QP671.C83 Es5 bite C.l G364653 T H E BAL ME LIBRARY lliiiiiiim 3 0 6 9 2 1 0 0 2 8 4 8 8 2 University of Ghana http://ugspace.ug.edu.gh PROTEIN PROFILE AND M ITOCHONDRIAL DNA ANALYSIS OF CLARIAS GARIEPINVS SURVIVING IN THE KORLE LAGOON. A THESIS SUBMITTED BY JUSTICE CASTRO ESOUN - NYARKOH BSc. (BIOCHEMISTRY) TO THE DEPARTMENT OF BIOCHEMISTRY, FACULTY OF SCIENCE, UNIVERSITY OF GHANA, LEGON, GHANA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF PHILOSOPHY (M.PHIL) DEGREE IN BIOCHEMISTRY S E P T E M B E R 2 0 0 0 University of Ghana http://ugspace.ug.edu.gh DECLARATION I declare that the experimental work for this thesis was carried out by me in the Department o f Biochemistry and Noguchi Memorial Institute for Medical Research both o f the University o f Ghana, Legon, under the supervision o f Professor Marian Ewurama Addy and Dr. Alex Kwadwo Nyarko. SIGNATURE M R JUSTICE CASTRO ESOUN-NYARKOH (STUDENT) SIGNATURE. O PROFESSOR MARIAM EW URAMA ADDY (PRINCIPAL SUPERVISOR) SIGNATURE... DR. ALEX KW ADW O NYARKO (CO-SUPERVISOR) University of Ghana http://ugspace.ug.edu.gh DEDICATION This thesis is dedicated to Rev. and Mrs. R.A. Addison, National Head o f the Church of Pentecost, Nigeria. University of Ghana http://ugspace.ug.edu.gh ACKNOW LEDGEM ENT First o f all, I wish to express my heart felt gratitude and utmost thanks to the Almighty God through His Son Jesus Christ my Lord and Saviour for bringing me this far in my education and providing me with all my needs. I wish to extend my sincere thanks to my supervisors, Professor Marian Ewurama Addy and Dr. Alex K. Nyarko for their keen interest, guidance, relentless efforts, constructive criticisms, error detection, correction o f manuscripts and invaluable suggestions where necessary in the course o f the execution o f this work. I am grateful to Dr. S.T. Sackey for providing me with RNase and Proteinase k used for the extraction and purification o f DNA. Special thanks also goes to the entire technicians and laboratory assistants o f the Department o f Biochemistry for their immense support and assistance during the laboratory work. I am grateful to the Noguchi Memorial Institute for Medical Research (NMIMR), Legon for making available to me their facilities. I wish to specifically acknowledge Dr. George Armah, Head o f Electron Microscopy Unit, Mr. Mark Ofosu-Hene, Research Assistant o f the Clinical Pathology Unit, Dr. K.M. Bosompem o f the Parasitology Unit and Miss Susuana Damanka o f the Electron Microscopy Unit. I am also grateful to Mr. Christopher Renner, a monitoring officer o f the Environmental Protection Agency (EPA), for his immense help. Mention must be made o f Mr. I. Ahia o f the Ministry o f Food and Agriculture, Fisheries Department for providing the fish from the M inistry o f Food and Agriculture Aquaculture Demonstration Centre, Ashiaman. My appreciation also goes to Mr. Amoah, National Service Personnel at the Department o f Computer Science for assisting me in the statistical analysis. 1 also wish to acknowledge the prayers and moral support offered me by the brethren o f Pentecost Student and Associate (PENSA), especially the support and encouragement offered by Professor G.T. Odamtten, Head o f the Department o f Botany and Patron o f PENSA. University of Ghana http://ugspace.ug.edu.gh My sincere thanks goes to Miss Justina Aidoo and Miss Juliana Quartey o f Apam for their financial contribution and moral support offered me, not forgetting my dear mother Madam Rebecca Quaye and father Mr Christopher Esoun-Nyarkoh. I wish to acknowledge the support and directions as well as encouragement o f Mr. Kwame Sefah, Akwesi Agyeman, Kwabena Amposah Manager, Tobias Hammond, Mr. F.C. Mills- Robertson (Ph.D student), Mr. Charles Brown (Ph.D student), John Dadzie-M ensah and Mrs. B.M. Ogoe and all other postgraduate students o f the Department o f Biochemistry, Legon. IV University of Ghana http://ugspace.ug.edu.gh ABSTRACT Studies have established the presence o f some species including fish, in highly polluted water bodies. In order to develop a better understanding o f their ability to survive in such environments, this work was undertaken to ascertain the extent o f microsomal cytochrome P450 induction, the modification o f other proteins and the impact o f the pollutants on the genetic material o f the surviving organisms. In these studies, hepatic microsomal and cytosolic fractions were prepared from Clarias gariepinus, a fish species surviving in the highly polluted Korle lagoon. The same fractions were also prepared from fish samples obtained from the W eija Lake and fishponds at the Ministry o f Food and Agriculture aquaculture demonstration centre (MOFA). The microsomal and cytosolic protein concentrations as well as activities of pollution-induced enzymes were assessed using ethoxyresorufin-O-deethylase (EROD), pentoxyresorufm-O-deethylase (PROD) and p-nitrophenol (PNP) hydroxylase assays. The values were compared among fish samples from the various water bodies before and after acclimatization to laboratory conditions. Cytosolic and microsomal protein profiles were obtained by separating the protein using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Mitochondrial DNA was extracted and purified by RNase digestion and the G+C base composition was determined. In samples both before and after acclimatization to laboratory conditions, cytosolic and microsomal protein concentrations were significantly higher for fish from the Korle lagoon compared to fish from MOFA and W eija Lake. The levels o f EROD activities before and after acclimatization to laboratory conditions were significantly higher in fish obtained from the Korle lagoon compared to those obtained from W eija Lake and MOFA. The levels o f PNP hydroxylase activities before acclimatization to laboratory conditions were significantly higher in fish obtained from the Korle lagoon compared to those obtained from W eija Lake but not those from MOFA. After depuration, the PNP hydroxylase activities were significantly higher for fish from the Korle lagoon compared to samples from both the Weija Lake and MOFA. EROD indicating CYP induction by polycyclic aromatic hydrocarbons (PAH), was approximately ten times higher in the samples from the Korle lagoon compared to the samples from the two other water bodies, even after acclimatization to laboratory conditions. PROD activity, an indication of University of Ghana http://ugspace.ug.edu.gh CYP induction by phenobarbital and its related compounds was significantly lower in the Korle lagoon samples before and after acclimatization. These results indicate the induction o f CYP 1A and CYP 2E by polycyclic aromatic hydrocarbons (PAH) and alcohols respectively in fish obtained from the Korle lagoon while induction o f CYP 2B isozyme was depressed. The pattern o f cytosolic and microsomal protein profile and the corresponding scan for samples from the various water bodies were similar but the intensity o f bands and heights o f peaks for fish obtained from the Korle lagoon were much higher even after depuration. Comparison o f G+C base composition o f mitochondrial DNA for C. gariepinus from the water bodies did not show any statistically significant differences. The study has shown that the induction o f higher amounts o f particular isozymes o f cytochrome P450 such as the 1A and 2E isozymes to an extent that the levels are maintained even in the absence o f the inducer might be the property enabling this organism to survive in the highly polluted Korle lagoon. VI University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS Declaration................................................. — ............ — i D edication................ — ........................................................ ii Acknowledgement — .......................................................... .......... ............... ....... ............. ................ iii Abstract--------------------------------------- ------------------------ --------------------------------------------------v Table o f contents---------------------------------------------------------------------------------------- ------------ vii Abbreviations...........................................................................................................................................ix List o f figures----------------------------------------------------------------------------------------------------------xi List o f tab les.............. .............................................................................................................................xii C H A PT E R ONE INTRODUCTION AND LITERATURE REVIEW ------------------------------------------------------ 1 General Introduction ...................... ....... ................... .......... .............................. .......................... 1 Literature R eview ------------ — ....................................................................- 6 Pollution o f Water B o d ies — ....................................................... 6 Biomarkers o f Pollution------------------------------------- ---------- ----- ---------------------------------- 10 Cytochrome P-450 - Dependent M onooxygenase------------------------------------------------------ 11 Genotoxicity — ........... - ............. - ............. — ............— .......... — .........— ...................... ig Protein Profile — ........... — .........— ..........—............— .......... — ______________ 22 Page vii University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO MATERIALS AND M E T H O D S -....................................- ....... — ....... -....... -------- 24 Chemicals and Reagents --------------- ------------------------ ------------ — ....... -................ — ....... — 24 Pre-treatment o f fish sam ples------------------------------ 25 Preparation o f microsomes and other subcellular fractions---------------------------------------------- 25 Monooxygenase assays----------------------------------------------------------------------- 28 Sodium dodecyl sulphate-polyacrylamide gel electrophonesis (SD S-PA G E ) — .........32 DNA extraction and analysis..............................— ............................. 34 CHAPTER THREE RESULTS ......................... -........................ — .................. 37 Total p ro te in ............................... -.................................................... ................................- ................ - 37 Total microsomal CYP-450 monooxygenase activity................... ............. ............. — .................41 Protein profile----------------------- — .......................— 52 Mitochondrial DNA ................................................-................................................. 60 CHAPTER FOUR DISCUSSION AND CO NCLUSION ............... 64 REFERENCES--------------------------------------------------- - 78 APPENDIX 1..................................................................................................... ............................ -— 89 APPENDIX 2 ........ -.............................92 APPENDIX 3 ............... 93 viii University of Ghana http://ugspace.ug.edu.gh AhR Aromatic hydrocarbon receptor APS Ammonium persulphate BNF p-naphthoflavone CYP-450 Cytochrome P-450 DDE Dichlorodiphenyldichloroethane DDT Dichlorodiphenyltrichoroethane DMSO Dimethylsulfoxide DTT Dithiothreitol EMS Ethylmethanesulfonates EDTA Ethylenediaminetetraacetic acid ER Endoplasmic reticulum EROD Ethoxyresorufin-0 -deethylase GST Glutathione-S-transferase 3-MC 3-methylcholanthrene MOFA Ministry o f Food and Agriculture aquaculture demonstration centre mtDNA Mitochondrial deoxyribonucleic acid PAGE Polyacrylamide gel electrophoresis PAHs Polycyclic aromatic hydrocarbons ABBREVIATION ix University of Ghana http://ugspace.ug.edu.gh PCB Polychlorinated biphenyl PCDD Polychlorinated dibenzo-p-dioxin PNP Paranitrophenol PROD Pentoxyresorufin-O-deethylase SDS Sodium dodecyl sulphate TEMED N,N,N',N'-tetramethylethylenediamine Tris Tris (hydroxymethyl) aminomethane University of Ghana http://ugspace.ug.edu.gh 1. Korle lagoon and associated channels-------------------------------- 7 2. Electron transport system in xenobiotic-metabolizing cytochrome P450 m onoxygenase------------------------------------------------------- ' ** 3. Hydroxylation reaction involved in EROD assay— ......-....... ----------------------------- 15 4. The metabolic activation o f benzo(a)pyrene, a polycyclic aromatic hydrocarbon that is a powerful carcinogen------------------------------------- ----------- ----- 21 5. Flow chart for the preparation o f microsomes and other subcellular fractions 27 6. Mean total heptic microsomal protein concentration for Clarias gariepinus before and after acclim atization...........................— .......... — ....................... 39 7. Mean total heptic cytosolic protein concentration for C. gariepinus before and after acclim atization .............................. 42 8. Mean specific EROD activity for C. gariepinus before and after acclim atization— 44 9. Mean specific PROD activity for C. gariepinus before and after acclim atization— 47 10. Mean specific PNP hydroxylation activity for C. gariepinus before and after acclimatization..................... 50 1 la. SDS-PAGE pattern o f cytosolic protein profile for C. gariepinus obtained from the various water bodies................ -............................... 53 l ib . Electrophoregram scan o f cytosolic protein profile under denaturation conditions for C.gariepinus from the various water bodies...................... - 54 12a. PAGE o f cytosolic protein profile for C. gariepinus obtained from the various w aterbodies............................. -......................................................... 56 12b. Electrophoregram scan o f cytosolic protein profile under non-denaturation conditions for C. gariepinus from the various water bod ies ............................................ 57 13a. SDS-PAGE o f microsomal protein profile for C. gariepinus obtained from the various water bodies —............ 58 13b. Electrophoregram scan o f microsomal protein profile under denaturation conditions for C. gariepinus from the various water bod ies --------- 59 14. Thermal denaturation curves o f mtDNA for C. gariepinus obtained from the various w aterbodies ------ — .................. 6 1 LIST OF FIGURES Page xi University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES 1. Student's t-test o f pairs o f data on microsomal protein concentration for C. gariepinus from different water bodies-------------------------------------- 40 2. Student's t-test o f pairs o f data on cytosolic protein concentration for C. gariepinus from different water bodies----------------------------------------------------- 43 3. Student's t-test o f pairs o f data on mean specific EROD activity for C. gariepinus from different water bodies................... 45 4. Student's t-test o f pairs o f data on mean specific PROD activity for C. gariepinus from different water bodies................................ - 48 5. Student's t-test o f pairs o f data on mean specific PNP hydroxylation activity for C. gariepinus from different water b o d ies---------------------------------------- 51 6. G+C base content o f the mitochondrial DNA for C. gariepinus from different water bodies..................... 62 7. Student's t-test o f pairs o f data on mean G+C base content................................................ 63 Page xii University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION AND LITERATURE REVIEW GENERAL INTRODUCTION The increase in population and its related human activities such as agriculture, urbanisation and industrialisation have resulted in the production of large volumes o f waste (pollutants), which are discharged directly into the atmosphere, streams, rivers, lakes and lagoons without any form of pre-treatment. Water is the basic element for life but the presence o f excessive pollutants in the water bodies has caused most fresh water bodies to become detrimental to the health of living organisms. Thus the aquatic environment acts as sinks or reservoir for the deposition of numerous chemicals o f natural and anthropogenic origin. Most toxic metal and chemical pollutants enter surface and underground water through direct waste discharge and run off, as well as percolation. The impact o f these contaminants on aquatic organisms and the ecosystem results in changes in community, composition, species abundance and population structure o f fish (Munkittrick and Dixon, 1989). Research has shown that there is reproductive impairment, tumour formation and other pathological anomalies as well as behavioural or physiological changes associated with exposure of individual organisms to toxic chemicals (Leatherland and Sunstegard, 1984). High incidence of tumours, chromosomal and mitotic aberrations, genetic and physiological disorders in fishes have been reported in perch (Perea fluviatilis) in areas within Swedish Baltic coastal waters contaminated with polycyclic aromatic hydrocarbons (PAHs) (Ericson et al., 1995). Aquatic life in the Great Lakes has also been greatly affected by large amount of toxic chemicals 1 University of Ghana http://ugspace.ug.edu.gh present in industrial waste discharged directly into the lake without any form o f pre-treatment. Genetic impact studies conducted revealed great variation between the organisms found in the Great Lakes and their counterparts found in relatively pristine environment (Murdoch and Hebert, 1994). The ultimate effect of pollution in aquatic ecosystem is the loss of biodiversity and some water bodies are sometimes referred to as "dead water". Fortunately, some organism, including bacteria, protozoa ciliates, algae, molluscs and some species o f fish are found in such polluted ecosystem (Velz, 1970). Some o f the surviving fish found in highly polluted water bodies include perch along the Swedish Baltic coast, brown bullhead in the contaminated areas in the Great Lakes (Murdoch and Hebert, 1994), and catfish in the Korle Lagoon (personal communications with inhabitants around the lagoon). Such organisms may have acquired resistance to the pollutants either by the synthesis of new gene products as a result o f toxic-induced mutation in the gene structure, adaptation or survival conferred to them by the presence of plasmids which may synthesize proteins necessary for the breakdown o f the pollutants. Ghana and Africa as a whole were thought to be free from industrial pollution. The recent increase in population growth accompanied by intensive formal education, urbanisation, industrialisation and bad waste management policies, as well as improper siting o f industrial installation, have resulted in pollution of both fresh and marine water bodies (Biney, 1991). Current assessment of environmental pollution in Ghana has revealed that large volumes of industrial liquid effluent from the brewery, pharmaceutical, textile, food processing, chemical, metal, paper and wood processing industries are not subjected to any form o f pre-treatment before being discharged into the environment. This has caused an extensive pollution of most streams and lagoons in the cities, severely destroying the ecosystem. University of Ghana http://ugspace.ug.edu.gh The Odaw river and Korle lagoon in Accra, Chemu lagoon in Tema, and Aboabo, Sisai and Subin rivers in Kumasi are the most affected water bodies in Ghana (Biney and Amuzu, 1995). About 22 million cubic metres of industrial waste are discharged into some water bodies in Ghana resulting in 40,000 kg biochemical oxygen demand ( BOD) daily as opposed to a maximum permissible levels o f 50kg BOD daily. The major offenders are the food and beverage industries, which discharge about 5 million cubic metres o f waste. The chemical industries follow with 500,000 cubic metres o f waste, while the metal industries produce 2,500 cubic metres. These industries thus, destroy life in some lagoons in Ghana (Biney, 1991). Industrial liquid wastes, therefore, need some pre-treatment before being discharged into the aquatic ecosystem. Pollution destroys many aquatic lives, however other organisms survive in such polluted environment. Characterization of surviving organisms in such environs has shown that biotransfomation of pollutant in the water bodies is important for survival o f the organism. Among microbes such as Alcaligens, Pseudomonas, Acinetobactor and Flavobacterium, which are normally found in water bodies that received effluent from US Navy ships, only Alcaligens was identified to play a role in the complete denitrification o f waste water discharge from US Navy ship boiler tubes which contain high levels of sodium nitrite (Arquiaga et al., 1993). High chitin and melanin found in the cell walls of fungi have been shown to have high metal biosorptive ability that can be used to combat metal pollution from industrial source (Gadd and White, 1993). Filamentous fungi biotransform telluride and selenium into volatile substances (Gharieb et al., 1999) and specific fungi are known to biodegrade PAH, plant polymers, insecticide, herbicide and radionuclei pollutants (Extended Summaries, 1998). 3 University of Ghana http://ugspace.ug.edu.gh Characterisation o f organisms surviving in highly polluted environment have been useful in pollution studies because such studies have led to the utilisation o f microbes (bacteria and fungi) and some plant species in biodegradation, bioremediation and biotransformation o f industrial and domestic waste in some developed countries. Hydrocarbon utilising microbes were used in the biodegradation and bioremediation of petroleum pollutants in USA during the Alaska oil spills (Atlas, 1995). Mixed microbes entrapped in cellulose triacetate have been used in the treatment of organic waste effluent from food industries (Hsu and Lin, 1996). Mesophilic and thermophilic microbes have been used to digest and to treat waste effluent from coffee processing industries (Dinsdale et al., 1996). Sasa et a i , (1995) reported the use of microorganisms in the biological treatment o f wastewater. Water hyacinth, inoculated with Bacillus maratorium, was used to biodegrade and biotransform industrial waste containing phenol and petroleum products into utilisable resources. For example, toxic hazardous waste can be used by the water hyacinth for its anabolic processes while the plant can be harvested and used as foliage to feed pigs (Shijun and Jingsong, 1989). Therefore organisms found in Ghanaian polluted water bodies could be exploited in the treatment o f industrial waste instead of importing (micro) organisms or chemicals from developed countries for the purpose. In order to develop a better understanding of the ecology of organisms that can biodegrade and biotransform pollutants in our water bodies it is important to assess and characterise them biochemically. The main objective of this research is to characterize an organism living in a highly polluted water body by comparing its pollution-specific enzyme activities, protein and DNA profiles with those of the same organism living in a relatively pristine water body. 4 University of Ghana http://ugspace.ug.edu.gh In Ghana, most o f the highly polluted water bodies, especially Korle and Chemu lagoons in Accra and Tema respectively, have lost almost all edible fish species (Biney, 1991). Investigation conducted among the indigenous people living around the Korle lagoon suggests that besides some crab species (Molluscs), catfish (Clarias sp) are still surviving in the lagoon. Therefore it was worthwhile to subject this species of catfish to biochemical and genetic analysis to assess the impact o f pollution and to determine biochemical characteristics that enable this particular fish to survive in the polluted environment. The specific objectives are to: i) Identify an organism surviving in a heavily polluted water body; ii) Assess the extent o f protein induction due to pollution in this organism; iii) Determine the protein profile o f the organism from its natural habitat; iv) Determine G+C base composition of the genetic material of the organism; v) Compare the extent of protein induction, protein profile and the G+C base content of the same species o f organism from aquatic environment, with different pollution histories. 5 University of Ghana http://ugspace.ug.edu.gh LITERATURE REVIEW Pollution of Water Bodies The impact of pollution on aquatic life worldwide has led to the loss o f valuable and edible fish species, but there are some, which have survived in such polluted water bodies. Examples include perch found in creosote contaminated river Angermanalven (Sweden) (Ericson et al., 1999) and PAH contaminated Swedish sea (Ericson et al., 1998), Northern pike (Esox Vucius) found in PAH contaminated Baltic sea (Ericson et al., 1998) and the brown bullhead in the Great lakes contaminated with PAH and other industrial waste effluent water (Murdoch and Hebert, 1994). The Korle lagoon in Ghana covers a total area o f 25 km2 (See site plan in fig. 1). It is located to the South West of the central business district o f Accra and stretches about 2.8 km inland and drains a total catchment area of about 400 km2. Majority of the drains in Accra enters the lagoon. The Odaw River is the main tributary o f the lagoon. Other channels, flanking the Eastern and Western sides of the lagoon, also drain into it. Most o f the manufacturing industries in Accra are sited in its catchment and tributaries (Biney, 1991). Therefore, all waste effluents from these manufacturing industries are carried into the lagoon. Effluents from industries sited in the catchment area are discharged into the lagoon and this has caused severe and extensive pollution of the lagoon. Effluents and other wastes from artisanal workshop, which are inappropriately sited around the catchment area, are also discharged without any form of pre-treatment into the lagoon. Thus, the general sanitation around the entire lagoon is very poor. 6 University of Ghana http://ugspace.ug.edu.gh Fig I Korle Lagoon and associated channels. ®- Site of sample collection 1 University of Ghana http://ugspace.ug.edu.gh The major pollutants originate from industries, which deal with food, and beverage processing (breweries being the largest contributor), textile, metal and metalloplastic, chemical, paper and pulp. Other sources of major pollutants are domestic, commercial, schools and hospitals, specifically the Korle Bu Teaching Hospital. Solid waste such as paper, food leftovers, scrap metals, old batteries, broken glasses, plastic, textile products, wood cutting, cans and constructional waste are deposited in the lagoon, and this has resulted in the silting of the lagoon (Biney and Asmah, 1994). The lagoon, until recently, supported both fin and shellfish life. When it was clean, it supported Caranx hippos (Horse mackerel), Tilapia melanotheron, Penaeas notalis (Shrimps), and Meriophthalmus papilia (mud skippers) as some o f its aquatic organisms (Biney and Amuzu, 1995). It is on record that species caught by fishermen using dragnets in the vicinity o f the outlet of the lagoon to the sea included sardinella, anchovies, mugil, barracuda, sea bream crab and octopus (Biney and Asmah, 1994). Thus, the lagoon served as economic livelihood for the indigenous settlers who also used the fish to supplement their protein intake. The present grossly polluted state o f the lagoon has almost completely wiped off the fish species, thus depriving the people of their livelihood. The only obvious life around the lagoon is the avian fauna, mainly wild duck; herons and egrets, which are mostly palearctic migrants. The main vegetation includes tufts o f grasses (Paspalluum sp and Sporobolus sp) clumps o f Euphorbia sp, Agara sp and mangrove with Avicennia germinans being the dominant species (Biney et al„ 1994). A study conducted in Ghana (IMDC, 1993) described the Odaw river and the Korle lagoon as the most polluted surface water bodies in Accra. Effluent from the breweries and food processing industries as a whole have high content of organic matter with high BOD that depletes dissolved 8 University of Ghana http://ugspace.ug.edu.gh oxygen and cause euthrophication of the lagoon and hence contributes to the loss o f aquatic life (IMDC, 1993). These water bodies have high BOD level, low dissolve oxygen, elevated levels of phosphates, nitrates, and ammonia compared to non-polluted water bodies. Anoxic conditions prevailing in these water bodies generate methane and hydrogen sulphide gases which give rise to the characteristic smell and odour in the environs o f the lagoon (IMDC, 1994). Compared to the natural seawater background, the polluted lagoon also has high levels o f heavy metal including Hg, As, Cd, Pb, Zn, Ni, Sn, Se, and Cr. Hg, Pb, and Sn contamination are believed to be responsible for the loss of aquatic fauna (Biney et al., 1994). Typical biochemical, physiological and morphological symptoms in most fish found in contaminated water are fin erosion, reduced gonad weight, liver enlargement, induction of liver ethoxyresorufm-O-deethylase (EROD) activity, changes in carbohydrate metabolism and disturbed ion balance. Other abnormalities include stimulated red blood cell production and altered white blood cells, indicating weakened immune system (Andersson et al., 1988). Most of these disturbances were detected in perch and other fish studied in Finland, Canada and USA that were exposed to bleached pulp mill effluents (Lindstrom-Seppa and Oikari, 1989; Munkittrick et al., 1991; Adams et al., 1996). Research has also shown that although the effect of toxins on behaviour, physiology and the population structure may be mitigated within few generations after the reduction of the pollutants, the genetic consequence o f exposure to the pollutants usually persists. These include the introduction o f contaminant-induced mutation and culling o f genetic variation due to natural selection or population collapse (Eisenstedt et al., 1982). The biological organisation o f organisms in aquatic ecosystems affected by the effects of exposure to toxic environmental pollutants can be studied and classified at three different levels. 9 University of Ghana http://ugspace.ug.edu.gh It can be studied through biochemical effects at the molecular and cellular levels, physiological and pathological effect at the tissue, organ and organism level and ecological effect on the population, community and the entire ecosystem level. Studies on the effects o f xenobiotics have shown that their biochemical effect can be transmitted to the physiological and pathological level, which eventually appears at the ecological level. Biomarkers of Pollution Biomarkers are indicators o f biological responses, which result from the interaction of xenobiotics (toxic pollutants) and the physiological systems of an organism exposed to such pollutants. The biological responses are related to the type of toxic pollutant that an organism is exposed to (Peakall, 1994). These biological indicators or biochemical markers have been developed and used as information tools to assess the extent and effect of chemicals and the susceptibility of aquatic organisms to such pollutants. Biomarkers may be used to elucidate the cause-effect and dose-effect relationships in health risk assessment for environmental pollution monitoring purpose (McCarthy and Shugart, 1990). They are specific and sensitive biological responses, which confirm the presence of pollutants in the environment, and serve as early warning indicators in surviving organisms. These indicators may predict future harm and therefore, enable corrective or preventive measures to be taken to avert destruction of biodiversity. Biomarkers also enable governments to legislate on industrial waste disposal, treatment and recovery o f hazardous waste to avoid possible extinction of aquatic organisms and further damage to the environment. Several biochemical assay systems are based on carbohydrate, protein, fatty acid metabolism and blood chemistry (EIFAC, 1975; ICES, 1978; Stegeman, 1981). The most prominent and 10 University of Ghana http://ugspace.ug.edu.gh advanced biochemical methods depend on the induction o f protein biomarkers such as metallothioneins (Olsson, 1978) and the cytochrome P-450 (CYP-450) monooxygenase system (EIFAC 1975; Stegeman, 1981). Cytochrome P-450 - Dependent Monooxygenase Many exogenous chemical compounds and pollutants induce CYP-450 systems. These compounds (xenobiotics) include polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), dichlorodiphenyltrichloroethane (DDT) and dibenzofuran (Andersson et al., 1985). Xenobiotics are classified as carcinogens, mutagens and clastogens (Houk, 1992). Field investigations have revealed that most o f the biochemical and physiological effects observed in fish exposed to toxic effluents disappear or show a weak response. For example, gonad size and hematocrite value revert to normal after sometime. However, certain other parameters such as delay in sexual maturity and EROD activity indicate an incomplete recovery from toxic substances, which may be biotransformed into genotoxic substances (Larsson et al., 1999). CYP 450 detoxifies xenobiotics in eukaryotes. Detoxification o f xenobiotics in eukaryotes occurs in the liver in two phases. Phase I reactions involve the conversion of certain hydrophobic compounds to a hydrophilic form through reactions including epoxidation, hydroxylation, deamination and deethylation (Gillette, 1966). Phase II reactions involve the conjugation of the product(s) of phase I with endogenous polar compounds (Smith, 1968) which facilitates their subsequent removal. The enzyme involved in phase I reaction, the cytochrome P-450 monooxygenase is found in the endoplasmic reticulum (ER). 11 University of Ghana http://ugspace.ug.edu.gh This enzyme system is a member of the haemoprotein superfamily that catalyses the biotransformation of both endogenous and xenobiotic compounds. Steriods, prostaglandins, bile salts and fatty acids are some examples of endogenous compounds, which are biotransformed by the monooxygenase enzyme complex. Some xenobiotics metabolized by the enzyme include PAH, PCB, DDT, polychlorinated dibenzo-p-dioxin (PCDD), pesticides, dioxins, petroleum products, hexachlorobenzenes, drugs and food additives (Nelson et al., 1996). The monooxygenases are a family of enzymes that are membrane bound (integral protein). They are bound to ER and upon disruption of the cell, the ER fragments into small vesicles called microsomes, which can be separated from other organelles through a series o f differential centrifugation. The enzyme is found in the ER o f the liver, kidney, lungs, spleen and gastrointestinal tract in vertebrates. It can also be found in the nuclear membrane of hepatocytes and mitochondria o f the adrenal cortex (Watanuki et al., 1978). Ambike et al. (1970) established the presence o f the enzyme in the fungus Claviceps purpurea and their studies showed a direct correlation between alkaloid production and enzyme level. Their studies identified the involvement o f the monooxygenase in the hydroxylation of anisole in yeast and bacteria. Studies conducted on the CYP 450 have established that there are several forms of the protein and each isoform is substrate specific (Klotz et al., 1986). The characterisation of the various isoforms was based on the amino acid sequence, which were either determined directly or from the gene sequence (Nebert et al., 1991). CYP 450 gene has multiple sub-families with each sub­ family also having more than one member. There are four main families of CYP 450 in vertebrates. These include P450 I which can be induced by PAH, PCB, isosafrole and 13- naphthaflavone (B-NF); P450 II which is induced by phenobarbital, ethanol (alcohols) and acetone; P 450 III is induced by steroids and macrolide antibodies; and P-450 IV is induced by 12 University of Ghana http://ugspace.ug.edu.gh clofibrate. These gene-families represent different subunits o f CYP 450 (Nelson et al., 1996). Lately the nomenclature has been expanded to include chromosomal localisations that respond to different inducing agents differently. The essential components o f the drug metabolizing enzyme system are cytochrome P450, NADPH-dependent cytochrome P450 reductase and phospholipids (Gibson and Schenckman 1978). Phosphatidylcholine and traces of phosphatidylinositol and phosphatidylethanolamine are the main phospholipids found in the membrane bound cytochrome P450 monooxygenase enzyme complex. The NADPH dependent cytochrome P450 reductase is a hexamer that consists of FAD and FMN prosthetic groups. The cytochrome P-450 is an oligomeric complex, which contains iron protoporphyrin IX as its prosthetic group. This complex has maximum absorption at wavelength o f 450nm when reduced by NADPH or dithionite and complexed with carbon monoxide (Yasukochi and Masters, 1976). The key step in the oxygenation reaction is the insertion of one atom o f molecular oxygen into the substrate to produce an unstable intermediate, which breaks down to yield the final polar product. The overall monooxygenase reaction can be summarised by the equation: RH + 0 2 + NADPH + YC ROH + H20 + NADP+ In the above reaction, a lipophilic xenobiotic RH is converted to a hydrophilic product ROH. This product ROH can then be conjugated by phase II enzymes such as glutathione-S-transferase (GST) for subsequent elimination. In the metabolism o f xenobiotics, NADPH cytochrome P-450 reductase, which is a flavoprotein, catalyses the transfer o f electrons from NADPH to the cytochrome P-450. The monooxygenase functions as an electron transport chain, and is shown in figure 2 13 University of Ghana http://ugspace.ug.edu.gh NA D PH - i f NADP+ Oxidized Flavoprotein reduced ' Fe2+-XH Cytochrome P-450 / XH Fe3+ o2 rc XOH + H20 Figure 2: Electron transport system in xenobiotic-metabolizing cytochrome P-450 monooxygenase. XH=Xenobiotic, TC = Ternary complex, XOH= Oxygenated xenobiotic. The CYP 450 system catalyzes a variety of reactions in the metabolism of xenobiotics. These include: Aromatic hydroxylation Ph—H [Q]_____ ^ Ph— OH Aliphatic hydroxylation r — c h 3 roi ^ R— CH2—OH N—dealkylation R—NHCH3 — [O]-------- ► [R— NHCHz—OH] -------------------► RNH2 + HCHO O— dealkylation R—OCH3 TOl » [R— OCH2OH] ----------------- ►R— OH + HCHO In this research, one o f the assays used is EROD which reflects O-dealkylation reaction and is specific for CYP 450 IA induced by PAH. The EROD assay is shown figure in 3: 14 University of Ghana http://ugspace.ug.edu.gh Ethanal NADPH, EROD Ethoxyresorufin Resorufin Fig 3. Hydroxylation reaction involved in EROD assay EROD activity is measured by means of spectrofluorometer, which is specific and very sensitive. The assay evaluates the conversion of ethoxyresorufin into a fluorescent metabolite, resorufin. Resorufin fluoresces at specific excitation and emission wavelength that are different from those o f the substrate (Burke et al., 1985). Pentoxyresorufin O-deethylase (PROD) assay was also used to assess induction of CYP 450 2B usually induced by phenobarbital and its related compounds, thus indicating whether some of the pollutants in the water bodies belonged to this class. In addition, p-nitrophenol hydroxylation (PNP) assay was used as a marker for aromatic hydroxylation reaction, specific for CYP450 2E which is induced by ethanol. This induction can be enhanced by higher alcohols which are associated with increase in hepatic drug metabolism, acetaminophen hepatoxicity and cancer (Lieber, 1988). 15 University of Ghana http://ugspace.ug.edu.gh Genotoxicity The DNA constitution of the genome of a particular species of organism remains very much the same and only few changes occur due to recombination that occur in the nucleus. In eukaryotes the changes that occur in the base sequence are at a very slow rate as opposed to prokayotes. Physical and chemical agents facilitate the changes by causing mutation of the genetic material. The damaged DNA sequence may be corrected in some areas by an in-built repair mechanism although most of the DNA damage cannot be directly corrected (Darnell et al., 1990). The effect o f genotoxic substances on higher organisms may not become obvious until a long time after the exposure. Chemical modification of DNA is generally accepted to be a critical initiating step in chemical carcinogenesis and cytotoxicity. Following the initial exposure, future generations can be affected through reduced embryonic viability and genetic disorders. There are several reports o f liver lesions and other histopathological abnormalities in the benthic fish species found in industrially contaminated areas (Vethaak et al., 1992; Myers et al., 1994). Furthermore, exposure to mutagens increases germline mutation rates, which may decrease fitness o f affected species (Wiirgler and Kramers, 1992). Xenobiotics (pollutants) present in industrial effluent discharged into water bodies without any form of pre-treatment are usually carcinogenic and have a detrimental effect on most aquatic organisms. Due to their lipophilic nature, they accumulate in tissues such as gonad, liver and kidney. In the gonad, they induce heritable mutations in the germ line cells, and this is the main cause of variations in organisms in contaminated water. There are a number of factors other than pollution that may affect the genetic diversity of organisms. These include radiations and geographical location. Indeed some major historical and 16 University of Ghana http://ugspace.ug.edu.gh geographical events have been implicated as factors influencing the evolutionary processes leading to the extant patterns of interspecific genetic diversification and systematic relationships (Brundin, 1965; Bot et al., 1989). However, industrial and domestic pollutants discharged into the environment have been a major historical and current factor influencing the evolutionary processes leading to intra and interspecific genetic diversification. PAHs are the main component o f industrial waste and are easily accumulated in aquatic organisms, especially fish, due to their lipophilic nature. Highly reactive intermediates are produced during the biotransformation of PAH in the liver microsomes, which bind to the biological macromolecules such as proteins and nucleic acids (Millar and Millar, 1947). Several studies have demonstrated an elevated level o f DNA adduct in fish species found in areas contaminated with PAHs (Dunn et al., 1987; Ericson et al., 1998). Laboratory exposure of fish to PAH have also shown hepatic DNA adducts (French et al., 1996). The genetic patterns among the Atlantic cod {Gadus morhua) from different geographical regions have been studied and used to classify the genetic population structure and the gene flow in that fish species (Pogson et al., 1995). van der Meer et al., (1992) have reviewed the molecular mechanism o f genetic adaptation to xenobiotic compounds by microbes in their natural habitat. The mitochondrial DNA (mtDNA) composition was used to characterise three Chlamydomonas species isolated from soil in relation to metal resistance (Spanier et al., 1992). DNA adducts serve as a biomarker for exposure to PAH, and adduct levels in livers of fish correlate with the degrees o f PAH contamination (Stein et al., 1992) Also, DNA adducts being cellular reaction products are the integrated outcome of several pharmacokinetic processes, such 17 University of Ghana http://ugspace.ug.edu.gh as uptake, distribution, metabolism, excretion, DNA repair and cellular turnover (van der Oost et al., 1994). There are certain chemicals, which interact with DNA to induce mutations. These chemicals fall into two classes. The first group o f compounds, which include dimethylsulfate, nitrogen mustard and methylnitrosourea, acts directly on DNA without any metabolic activation. The second group o f compounds are chemically inert (carcinogens), which require metabolic activation. The indirect acting carcinogens include benzo(a)pyrene, dibenz(a,h)anthracene, 2-naphthylamine, dimethyl nitroamine, vinyl chloride, aflatoxin B (Aspergillus flavus) and 2-acetylaminofluorene. These chemicals are metabolised by microsomal CYP 450 that is involved in detoxification of noxious chemicals into highly reactive electrophiles. Figure 4 shows the metabolic activation of benzo(a)pyrene, an inert PAH that is metabolized into a reactive carcinogenic intermediate. This reactive intermediate or electrophile reacts with negatively charged centres in DNA, RNA and protein that lead to the modification of both free and circular DNA, for subsequent phenotypic expression. Cells or organisms exposed to chemical carcinogens often induce permanently altered state in circular DNA (Darnell et al., 1990). While some mutations have detrimental effect on the organism, others show no observable effect, because they either occur in parts of the DNA that do not encode vital information, or have no effect on the coded information. Some mutations are beneficial because they enable the organism to adapt to the environment, an example is haemoglobin AS. Mitochondrial monooxygenase in rat liver has been shown to be capable o f activating carcinogen, which then modifies mtDNA. For example, aflatoxin B l, a hepatic carcinogen is metabolized by rat liver monooxygenase into an electrophilic reactive form, which preferentially 18 University of Ghana http://ugspace.ug.edu.gh covalently modifies mtDNA (Niranjan et al., 1982). Similarly, studies using tissue culture cells, indicate that bioactivated benzo(a)pyrene also causes modification of both mtDNA and nuclear DNA but affects mtDNA more (Allen and Coombs, 1980). Carcinogenic alkylating agents modify mtDNA by a factor o f about five times greater than the nuclear DNA from the same cell (Wunderlich et al., 1970). Furthermore, evidence suggests that mtDNA is the genetic target o f lipophilic contaminants (Allen and Coombs, 1980) that form DNA adducts, thus enhancing the probability o f mutation (Eisenstadt et. al., 1982). Maternal transmission of mtDNA ensures that variation can be generated only by mutation unlike the nuclear genome in which variation can be introduced through recombination (Murdoch and Hebert, 1994). Mutations accumulate in mtDNA at a more rapid rate than in the nuclear DNA (Brown et al., 1979), suggesting that the mtDNA replication enzyme complex lacks the editing function. These factors alone could contribute greatly to a high mutation rate in addition to the fact that mtDNA has a higher turnover rate than nuclear DNA in tissues (Clayton et al., 1974; Niranjan et al., 1982). At the population level, natural levels of mtDNA diversity may be drastically reduced by either strong selection pressures or population collapse associated with environmental degradation. Degradation may be due to contaminant input, reduced water and/or habitat alteration. When the selective pressure is reduced (or the effective population rebounds) the genetic diversity will be restored through the process o f immigration of variant genotype and mutation. When the fish species have low immigration and therefore low rate of gene flow, such geographically separated population will remain genetically distinct. The renewal of genetic diversity through immigration or mutation may, therefore, be a slow process requiring many generations. At the individual level female fish exposed to genotoxic compounds during oogenesis may incorporate 19 University of Ghana http://ugspace.ug.edu.gh mutated mtDNA molecules into their eggs, and hence produce offspring that are heteroplasmic for mtDNA. Such variations will reflect in the population after sometime due to random assortment o f mtDNA molecules at reproduction, which ensure the transition from heteroplasmy to fixation within individuals o f newly arisen mutation (Murdoch and Hebert, 1994). Studies on rapid evolution o f animal mtDNA revealed that the degree of divergence of higher animals were at the site o f recognition and cleavage of restriction endonuclease (Brown et al., 1979). Bogenhagen and Clayton (1974) have inferred that organisms with gene mutation in mitochondria survive because the mitochondrion is polyploid and each cell contains at least one copy of the mitochondrial genome. Hence, a mutation inactivating a gene in one genome might have little or no effect on the fitness of the organism. DNA adducts in both nuclei and mitochondria have been studied and used to classify organisms in relation to geographical and geological patterns as well as the effect o f pollution. Taylor and Dodson (1995) studied the molecular diversity of Holarctic fish in relation to biogeographic events. Ericson et al., (1999) used DNA adducts as a biomarker to study perch (Perea fluviatilis) in relation to creosote contamination. The effect o f PAH on the DNA and histopathological abnormalities as well as organosomatic indices in perch and northern pike (Esox lucius) have been studied and classified (Ericson et al., 1998). Unlike the perch and Northern pike the mtDNA of catfish has not been investigated. Such studies could help in determining how pollutants in water bodies in Ghana affect DNA of organisms that are found in these water bodies. 20 University of Ghana http://ugspace.ug.edu.gh Benzo(a)pyrene 7,8-Dihydrodio l 4 ,5-D ihydrod io l (Nonreactive P-450 m on o o xyg en a se 7 ,8-D io l-9 ,10-epoxide (Ult imate carcinogen: highly reactive e lectrophile) Fig 4: The metabolic activation o f benzo(a)pyrene, a polycyclic aromatic hydrocarbon that is a powerful carcinogen. 21 University of Ghana http://ugspace.ug.edu.gh DNA techniques used in the classification of organisms include G+C base composition. This has been used to classify both mitochondrial and nuclear DNA. Pot et al., (1989) used this technique among others to study and classify the intra- and inter-generic relationships o f the genus Oceanospirillum into five species. De Ley (1970) has also used this method to examine the association between melting point, bouyant density and chemical base composition o f DNA in bacteria. Stanley et al., (1992) characterised Campylobacter helveticus sp isolated from domestic animals faeces based on their DNA base composition and total protein profile. In their study, they showed that the isolates had the same base composition while their protein profile, rRNA gene profile and genomic DNA homology were different in each isolate. Thus, since organisms of the same genus and species are expected to have the same genomic DNA chemical composition any differences may be attributed to mutation that have occurred over a period of time. Protein profile Proteins are gene products and any influence o f xenobiotic (environmental pollutant) on the genetic material is expected to be reflected in the gene product. Environmental pollution can result in the production of non-functional proteins, isozymes or proteins, which may have different molecular weight, compared to those o f natural gene products. Protein resolution is performed on polyacrylamide-gel electrophoresis (PAGE). This technique separates charged molecules such as nucleic acid and proteins on the basis of their movement though a polymatrix gel under the influence of an electric field. The technique employs a discontinuous buffer system in the presence of denaturing anionic detergent, sodium dodecyl sulphate (SDS). The SDS-PAGE technique separates proteins by a more conservative parameter of molecular weight. PAGE of whole cellular protein has been used to characterise organisms. University of Ghana http://ugspace.ug.edu.gh SDS-PAGE of whole cell proteins have been shown to be relatively simple, easy, reproducible and reliable procedure for identification. The outer membrane protein profile o f Serratia marcescens has been studied and used to characterise the organism into 3 major groups (Larsen and Biedermann, 1993). This protein profile technique was used by Bouchara et al., (1993) to study inducible proteinase synthesis in Aspergillus fumigatus in the presence o f other protein. The studies reviewed on the effect of pollution on the induction o f CYP, DNA and protein profile provide the rationale for the present research reported in this thesis. 23 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO MATERIALS AND METHODS F ish S a m p le Catfish (between 500g and 600g) used for this research were obtained from the Korle Lagoon between the old Fadama street and the Korle Bu bridge in Accra, the Weija the Lake and Ministry of Food and Agriculture aquaculture demonstration centre (MOFA) at Ashiaman, all in the Greater Accra Region. The fish from each locality were divided into two and one part kept for 21 days in aquaria under laboratory conditions. The samples from the Weija Lake and MOFA were used as fish from relatively pristine (pollution free) aquatic environment. These provide baseline values for comparison in this project. The fish kept in the aquarium were fed with feed prepared by MOFA Fishery Department. Every catfish used in this project was sent to the Department of Zoology for identification. Chemicals and Regents Ethylenediaminetetracetic acid (EDTA), bovine serum albumin (BSA), resorufin, 7-ethoxyresorufin, 7-pentoxyresorufin, p-nitrophenol, nicotinamide adenine dinucleotide phosphate (NADPH), Tris(hydroxymethyl) aminomethane (Tris), acrylamide, sodium acetate N, N'-methylenebisacrylamide, sodium dodecyl sulphate (SDS), dimethylsuphoxide (DMSO), Proteinase K, RNase and SDS molecular weight markers were obtained from Sigma Chemical Company, (St. Louis, MO, USA). Sodium dihydrogen phosphate, potassium chloride, Folin- Ciocalteau phenol reagent and magnesium chloride were obtained from Hopkins and Williams, England. Chemicals obtained from Fluka were dithiothreitol (DTT), sodium hydroxide, sodium potassium tartrate, ammonium persulphate, NNN^N'-tetram ethylethylenediam ine (TEMED), 24 University of Ghana http://ugspace.ug.edu.gh Coomassie brillant blue R250, phenol, trisodium citrate and sodium chloride. Copper sulphate pentahydrate, sodium carbonate, glycine, methamol, glacial acetic acid, CX-amylase, ethanol, chloroform and isoamylalcohol were obtained from BDH Limited, England. All chemicals were of analytical grade. METHODS Pre-treatment of fish samples Catfish were caught with gill nets from their natural environment and immediately put in cages of water and transported to the Research Laboratory o f the Department o f Biochemistry. The catfish in each set were divided into two. Half from each set were kept in an aquarium while the other half was stunned by a blow to the head and the livers were excised and the gallbladder carefully removed. The livers were stored at -80°C in a homogenising buffer (Appendix 1), until they were used. Those kept in the aquaria for depuration were killed and the liver removed after 21 days. A total of 30 fish, comprising 14 from the Korle lagoon and 8 each from the Ministry of Food and Agriculture aquaculture demonstration centre (MOFA) and Weija Lake respectively, were used for the studies. Preparation of microsomes and other subcellular fractions. The frozen liver tissues were thawed on ice, weighed, diced with a pair o f scissors and homogenized in a volume of ice-cold homogenization buffer with a Glas-Col homogenizer. The 25 University of Ghana http://ugspace.ug.edu.gh homogenates were diluted with the homogenization buffer to give 4ml-homogenization buffer per gram o f liver tissue. The homogenates were centrifuged at 10,000g for 10 minutes at 4°C in a Hitachi 20 PR-52D centrifuge with RPR20-2-1128 rotor. The pellets were discarded. The supernatant fractions were further centrifuged at 16,800g for 10 minutes at 4°C. The pellets, which contained mitochondria, were stored at -20°C for DNA extraction. The supernatant fractions were centrifuged at 105,000g for 60 minutes using Hitachi 80P-7 preparative ultracentrifuge with RP65T 453 rotors. The supernatant fractions were aliquot into labelled eppendorf tubes and stored at -20°C for further analysis. The pellets were washed to remove pigments from the microsomes and centrifuged at 105,000g. The washed pellets were suspended in storage buffer pH 7.6 (see appendix 1) in a ratio o f lg liver weight to 2ml buffer (2m l/lg liver weight). The re-suspended microsomal pellets were distributed into labelled 1.5ml eppendorf tubes and stored at - 80°C. 26 University of Ghana http://ugspace.ug.edu.gh Liver Homogenate 10,000g at 4°C for 10 min (Washed) (Stored at -20°C for further analysis) Pellet Supernatant (discarded) (Microsomes) Fig 5: Flow chart fo r the preparation o f microsomes and other subcellular /ractions. 27 University of Ghana http://ugspace.ug.edu.gh Protein Determination Microsomal and cytosolic protein concentrations were determined by the method of Lowry et al., (1951). The method depends on coloured complexes emanating from a reaction between alkaline copper-phenol reagent and tyrosine and/or tryptophan, and absorbance measured at 750nm. Prior to the determination, a standard curve o f bovine serum albumin (BSA) was prepared. In this, lm g o f BSA was dissolved in 10ml o f 0.5M NaOH, which constituted the stock o f lOOug/ml-protein solution. Various concentrations between 0 and 50ug/ml (0, 10, 20, 30, 40, 50) were prepared. To 1ml of each diluent, 5ml o f alkaline copper phenol reagent (see appendix) were added, mixed thoroughly by vortexing, and allowed to stand for 10 minutes. To each mixture, 0.5ml o f Folin reagent was added, mixed immediately and allowed to stand for 30 minutes. The absorbances were read at 750nm in a double beam spectrophotometer, (Schimadzu UV-190). The blank contained all reagents except the BSA. A standard curve of absorbance against BSA concentrations (ug/ml) was plotted and used to determine the actual microsomal and cytosolic protein concentrations of the fish samples. The microsomal and cytosolic fractions were diluted 1:20, and 1:25 respectively with 0.5M NaOH. The alkaline copper phenol reagent and Folin reagent were added as described above for BSA standard curve. The absorbance was measured and the protein concentration read from the BSA standard curve (shown in appendix 2). 28 University of Ghana http://ugspace.ug.edu.gh Monooxygenase Assays 7-ethoxyresorufin-O-deethylase (EROD) assay: The 7-ethoxyresorufin-O-deethylase (EROD) assay was determined spectrofluorometrically as described by Burke et al., (1985). The method essentially measures the overall cytochrome-P- 450 monooxygenase activity due to the induction of CYP 1A protein by PAH. The excitation and emission spectra of resorufin and 7-ethoxyresorufm (7-ER) were obtained using the SFM-25 spectrofluorimeter, a total volume o f 2ml solution, made up o f 1990|il 0.1M NaH2P0 4 buffer pH7.6 (see appendix 1) and 10 (xl o f 0.41mM 7-ER or 0.85mM resorufin. The excitation and emission wavelengths were set at 510nm and 585nm respectively. Each reaction was performed in a 2ml-reaction mixture, which was made up of 1930^1 of 0.1 M NaH2P0 4 assay buffer pH 7.6, 50pl of microsomal preparation with a protein concentration of 0.2mg/ml and 10 |il 0.41nM 7-ER. The reaction mixture was incubated for 5 minutes at 37°C and the fluorescence measured at 510 and 585 excitation and emission wavelength respectively for 30 seconds. A lOjul aliquot o f 50mM NADPH was stirred into the mixture to start the deethylation reaction. The progressive increase in fluorescence (as 7-ER was converted to resorufin) was recorded for 3 minutes. An internal resorufin standard o f known concentration was added and the sudden increase in fluorescence recorded for another minute. The reactions were performed under subdued light at room temperature. 29 University of Ghana http://ugspace.ug.edu.gh 7-Pentoxyresorufin-O-deethylase (PROD) assay. PROD activity was determined from the rate of formation o f resorufin from 7-pentoxyresorufin (PR). The assay was carried out as described above for EROD except that 10|il o f 0.4 ImM 7-PR was used instead of 7-ER. Both the EROD and PROD activities were calculated using the linear change in fluorescence with time as a result of the addition of NADPH. The specific enzyme activity was estimated by the formula: Specific enzyme activity (nmol resorufin formed) = F x C x D (nmol m in'1 m g'1 microsomal protein) t x S x P C = Concentration of internal resorufin standard D = Dilution factor for sample (2000/50=40) F = Fluorescence o f sample reaction P = Concentration of microsomal protein (mg) S = Spike or fluorescence o f internal resorufin standard t = time over which fluorescence was measured. p-Nitrophenol (PNP) assay The PNP hydroxylation activity was determined as described by Reinke and Moyer (1985). Briefly, the assay involves the formation of 4-nitrocatechol from p-nitrophenol by p-nitrophenol hydroxylase. The 4-nitrocatechol is measured spectrophotometrically (Shimadzu CL-720) under alkaline condition.. Each reaction assay was performed in 1ml reaction mixture (made up of 500^1 of 0.1M Tris buffer pH 7.4, 50^1 of lOmM NADPH, 300nl o f 0.0167M MgCl2 and 100|il 30 University of Ghana http://ugspace.ug.edu.gh microsomal preparation, to give a final concentration of 0.05M Tris buffer, 5mM MgCl?, 0.4g/ml NADPH and 0.3mg microsomal protein). The reaction was initiated by the addition of 50 |il of 2.0 x 10'6 PNP to each reaction mixture. Incubation was carried out for 10 minutes in a shaking water bath at 37°C. The reaction was terminated by the addition of 0.5ml o f 0.6N HCIO4 to the incubation mixture. The mixture was centrifuged at 610g using Hitachi SCT 5BA centrifuge to remove precipitated proteins. An aliquot of 1ml o f the supernatant fraction was mixed with 100^1 of 10M NaOH. The absorbance of the mixture was read at 546nm. The amount of 4- nitrocatechol formed was calculated using the molar extinction coefficient o f 10.28mM'' cm '1. The specific enzyme activity was estimated using the formula: 4-nitrocatechol formed (|imol/min/mg microsomal protein) = A x D E x t x P A = Absorbance o f 4-nitrocatechol formed D = Dilution factor for sample E = Extinction coefficient (10.28mM_l cm"1) t = time over which reaction was performed P = Concentration of microsomal protein used 31 University of Ghana http://ugspace.ug.edu.gh Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Gel Preparation The SDS-PAGE was performed using slight modification o f the procedure o f Laemmli (1970). The gel cassettes were assembled using clean glass plates 12cm by 14cm as instructed by the manufacturer. A spacer o f 1.5mm was used to obtain a gel o f 1.5mm thickness. A separation gel solution (24ml) o f final concentration o f total acrylamide content o f 10% with 2.67% N, N-methylenebisacrylamide cross linker content was prepared as follows: A volume of 8ml acrylamide monomer solution, 5ml separating gel buffer (Tris-HCl pH 8.8) and 0.2ml of 10% SDS solution were added to 6.67ml deionized H 2O and mixed. The gel was polymerized chemically by the addition o f 0.1ml 10% w/v of ammonium per sulphate (APS) and 10|il of N,N,N1 ,N'-tctramcthylethylencdiamine (TEMED). The final concentration of other components in the gel solution were 0.375M Tris-HCl, pH 8.8, 0.1% w/v SDS, 0.05% APS and 0.05% TEMED. (See appendix 1 for stock solution preparation). By means of a dropping pipette, the separation gel solution was dispensed into the gel cassette gently, avoiding air bubbles. It was overlaid with deionized H2O and allowed to polymerize under fluorescent light. The deionized H2O was drained by inverting the gel cassette after polymerization. The separating gel was overlaid with a stacking gel o f total acrylamide content o f 5% with crosslinker content o f 2.67%, prepared by adding 830^1 of acrylamide monomer solution, 1 2 5 0 jj,1 of stacking gel buffer and 50^1 of 10% SDS to 2 8 50 |il deionized H20 and mixing. The concentration of other components were 0.125M Tris-HCl pH 6.8, 0 .1% SDS, 0.05% APS and 0.1% TEMED. The gel was polymerized chemically by the addition of 25^1 of APS and 5ul 32 University of Ghana http://ugspace.ug.edu.gh TEMED, the stacking gel solution being immediately dispensed gently onto the separation gel and the PTFE comb (14 wells) inserted, care being taken to avoid any air bubbles below or on the side of the teeth of the comb. The gel was allowed to polymerize under fluorescent light. After polymerization, the comb was gently removed (to avoid disturbance of the stacking gel) and the wells washed several times with deionized H2O and filled with tank buffer (see appendix 1). The sandwich plate with the gel was placed in the electrophoretic tank and filled with electrode running (tank) buffer pH 8.3. Sample preparation and application A sample buffer comprising 0.0625M Tris HC1 pH 6.8, 2% SDS, 10% glycerol and 5% dithiotreitol (DTT) with 0.001% bromophenol blue was prepared (see appendix 1). The samples (cytosolic and microsomal fractions) from the Korle Lagoon were diluted with sample buffer in the ratio 1:4 while those from Weija Lake and MOFA were diluted in the ration of 1:2 to give approximate protein concentration of 20ug/ml. The proteins were completely denatured by immersing the samples (in eppendorf tubes) in boiling water for 3 minutes. By means of a micro syringe and needle, 20^1 of samples mixed with the sample buffer were loaded onto the wells. SDS molecular weight markers ranging between 14000 dalton and 66000 dalton were also loaded. The gels were subjected to electrophoresis using an initial current of 20mA. The current was increased to 50mA after the tracking dye had moved from the stacking gel to about a centimeter into the separation gel. The run was terminated when the tracking dye reached the bottom o f the gel. The gels were carefully removed and simultaneously fixed and stained overnight in a staining solution containing 0.10% Coomassie brillant blue (appendix I). The gels were 33 University of Ghana http://ugspace.ug.edu.gh destained in the same staining solution but without the Coomassie brillant blue. The process of destaining was repeated several times on a shaker until the background gel became clear. The gels were stored in 20% glycerol and later photographed. The gels were scanned on a Cosmo densitometer. DNA extraction and analysis Mitochondrial DNA was extracted by the method of Ericson et al., (1998). The mitochondrial pellets (about lOOmg) were suspended in 700(il of 50mM Tris and 20mM EDTA buffer pH 8.0, with 0.5% SDS. They were incubated with 35|xl of 20mg/ml proteinase K (final concentration lmg/ml) for 3 hours at 37°C. Proteins were removed by sequential extraction with phenol, phenol-chloroform-isoamylalcohol (25:24:1) and chloroform-isoamylalcohol (24:1). The nucleic acids were precipitated by adding 0.1 volume of 5M NaCl and 1 volume o f -20°C absolute ethanol. The mixture was kept on ice for 30 minutes and centrifuged. The supernatant fractions were discarded and the nucleic acid pellets suspended in 700jnl of 50mM Tris-HCl and 20mM EDTA at pH 8.0. The suspended pellets were incubated with 5jj.1 of 20mg/ml RNase and 20|ig of oc-amylase (for hydrolysis o f liver glycogen) for 30 minutes at 37°C. Protein extraction with the organic solvents was repeated. The DNA was precipitated by the addition o f 0.1 volume 5M NaCl and 1 volume -20°C ethanol and kept on ice for 30 minutes. The DNA solutions were centrifuged at 500g and pellets dissolved in lOmM Tris and ImM EDTA buffer pH 7.4. The purity of the DNA was assessed by measuring UV absorbance at 260nm and 280nm using double beam spectrophotometer (Schimadzu UV-190). The ratio o f UV absorbance at 260m and 280nm (A260/A280) was used to assess the purity of the DNA. The samples were kept at -20°C for further analysis. 34 University of Ghana http://ugspace.ug.edu.gh G+C base content The G+C base content o f the DNA was determined using the ultraviolet absorbance- temperature profile method developed by Mandel and Marmur (1968). The DNA sample was diluted with standard saline citrate (see appendix 1) to obtain a final DNA concentration o f 20ug/ml. The initial absorbance A 25 was measured at 25°C at 260nm wavelength using a double beam spectrophotometer (Schimadzu UV-190). Based on a preliminary rough determination of melting temperature of the DNA, the temperature o f the chamber was raised to 60°C by means of a temperature regulated water bath. The absorbance was measured at 60°C and the temperature gradually increased by intervals of 2°C. The absorbance was measured with increasing temperature until there was no significant change in absorbance with temperature increase. The absorbance was corrected for concentration dilution caused by solvent expansion using tables provided by Mandel and Marmur (1968). The relative absorbance was determined using A /A 25; where: At = Absorbance at temperature t A2s = Absorbance at 25°C (room temperature). The melting temperature Tm (50lh percentile) was obtained from a graph of relative absorbance against temperature. The G+C base content was calculated using the linear relationship: G+C = (Tm - 69.3) 2.44. 35 University of Ghana http://ugspace.ug.edu.gh Statistical Analysis The Student's t-test was used to test for the level o f significance between means obtained for catfish from the different waterbodies. Also, the differences between the means obtained before and after acclimatization to laboratory conditions for 21 days for sample from the same water body was evaluated. P values less than 0.05 were considered significant. In the calculation o f the t-values, two-sample assuming unequal variances were used. 36 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE RESULTS The catfish obtained from the Korle lagoon as the only surviving fish in this highly polluted water body was identified as Clarias gariepinus at the Department of Zoology, University of Ghana. The biochemical parameters measured as markers of pollution included total cytosolic and microsomal protein concentrations and the activities o f 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-deethylase (PROD) and paranitrophenol hydroxylase (PNP). These microsomal protein activities indicated the type of CYP-450 induced. The establishment of a protein profile involved the separation of cytosolic and microsomal proteins using both denatured and native gels. The DNA analysis involved the determination of G+C base content of the mitochondrial (mt) DNA to determine whether the effect of pollution in the Korle lagoon had changed the base composition o f the mtDNA. Total Protein Microsomal protein concentration The results of total microsomal protein concentration for C. gariepinus obtained from the various water bodies of different pollution histories before and after acclimatization under laboratory conditions for 21 days are presented in fig. 6. The results show that total microsomal protein concentration in the fish from the Korle lagoon is higher than the level found in fish obtained from the Weija Lake and the aquaculture demonstration centre o f the Ministry of Food and 37 University of Ghana http://ugspace.ug.edu.gh Agriculture (MOFA). The fish from MOFA had the least concentration o f microsomal protein. The result showed that the protein concentration o f fish obtained from the Korle lagoon was approximately 90% and 100% more than that of fish from the Weija Lake and MOFA respectively, before acclimatization to the laboratory conditions. The results further showed a decrease in microsomal protein concentration after acclimatization to laboratory conditions for 21 days. The decrease in protein concentration for fish from the Korle lagoon was 32%, that for fish from the Weija Lake was 58% and that from MOFA was 44%. The protein concentration after acclimatization for fish from the Korle lagoon was 300% and 260% higher than those from Weija Lake and MOFA respectively; that for fish from MOFA was 16% higher than that for fish from Weija Lake. The statistical analysis, using the Student's t-test as shown in table 1, indicates a significant difference in microsomal protein concentration between fish from the Korle lagoon and those from the Weija Lake and MOFA, before and after acclimatization. In contrast, no statistically significant differences were observed between protein concentrations o f fish from Weija Lake and MOFA before and after acclimatization to laboratory conditions. Table 1 further shows a significant difference in microsomal protein concentration before and after acclimatization to laboratory condition for fish from the various water bodies. 38 University of Ghana http://ugspace.ug.edu.gh KORLE W EIJA WATER BOOIES MOFA ^ Non-acclimatized ® Acclimatized Fig 6 : Mean total hepatic microsomal protein concentration fo r C. sariepinus before and after acclimatization 39 University of Ghana http://ugspace.ug.edu.gh Table 1: Student’s t-test o f pairs o f microsomal protein concentration for C. gariepinus from different water bodies. Source o f C. gariepinus t-statistics t-tabulated P Non acclimatized Korle lagoon and Weija Lake 3.728 2.145 <0.05 Korle lagoon and MOFA 4.842 2.228 <0.05 Weija Lake and MOFA 1.398 2.571 n.s Acclimatized Korle lagoon and Weija Lake 6.738 2.571 <0.05 Korle lagoon and MOFA 5.067 2.262 <0.05 Weija Lake and MOFA -0.689 2.447 n.s Non acclimatized and acclimatized Korle lagoon 2.460 2.131 <0.05 Weija Lake 5.673 2.571 <0.05 MOFA 4.000 2.571 <0.05 P value <0.05 for significant level n.s no significant differences P - significance level. 40 University of Ghana http://ugspace.ug.edu.gh Cytosolic Protein Concentration The results o f total cytosolic protein concentration for C. gariepinus are shown in fig. 7. indicating a high cytosolic protein concentration in the fish obtained from the Korle lagoon before and after acclimatization to laboratory conditions for 21 days. The protein concentration of fish from the Korle lagoon was approximately 300% higher than those obtained from the Weija Lake and MOFA, before acclimatization to laboratory conditions. The results further showed a small decrease in protein concentration for fish from Weija and MOFA after acclimatization to laboratory whereas the decrease after acclimatization was 20% for fish from the Korle lagoon. The protein concentration o f fish from the Korle lagoon was more than those from Weija and MOFA by more than 200% after acclimatization. The summary of the statistical analysis is shown in table 2. The table shows a significant difference in cytosolic protein concentration between fish from the Korle lagoon and those from Weija Lake and MOFA before and after acclimatization. Comparing fish from the wild and those acclimatized to laboratory conditions, no significant differences were observed for the cytosolic protein concentrations for fish from the various water bodies. Total Microsomal CYP-450 Monooxygenase Activity Ethoxyresorufin-O-deethylase (EROD) activity. The use of the EROD assay was to indicate the presence of CYP 1A induction by polycyclic aromatic hydrocarbon (PAH) compounds.The results for EROD assay before and after acclimatization are shown in fig. 8. The results indicate that EROD activity in rom the Korle lagoon before and after acclimatization was approximately 1000% or 10 41 University of Ghana http://ugspace.ug.edu.gh Cy to so lic pr oW n co nc en tr at io n (u g/ m l) Fig 7: Mean total cytosolic protein concentration fo r C. eariepinus before and after acclim atization to laboratory conditions \2 University of Ghana http://ugspace.ug.edu.gh Table 2: Student's t-test o f pairs o f data on cytosolic protein concentration for C. gariepinus from different water bodies. Source o f C. gariepinus t-statistics t-tabulated P Non acclimatized Korle lagoon and Weija Lake 6.708 2.365 <0.05 Korle lagoon and MOFA 6.818 2.365 <0.05 Weija Lake and MOFA 0.063 2.262 n.s Acclimatized Korle lagoon and Weija Lake 10.254 2.450 <0.05 Korle lagoon and MOFA 10.415 2.570 <0.05 Weija Lake and MOFA 0.097 2.365 n.s Non-acclimatized and acclimatized Korle lagoon 2.155 2.201 n.s Weija Lake 0.821 2.262 n.s MOFA 0.619 2.262 n.s 43 University of Ghana http://ugspace.ug.edu.gh M ea n sp ec if ic ER O D ac tiv ity (n m ol /m ln /m g m ic ro so p ro te in ) □ Non-acclimatized ■ Acclimatized Fig 8: Mean specific EROD activity fo r C. eariepinus before and after acclimatization. 44 University of Ghana http://ugspace.ug.edu.gh Table 3: Student's t-test o f pairs o f data on mean specific EROD activity for C. gariepinus from different water bodies. Source o f C. geriepinus t-statistics t-tabulated P Non acclimatized Korle lagoon and Weija Lake 15.833 1.998 <0.05 Korle lagoon and MOFA 15.101 1.997 <0.05 Weija Lake and MOFA -3.388 2.004 <0.05 Acclimatized Korle lagoon and Weija Lake 18.500 2.064 <0.05 Korle lagoon and MOFA 17.289 2.064 <0.05 Weija lake and MOFA -0.968 2.110 n.s Not acclimatized and acclimatized Korle lagoon 3.287 1.988 <0.05 Weija Lake 0.243 2.045 n.s MOFA 2.547 2.020 <0.05 45 University of Ghana http://ugspace.ug.edu.gh times higher compared to the EROD activity recorded for fish from Weija Lake and MOFA The statistical analysis shown in table 3 indicates significant differences in EROD activity for fish from the Korle lagoon, and those from the Weija Lake and MOFA before and after acclimatization to laboratory condition. There was also a significantly higher EROD activity in the MOFA fish samples compared to those from the Weija Lake before acclimatization. However, the difference in EROD activity after acclimation was not statistically significant. The EROD activity was higher in fish from wild than after acclimatization. The 25% and 22% reduction in EROD activity for fish from the Korle lagoon and MOFA respectively were statistically significant. In contrast, the 8% reduction in EROD activity in the fish from the Weija Lake before and after acclimatization was not statistically significant. Pentoxyresorufin-O-deethylase (PROD) activity PROD activity is an indication of CYP 2B induction by phenobarbital and its related compounds. The results for mean specific PROD activity before and after acclimatization to laboratory conditions for 21 days are presented in fig. 9. For the non acclimatized samples, PROD was higher for fish from MOFA with those from the Korle lagoon showing minimum activity. The enzyme activity in fish from MOFA was about 35% higher than those from the Korle lagoon and about 20% higher than those from the Weija Lake. PROD activity observed for fish from the various water bodies were statistically significant for fish from the wild as shown in table 4. The highest PROD activity was not observed in fish obtained from the Korle lagoon which is thought to be the most highly polluted in this study. This observation is in contrast to that 46 University of Ghana http://ugspace.ug.edu.gh M ea n sp e ci fi c P R O D ac ti vi ty (p m o l/ m ln /m g m ic ro s o m a l p ro te in ) 27.1 19.3 KORLE WEIJA WATER BODIES MOFA □ Non-acclimatized Acclimatized Fig 9: Mean specific PROD activity fo r C. gariepinus before and after acclimatization to laboratory conditions. 47 University of Ghana http://ugspace.ug.edu.gh Table 4: Student's t-test o f pairs o f data on mean specific PROD activity for C. gariepinus from different water bodies. Source o f C. gariepinus t-statistics t-tabulated P Non acclimatized Korle lagoon and Weija Lake 2.949 2.093 <0.05 Korle lagoon and MOFA 4.626 2.086 <0.05 Weija Lake and MOFA 2.242 2.101 <0.05 Acclimatized Korle lagoon and Weija Lake 7.078 2.145 <0.05 Korle lagoon and MOFA 7.078 2.145 <0.05 □ Weija Lake and MOFA 0.032 2.002 n.s Non acclimatized and acclimatized Korle lagoon 2.242 2.060 <0.05 Weija Lake 1.931 2.228 n.s MOFA 2.452 2.160 <0.05 48 University of Ghana http://ugspace.ug.edu.gh for the EROD activity. The results showed a 31% lower PROD activity for fish obtained from the Korle lagoon than those from Weija. The PROD activity was lower after acclimatization to laboratory condition for 21 days. The reduction in activity was 17%, 13% and 29% respectively for fish obtained from the Korle lagoon, Weija Lake and MOFA. PROD activity after acclimatization was comparable in fish from Weija and MOFA. Activity o f the enzyme is however lower in fish from the Korle lagoon. While the reduction in PROD activity was statistically significant for fish obtained from the Korle and MOFA after acclimatization, that for fish from Weija Lake was not. Furthermore, the difference in PROD activity between the Korle lagoon samples and those from the Weija Lake and MOFA was statistically significant, but that for samples from Weija Lake and MOFA were not. Paranitrophenol hydroxylase (PNP) activity The PNP enzyme system is specific for CYP-2E isozyme induced by ethanol. The induction response is enhanced by higher carbon chain alcohols such propanol, butanol and pentanol. The results of PNP hydroxylase enzyme activity o f microsomes prepared from fish samples from different water bodies are shown in fig. 10. The statistical analysis of the result is shown in table 5. The activity of PNP hydroxylase was high in fish obtained from the Korle lagoon and MOFA compared to fish from the Weija Lake. The activity for the Korle lagoon samples were higher although not statistically different from those from MOFA. PNP activities for Korle lagoon and MOFA samples were more than 100% 49 University of Ghana http://ugspace.ug.edu.gh □ Non-acclimatized H Acclimatized Fig 10: Mean specific PNP hydroxylase activity fo r C. sariepinus before and after acclimatization to laboratory conditions 50 University of Ghana http://ugspace.ug.edu.gh Table 5: Student's t-test o f pairs o f data on mean specific PNP activity for C. gariepinus from different water bodies. Source o f C. gariepinus t-statistics t-tabulated P Non acclimatized Korle lagoon and Weija Lake 3.195 2.068 <0.05 Korle lagoon and MOFA 0.885 2.042 n.s Weija Lake and MOFA -3.089 2.120 <0.05 Acclimatized Korle lagoon and Weija Lake 8.314 2.365 <0.05 Korle lagoon and MOFA -7.700 2.262 <0.05 Weija lake and MOFA -5.467 2.262 <0.05 Non acclimatized and acclimatized Korle lagoon 0.175 2.055 n.s Weija Lake 3.403 2.365 <0.05 MOFA 4.625 2.201 <0.05 51 University of Ghana http://ugspace.ug.edu.gh higher than the activities obtained for the Weija Lake samples. The differences were statistically significant.For the acclimatized samples, the PNP hydroxylase activity for fish from the Korle lagoon was significantly higher, than those from Weija Lake (at least 450%) and MOFA (at least 36%). The PNP activities for the acclimatized Weija samples were significantly lower than that of MOFA samples by 20%. Comparing fish from the wild and those acclimatized to the laboratory conditions, the results indicated 50% and 67% reduction in activity for fish in Weija Lake and MOFA respectively. These decreases in PNP hydroxylase activity were statistically significant. In contrast, the percentage change in enzyme activity for the fish obtained from the Korle lagoon, was not statistically significant. Protein Profile The SDS-PAGE electrophoregram of cytosolic protein under denaturation condition is shown in fig. 1 la. All the fish samples from the various water bodies showed similar patterns of protein bands, even though the protein bands in the sample from Weija appear faint. However, there are many more protein bands in the 3 samples from the Korle lagoon. Fig. l ib shows the scan of the electrophoregram presented in fig. 11a. The pattern of peaks corresponds to the protein bands. The electrophoregram indicated that the intensity and number o f the protein bands of the Korle lagoon samples were higher than those from Weija Lake and MOFA (fig 11a). The pattern of protein peaks in fig 1 lb identified proteins, which were very faint and could not be seen in fig. 11a. The cytosolic protein profile for sample o f fish from the Korle lagoon shows very high protein peaks that exceeded the set range for the densitometer used. The peaks recorded for the Weija and MOFA samples were not as high as those from the Korle lagoon samples. Furthermore these peaks correspond to proteins, which did not appear in fig. 11 a. The peaks for MOFA samples were higher than those from Weija samples but lower than those from the Korle University of Ghana http://ugspace.ug.edu.gh Fig 11a: SDS-PAGE o f cytosolic protein from C. gariepinus obtained from the various water bodies. Cytosolic fraction o f 0.2mg/ml protein concentration was applied to each lane. Lane 1, standard molecular weight markers; lane 2,3 and 5,Korle lagoon samples; lane 4, Weija Lake sample and lane 6, MOFA sample. 53 University of Ghana http://ugspace.ug.edu.gh Fig 1 lb: Electrophoregram scan of cytosolic protein profile under denaturation conditions for C earieninus from the various water bodies D irection of scan: H igher m olecular w eight to lower m olecular w eight. O 0 54 University of Ghana http://ugspace.ug.edu.gh lagoon samples. The scanning of the electrophoregrams for the three samples showed similar protein profiles but differed in peak heights for fish from the various water bodies. Two distinct peaks B|< and Ck (Korle samples) were considerably higher than the corresponding peaks for the Weija ( Bw and Cw) and MOFA (, Bm and Cm) samples. Peak heights of B and C, like most o f the other peaks were consistent with the trend of protein concentration for the various water bodies, that is, highest for samples from the Korle lagoon. In contrast the peaks labelled A were not; the heights were approximately the same for the Weija and Korle samples with the samples from MOFA having the least value. The results in fig 12a display the electrophoregram for the cytosolic protein analysed under non denaturation condition. The native protein profile shows more intense protein for samples from the Korle lagoon compared to samples from the other two water bodies. The results of the electrophoregram scan for the cytosolic proteins under non-denaturation conditions are shown in fig. 12b. The height and the patterns o f peaks correspond to the results shown in fig. 12a. The pattern of cytosolic protein peaks shows two distinct protein peaks D and E. The peaks and Ek are considerably higher than their counterpart Dw, Dm and Ew- Em. The peak Ek exceeded the set range for the densitometer used while Em was the lowest peak among the E peaks. In contrast, Dm (MOFA) was higher than Dw (Weija), with the Dk (Korle) being the highest peak. The results presented in fig. 13a show the electrophoregram of the microsomal proteins under denaturation conditions. The major protein patterns are similar except for the relatively lower molecular weight proteins, which were absent in MOFA and Weija samples. The low molecular weight bands (32kD to 43kD) for the Korle lagoon samples 55 University of Ghana http://ugspace.ug.edu.gh Fig 12a: Page o f cytosolic protein from C. gariepinus obtained from the various water bodies. Cytosolic fraction o f 0.2 mg/ml protein concentration was applied to each lane. Lane 1; standard molecular weight markers; lane 2 and 3. Korle lagoon samples; lane 4, Weija Lake sample and lane 5, MOFA sample. 56 University of Ghana http://ugspace.ug.edu.gh nti CTQ* K>cr g- ft- 5* ^ to/TV ^''t £ o"tTO >! a 2 Co TO C a '*>. Oto ♦.2r-TO § S'c a 5 7 University of Ghana http://ugspace.ug.edu.gh 76.0 66.0 ► > W j r 36.0 f . 35.8 ---------------i­ *- 21 0 34 7 32.0 29.0 24.0 142 kD Fig 13a: SDA-PAGE o f microsomal protein from C. eariepinus obtained from the various water bodies. M icrosomal fraction o f 0.2mg/ml protein concentration was applied to each lane. Lane 1, standard molecular weight markers; lane 2 and 3, Korle lagoon samples; lane 4, Weija Lake sample; and lane 5, MOFA sample. 58 University of Ghana http://ugspace.ug.edu.gh Fig 13b. Electrophoregram scan of m icrosom al protein profile under denaturation conditions for C. varieninus from the various w ater bodies. ̂ D irection of scan: lower m olecular w eight to higher m olecular w eight. 59 University of Ghana http://ugspace.ug.edu.gh are o f higher intensity than those for MOFA and Weija samples. A scan of the electrophoregram is shown in fig 13b. The peaks for the Weija and MOFA samples were lower than those from the Korle lagoon samples. Compared to the Korle lagoon samples, there were virtually no peaks for the lower molecular weight proteins for the MOFA and Weija samples. Three conspicuous protein bands (about 53.5kD, 58.5kD and 78kD molecular weight) were observed in the profile for the Korle lagoon samples. These bands corresponded to peaks H, G and F respectively in fig 13b. The peak Hk for Korle sample was the highest while its corresponding peak Hm for MOFA sample was the lowest. Generally, the protein bands for samples from the Korle lagoon were more intense than those from MOFA and Weija Lake. Figs 13a and 13b show that the samples from the Korle lagoon have higher microsomal protein concentration than those from MOFA and the Weija Lake. MOFA samples had the lowest concentration and number of proteins in the microsomes. Mitochondrial DNA The temperature profile for the mtDNA denaturation is shown in fig. 14. All the fish samples obtained from the various water bodies showed similar pattern of increasing relative absorbance with increasing temperature. However, fish obtained from the Korle lagoon showed higher relative absorbance than those from the Weija Lake and MOFA. The mean melting temperature (50th percentile) of the Korle lagoon samples was 93.2°C while fish obtained from Weija Lake and MOFA were 93.0°C and 92.9°C respectively. 60 University of Ghana http://ugspace.ug.edu.gh R e la ti v e A b s o r b a n c e TEM PERATURE MOFA— W3JA KORLE Fig 14: Thermal denaturation curves o f mtDNA fo r C. eariepinus obtained from the various water bodies. 61 University of Ghana http://ugspace.ug.edu.gh G+C base content of the purified mtDNA isolated from the various samples after homogenization was determined using ultraviolet absorption. The results are shown in table 6 and results for t-test o f significance is shown in table 7. Table 6: G+C base content o f the mitochondrial DNA for C. gariepinus from different water bodies. Source of C. gariepinus LTV Absorbance Ratio G+C content (Mean ± SD) mol% 260nm 280nm 260/280 Korle lagoon 0.608 0.349 1.74 58.21 ±2.10 Weija Lake 0.176 0.101 1.75 57.93 + 1.80 MOFA 0.249 0.138 1.80 57.69+ 1.85 62 University of Ghana http://ugspace.ug.edu.gh Table 7: Student's t-test o f pairs o f data on mean G+C base content. Source of C. gariepinus t-statistics t-tabulated P Korle lagoon and Weija Lake 0.893 2.101 n.s Korle lagoon and MOFA 1.506 2.101 n.s Weija Lake and MOFA 0.668 2.101 n.s The samples from the Korle lagoon had the highest G+C base content but statistically there was no significant difference between G+C base content o f mitochondrial DNA in fish samples from the various sources. 63 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR DISCUSSION AND CONCLUSION Pollution affects aquatic life worldwide, leading to the loss o f valuable and edible fish species. In Ghana, the impact o f pollution on the Korle lagoon and other heavily polluted water bodies has been documented (Biney, 1991). The studies reported in this thesis were designed to examine some of the biochemical and molecular changes that facilitate the survival of an organism in this lagoon. Thus, the first objective o f this study was to identify an organism, preferably eukaryote, surviving in the Korle lagoon. The presence of Clarias gariepinus as the only surviving fish species in the highly polluted Korle lagoon is in contrast to the report o f the Korle lagoon ecological studies conducted by Biney and Amuzu (1995). According to that report, the grossly polluted state of the Korle lagoon had resulted in the complete loss of all fish species in the main lagoon body where the C. gariepinus for this study was found. The identification o f C. gariepinus in the Korle lagoon suggests that other fish species might have adapted to the effect of toxic xenobiotics and are surviving in the lagoon. Therefore, further ecological studies of the main Korle lagoon need to be conducted in order to identify all fish species that have adapted to the toxic xenobiotics in the Korle lagoon. Enzyme activities that are specific to inducible proteins were measured in order to ascertain the extent o f protein induction due to pollution by xenobiotics discharged into the Korle lagoon. Enzyme activities measured for samples from the Korle lagoon were compared with those 64 University of Ghana http://ugspace.ug.edu.gh obtained from relatively less polluted water bodies. The same comparison was for activities measured after acclimatization in the laboratory tanks filled with clean water. Cytosolic and microsomal protein concentrations were also measured. The present study indicates that there were significantly higher cytosolic and microsomal protein concentrations in fish samples from the highly polluted Korle lagoon compared to those from the Weija Lake and MOFA. This is likely to be due to the presence o f excessive amounts of pollutants in the Korle lagoon. These findings are consistent with previous investigations of protein induction in Oreochromis niloticus in response to xenobiotic pollutant discharged by the Akosombo Textile Limited (ATL) into the Volta Lake (Addy et al., 1995), and also in Sarotherodon melanotheron present in the Fosu lagoon (Renner, 1998). These investigations clearly demonstrate that induction o f both cytosolic and microsomal hepatic proteins occur in fish that are obtained from polluted water bodies compared to fish found in relatively pristine water bodies. The mean total microsomal protein concentration for fish sample from the Korle lagoon was almost twice those from Weija Lake and MOFA. The total microsomal protein content of fish samples from the wild gave an indication o f more protein induction in fish from the Korle lagoon, which reflects the extent o f pollutants discharged into the lagoon. The differences in microsomal protein concentrations between Korle lagoon sample and the other samples after acclimatisation to laboratory condition for 21 days were significant. When fish from Weija lake and MOFA were acclimatised in tap water for 21 days in the laboratory, the decrease in microsomal protein concentrations was over 40%. Samples from the Korle lagoon showed the smallest decrease (32%) in microsomal protein. Inspite o f the decrease, the protein concentration for fish from the Korle lagoon remained high after acclimatisation. Lech et al.. 65 University of Ghana http://ugspace.ug.edu.gh (1982) reported that microsomal protein induction due to xenobiotics was reversed when feral fishes were acclimatised in tap water for 7 days. The results obtained in this study indicate that a much longer period of time in tap water is required for total depuration. It is also possible that in the case o f fish from Korle lagoon, the pollution induction process cannot be reversed completely. Further studies need to be conducted to ascertain the time required for complete reversal o f induction in the absence of an inducer, if this is possible. There was no significant difference between protein concentration for fish from MOFA and Weija Lake after acclimatization to laboratory conditions. In each case, however, there was a significant decrease after depuration, indicating that those water bodies are also polluted but not to the same extent as the Korle lagoon. The results of cytosolic protein concentrations o f fish from the various water bodies showed trends similar to those of the microsomal protein concentrations, indicating induction of cytosolic proteins in the highly polluted Korle lagoon. Values for the Weija and MOFA samples were the same, comparing pre- and post- acclimatization results; the reduction in cytosolic protein was not statistically significant for both samples.