern Research”. Department of Zoology, University of North Bengal (oral presentation). 6. Dawa Bhutia, Benoy K. Rai, Joydeb Pal. 2014. “Effects of cypermethrin on the activities of hepatic cytochrome P450 in Channa punctatus (Bloch)”, UGC sponsored National seminar on “Biological Research in Human Welfare”. Department of Zoology, University of North Bengal (poster presentation). 7. Dawa Bhutia, Benoy K. Rai, Joydeb Pal. 2015. Hepatic Cytochrome P450 in Freshwater Teleost, Channa punctatus (Bloch) as Biomarkers of Cypermethrin Toxicity. National Conference on “Applied Zoology in Sustainable Development: An Update”. Department of Zoology, University of North Bengal (oral presentation).
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Appendix C
REPRINTS OF PUBLICATIONS
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130
Vol.58, n.1: pp. 130-135, January-February 2015 BRAZILIAN ARCHIVES OF http://dx.doi.org/10.1590/S1516-8913201400049 ISSN 1516-8913 Printed in Brazil BIOLOGY AND TECHNOLOGY AN INTERNATIONAL JOURNAL
Hepatic Cytochrome P450 as Biomarkers of Cypermethrin Toxicity in Freshwater Teleost, Channa punctatus (Bloch)
Dawa Bhutia, Benoy Kishore Rai and Joydeb Pal * Ecology and Toxicology Laboratory; Department of Zoology; University of North Bengal; Siliguri, West Bengal - India
ABSTRACT
In this study, Channa punctatus was treated with sub-lethal concentration of cypermethrin (6.6 µg/L) for 5, 10 and 15 days and its effect on total CYP 450 and the activity of hepatic CYP450 isoforms measured. Total CYP450 content and CYP1A mediated EROD activity was significantly induced (p<0.05) in all three treated groups compared to control whereas only 15 days treated group showed significant induction in CYP2B mediated N,N- dimethylaniline demethylase activity. CYP2E1 mediated aniline hydroxylase activity showed only a marginal increase while there was inhibition of CYP3A4 mediated erythromycin demethylase activity. Liver somatic index (LSI) also showed a marginal increase in all the treated groups. Results showed differential induction of CYP1A, CYP2B, CYP2E1 and inhibition of CYP3A4 isoform due to cypermethrin treatment in C. punctatus . The study clearly showed CYP1A isoform as the most responsive and important biomarker for monitoring the aquatic pollution.
Key words: cypermethrin, cytochrome P450, fish , Channa punctatus, biomarker
INTRODUCTION such as chlorinated hydrocarbons, organophosphates and carbamates. Cypermethrin Pesticide usage is a critical concern which may is a type of cyanophenoxybenzyl pyrethroid and is have an adverse effect on the delicate ecosystem categorized as restricted use pesticide by US EPA because of their toxicity, persistency and tendency because of its high toxicity to fish (Saha and to concentrate in organisms as they move up in the Kaviraj 2009). food chain, increase their toxicity to fish, birds Fish, as a bioindicator species, plays an and other wildlife and, in turn to man. While the increasingly important role in the monitoring of pesticides are instrumental in achieving significant water pollution because it responds with great increase in crop productivity, they also cause sensitivity to the changes in the aquatic serious ecological hazards to the non-target environment. Biochemical markers are animals especially the fish, which forms an measurable responses to the exposure of an important part of food chain for various animals organism to xenobiotics. Regarding biochemical including human beings (Sharma and Ansari responses in fish to aquatic pollutants, cytochrome 2011). P450 (CYP 450) is known to play a major role in Synthetic pyrethroids have been introduced over the oxidative metabolism/biotransformation of a the past two decades for agricultural and domestic wide range of both endogenous and exogenous use as replacements for more toxic pesticides, compounds and is considered one of the most
*Author for correspondence: [email protected]
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important phase I biotransformation enzymes to be inactive after 72 h (Saha and Kaviraj 2008) (Siroka and Drastichova 2004). The families and only water for the control group. responsible for the metabolism of xenobiotics are Homogeneity was maintained in all the groups by CYP1 to CYP3, and to a much lesser extent providing similar experimental conditions. At the CYP4, whereas cytochrome P450 enzymes end of the experiment, livers were excised, belonging to other families are concerned with the weighed and the liver somatic index (LSI) was metabolism of endogenous substrates (Ioannides determined as percentage ratio of liver weight to and Lewis 2004). body weight. The liver somatic index was CYP 450 is very sensitive to a wide range of calculated by using the formula: LSI = (liver xenobiotics and is usually the first detectable and weight/total live weight) X 100. As the liver quantifiable response to environmental change, samples were too small to be processed including a change in the chemical environment. individually for enzyme activity, they were pooled Understanding of toxicological mechanisms have (eight fish livers each) before homogenization. provided an advancement in biological tools for environmental assessments and CYP 450 family is Microsome isolation widely studied as a biomarker for environmental Microsomes were isolated using the procedure contamination in aquatic ecosystems. These tools, described by Chang and Waxman (1998). Livers called biomarkers, are biological responses at were perfused with a large volume of ice cold molecular, cellular, or organism levels and have perfusion buffer (1.15% KCl, 1mM EDTA, pH accordingly been classified as biomarkers of 7.4) to get rid of unwanted tissues, fat bodies and exposure, effect, and susceptibility (Moore et al. blood and homogenized in four volumes of 2004). homogenization buffer (1.15% KCl, 1 mM EDTA The effects of cypermethrin on commercially and 0.05 M Tris, pH 7.4) using a teflon important Indian species of fish are poorly homogenizer. The homogenate was centrifuged in documented. The objective of the present study a cooling centrifuge at 12000 g for 20 min. The was to evaluate if activities of hepatic cytochrome supernatant was subjected to centrifugation at P450 could be evaluated as potential biomarkers 100000 g for 60 min at 4ºC in a super speed of cypermethrin toxicity to fish. vacuum centrifuge. The resultant pellet were resuspended in two volumes of resuspension buffer containing 0.05 M Tris, 1mM EDTA and MATERIALS AND METHODS 20% Glycerol v/v, pH 7.4 as the hepatic microsomal fraction. Fish Specimens of Channa punctatus were collected Enzyme assay from the local fish market. Fish were acclimatized Sectral analysis of CYP 450 was based on the in the laboratory for a period of two weeks. After mepthod described by Omura and Sato (1964). that, healthy fish (male and female) weighing Protein in the microsomal fraction was estimated approximately 35± 5 g were transferred to glass as described by Lowry et al. (1951) using bovine aquarium with 50 L capacity in controlled light serum albumin as standard. Ethoxyresorufin O- (12 hr light/12 hr dark) and aeration conditions. deethylase (EROD) activity in the liver They were fed regularly with small pieces of microsome samples was determined chopped fish at fixed rate during the course of the spectrophotometrically by the method of Klotz et experiment. al. (1984). NNDMA activity was determined by the method Experimental design of Schenkman et al. (1967) with minor Fish were randomly taken in six groups (eight fish modifications to detect CYP2B activity. Reaction in each aquarium) each of control and treated. A mixture consisted of 100 mM N, N- sub-lethal concentration of 6.6 µg/L cypermethrin dimethylaniline, 150 mM MgCl 2, 100 mM based on acute toxicity data (not published) semicarbazide and microsomes 1.5-2.0 mg. The generated from the laboratory experiments was mixture was pre-incubated for 5 min at 32ºC and used to treat the experimental fish. The water was the reaction started by adding 10mM NADPH. renewed every 48 h with fresh pesticide for the Following aerobic incubation for another 30 min treated groups as cypermethrin has been reported the reaction was terminated by
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adding 0.5 mL each of 25% zinc sulfate and RESULTS saturated barium hydroxide. Formaldehyde formed during the assay was measured by the Total CYP 450 was significantly induced in all the method of Nash (1953) at 412 nm. three treated groups (5, 10 and 15 days) with Aniline hydroxylase activity (CYP2E1) was higher induction seen in the groups with determined by measuring the amount of p- increasing number of days when compared to aminophenol formed at 630 nm. The method was control (Table 1). The carbon monoxide modified from Imai et al. (1966) by using aniline difference spectra of dithionite reduced liver (10 mM) as substrate and NADPH (10 mM) microsomes of C. punctatus were studied at 1 min instead of an NADPH generating system. In interval over a period of 5 min (Fig. 1). The addition, the incubation mixture consisted 100 maximum absorbance was observed at 1 and 2 mM MgCl 2, 120 mM tris (pH 7.4) and 1.5-2.0 mg min and then gradual decrease in absorbance was microsomal protein. After pre-incubation for 5 seen with increase in time interval. min at 32ºC, the reaction was initiated by the The LSI values were higher in all the treated addition of NADPH and incubated for 30 min. groups (Table 1). EROD (CYP1A) activity was Erythromycin demethylase (CYP3A4) activity significantly induced in all the three treated was measured following the method of groups with highest induction shown by the 15 Werringloer (1978) at 412 nm. The reaction was days treated group as expected. The activity incubated at 32ºC for 10 min. Formaldehyde increased with the exposure time (Fig. 2). formed as the end product was measured by the Five and 10 days treated groups showed no method of Nash (1953) at 412 nm. All the assays significant increase in N, N-dimethylaniline (N, were done in duplicate. N-DMA) demethylase (CYP2B) activity. The activity was significantly induced only in the 15 Statistical analysis days treated group when compared to the control Data were expressed as mean ± standard deviation (Fig. 3). and were analyzed using one-way analysis of No significant increase in aniline hydroxylase variance (ANOVA) followed by Dunnet’s test. (CYP2E1) activity was observed in 15 days The statistical significance was tested at 5% treated group whereas 5 and 10 days treated levels. groups showed no induction (Fig. 4).
Table 1 - Total CYP 450 content and liver somatic index (LSI) of Channa punctatus . Parameter control 5 days 10 days 15 days F-value p-value Total CYP450 content (nmol/mg 0.276±0.089 0.359±0.020* 0.382±0.042* 0.425 ±.053* 6.190 0.003 protein) Liver Somatic Index [LSI %] 0.914±0.113 0.985±0.096 1.009±0.120 1.042±.109 1.924 0.155 values are Mean ± SD (n=6). * significantly different from control
Figure 1 - Carbon monoxide difference spectra of dithionite reduced liver microsomes of Channa punctatus at 1 min interval over a period of 5 min.
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All the three treated groups showed an inhibition in Erythromycin demethylase (CYP3A4) activity (Fig. 5). But only 10 and 15 days treated groups resulted in significant difference with respect to the control. The percentage of inhibition is shown in parenthesis.
Figure 2 - CYP1A mediated EROD activity in liver of Channa punctatus. * indicates significant difference from control (p<0.05).
Figure 5 - CYP3A4 mediated erythromycin demethylase (ERND) activity in liver of Channa punctatus. *indicates significant difference from control (p<0.05).
DISCUSSION
Hepatic CYP 450 expression of fish can be induced by exposure to xenobiotics because specific CYP1A expression is highest in this
organ. Most lipophilic chemicals, including drugs, Figure 3 - CYP2B mediated N, N-dimethylaniline pesticides, carcinogens, environmental pollutants demethylase (N, N-DMA) activity in liver and naturally occurring compounds undergo of Channa punctatus. *indicates enzyme-mediated oxidative, hydrolytic or significant difference from control conjugative biotransformation in liver and in extra (p<0.05). hepatic tissues, yielding more polar metabolites
that can be easily excreted. Thus, metabolism plays a critical role in determining both the efficacy and the residence time of drugs in the body as well as in modulating the response to toxic chemicals (Akdogan and Sen 2010). Two soret peaks were seen in the dithionite reduced spectra of liver microsomes (Fig. 1). These peaks consisted of the characteristic absorbance at 450 nm for the reduced CYP 450- CO complex and the other of lesser magnitude at around 420-425 nm, which could be possibly due to the absorbance of contaminating hemoglobin since tissues could not be adequately perfused (Klemz et al. 2010). Liver somatic index (LSI) has frequently been Figure 4 - CYP2E1 mediated aniline hydroxylase (AH) used as a biomarker for examining fish exposed to activity in liver of Channa punctatus. contaminants. LSI values are generally elevated in
Braz. Arch. Biol. Technol. v.58 n.1: pp. 130-135, Jan/Feb 2015 134 Bhutia, D. et al.
the vertebrates experiencing induction of hepatic in vitro transformation of toxic aniline to nontoxic microsomal P450 for the detoxification of organic derivative p-aminophenol (Zen and Korkmaz compounds and LSI is generally used to determine 2009). Although, CYP2E1 is responsible for the the physiological status of the fish (Shailaja et al. metabolism and potential bioactivation of a number 2006). Increase in LSI is commonly seen in fish of low-molecular-weight pharmaceutical and other exposed for long periods of time to organic xenobiotic compounds, including acetaminophen, contaminants in the laboratory and field. The ethanol, isoniazid, halogenated anesthetics, increase in LSI can be due to hyperplasia acetone, and benzene (Omiecinski et al. 1999), a (increased cell number) and/or hypertrophy study by Bhutia et al. (2013) has reported its role in (increased cell size). It may be associated with metabolism of pesticide with long-term exposure. increased capacity to metabolize xenobiotics so it This study showed the inhibition of CYP3A4 could be considered as adaption to the presence of activity which was somewhat surprising given that pollution rather than a dysfunction (Heath 1995). CYP3A is one of the most abundant P450 isoform Enzyme activity increases with increase in in fish liver possessing a broad range of substrate exposure period to the xenobiotics as it brings specificity (Hegelund and Celander 2003) and about the physiological differences within the accounts for the metabolism of almost 50% of the organism (Arellano-Aguilar et al. 2009). Each currently used pharmaceutical agents (Parkinson isoform of the cytochrome P450 participates in the 1996). This could also be the reason for high metabolism of many different compounds, but one toxicity of pyrethroid insecticide in fish species as substrate can also be metabolized by several CYP3A4 was reported to be induced in Labeo different isoforms (Siroka and Drastichova 2004). rohita (Rai et al. 2010) and channel catfish CYP1A subfamily is the most studied CYP 450 (Stuchal et al. 2006) after exposure to isoform and is responsible for a wide range of chlorpyriphos and methoxychlor. xenobiotic biotransformation whose catalytic The present study clearly demonstrated the activity is expressed as activity of EROD. In this usefulness of CYP 450 as an important biomarker study, the EROD activity was induced in all the for monitoring the aquatic pollution. Of all the treated groups, probably due to EROD`s higher CYP 450 isoforms (CYP1A, CYP2B, CYP2E1 detection sensitivity. Well-established inducers of and CYP3A4), CYP1A was the most sensitive CYP1A and EROD are organic contaminants isoform towards cypermethrin toxicity and in belonging to PCBs, PAHs, PCDDs and PCDFs. accordance to various other studies as one of the However, induction by pesticide compounds has most established biomarker of CYP 450 family. also been reported (Haluzova et al. 2011). Bhutia et al. (2010) reported the absence of CYP2B in fish, H. fossilis as it did not respond to ACKNOWLEDGMENTS the mammalian specific CYP2B inducer, Phenobarbital. However, Stegeman et al. (1997) The author (Dawa Bhutia) is highly grateful and reported the presence of CYP2B like activities in would like to thank University Grants commission, four tropical fish species using catalytic activity New Delhi, India for providing UGC-Meritorious assay and detection of the CYP2B protein by an Fellowship during the course of this work. The immunoblot assay. Similarly, Bhutia et al. (2013) authors would also like to thank the Head of also reported the induction of CYP2B like Department of Zoology, N.B.U. for the laboratory activities in fish, H. fossilis after exposure to facilities provided during the course of the work. pesticide, cypermethrin. It is likely that some P450s differ in their functions among the mammals and fish and follow different REFERENCES mechanisms of induction. In consequence, Akdogan HA, Sen A. Characterization of drug whenever P450 enzymes are used as biomarkers in metabolizing enzymes and assessment of aging in the monitoring programs, one should be cautious with gilthead seabream ( Sparus aurata ) liver. Vet Med. metabolic differences, even among fish species. 2010; 55(9): 463-471. CYP2E1 mediated p-hydroxylation has been Arellano-Aguilar O, Montoya RM, Garcia CM. shown to be a possible pathway for the Endogenous Functions and Expression of metabolism of xenobiotics in the liver of fish Cytochrome P450 Enzymes in Teleost Fish: A species. This activity can be detected by studying Review. Rev Fish Sci. 2009; 17(4): 541-556.
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Bhutia D, Rai BK, Pal J. Multiple forms of Cytochrome Parkinson A. Biotransformation of xenobiotics. In: P450 family in liver of fresh water teleost fish Klaassen CD, Editor. Casarett and Doull’s Heteropneustes fossilis (Bloch). NBU J Anim Sci. Toxicology: The Basic Science of Poisons, 5th ed . 2010; 4: 28-35. New York: McGraw-Hill; 1996. p. 113-186. Bhutia D, Rai BK, Pal J. Detection of Multiple Rai BK, Bhutia D, Pal J. Cytochrome P450 in liver of Cytochrome P450 in Hepatic tissue of Indian major carp, (Labeo rohita, Ham.) following Heteropneustes fossilis (Bloch) Exposed to sub-lethal exposure of chlorpyriphos. NBU J Anim Cypermethrin. Proc Zool Soc. 2013; 66(1):14-19. Sc. 2010; 4: 22-27. Chang TKH, Waxman DJ. Enzymatic analysis of Saha S, Kaviraj A. Acute toxicity of synthetic cDNA-expressed human CYP1A1, CYP1A2, and pyrethroid cypermethrin to some freshwater CYP1B1 with 7-ethoxyresorufin as a substrate. In: organisms. Bull Environ Contam Toxicol . 2008; Phillips IR, Shephard EA. Editors. Methods in 80(1): 49-52. Molecular Biology: Cytochrome P450 Protocols , Vol. Saha S, Kaviraj A. Effects of cypermethrin on some 107 , Totowa: Humana Press Inc.; 1998. p. 103-109. biochemical parameters and its amelioration through Haluzova I, Modra H, Blahova J, Havelkova M, Siroka dietary supplementation of ascorbic acid in Z, Svobodova Z. Biochemical markers of freshwater catfish Heteropneustes fossilis. Chem. contamination in fish toxicity tests. Interdiscip 2009; 74(9): 1254-1259. Toxicol. 2011; 4(2): 85-89. Schenkman JB, Remer H, Stabrook RW. Biochemical Heath A G. Water Pollution and Fish Physiology. assay of cytochrome P-450. Mol Pharmacol. 1967; 3: Second edition. Florida: CRC Press Inc.; 1995. 113-126. Hegelund T, Celander MC. Hepatic versus extrahepatic Shailaja MS, Rajamanickam R, Wahidulla S. Increased expression of CYP3A30 and CYP3A56 in adult formation of carcinogenic PAH metabolites in fish killifish ( Fundulus heteroclitus ). Aquat Toxicol. promoted by nitrite. Environ Pollut. 2006; 143(1): 2003; 64(3): 277-291. 174-177. Imai Y, Ito A, Sato R. Evidence for biochemically Sharma DK, Ansari BA. Effect of Deltamethrin and a different types of vesicles in the hepatic microsomal Neem Based Pesticide Achook on Some Biochemical fraction. J Biochem. 1966; 60(4): 417-428. Parameters in Tissues Liver, Ovary and Muscle of Ioannides C, Lewis DFV. Cytochromes P450 in the Zebrafish, Danio rerio (Cyprinidae), Res. J Chem Sci. Bioactivation of Chemicals. Curr Top Med Chem. 2011;1(4): 125-134. 2004; 4: 1767-1788. Siroka Z, Drastichova J. Biochemical Markers of Klemz C, Salvo LM, Neto J da CB, Bainy ACD, Assis Aquatic Environment Contamination -Cytochrome HC da S da. Cytochrome P450 Detection in Liver of P450 in Fish. A Review. Acta Veterinaria Brno. the Catfish Ancistrus multispinis (Osteichthyes, 2004; 73: 123-132. Loricariidae) Braz Arch Biol Technol. 2010; Stegeman JJ, Woodin BR, Singh H, Oleksiak 53(2):361-368. MF,Celander M. Cytochromes P450 (CYP) in Klotz AV, Stegeman JJ, Walsh C. An alternative 7- tropical fishes: catalytic activities, expression of ethoxyresorufin O-deethylase activity assay: a multiple CYP proteins and high levels of microsomal continuous visible spectrophotometric method for P450 in liver of fishes from Bermuda. Comp Biochem measurement of cytochrome P450 monooxygenase Physiol C Pharmacol Toxicol Endocrinol . 1997; activity. Anal Biochem. 1984; 140(1): 138-145. 116(1): 61-75. Lowry OH, Rosenbrough NS, Farr AL, Randall RJ. Stuchal LD, Kleinow KM, Stegeman JJ, James MO. Protein measurement with the Folin phenol reagent. J Demethylation of the pesticide methoxychlor in liver Biol Chem. 1951; 193(1): 265-275. and intestine from untreated, methoxychlor-treated, Moore NM, Depledge MH, Readman JW, Leonard and 3-methylcholanthrene treated channel catfish DRP. An integrated biomarker-based strategy for (Ictalurus punctatus ): Evidence for roles of CYP1 ecotoxicological evaluation of risk in environmental and CYP3A family isozymes. Drug Metab Dispos. management. Mutat Res. 2004; 552: 247–268. 2006; 34(6): 932-938. Nash T. The colorimetric Estimation of Formaldehyde Werringloer J. Assay of fomaldehyde generated during by means of the Hantzsch Reaction. Biochem J. 1953; microsomal oxidation reactions. Methods Enzymol. 55(3): 416-421. 1978; 52: 297-302. Omiecinski CJ, Remmel RP, Hosagrahara VP. Concise Zen T, Korkmaz H. The effects of Urtica Dioica L. leaf Review of the Cytochrome P450s and their Roles in extract on aniline 4-hydroxylase in mice. Acta Pol Toxicology. Toxicol Sci. 1999; 48(2): 151-156. Pharm. 2009; 66(3): 305-309. Omura J, Sato R. The carbon monoxide binding pigment of liver microsomes. I. Evidence for its heme protein nature . J Biol Chem. 1964; 239(7): 2370- Received: April 02, 2014; 2378. Accepted: June 11, 2014.
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E
Proc Zool Soc (Jan-June 2013) 66(1):14–19 T H Y DOI 10.1007/s12595-013-0063-6 T
K O LK ATA RESEARCH ARTICLE
Detection of Multiple Cytochrome P450 in Hepatic Tissue of Heteropneustes fossilis (Bloch) Exposed to Cypermethrin
Dawa Bhutia • Benoy K. Rai • Joydeb Pal
Received: 2 July 2012 / Revised: 13 February 2013 / Accepted: 17 March 2013 / Published online: 28 March 2013 Ó Zoological Society, Kolkata, India 2013
Abstract Cypermethrin (alpha-cyano-3-phenoxybenzyl Keywords Pesticide Fish Biomarker EROD ester of 2,2-dimethyl-3-(2,2-dichlorovinyl) cyclopropane Aquatic pollution carboxylic acid) is a synthetic pyrethroid. It is one of the most widely used pesticide in commercial agricultural applica- tions because of its high effectiveness against target species. Introduction Beside its target toxicity it is also highly toxic to other non- target species like fish, bees and aquatic insects. The aim of Contamination of the environment with anthropogenic this study was to detect the presence of cytochrome P450 compounds is a widespread phenomenon and occurs both (CYP 450) in the hepatic microsomes of Heteropneustes from fixed and diffused source. Pesticides carried away by fossilis upon exposure to cypermethrin. The 96 h LC50 value rains and floods to larger water bodies like ponds and rivers for each exposure route was calculated and two groups were alter the physico-chemical properties of water. Cyper- treated, with one group receiving a single IP (intraperitoneal) methrin, the alpha-cyano-3-phenoxybenzyl ester of 2,2- injection for 96 h (0.030 mg/kg body weight) and the other dimethyl-3-(2,2-dichlorovinyl) cyclopropane carboxylic group with 1/3 sub-lethal concentration (1.2 lg/l) of the acid is a widely used pesticide based on pyrethroids and
LC50 value in water for 15 days. Activities of the enzymes account for almost over 30 % of the global pesticide use ethoxyresorufin-o-deethylase (EROD), N,N-dimethylani- (Dahamna et al. 2011). Although synthetic pyrethroids are line demethylase, aniline hydroxylase and erythromycin less persistent and less toxic to mammals and birds, they demethylase mediated respectively by the isozymes CYP1A, are highly toxic to a number of non-target organisms such CYP2B, CYP2E1 and CYP3A4 were studied. The liver as bees, freshwater fish and other aquatic organisms even at somatic index (LSI) was also calculated to determine the a very low concentration (Oudou et al. 2004). physiological status of the fish. Activities of CYP1A, Biomarkers so called biological responses to xenobiotics CYP2B and CYP2E1 enzymes increased significantly while have been studied as environmental pollution indicators that of CYP3A4 enzyme inhibited as compared to control. and used as early warning systems of environmental con- Total CYP 450 content was also significantly induced in both tamination (Walker et al. 1996). The biochemical marker the treated groups. The increase in activities of CYP P450 that is best studied so far is the stimulation of cytochrome isozymes could be used as a biomarker to indicate the pol- P450-dependent monooxygenases (CYP 450) which is also lution of an aquatic environment by the pesticide. considered the most dominant enzyme system responsible for oxidation processes in phase I biotransformation. Payne and Penrose (1975) were among the first to make use of this enzyme system as a biomarker, reporting elevated CYP 450 activity in fish from petroleum-contaminated sites in the aquatic environment and members of CYP 450 gene & D. Bhutia B. K. Rai J. Pal ( ) families 1–4 are considered prominent in xenobiotic Department of Zoology, Ecology and Toxicology Laboratory , University of North Bengal, Siliguri 734013, West Bengal, India metabolism. Thus, CYP 450 enzymes are important in the e-mail: [email protected] mechanisms underlying chemically induced toxicity or 123 Proc Zool Soc (Jan-June 2013) 66(1):14–19 15 disease and chemical–ecological interactions. Fish species The fish were sacrificed by a single blow on the head are useful as bioindicators, since they are promptly exposed and then livers were excised, weighed and the liver somatic to diverse anthropogenic water contaminants and in the index (LSI) was determined as percentage ratio of liver fish, CYP 450 has primarily been studied as a biomarker weight to body weight. The liver somatic index was cal- indicating pollution of the aquatic environment by indus- culated by using the formula: LSI = (liver weight/total live trial or agricultural sewage. However, responses to xeno- weight) 9 100. As the liver samples were too small to be biotics in fish may differ from those in other species processed individually for enzyme activity, they were (Siroka and Drastichova 2004). pooled before homogenization. This work was aimed to evaluate the effects of cyper- Microsomes were isolated using the procedure described methrin on CYP1A, CYP2B, CYP2E1and CYP3A4 by Chang and Waxman (1998), with minor modifications. enzymes as biomarkers in Heteropneustes fossilis. Stan- Livers were perfused with a large volume of ice cold dardization of biomarkers for detection of sub-lethal effects perfusion buffer (1.15 % KCl, 1 mM EDTA, pH 7.4) to get of cypermethrin in this species under laboratory condi- rid of unwanted tissues, fat bodies and blood and homog- tions will enable biomonitoring of cypermethrin in the enized in four volumes of homogenization buffer (1.15 % environment. KCl, 1 mM EDTA and 0.05 M Tris, pH 7.4) using a Teflon homogenizer. The homogenate was centrifuged in a cool- ing centrifuge at 12,0009g for 20 min. The supernatant Materials and Methods was ultra centrifuged at 100,0009g for 60 min at 4 °Cina super speed vacuum centrifuge. The pellet were resus- Heteropneustes fossilis were collected from non-contami- pended in two volumes of resuspension buffer containing nated local ponds of Gossaipur of Darjeeling district. 0.05 M Tris, 1 mM EDTA and 20 % Glycerol v/v, pH 7.4 Fishes were acclimatized in the laboratory for a period of to obtain the hepatic microsomal fraction. 2 weeks. After 2 weeks, healthy fish (male and female) Analysis of CYP 450 was based on the method descri- weighing approximately 35 ± 5 g were transferred to glass bed by Omura and Sato (1964) with minor modifications. aquarium with 50 l capacity in controlled light (12 h light/ The microsomal preparation was diluted in 0.1 M phos- 12 h dark), and aeration conditions. The fishes were fed phate buffer and saturated with carbon monoxide for 40 s. regularly with small pieces of chopped fish during accli- The sample was equally divided between two cuvettes. The matization and feeding stopped during the course of baseline (400–500 nm) was recorded and a few milligrams treatment. of sodium dithionite were added to only one of the cuvette
The 96 h LC50 value was calculated using probit anal- in order to reduce CYP450 and the absorbance measured in ysis (Finney 1971). Fish were randomly taken in 5 groups a spectrophotometer. (10 fishes in each aquarium of 20 l capacity). The 96 h Protein in the microsomal fraction was estimated as
LC50 value for H. fossilis was determined as 3.783 lg/l described by Lowry et al. (1951). The values of protein (data not published). One group of the experimental fish were determined from the standard curve prepared from (5–7 fish) was treated with sub lethal concentration OD values against different concentrations of bovine serum
(1.2 lg/l) of cypermethrin, Ripcord 10 % EC (1/3 of LC50 albumen standard. value) for 15 days. The water was renewed every 2 days EROD (ethoxyresorufin O-deethylase) was determined and the desired concentration of the pesticide was main- by spectrophotometric method of Klotz et al. (1984). The tained before introducing the treated group while only reaction mixture consisted of (0.1 M) NaCl, (2 lM) water was changed for the control groups. 7-ethoxresorufin, (0.1 M) tris buffer-pH 7.8 and 100–200 lg Similarly, another group was administered with a of microsomes. After pre-incubation at 30 °C for 5 min the single IP (intraperitoneal) injection of 0.03 mg/kg body reaction was initiated by the addition of (0.01 M) NADPH weight of the pesticide. The 96 h IP LC50 value was and the reaction was stopped following 10 min of incubation determined as 0.090 mg/kg body weight (data not pub- at the same temperature with 0.5 ml of ice cold trichloro- lished). The pesticide was diluted with distilled water acetic acid. After incubation, the amount of resorufin formed and the positive (?VE) control fishes received only an IP was measured at 572 nm. injection of distilled water in the same volume as the Procedure of Schenkman et al. (1967) was adopted with injected group. Homogeneity was maintained in all the minor modifications to determine the N,N-dimethylaniline groups by providing similar experimental conditions. (N,N-DMA) demethylase and aniline hydroxylase activity. Two treated and the ?VE control groups were studied The reaction mixture for N,N-DMA activity consisted of for the experiment to determine the stress level, speci- the (0.1 M) phosphate buffer-pH 7.4, (0.1 M) N,N-
ficity and interaction of the pesticide with respect to the dimethylaniline, (0.15 M) MgCl2, (0.1 M) semicarbazide control. and microsomes (1.0 mg). The mixture were incubated for 123 16 Proc Zool Soc (Jan-June 2013) 66(1):14–19
five minutes at 32 °C and the reaction started by adding Results and Discussion (0.1 M) NADPH instead of NADPH generating system (NADP, glucose-6-phosphate, glucose-6-phosphate dehy- Both the treated groups showed significant stimulation drogenase and MgCl2). Following aerobic incubation for (p \ 0.05) with respect to the control in the total CYP450 another 30 min the reaction was terminated by adding content (Table 1). The 15 days sub-lethal treated group 0.5 ml each of 25 % zinc sulfate and saturated barium showed higher stimulation (0.523 nmol/mg protein) than hydroxide. After centrifugation at 10,0009g for 10 min, the 96 h IP treated group (0.509 nmol/mg protein). The 1 ml of the supernatant was mixed with 2 ml of double ?VE control group did not show any significant difference strength Nash reagent and incubated at 60 °C for 30 min. with the control group. The increase in CYP 450 content Formaldehyde formed as the end product of N,N-dimeth- after the treatment positively correlated with the increase in ylaniline demethylase activity was measured by the method the enzymatic activity. of Nash (1953) at 412 nm. Both the treated groups and the ?VE control group The reaction mixture for aniline hydroxylase activity showed marginal increase in LSI with respect to that of the consisted of (0.1 M) aniline, (0.1 M) MgCl2, (0.12 M) tris- control but the means were not statistically significant. pH 7.4 and 1.0 mg of microsomal protein. The reaction When all the groups were considered, the 96 h IP treated mixture temperature was raised to 32 °C by incubation in a group showed higher LSI value than the 15 days sub-lethal water bath for 5 min and the reaction was started by adding treated and the control groups (Table 1). (0.1 M) NADPH. The reaction was terminated after 30 min Table 2 shows the enzymatic activity reflecting stimu- by adding 0.5 ml of ice cold trichloroacetic acid. The lation of CYP1A, CYP2B and CYP2E1 and inhibition of precipitate was removed by centrifugation at 10,0009g for CYP3A4 isozymes. The treated groups, i.e. 96 h IP treated 10 min and 1 ml of supernatant was added to 1 ml of a and the 15 days sub-lethal treated showed similar trend in solution containing 2 % phenol in (0.2 M) NaOH and 1 ml the enzymatic activities when compared with the control in of (1 M) Na2CO3. After 30 min of incubation at 32 °C, the all the 4 enzyme activities studied—EROD, Aniline p-aminophenol formed as the end product of aniline hydroxylase, N,N-dimethylalinile demethylase and Eryth- hydroxylase activity was measured at 630 nm. romycin demethylase. Similar results were also displayed Erythromycin demethylase activity was measured fol- by the control and the ?VE control group. No significant lowing the method of Werringloer (1978) at 412 nm. The difference in the enzyme activities was seen between the reaction mixture consisted of (0.05 M) phosphate buffer- control and the ?VE control (injected with distilled water pH 7.25, (0.01 M) erythromycin, (0.15 M) MgCl2 and alone) and also between 15 days sub-lethal and 96 h IP microsomes (1 mg). Reactions were initiated by the addi- treated groups following injection with cypermethrin. tion of (0.1 M) NADPH, and the mixture was incubated for EROD activity was significantly stimulated (p \ 0.01) 10 min at 32 °C. The amount of formaldehyde formed was by both the treatment regimes of cypermethrin. The measured follwing the method of Werringloer (1978)at 15 days treated group showed higher stimulation than the 412 nm. The reaction was terminated by adding 0.5 ml of 96 h IP treated group. An increase of almost 1-fold in 20 % trichloroacetic acid. All assays were done in dupli- the activity was seen in both the treated group compared to cate and repeated twice. the control and the ?VE control (Table 2). Both the Differences between groups of animals or treatments treated group showed stimulation in N,N-dimethylaniline were analysed using one-way analysis of variance demethylase activity compared to the control. The 15 days (ANOVA) followed by Least Significant difference (LSD) treated group of H. fossilis showed the highest stimulation test. The statistical significance was tested at 1 and 5 % with an increase of 225 % while 167 % increase was seen levels. in 96 h treated group (Table 2). Aniline hydroxylase
Table 1 Total CYP 450 content and liver somatic index (LSI) of H. fossilis Parameter Control ?ve Control 96 h IP treated 15 days sub-lethal F-value p value (n = 7) (n = 5) (n = 5) treated (n = 6)
CYP 450 content (nmol/mg protein) 0.313 ± 0.061 0.294 ± 0.133ns 0.509 ± 0.069* 0.523 ± 0.234* 4.233 .019 Liver somatic index [LSI %] 0.988 ± 0.140 1.108 ± 0.098ns 1.192 ± 0.296ns 1.029 ± 0.227ns 1.140 .358 Values are Mean ± SD ?v positive, ns not significant * Significantly different (p B 0.05)
123 Proc Zool Soc (Jan-June 2013) 66(1):14–19 17
Table 2 EROD, N,N-dimethylaniline (N,N-DMA) demethylase, aniline hydroxylase (AH) and erythromycin demethylase (ERND) activity of hepatic microsomes in H. fossilis Parameter Control (n = 7) ?ve control (n = 5) 96 h IP treated (n = 5) 15 days sub-lethal treated (n = 6) F value p value
ERODa 34.450 ± 3.357 33.886 ± 3.592ns 61.897 ± 9.968** 63.661 ± 10.766** 20.412 0.000 N,N-DMAb 0.112 ± 0.064 0.154 ± 0.115ns 0.300 ± 0.058* 0.364 ± 0.164** 7.634 0.002 AHb 0.104 ± 0.094 0.169 ± 0.061ns 0.375 ± 0.163* 0.249 ± 0.045* 7.185 0.002 ERNDb 0.743 ± 0.168 0.770 ± 0.120ns 0.504 ± 0.166* 0.505 ± 0.142* 4.398 0.021 Values are Mean ± SD * Significantly different (p B 0.05), ** (p B 0.01), p value indicates significant difference between the control and different treated groups ?ve positive, n not significant with the control a Expressed as (pmol/mg protein/min) b Expressed as (nmol/mg protein/min) activity was significantly stimulated (p \ 0.05) in both the thus adding to the absorbance at 420 nm. Two soret peaks treated group. The 96 h IP treated group of H. fossilis were seen in the dithionite reduced spectra of liver showed the highest stimulation with an increase of 260 % microsomes (Fig. 1). These peaks consisted of the char- than that of 15 days treated group with 139 % when acteristic absorbance at 450 nm for the reduced CYP450- compared with the control. No stimulation in the CYP3A4 CO complex and the other of lesser magnitude at around was shown by both the treated groups of fish studied. Also 420–425 nm which may be possibly due to the absorbance there was no significant difference in the activities of CYP of contaminating hemoglobin or CYP 420 (Klemz et al. 450 isozymes between the control and the ?VE control. 2010). The carbon monoxide difference spectra of dithionite LSI has frequently been used as a biomarker for reduced liver microsomes of H. fossilis were studied at examining fish exposed to environmental contaminants and 1 min interval over a period of 5 min. The maximum also to determine the physiological status of the fish. LSI absorbance was noticed at 1–2 min and then a gradual values are generally elevated in vertebrates experiencing decrease in absorbance was seen with increase in time stimulation of hepatic microsomal CYP 450 and the ele- interval. This time interval corresponds to the length of vation in LSI may be due to the altered allocation of energy incubation required for maximum reduction of the enzyme reserves for detoxification of organic compounds (Miller by sodium dithionite. The CYP 450 converts to a stable et al. 2009). denatured state CYP 420 under long periods of incubation The biochemical responses assessed in the present study are the earliest indicators of exposure to stressors that can be detected in an organism. CYP 450 metabolizes and biotransforms most lipophilic xenobiotics including drugs, pesticides, carcinogens and environmental pollutants into more polar metabolites that can be easily excreted from the body and are useful as early warning indices of environ- mental alteration, so that remedial actions may be taken by resource protection managers in time to prevent permanent decline in fish populations (Thomas 1990). It has been detected that variability of enzyme activity is higher if the organism in test is exposed for longer duration to the xenobiotic or pollutants as it brings about the physiological differences within the organism (Arellano- Aguilar et al. 2009). This may be the reason that the activities of CYP450 is slightly higher in 15 days sub- lethal treated compared to the 96 h IP treated group. Although an IP injection of test agents is not an environ- mentally relevant route of exposure, however, for phar- macological considerations, it is a common practice of Fig. 1 Carbon monoxide difference spectra of dithionite reduced liver microsomes of H. fossilis at 1 min interval over a period of exposure to chemical in animals as it allows a direct effect 5 min on the target organs (Rice and Roszell 1998). Fish can also 123 18 Proc Zool Soc (Jan-June 2013) 66(1):14–19 be subjected to desired concentrations of pesticides through CYP450 mediated enzyme activities in H. fossilis could injection to cut down the time of exposure and also for serve as a useful biomarker of cypermethrin pollution in understanding the effects on biotransformation enzymes the aquatic environment. (Assis et al. 2009). CYP 450s, principally the CYP1A isoform have been Acknowledgments The authors are highly grateful and would like used to detect the presence of pollutants in aquatic envi- to thank UGC (University Grants commission), New Delhi, India for providing the financial support during the course of this work. The ronment due to its high affinity to xenobiotics, such as authors would also like to thank the Department of Zoology, N.B.U. polycyclic aromatic hydrocarbons (PAHs) and polychlori- for the help provided during the working period. nated biphenyls (PCBs) and have been so far proved to be the most sensitive indicators (Schlenk and Di Giulio 2002). Although the evaluation of CYP1A stimulation by EROD References activity is a common tool for quantifying environmental exposure to aryl hydrocarbon receptor (AhR) ligands, a Arellano-Aguilar, O., R.M. Montoya, and C.M. Garcia. 2009. number of studies have been conducted that establishes Endogenous functions and expression of cytochrome P450 CYP1A stimulation in response to pesticides. Lemaire enzymes in teleost fish: A review. Reviews in Fisheries Science 17: 4541–4556. et al. (2010) have reported induced level of EROD activity Bhutia, D., B.K. Rai, and J. Pal. 2010. Multiple forms of Cytochrome in liver microsomes of roundnose grenadier in response to P450 family in liver of fresh water teleost fish Heteropneustes DDT exposure. Similar results have been confirmed in fossilis (Bloch). North Bengal University Journal of Animal fishes exposed to deltamethrin (Assis et al. 2009) and Sciences 4: 28–35. Chang, T.K.H., and D.J. Waxman. 1998. Enzymatic analysis of chlorpyriphos (Rai et al. 2010). cDNA-expressed human CYP1A1, CYP1A2, and CYP1B1 with Earlier studies have reported that Phenobarbital, specific 7-ethoxyresorufin as a substrate. In Methods in molecular inducer of CYP2B in mammals have not been found to biology: Cytochrome P450 protocols, vol. 107, ed. I.R. Phillips, induce CYP2B in fish (Bhutia et al. 2010), although and E.A. Shephard, 103–109. Totowa: Humana Press Inc. Dahamna, S., A. Belguet, D. Bouamra, A. Guendouz, M. Mergham, CYP2B like activities do occur in fish as reported by and D. Harzallah. 2011. Evaluation of the toxicity of cyper- Stegeman et al.(1997). It may be possible that pesticides do methrin pesticide on organs weight loss and some biochemical induce CYP2B like activities (Price et al. 2008) in fish. and histological parameters. Communications in Agricultural Though specific CYPs are necessary to metabolize xeno- and Applied Biological Sciences 76: 915–921. de Assis, H.C.S., L. Nicareta, M.L. Salvo, C. Klemz, H.J. Truppel, biotics, pesticides have the ability to induce one or more and R. Calegari. 2009. Biochemical biomarkers of exposure to forms of CYP 450 simultaneously. deltamethrin in freshwater fish, Ancistrus multisipinis. Brazilian Aniline hydroxylation by cytochrome P450-dependent Archives of Biology and Technology 52: 1401–1407. enzyme systems has been shown to be a possible way for Finney, D.J. 1971. Probit analysis, 3rd ed. Cambridge, London: Cambridge University Press. metabolism in the liver of fish species. Aniline hydroxylase Klemz, C., L.M. Salvo, J.C.B. Neto, A.C.D. Bainy, and H.C.S. de activity is suggested to be mainly catalyzed by enzymes Assis. 2010. Cytochrome P450 detection in liver of the catfish belonging to the CYP450 2E1 sub-family in fish as in Ancistrus multispinis (Osteichthyes, Loricariidae). Brazilian mammals and studies have reported an stimulation of Archives of Biology and Technology 53: 361–368. Klotz, A.V., J.J. Stegeman, and C. Walsh. 1984. An alternative CYP2E1 activity upon exposure to pesticides carbaryl 7-ethoxyresorufin O-deethylase activity assay: a continuous (Tang et al. 2002) and parathion (Mutch et al. 2003). visible spectrophotometric method for measurement of cyto- Both the treated groups showed significant inhibition in chrome P450 monooxygenase activity. Analytical Biochemistry erythromycin demethylase activity although CYP3A is 140: 138–145. Lemaire, B., G.I. Priede, A.M. Collins, M.D. Bailey, N. Schtickzelle, reported to be induced in Labeo rohita exposed to chlor- P.J. Thome, and F.J. Rees. 2010. Effect of organochlorines on pyriphos (Rai et al. 2010). It is seen that CYP3A4 is the cytochrome P450 activity and antioxidant enzymes in liver of predominant isoform responsible for metabolizing pyre- roundnose grenadier Coryphaenoides rupestris. Aquatic Biology throid pesticides in mammals (Scollon et al. 2009), thus, a 8: 161–168. Lowry, O.H., N.S. Rosenbrough, A.L. Farr, and R.J. Randall. 1951. potential explanation for the high pyrethroid toxicity in fish Protein measurement with the Folin phenol reagent. Journal of can be due to the inhibition of CYP3A4 isoform in the liver Biological Chemistry 193: 265–275. and other tissues. Miller, L.L., J.B. Rasmussen, V. Palace, and A. Hontela. 2009. The The present study demonstrates evidence of CYP 450 physiological stress response and oxidative stress biomarkers in rainbow trout and brook trout from selenium-impacted streams in a related xenobiotic metabolism in the liver of H. fossilis coal mining region. Journal of Applied Toxicology 29: 681–688. exposed to cypermethrin. The CYP 450 mediated catalytic Mutch, E., A.K. Daly, J.B.S. Leathart, P.G. Blain, and F.M. Williams. activity may also be a very efficient adaptive strategy of the 2003. Do multiple P450 isoforms contribute to parathion species to increase tolerance to the pesticides and guarantee metabolism in man? Archives of Toxicology 77: 313–320. 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123 Research Article NBU J. Anim. Sc. 4: 28 – 35 (2010)
MULTIPLE FORMS OF CYTOCHROME P450 FAMILY IN LIVER OF FRESHWATER TELEOST FISH, Heteropneustes fossilis (Bloch)
Dawa Bhutia, Benoy K Rai and Joydeb Pal*
Ecology and Toxicology Laboratory, Department of Zoology, University of North Bengal, District Darjeeling-734013
Received: November 13, 2010 Accepted: December 13, 2010
ABSTRACT Cytochrome P450 (CYP450) is a very large and diverse super family of heme proteins found in all domains of life. Among the diverse family of Cytochrome P450, family 1, 2, and 3 are of prime importance for the oxidation of xenobiotic compounds. Except for cytochrome P450 1A (CYP1A) information on other CYP450 families in fish is limited as compared to mammals. In the present study, CYP1A, CYP2E1, CYP2B and CYP3A-like activity have been reported from liver microsomes of fish, Heteropneustes fossilis. CYP1A-dependent EROD activity, CYP2B-dependent N, N-dimethylaniline demethylase activity, CYP3A-dependent erythromycin demethylase activity and CYP2E1-dependent aniline hydroxylase activity were detected in liver microsomes. Induction of specific enzyme activities upon treatment with inducers such as naphthalene, phenobarbitone, deflazacort and acetone, specific for the CYP450 family was found except for CYP2B.
Key words: Cytochrome P450, Heteropneustes fossilis, CYP1A, CYP2E1, CYP2B, CYP3A.
INTRODUCTION classified within the same family and exhibiting 55% sequence similarity are grouped into the same Cytochrome P450 is a very large and diverse subfamily (Nebert and McKinnon, 1994; Nelson et super family of heme proteins found in bacteria, al., 1996). archaea and eukaryotes. The term cytochrome P450 Cytochrome P450, the most important group refers to a family of heme proteins present in all of phase I biotransformation enzymes, are involved mammalian cell types, except mature red blood cells in detoxification and elimination of foreign compounds and skeletal muscle which catalyzes oxidation of a from the body (Anzenbacher and Anzenbacherová, wide variety of structurally diverse compounds, such 2001). Fish plays an important role in monitoring of as steroids and fatty acids (including prostaglandins water pollution because it responds with great and leukotriens) and exogenous compounds, such as sensitivity to changes in aquatic environment (Saha drugs, food additives or industrial by-products that and Kaviraj, 2003). The induction of cytochrome P450 enter the body through the skin (Okita and Masters, isoforms can be used as biochemical markers of 1997). Several hundred CYP450s have been pollution in fish exposed to toxic compounds in the characterized in different species of organisms that aquatic system. Biochemical markers are measurable have been studied (Nelson et al., 1996). A responses to the exposure of an organism to standardized nomenclature has been adopted based xenobiotics. In fish the cytochrome P450 has primarily on sequence similarities to categorize them primarily being studied as biomarker indicating into various families and subfamilies. P450 proteins pollution of the aquatic environment by industrial or exhibiting 40% similarity in amino acid sequence are agricultural sewage. Cytochrome P450 responds to
*Author for correspondence: Prof. Joydeb Pal, [email protected]
28 Multiple Forms of Cytochrome P450 Family in Liver of Freshwater Teleost Fish water contamination when it is too low or when the Dose treatment contaminant is no longer dissolved in water but persist Fishes were given a single intraperitoneal only in residues of living matter (Machala et al., injectin of 20 mg/kg bodyweight of naphthalene, 1997). phenobarbitone and deflazacort and 2 ml/kg body In fish, cytochrome P450 1A (CYP1A) weight of acetone. After an exposure of 72 hours, activities are often used as a marker to determine the enzyme activities were studied with respect to the quantities of persistent organic pollutants control groups. (Havelkova et al., 2007). Induction of cytochrome P450 1A in fish has been widely used as a sensitive Preparation of Microsomes and convenient early warning signal of exposure to The procedure for preparation of aryl hydrocarbon agonists such as polychlorinated microsomes was based on the methods reported by biphenyls, dioxins, furans, organochlorine pesticides Nilsen et al. (1998), Chang and Waxman (1998), with and polyaromatic hydrocarbons (Payne et al.,1987; minor modifications. A sample of liver tissues (0.5 to Goksoyr and Forlin, 1992; Beyer et al., 1996; 2.0g) were placed in ice cold homogenization buffer Jonsson, 2003). In mammals, phenobarbital (PB) is containing 1.15% KCl, 1mM EDTA and 0.05M Tris, an in vivo inducer of the cytochrome P450 2B pH 7.4 (4 ml per gram of tissue) and homogenized. (CYP2B) family, whereas in teleosts PB induction The homogenized tissues were centrifuged in a cooling of cytochrome P450 is unclear (Sadars et al., 1996). centrifuge (REMI C 24) at 12,000xg for 20 min at a Cytochrome P450 3A (CYP3A) plays an important temperature of 4oC. The supernatant (post role in the metabolism of endogenous substances and mitochondrial supernatant, PMS) were carefully xenobiotics including pharmaceuticals. Cytochrome harvested, without disturbing the pellet and further P450 2E1 (CYP2E1) metabolises many endogenous centrifuged in a super speed vacuum centrifuge or exogenous small molecules such as acetone, (VS35SMTi) at 1,00,000xg for 60 minutes at 4oC. glycerol, ethanol, acetaminophen, carbon tetrachloride, The supernatant were discarded (cytosol fraction) halothane or nitrosamines (Gonzalez, 2005). and the microsomal pellet were resuspended in a In the present study the fish, resuspension buffer containing 0.05M Tris, 1mM Heteropneustes fossilis were treated with specific EDTA and 20% Glycerol, pH 7.4 (2 ml per gram of inducers to study whether fish cytochrome P450 original tissue weight). The fraction were maintained shows similar activities like that of their mammalian on ice for immediate analysis or stored at -20oC for counterpart. future use.
MATERIALS AND METHODS Protein Estimation Protein in the microsomal fraction was Collection and maintenance of healthy fish estimated as described by Lowry et al. (1951) using Healthy Heteropneustes fossilis weighing bovine serum albumin as standard protein. an average of 35±2 gm were collected from commercial ponds of Sonapur of Darjeeling district. Spectral analysis of cytochrome P-450
After a bath in 0.1% KMnO4 solution for 1 min to Spectral analysis of cytochrome P450 in the avoid any infection, the fishes were kept in glass hepatic microsomal fraction was done using the aquaria measuring 90x35x35 cm and the depth of method described by Omura and Sato (1964). Briefly water was maintained at 20cm. The fishes were fed diluted microsome preparation was mixed with a few regularly with chopped fishes. The fishes were milligrams of sodium dithionite in order to reduce acclimatized for at least fifteen days under laboratory CYP450. The sample was equally divided between conditions before the start of experiment. two cuvettes. The baseline (400nm to 500nm) was
29 Dawa Bhutia, Benoy K Rai and Joydeb Pal recorded following which only one of the cuvette was Determination of erythromycin demethylase saturated with carbon monoxide for 30 seconds and activity the absorbance was measured in a spectrophotometer Erythromycin demethylase assay was (RAY LEIGH, UV-2601). The data was used to carried out by the procedure of Werringloer (1978). construct a graph using microsoft office excel 2007. Formaldehyde formed during the assay was measured by the method of Nash (1953). The Determination of N, N-dimethylaniline reaction mixture consisted of 0.1 M potassium demethylase activity phosphate buffer ((pH 7.2), 0.01 M erythromycin,
Demethylation assay was carried out by the 0.15 M MgCl2. Reactions were initiated by the procedure of Schenkman et al. (1967). addition of 0.01 M NADPH, and the mixture was Formaldehyde formed during the assay was incubated for 10 min at 30 ± 2 oC. The reaction was measured by the method of Nash (1953). Microsomal terminated by adding 0.5 ml of 20% trichloroacetic suspension (0.5 ml), was mixed with a reaction acid. Precipitated proteins were removed by mixture consisting of the substrate 0.1 M (N, N- centrifugation at 10,000 g for 10 min and 1 ml of dimethyl aniline), 0.15M MgCl2, 0.1 M semicarbazide. supernatant were added to 1 ml of Nash reagent and After pre-incubation at 30±2 oC for five min the measured at 412 nm. reaction was started by adding 40µl NADPH and incubated for 30 minutes at the same temperature. Determination of EROD (ethoxyresorufin O- The reaction was stopped by the addition of 0.5 ml deethylase) activity of each of 25% zinc sulfate and saturated barium EROD (ethoxyresorufin O-deethylase) hydroxide followed by centrifugation at 10,000xg for assay was carried out by the procedure of Klotz et 10 mins. Aliquot of clear supernatant (1ml) was mixed al. (1984). The reaction mixture consisted of 0.1 M with 2ml of Nash reagent and incubated at 60 oC for NaCl, 2 µM 7-ethoxresorufin, 0.1 M tris buffer (pH 30 minutes and the absorbance measured at 415nm. 7.4). The reaction was initiated by the addition of The experiment was run in duplicates and repeated. 0.01 M NADPH. After incubation at 30 ± 2 oC for The average values were considered. 10 min the reaction was terminated by the addition of 20% trichloroacetic acid and centrifuged at 20,000 Determination of aniline hydroxylase activity g for 10 min and the activity measured at 572 nm. Aniline hydroxylase assay was carried out by the procedure of Schenkman et al. (1967). The Statistical analysis reaction mixture consisted of 0.5 ml microsomal Data was analyzed (Students t- test, paired suspension, 0.1 M aniline, 0.1M MgCl2, 0.12 M tris two sample for means) using Microsoft Analysis (pH 7.4) and preincubated at 30±2 oC for 5 min. The ToolPak. reaction was started by adding 0.01 M NADPH following incubation at the same temperature. The RESULTS AND DISCUSSION reaction was terminated by adding 0.5 ml of 20% Lester et al. (1993) and Lewis (2001) trichloroacetic acid(TCA). Precipitated proteins were reported high levels of CYP 450 in the fish liver, removed by centrifugation at 10,000g for 10 min and accounting for 1 to 2% mass of hepatocytes. A 1 ml of supernatant were added to 1 ml of a solution difference spectrum with a peak absorbance at 450 containing 2% phenol in 0.2 M NaOH and 1 ml of was observed upon dithionite reduction followed by
Na2CO3. After 30 min incubation, the resultant blue bubbling of CO, showing the presence of cytochrome colour of p-aminophenol formed was measured at P450 in the microsomal fraction. The highest content 630 nm. of total CYP 450, 0.440 nmole/mg protein was recorded with deflazacort treated fish compared to
30 Multiple Forms of Cytochrome P450 Family in Liver of Freshwater Teleost Fish
0.214 nmole/mg protein in control group. Overall the fish treated with different inducers showed a higher content of CYP 450 with respect to control (Table I). Figure 1 highlights the peak absorbance with the control showing a maximum peak at 450 nm (0.01208), naphthalene at 451 nm (450 = 0.03127; 451 = 0.03129), phenobarbitone at 452 nm (450 = 0.01884; 451 = 0.01929), acetone at 450 nm (0.02629) and that of deflazacort treated at 449 nm (450 = 0.03091; 449=0.03093). The determination of cytochrome P450 levels as a response of the organism to the presence of pollutants in the aquatic environment has been reported by many studies from Figure 1. Carbon monoxide difference spectra of all over the world (Payne et al., 1987; Curtis et al., dithionite reduced liver microsomes of 1993; Aas et al., 2001). An increase in the level of Heteropneustes fossilis treated with different CYP 450 was reported in hepatic microsomal inducers, (a) control, (b) naphthalene treated, fraction of Heteropneustes fossilis when treated with (c) phenobarbitone treated, (d) acetone treated and (e) deflazacort treated.
Table I. Total cytochrome P450 content administered with different inducers with respect to control. Values are mean of (n) number of experiments ±SD.
pyrethroid insecticide, cypermethrin (Sadat et al., of CYP1A in European eel (Anguilla anguilla L.). 2009). The induction of CYP1A is mediated by the Ah The fish treated with different inducers receptor (AhR), a xenobiotic-binding protein present showed an increase in EROD activity than that of in the cytosol (Lewis, 2001). The most important control. EROD activity was seen to be significantly inducers of fish CYP1A, which is considered most induced, 66.274 ± 23.059 (p ≤ 0.05) only in relevant to pollution of the aquatic environment, naphthalene treated fish (Table 2). Pacheco and include PAHs, nitrated polyaromatic hydrocarbons Santos (2002) reported that naphthalene, a class of (NPAHs), PCBs, dioxins and some pesticides PAH, have been found to be specific for the induction (Machala et al., 1997; Jung et al., 2001).
31 Dawa Bhutia, Benoy K Rai and Joydeb Pal
In this study, acetone treated fish showed can also be metabolized by CYP1A2. The enzyme significant induction in aniline hydroxylase activity, is inducible by ethanol, acetone, isoniazid, and by 0.277±0.008 (p ≤ 0.01) as compared to control and starvation in animal models (Lieber, 1997). Acetone the fish treated with different inducers but the fish at large dose induces CYP2E1 in rat (Tu et al., 1983). treated with naphthalene, phenobarbitone and The results indicate that acetone in fish, deflazacort showed down regulation in aniline Heteropneustes fossilis inducing CYP2E1 as in the hydroxylase activity as compared to control fish (Table case of rats. II). CYP2E1 is responsible for the metabolism and There were no significant difference in N, potential bioactivation of a number of low-molecular- N-dimethylaniline (DMA) demethylase activity weight pharmaceutical and other xenobiotic between the control and the treated groups (Table compounds, including acetaminophen, ethanol, II). Over the last decades, fish liver microsomes have isoniazid, halogenated anesthetics, acetone, and been shown to metabolize prototypical mammalian benzene (Lee et al., 1996). Chlorzoxazone and p- CYP2B substrates, including aldrin, benzphetamine, nitrophenol are routinely used as substrate probes to ethylmorphine, aminopyrine and alkoxyresorufins measure CYP2E1 activity, although chlorzoxazone (Elskus and Stegeman, 1989; Haasch et al., 1994).
Table II. EROD, Aniline hydroxylase, N, N-dimethylaniline (DMA) demethylase and erythromycin demethylase activities treated with different inducers. Values are the mean of (n) number of experiments ± SD.
*
* Significantly different from control (t-test, p ≤ 0.05), **(p ≤ 0.01).
32 Multiple Forms of Cytochrome P450 Family in Liver of Freshwater Teleost Fish
CYP2B1 and CYP2B2 are the primary members due to structural similarities. In fish they are regulated expressed in rats. In rodents, enzymes from this by the same receptors as in mammals, namely, subfamily are typically inducible by phenobarbital and through the pregnane X receptor (Bresolin et al., other barbiturates, and are inhibited by metyrapone 2005). (Mimura et al., 1993). In fish, however, an apparent The results from the current study showed lack of response to PB-type inducers has been that fish CYP 450 responds differentially to different observed (Haasch et al., 1994; Stegeman, 1981). xenobiotics and indicated presence of multiple forms Deflazacort (synthetic corticosteroid) treated of CYP 450 in fish, Heteropneustes fossilis. The fish showed significant induction of erythromycin CYP 450 family- CYP1A, CYP2E and CYP3A show demethylase activity of 3.129±0.545 (p ≤ 0.01) where similar biotransformation reaction as that reported as the other treated groups showed lower activity for various mammalian species but CYP2B did not than that of the control group (Table II). Induction of follow the trend though still a lot of studies have to cytochrome P450 3A expression by dexamethasone be conducted. has been shown to occur in fish (Tseng et al., 2005). The study also showed a significant negative ACKNOWLEDGEMENT regulation of erythromycin demethylase activity in The authors are grateful to the University acetone and phenobarbitone treated group. Bork et Grants Commission for the financial support provided al. (1989) reported that CYP3A enzymes of during the course of work. mammals and fish exhibit similar catalytic properties
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Aas, E., Beyer, J., Jonsson, G., Reichert, W.L. & Andersen, A. 2005.Expression of PXR, CYP3A and MDR O.K. 2001: Evidence of uptake, biotransformation 1genes in liver of zebra fish. Comp. Biochem. and DNA binding of polyaromatic hydrocarbons Physiol. Toxicol. Pharmacol. 140:403-407. in Atlantic cod and corkwing wrasse caught in the Chang, T.K.H. & Waxman, D.J. 1998. Enzymatic analysis vicinity of an aluminium works. Mar. Environ. Res. of cDNA –expressed human CYP1A1, CYP1A2, and 52: 213-229 CYP1B1 with 7-ethoxyresorufin as a substrate. In Anzenbacher, P. & Anzenbacherová, E. 2001. Cytochromes Methods in Molecular Biology, Vol. 107: P450 and metabolism of xenobiotics. Cell. Mol. Life Cytochrome P450 Protocols, ed. I. R. Phillips and Sci. 58: 737–747. E. A. Sephard, pp 103-109, Humana Press Inc., Beyer, J., Sandvik, M., Hylland, K., Fjeld, E., Egaas, E., Totowa, NJ. Aas, E., Skare, J. & Goksoyr, A. 1996. Contaminant Curtis, L.R., Carpenter, H.M., Donohoe, R.M., Williams, accumulation and biomarker responses in flounder D.E., Hedstorm, O.R., Deinzer, M.L., Bellstein, (Platichthys flesus L.) and atlantic cod (Gadus M.A., Foster, E & Gates, R. 1993: Sensitivity of morhua L.) exposed by caging to polluted cytochrome P450-1A1 in fish as a biomarker for sediments in Sorfjorden, Norway. Aquat. Toxicol. distribution of TCDD and TCDF in the Willamette 36:75-98. River, Oregon. Environ. Sci. Technol. 27: 2149-2157 Bork, R.W., Muto, T., Beaune, P. H., Srivastava, P.K., Elskus, A. A. & Stegeman, J. J. 1989. Further consideration Lloyd, R.S. & Guengerich, F.P.1989. of phenobarbital effects on cytochrome P-450 Charactersition of m RNA species related to human activity in the killifish, Fundulus heteroclitus. liver cytochrome P 450 nifedipine oxidase and the Comp. Biochem. Physiol., 92C, 223. regulation of catalytic activity. J.Biol.chem. Goksoyr, A. & Forlin, L. 1992. The cytochrome P450 264:910-919. system in fish: aquatic toxicology and Bresolin, T., De Freitas Rebelo, M. & Celso Dias Bainy, environmental monitoring. Aquat. toxicol. 22:287-
33 Dawa Bhutia, Benoy K Rai and Joydeb Pal
312. Machala, M. 1997: Specificke markery mechanismU Gonzalez, F.J. 2005. Role of cytochromes P450 in chemical toxicity xenobiotik. Program asbornik konference toxicity and oxidative stress: studies with CYP2E1. ERA [Specific markers for toxicity mechanisms in Mutat. Res. 569:101–110. xenobiotics. Abstracts of the ERA ’97 Conference], pp. 211-219(In Czech). Haasch, M. L. Graf, W. K., Quardokus, E.M. Mayer, R. T. & Lech, J.J. 1994. Use of 7-alkoxyphenoxazones, Machala, M., Nezveda, K., Petrivalsky, M., Jarosova, A., 7-alkoxycoumarins, and 7-alkoxyquinolines as Piacka, V. & Svobodova, Z. 1997. Monooxygenase fluorescent substrates for rainbow trout hepatic activities in carp as biochemical markers of pollution microsomes after treatment with various inducers, by polycyclic and polyhalogenated aromatic Biochem. Pharmacol. 47: 893-903. hydrocarbons: Choice of substrates and effects of temperature, gender and capture stress. Aquat. Havelkova, M., Randak, T., Zlabek, V., Krijt, J., kroupova, Toxicol. 37: 113-123. H., Pulkrabova, J. & Svobodova, Z. 2007. Biochemical markers for assessing aquatic Mimura, M., Baba, T., Yamazaki, H., Ohmori, S., Inui, Y., contamination. Sensors. 7:2599-2611. Gonzalez, F. J., Guengerich, F. P. & Shimada, T. 1993. Characterization of cytochrome P-450 2B6 in Jonsson, E.M. 2003. A gill filament EROD assay: human liver microsomes Drug Metab. Dispos. 21: development and application in environmental 1048 –1056. monitoring. Ph. D. Thesis, Uppsala University, Sweden. Nash, T. 1953. The colorimetric Estimation of Formaldehyde by means of the Hantzsch Reaction. Jung, D. K., Klaus, T., Fent, K. 2001: Cytochrome P450 Bio chem. J. 416-421. induction by nitrated polycyclic aromatic hydrocarbons, azaarenes, and binary mixtures in Nebert, D. W. & McKinnon, R. A. 1994.Cytochrome P450: fish hepatoma cell line PLHC-1. Environ. Toxicol. Evolution and functional diversity. Prog.Liver Chem. 20: 149-159. Dis.12:63-97. Klotz, A. V., Stegeman, J. J. & Walsh, C. 1984. An Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. alternative 7-Ethoxyresorufin O-deethylase J., Feyereisen, R., Waxman, D. J., Waterman, M. Activity assay: a continuous visible R., Gotoh, O.,Coon, M. J., Estabrook, R. W., spectrophotometric method for measurement of Gunsalus, I. C. & Nebert, D. W. 1996. P450 Cytochrome P450 Monoxygenase Activity. J. Anal. superfamily: Update on new sequences, gene Biochem.140:138-145. mapping, accession numbers, and nomenclature. Pharmacogenetics. 6:1-42. Lee, S. S., Buters, J.T., Pineau, T., Fernandez-Salguero, P. & Gonzalez, F.J. 1996. Role of CYP2E1 in the Nilsen, B. M., Berg, K. & Goksoyr, A. 1998. Induction of hepatotoxicity of acetaminophen. J. Biol. Chem. cytochrome P4501A (CYP1A) expression in fish. . 271:12063–12067. In Methods in Molecular Biology, Vol. 107: Cytochrome P450 Protocols, ed. I. R. Phillips and Lester, S.M., Braunbeck, T.A, Teh, S.J., Stegeman, J.J., E. A. Sephard, pp103-109. Humana Press Inc., Miller, M.R. & Hinton, D.E. 1993.Hepatic cellular Totowa, NJ. distribution of cytchrome P450 1A in rainbow trout (Oncorhynchus mykiss): an immunohisto and Okita, R. T. & Masters, B. S. S. 1997. Biotransformation: cytochemical study. Cancer Res. 53: 3700-3706. The cytochrome P450. In: Text Book of Biochemistry with Clinical Correlations. Ed. T.M. Lewis, D.F.V. 2001. Guide to cytochrome P450 structure Devlin. Wiley-liss, New York pp.981-999. and function. Taylor and Francis Inc., London, p215. Omura, J. & Sato, R. 1964. The carbon monoxide binding pigment of liver microsomes. I. evidence for its heme Lieber, C.S. 1997. Cytochrome P-4502E1: Its physiological protein nature. J. Boil. Chem. 239: 2370-2378. and pathological role. Physiol. Rev. 77: 517–544. Pacheco, M. & Santos, M. A. 2002. Naphthalene and â- Lowry, O. H., Rosenbrough, N. S., Farr, A. L. & Randall, naphthoflavone effects on Anguilla anguilla L. R. J. 1951. Protein measurement with the Folin hepatic metabolism and erythrocytic nuclear phenol reagent. J. Biol. Chem. 193: 265-275.
34 Multiple Forms of Cytochrome P450 Family in Liver of Freshwater Teleost Fish
abnormalities. Environ. Int. 28: 285-293. Schenkman, J.B., Remer, H & Stabrook, R.W. 1967. Payne, J. F., Fancey, L. L., Rahimtula, A.D. & Porter, E.L. Biochemical assay of cytochrome P-450. Mol. 1987. Review and perspective on the use of mixed Pharmacol. 3:113-126. function oxygenase enzymes in biological Stegeman, J. J. 1981. Polynuclear aromatic hydrocarbons monitoring. Comp.Biochem. Physiol. 86: 233-245. and their metabolism in the marine environment. In Sadars, M. D., Ashs, R., Sundqvist, J., Olsson, P. & Polycyclic hydrocarbons and cancer, ed. H.V. Andersson, T. B. 1996. Phenobarbital Induction of Gelboin,Vol. 3, pp 1-60, Academic Press, New York. CYP1A1 Gene Expression in a Primary Culture of Tseng, H. P., Hseu, T.H., Buhler, D.R., Wang, W.D. & Hu, Rainbow Trout Hepatocytes. J. Biol. Chem. 271: C.H. 2005. Constitutive and Xenobiotics induced 17635–17643. expression of a novel CYP3A gene from zebrafish Sadat, A., Bhutia, D., Rai, B.K. & Pal, J. 2009. Cytochrome larva.Toxicol. Appl. Pharmacol. 205: 247-258. P450 in Cypermethrin treated freshwater catfish, Tu, Y.Y., Peng, R., Chang, Z. F. & Yang, C.S. 1983. Heteropneustes fossilis. NBU J. Anim. Sci. 3: 54- Induction of a high affinity nitrosamine 60. demethylase in rat liver microsomes by acetone Saha, S. & Kaviraj, A. 2003. Acute Toxicity of Synthetic and isopropanol. Chem-Biol Interact. 44:247-260. Pyrethroid Cypermethrin to Freshwater Catfish Werringloer J. 1978. Assay of fomaldehyde generated Heteropneustes fossilis (Bloch). Inter. J. Toxicol. during microsomal oxidation reactions. Methods 22: 325- 328 Enzymol. 52: 297-302.
35 Channa punctata isolate CHANNA cytochrome p450 CYP1A (cyp1A) gene, partial cds GenBank: KP282054.1 FASTA Graphics Go to:
LOCUS KP282054 2511 bp DNA linear VRT 04-APR-2015 DEFINITION Channa punctata isolate CHANNA cytochrome p450 CYP1A (cyp1A) gene,partial cds. ACCESSION KP282054 VERSION KP282054.1 KEYWORDS . SOURCE Channa punctata (spotted snakehead) ORGANISM Channa punctata Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;Actinopterygii; Neopterygii; Teleostei; Neoteleostei;Acanthomorphata; Anabantaria; Anabantiformes; Channoidei;Channidae; Channa. REFERENCE 1 (bases 1 to 2511) AUTHORS Bhutia,D., Rai,B.K. and Pal,J. TITLE Direct Submission JOURNAL Submitted (17-DEC-2014) Department of Zoology, University of North Bengal, Raja Rammohanpur, Siliguri, West Bengal 734013, India COMMENT ##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END## FEATURES Location/Qualifiers source 1..2511 /organism="Channa punctata" /mol_type="genomic DNA" /isolate="CHANNA" /db_xref="taxon:304456" /tissue_type="liver" /country="India: West Bengal, Siliguri" /collection_date="11-Dec-2014" gene <1..>2122 /gene="cyp1A" mRNA join(<1..720,845..971,1074..1163,1270..1393,1517..1603, 1825..>2122) /gene="cyp1A" /product="cytochrome p450 CYP1A" CDS join(<1..720,845..971,1074..1163,1270..1393,1517..1603, 1825..2122) /gene="cyp1A" /codon_start=1 /product="cytochrome p450 CYP1A" /protein_id="AKA59071.1"
/translation="GLCRLPGPKPLPLIGNVLELRHKPYQSLTAMSKRYGHVFQIHIG
TRPVVVLSGSETVRQALIKQGEEFAGRPDLYSFQFINDGKSLAFSTDQSGVWRARRKL
AYSALRSFSNLESKNSEYSCVLEEHVSKEAEYLIKRLCTVMKADGSFDPVRHIVVSVA
NVICGICFGRRYSHDDQELLSLVTLADDFNQVAGSGNPADFIPILQYLPSRNMKNFMD
LNARFNSFVQKIVREHYATYDKDNIRDITDSLIDHCEDRKLDENFNVQVSDEKIVGIV
NDLFGAGFDTVTTALSWSVMYMVAYPEIQERLYDELKVNVGLERSPRLSDKPNLPFLE
AFILEMLRHSSFLPFTIPHCTTKDTSLNGYFIPKDTCVFINQWQINHDPELWKDPFSF
NPDRFLSADSTEVNKVEGEKVVAFGLGKRRCIGEVIARNEVYLFLAILIQKLEFHQMP
GVPLDMTPQYGLTMKHKPCHLRATMRAINEQ"
ORIGIN 1 gggctttgtc gtctccctgg cccaaagccc ttgcctctca tcgggaatgt gctggagctt 61 cgccacaaac cttatcagag tctcactgct atgagcaagc gttatggtca tgtcttccag 121 atccacattg gcacacgtcc tgtggttgtg ttgagtggca gtgagacggt tcgtcaggct 181 ctcatcaagc aaggggaaga gttcgcaggc agacctgact tgtacagctt tcaattcatc 241 aatgacggaa aaagtctggc tttcagtaca gatcagtctg gtgtctggcg tgctcgcaga 301 aagctggctt acagtgccct gcgctccttt tccaacctgg agagcaagaa ctcagagtac 361 tcctgtgttc tagaagaaca cgtcagtaaa gaggcagagt atctaatcaa acgactctgc 421 actgtcatga aggcagacgg cagcttcgac cccgtccgtc acattgtcgt ctctgtggca 481 aatgtcatct gcggaatttg ctttggccga cgctacagcc acgatgacca ggagctgctc 541 agcttagtga cccttgctga tgactttaac caggtggcgg gaagtgggaa ccctgctgac 601 ttcatcccca ttctccagta tctgcctagc agaaacatga agaattttat ggacctcaat 661 gctcgcttca acagctttgt gcaaaaaata gtcagagaac actatgccac ctacgacaag 721 gtacaccgca aactaaatca ctcatgaaat gagttcacgt tttctcaaag ccataatttc 781 cttattattt ttgtgtgtat ttgtatgtac gtatgtgatt gtgattgttc ttaacttttt 841 tcaggacaac atccgtgata tcacagactc cctcattgat cactgcgagg acaggaagct 901 ggatgagaac ttcaatgttc aggtgtcaga tgagaagatt gtgggaattg tcaatgacct 961 atttggggct ggtacgctca cttcctatgt actgaataaa cctattgttg atatggtgat 1021 gtctgcctaa agaaaacaaa agtgccctca cataattaac atctcccttt caggttttga 1081 cactgtcacc actgcattgt catggtcagt gatgtacatg gtggcttacc cagagataca 1141 agagaggctt tatgatgagc tgagtaagta cactgatttt ggattttaca gttctattgc 1201 aaaaccactg aaggaatgtg gactaaaatc caaaaaaacg catactgagc atggatgact 1261 ttttttcaga ggtcaatgtg ggtctggagc ggagcccgcg tctctctgat aaacccaatt 1321 taccatttct ggaggccttc atcctggaga tgttgcggca ctcttcattc ctgcccttta 1381 ctatcccaca ctggtaaggt tcaactcaaa aagggtgaaa atagagctgt taagtgcata 1441 cttagtttta caggaccaca caaaaacaga tgcatgggat cgtgaggaga ctaagccttt 1501 gtttttcttt tctcagcacc acaaaagaca cgtctctgaa tggctacttc attccaaaag 1561 atacctgtgt cttcatcaat cagtggcaga ttaaccatga tccgtaagtt ctttttgtta 1621 cattttaaaa actttaaaac aaagcatgag gtgtaccaac ctaacagtta cacagtaaaa 1681 aaactgaagc agaaaactac ttacataaaa ccattaaagg aataagtaca gaaataagtt 1741 tgtcctaatg gaatttggag ctaaagccaa agatttattg tgcttttgta tatgacttac 1801 atttcctttc tctacccctc tcagtgagct gtggaaagat ccattttcct tcaacccaga 1861 ccgcttcttg agcgctgata gcactgaggt caacaaggtg gaaggggaga aggtagtggc 1921 tttcggccta ggaaagcggc gctgcatcgg cgaggtcatt gcacgaaatg aagtctacct 1981 cttcttggca attcttatcc agaagctaga gttccaccaa atgcctgggg taccactgga 2041 catgacgcca caatatggtc tcacaatgaa acacaaaccc tgccacctga gagccacaat 2101 gcgagcaatc aatgagcagt gaaactattt atatatttac tgtattcaga atgaattact 2161 caacagttga taattgttca ctcaagaact gtaaggttca gtgaaacaag catctttctc 2221 agaatgtacg gcaatggttg ccaaatctga tacatagagc aaatggattt gaagcaaata 2281 ggtgaaattt gcttgcttgc ttgcagactg tcagatattt ctgggtttgt aagatgaggg 2341 gttcctctat atgatcgatg cgtcttgagg aagaataagc agatacttgg ttttcctgct 2401 gtgttttttt gctgtgctgg aaacgtaact gttcttatgt agaagttgta tagcacacaa 2461 actatgttgc ttcaatcaac tttgggacac attatgtttt actggatatg c //
Channa punctata isolate NBUC1 cytochrome p450 CYP1A (cyp1A) gene, partial cds GenBank: KP203843.1 FASTA Graphics Go to:
LOCUS KP203843 1041 bp DNA linear VRT 04-APR-2015 DEFINITION Channa punctata isolate NBUC1 cytochrome p450 CYP1A (cyp1A) gene, partial cds. ACCESSION KP203843 VERSION KP203843.1 KEYWORDS . SOURCE Channa punctata (spotted snakehead) ORGANISM Channa punctata Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Actinopterygii; Neopterygii; Teleostei; Neoteleostei;Acanthomorphata; Anabantaria; Anabantiformes; Channoidei;Channidae; Channa. REFERENCE 1 (bases 1 to 1041) AUTHORS Bhutia,D., Rai,B.K. and Pal,J. TITLE Direct Submission JOURNAL Submitted (26-NOV-2014) Department of Zoology, University of North Bengal, Raja Rammohanpur, Siliguri, West Bengal 734013, India COMMENT ##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END## FEATURES Location/Qualifiers source 1..1041 /organism="Channa punctata" /mol_type="genomic DNA" /isolate="NBUC1" /db_xref="taxon:304456" /tissue_type="liver" /country="India" /collection_date="04-Jul-2014" gene <1..>971 /gene="cyp1A" mRNA join(<1..720,845..>971) /gene="cyp1A" /product="cytochrome p450 CYP1A" CDS join(<1..720,845..>971) /gene="cyp1A" /codon_start=1 /product="cytochrome p450 CYP1A" /protein_id="AKA59068.1"
/translation="GLCRLPGPKPLPLIGNVLELRHKPYQSLTAMSKRYGHVFQIHIG
TRPVVVLSGSETVRQALIKQGEEFAGRPDLYSFQFINDGKSLAFSTDQSGVWRARRKL
AYSALRSFSNLESKNSEYSCVLEEHVSKEAEYLIKRLCTVMKADGSFDPVRHIVVSVA
NVICGICFGRRYSHDDQELLSLVTLADDFNQVAGSGNPADFIPILQYLPSRNMKNFMD
LNARFNSFVQKIVREHYATYDKDNIRDITDSLIDHCEDRKLDENFNVQVSDEKIVGIV
NDLFGA"
ORIGIN 1 gggctttgtc gtctccctgg cccaaagccc ttgcctctca tcgggaatgt gctggagctt 61 cgccacaaac cttatcagag tctcactgct atgagcaagc gttatggtca tgtcttccag 121 atccacattg gcacacgtcc tgtggttgtg ttgagtggca gtgagacggt tcgtcaggct 181 ctcatcaagc aaggggaaga gttcgcaggc agacctgact tgtacagctt tcaattcatc 241 aatgacggaa aaagtctggc tttcagtaca gatcagtctg gtgtctggcg tgctcgcaga 301 aagctggctt acagtgccct gcgctccttt tccaacctgg agagcaagaa ctcagagtac 361 tcctgtgttc tagaagaaca cgtcagtaaa gaggcagagt atctaatcaa acgactctgc 421 actgtcatga aggcagacgg cagcttcgac cccgtccgtc acattgtcgt ctctgtggca 481 aatgtcatct gcggaatttg ctttggccga cgctacagcc acgatgacca ggagctgctc 541 agcttagtga cccttgctga tgactttaac caggtggcgg gaagtgggaa ccctgctgac 601 ttcatcccca ttctccagta tctgcctagc agaaacatga agaattttat ggacctcaat 661 gctcgcttca acagctttgt gcaaaaaata gtcagagaac actatgccac ctacgacaag 721 gtacaccgca aactaaatca ctcatgaaat gagttcacgt tttctcaaag ccataatttc 781 cttattattt ttgtgtgtat ttgtatgtac gtatgtgatt gtgattgttc ttaacttttt 841 tcaggacaac atccgtgata tcacagactc cctcattgat cactgcgagg acaggaagct 901 ggatgagaac ttcaatgttc aggtgtcaga tgagaagatt gtgggaattg tcaatgacct 961 atttggggct ggtacgctca cttcctatgt actgaataaa cctattgttg atatggtgat 1021 gtctgcctaa agaaaacaaa a //
Channa punctata isolate NBUC2 cytochrome p450 CYP1A (cyp1A) gene, partial cds GenBank: KP231221.1 FASTA Graphics Go to:
LOCUS KP231221 1498 bp DNA linear VRT 04-APR-2015 DEFINITION Channa punctata isolate NBUC2 cytochrome p450 CYP1A (cyp1A) gene, partial cds. ACCESSION KP231221 VERSION KP231221.1 KEYWORDS . SOURCE Channa punctata (spotted snakehead) ORGANISM Channa punctata Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;Actinopterygii; Neopterygii; Teleostei; Neoteleostei;Acanthomorphata; Anabantaria; Anabantiformes; Channoidei;Channidae; Channa. REFERENCE 1 (bases 1 to 1498) AUTHORS Bhutia,D., Rai,B.K. and Pal,J. TITLE Direct Submission JOURNAL Submitted (02-DEC-2014) Department of Zoology, University of North Bengal, Raja Rammohanpur, Siliguri, West Bengal 734013, India COMMENT ##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END## FEATURES Location/Qualifiers source 1..1498 /organism="Channa punctata" /mol_type="genomic DNA" /isolate="NBUC2" /db_xref="taxon:304456" /tissue_type="liver" /country="India" /collection_date="03-Sep-2014" gene <1..>1473 /gene="cyp1A" mRNA join(<1..72,197..323,426..515,622..745,868..954, 1176..>1473) /gene="cyp1A" /product="cytochrome p450 CYP1A" CDS join(<1..72,197..323,426..515,622..745,868..954, 1176..1473) /gene="cyp1A" /codon_start=1 /product="cytochrome p450 CYP1A" /protein_id="AKA59069.1"
/translation="MDLNARFNSFVQKIVREHYATYDKDNIRDITDSLIDHCEDRKLD
ENFNVQVSDEKIVGIVNDLFGAGFDTVTTALSWSVMYMVAYPEIQERLYDELKVNVGL
ERSPRLSDKPNLPFLEAFILEMLRHSSFLPFTIPHCTTKDTSLNGYFIPKDTCVFINQ
WQINHDPELWKDPFSFNPDRFLSADSTEVNKVEGEKVVAFGLGKRRCIGEVIARNEVY
LFLAILIQKLEFHQMPGVPLDMTPQYGLTMKHKPCHLRATMRAINEQ"
ORIGIN 1 atggacctca atgctcgctt caacagcttt gtgcaaaaaa tagtcagaga acactatgcc 61 acctacgaca aggtacaccg caaactaaat cactcatgaa atgagttcac gttttctcaa 121 agccataatt tccttattat ttttgtgtgt atttgtatgt acgtatgtga ttgtgattgt 181 tcttaacttt tttcaggaca acatccgtga tatcacagac tccctcattg atcactgcga 241 ggacaggaag ctggatgaga acttcaatgt tcaggtgtca gatgagaaga ttgtgggaat 301 tgtcaatgac ctatttgggg ctggtacgct cacttcctat gtactgaata aacctattgt 361 tgatatggtg atgtctgcct aaagaaaaca aaagtgccct cacataatta acatctccct 421 ttcaggtttt gacactgtca ccactgcatt gtcatggtca gtgatgtaca tggtggctta 481 cccagagata caagagaggc tttatgatga gctgagtaag tacactgatt ttggatttta 541 cagttctatt gcaaaaccac tgaaggaatg tggactaaaa tccaaaaaaa cgcatactga 601 gcatggatga ctttttttca gaggtcaatg tgggtctgga gcggagcccg cgtctctctg 661 ataaacccaa tttaccattt ctggaggcct tcatcctgga gatgttgcgg cactcttcat 721 tcctgccctt tactatccca cactggtaag gttcaactca aaaagggtga aaatgagctg 781 ttaagtgcat acttagtttt acaggaccac acaaaaacag atgcatggga tcgtgaggag 841 actaagcctt tgtttttctt ttctcagcac cacaaaagac acgtctctga atggctactt 901 cattccaaaa gatacctgtg tcttcatcaa tcagtggcag attaaccatg atccgtaagt 961 tctttttgtt acattttaaa aactttaaaa caaagcatga ggtgtaccaa cctaacagtt 1021 acacagtaaa aaaactgaag cagaaaacta cttacataaa accattaaag gaataagtac 1081 agaaataagt ttgtcctaat ggaatttgga gctaaagcca aagatttatt gtgcttttgt 1141 atatgactta catttccttt ctctacccct ctcagtgagc tgtggaaaga tccattttcc 1201 ttcaacccag accgcttctt gagcgctgat agcactgagg tcaacaaggt ggaaggggag 1261 aaggtagtgg ctttcggcct aggaaagcgg cgctgcatcg gcgaggtcat tgcacgaaat 1321 gaagtctacc tcttcttggc aattcttatc cagaagctag agttccacca aatgcctggg 1381 gtaccactgg acatgacgcc acaatatggt ctcacaatga aacacaaacc ctgccacctg 1441 agagccacaa tgcgagcaat caatgagcag tgaaactatt tatatattta ctgtatca // Channa punctata isolate NBUC3 cytochrome p450 CYP1A (cyp1A) gene, partial cds GenBank: KP271996.1 FASTA Graphics Go to:
LOCUS KP271996 1108 bp DNA linear VRT 04-APR-2015 DEFINITION Channa punctata isolate NBUC3 cytochrome p450 CYP1A (cyp1A) gene,partial cds. ACCESSION KP271996 VERSION KP271996.1 KEYWORDS . SOURCE Channa punctata (spotted snakehead) ORGANISM Channa punctata Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;Actinopterygii; Neopterygii; Teleostei; Neoteleostei;Acanthomorphata; Anabantaria; Anabantiformes; Channoidei;Channidae; Channa. REFERENCE 1 (bases 1 to 1108) AUTHORS Bhutia,D., Rai,B.K. and Pal,J. TITLE Direct Submission JOURNAL Submitted (12-DEC-2014) Department of Zoology, University of North Bengal, Raja Rammohanpur, Siliguri, West Bengal 734013, India COMMENT ##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END## FEATURES Location/Qualifiers source 1..1108 /organism="Channa punctata" /mol_type="genomic DNA" /isolate="NBUC3" /db_xref="taxon:304456" /tissue_type="liver" /country="India: West Bengal" /collection_date="12-Nov-2014" gene <114..>719 /gene="cyp1A" mRNA join(<114..200,422..>719) /gene="cyp1A" /product="cytochrome p450 CYP1A" CDS join(<114..200,422..719) /gene="cyp1A" /codon_start=2 /product="cytochrome p450 CYP1A" /protein_id="AKA59070.1"
/translation="TTKDTSLNGYFIPKDTCVFINQWQINHDPELWKDPFSFNPDRFL
SADSTEVNKVEGEKVVAFGLGKRRCIGEVIARNEVYLFLAILIQKLEFHQMPGVPLDM
TPQYGLTMKHKPCHLRATMRAINEQ"
ORIGIN 1 actcaaaaag ggtgaaaata gagctgttaa gtgcatactt agttttacag gaccacacaa 61 aaacagatgc atgggatcgt gaggagacta agcctttgtt tttcttttct cagcaccaca 121 aaagacacgt ctctgaatgg ctacttcatt ccaaaagata cctgtgtctt catcaatcag 181 tggcagatta accatgatcc gtaagttctt tttgttacat tttaaaaact ttaaaacaaa 241 gcatgaggtg taccaaccta acagttacac agtaaaaaaa ctgaagcaga aaactactta 301 cataaaacca ttaaaggaat aagtacagaa ataagtttgt cctaatggaa tttggagcta 361 aagccaaaga tttattgtgc ttttgtatat gacttacatt tcctttctct acccctctca 421 gtgagctgtg gaaagatcca ttttccttca acccagaccg cttcttgagc gctgatagca 481 ctgaggtcaa caaggtggaa ggggagaagg tagtggcttt cggcctagga aagcggcgct 541 gcatcggcga ggtcattgca cgaaatgaag tctacctctt cttggcaatt cttatccaga 601 agctagagtt ccaccaaatg cctggggtac cactggacat gacgccacaa tatggtctca 661 caatgaaaca caaaccctgc cacctgagag ccacaatgcg agcaatcaat gagcagtgaa 721 actatttata tatttactgt attcagaatg aattactcaa cagttgataa ttgttcactc 781 aagaactgta aggttcagtg aaacaagcat ctttctcaga atgtacggca atggttgcca 841 aatctgatac atagagcaaa tggatttgaa gcaaataggt gaaatttgct tgcttgcttg 901 cagactgtca gatatttctg ggtttgtaag atgaggggtt cctctatatg atcgatgcgt 961 cttgaggaag aataagcaga tacttggttt tcctgctgtg tttttttgct gtgctggaaa 1021 cgtaactgtt cttatgtaga agttgtatag cacacaaact atgttgcttc aatcaacttt 1081 gggacacatt atgttttact ggatatgc //
Heteropneustes fossilis partial cyp4501a gene for Cytochrome P450 1A GenBank: LN736019.1 FASTA Graphics Go to:
LOCUS LN736019 534 bp DNA linear VRT 18-JAN-2015 DEFINITION Heteropneustes fossilis partial cyp4501a gene for Cytochrome P4501A. ACCESSION LN736019 VERSION LN736019.1 KEYWORDS . SOURCE Heteropneustes fossilis (stinging catfish) ORGANISM Heteropneustes fossilis Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Actinopterygii; Neopterygii; Teleostei; Ostariophysi; Siluriformes; Heteropneustidae; Heteropneustes. REFERENCE 1 AUTHORS Bhutia,D., Rai,B.K. and Pal,J. JOURNAL Unpublished REFERENCE 2 (bases 1 to 534) AUTHORS Bhutia,D. TITLE Direct Submission JOURNAL Submitted (23-DEC-2014) Ecotoxicology Laboratory, Department of Zoology, NBU, West Bengal, 734013, INDIA FEATURES Location/Qualifiers source 1..534 /organism="Heteropneustes fossilis" /mol_type="genomic DNA" /db_xref="taxon:93621" /tissue_type="liver" /country="India:West Bengal, Siliguri" /collection_date="05-Jun-2014" /PCR_primers="fwd_seq: cgagggtgagagttctgagt, rev_seq: cagcttcctgtcctcacagt" gene <1..>534 /gene="cyp4501a" CDS join(<1..383,478..>534) /gene="cyp4501a" /codon_start=3 /product="Cytochrome P450 1A" /protein_id="CEL26595.1"
/translation="EGESSEYSCALEEHISKEGLYLIERLHSVMKANGGFDPFRHIVV
SVTNVICGMCFGRRYSHDDHELLSLVNLSEEFNQVVGSGNPADFIPFLRLLPSTSMNK
FLAINQRFNVFMQKLVREHYETFNKDNIRDITDSLIDHCEDRKL" exon <1..383 /gene="cyp4501a" intron 384..477 /gene="cyp4501a" exon 478..>534 /gene="cyp4501a"
ORIGIN 1 tcgagggtga gagttctgag tattcctgtg ccctggagga acacatcagc aaagagggcc 61 tgtacctgat cgagaggctg cacagtgtta tgaaggccaa tggtggattc gacccgttcc 121 gtcacattgt ggtgtctgtg acaaacgtga tctgtggcat gtgctttggc cgacgctaca 181 gccacgacga ccacgagctg ttaagcctgg tgaacttaag cgaagagttc aaccaagtgg 241 tgggcagcgg aaacccggcc gacttcattc ccttcctgcg cctcctgccc agcacgagca 301 tgaataaatt cctggccatc aaccagcggt ttaacgtgtt catgcagaag ctggtcagag 361 agcattacga gacattcaat aaggttcgtg cacagtgtat acaaatttca tgacgagatt 421 ctgtcaaaat tcaggatgct cacactgtct tgtatcttct gattttttgt tttttaggac 481 aacatccgtg atatcactga ctctctcatc gatcactgtg aggacaggaa gctg //
Clarias batrachus isolate HM514 cytochrome p450 CYP1A (cyp1A) gene, partial cds GenBank: KP336485.1 FASTA Graphics Go to:
LOCUS KP336485 509 bp DNA linear VRT 04-APR-2015 DEFINITION Clarias batrachus isolate HM514 cytochrome p450 CYP1A (cyp1A) gene, partial cds. ACCESSION KP336485 VERSION KP336485.1 KEYWORDS . SOURCE Clarias batrachus (walking catfish) ORGANISM Clarias batrachus Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Actinopterygii; Neopterygii; Teleostei; Ostariophysi; Siluriformes; Clariidae; Clarias. REFERENCE 1 (bases 1 to 509) AUTHORS Bhutia,D., Rai,B.K. and Pal,J. TITLE Direct Submission JOURNAL Submitted (23-DEC-2014) Department of Zoology, University of North Bengal, Raja Rammohanpur, Siliguri, West Bengal 734013, India COMMENT ##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END## FEATURES Location/Qualifiers source 1..509 /organism="Clarias batrachus" /mol_type="genomic DNA" /isolate="HM514" /db_xref="taxon:59899" /tissue_type="liver" /country="India: West Bengal" /collection_date="16-Dec-2014" /PCR_primers="fwd_name: hm-f, fwd_seq: cgagggtgagagttctgagt, rev_name: hm-r, rev_seq: cagcttcctgtcctcacagt" gene <1..>509 /gene="cyp1A" mRNA join(<1..366,464..>509) /gene="cyp1A" /product="cytochrome p450 CYP1A" CDS join(<1..366,464..>509) /gene="cyp1A" /codon_start=1 /product="cytochrome p450 CYP1A" /protein_id="AKA59072.1"
/translation="EYSCALEEHISKEGLYLIERLHSVMKASGGFDPFSHIVVTVTNV
ICGMCFGRRYSHDDRELLSLVNLSEEFNQVVGSGNPADFIPFLRLLPSTSMKKFLAIN
ERFNVFMQRLVKEHYETYNKDNIRDITDSLIDHCE"
ORIGIN 1 gagtactcct gcgccctgga ggaacacatc agcaaggaag gcctgtacct gattgagagg 61 ctgcacagtg ttatgaaggc cagcggcgga ttcgacccgt tcagtcacat tgttgtgact 121 gtcacgaacg tgatctgcgg catgtgcttt ggtcgccgct acagccacga tgaccgggaa 181 ctgttaagcc tggtgaactt aagcgaagag ttcaaccaag tggtgggcag cggaaacccg 241 gctgacttca ttcccttcct gcgcctcctg cccagcacga gcatgaagaa attcctggcc 301 atcaacgagc gctttaacgt gttcatgcag aggctggtca aagagcatta cgagacatac 361 aataaggttc gtgcacactt tgatcaagtg tatccgaata gcgtgatgag attccatcaa 421 aaattagcat gttcacgctt tgtgtttttt tctttccctc caggacaaca ttcgtgatat 481 cacagactct ctcatcgatc actgtgagg //