THE MODULATION OF RAT LIVER MICROSOMAL CALCIUM ION-PUMPING ATPase BY DICOPHANE AND LOW PROTEIN INTAKE BY GBENGA ADEBOLA ADENUGA M.Sc. Biochemistry (Kharkov) A thesis in the Department of BiochemistPf Submitted to the Faculty of Basic Medica! Sciences in partial fulfilment of the requirements tor the degree of DOCTOR OF PHILOSOPHY of the UNIVERSITY OF IBADAN MAY 1992. ' UNIVERSITY OF IBADAN LIBRARY 2 ABSTRACT The effects of the üver tumour promoter, dicophane, with those of low protein intake (LPI) 2+ on the functional expression of rat liver microsomal Ca -ATPase w;ire compared. The effects of dicophane and LPI on the activity of the microsomal enzyme after carcinogenic initiation by pretreatment with aflatoxin B (AFB ), a genotoxic liver carcinogen, were also 1 2+ compared. The Status of membrane - bound Ca -ATPase of erythrocytes of humans having primary liver cancer (PLC) and kwashiorkor was assessed. 2+ The specific activüy of membrane - bound microsomal Ca -AT :iase of the livers of untreated rats was 4.543 £ 0.857 pmole P/mg protein/hr. at pH 8.0 and was insensitive to calmodulin. The specific activity of the enzyme was significantly ö-creased (P < 0.01) following subcutaneous administration of a single dose of 75mg dicop! ,ane/kg body wt.; the 2+ 24- affinity of the enzyme for Ca was however unaffected. Similarly, liver microsomal Ca - ATPase activity was significantly diminished following the ingestion of low protein diet by rats 2+ for 12 weeks. The mean Ca -ATPase activity of AFB -treated animal?, (in Ihn absence of dicophane) was not significantly different (P > 0.05) from that of AFEMreated rats which subsequently received dicophane. In contrast, liver microsomal Ca'+-ATPase activity of animals fed low protein diet prior to and after AFB ingestion was highe! ( P < 0.05) than that 2+ of animals which wem on low protein diet only. Basal activity of erytl n ocyte Ca -ATPase in paediatric Controls and those having kwashiorkor (protein-energy-mainutrition) were similar (P > 0.05); similar observations were made between normal adults and those suffering from PLC. Erythrocyte Ca ATPase of either PLC or kwashiorkor patients was however, some- what, less sensitive (15-40 %) to the stimulatory effect of calmodulin, an endogenous activator 24- of the Ca -pump. 24- These results suogest that liver microsomal Ca -ATPase could be a useful biochemical marker to determine the onset or occurrence of tumour promotion in liver cells. Finally, chronic dielary proteir malnutrition mimics the effect of Chemical liver tumour Promoters and could possibly enhance the development of human PLC particularly n those areas of the tropics where malnulntion is prevalent. Future confirmatory experiments are however re- quired to fully justify this postulate. UNIVERSITY OF IBADAN LIBRARY ACKNOWLEDGEMENTS My very sincere appreciation and thanks go to my research Supervisor, Professor Enitan Abisogun Bababunmi for giving me a very sound training in Biomembrane Research and Biochemical Toxicology. I thank him for providing me with chemicals/reagents as and when due and for generously sharing with me current research publicat;ons which he normally brings back from hk> very numerous trips to other laboratories overseas. He arranged my trip to the University of Liverpool where some of the experiments reparted in this thesis were performed; and alsc made it possible for me to attend the 15th International Cancer Congress in Hamburg, Germany. I also want to thank him for finding the time to read the thesis in spite of his very tight schedule. I thank him most sincerely for his guidance and perseverance throughout the cou'se of this work and during the writing of the thesis. Professor Emerole, Professor Uwaifo and Dr. Fafunso all served one time or the other as Head of Department during the period of this investigation. I thank them all for making Chemicals and equipment in the Department available for this work ! am equally grateful to other Lecturers in the Department - Professor Maduagwu, Drs. Faparunsi, Jeyakumar and Nwankwo. I thank Professor O.O. Olorunsogo for the very many biochemical techniques learnt from him. I also want to thank him for reading this thesis and for his brotheriy advice and concem. Dr. C.O. Bewaji of the University of llorin meticulously read this thesis in spite of his very tight schedule. I want to thank him for his constructive criticism and advice. I am indebted to Professor Ralph G. Hendrickse of the Univeiri y of Liverpool, U.K., for allowing me to use the excellent research facilities in his laboratories to carry out some of the experiments reported in this thesis. I am grateful to Dr. Peggy Maxwell, Rick Young, Cathy Harrison and my Cameroonian friend, Tony Ndifor - all of the Liverpool School of Tropical Medicine for their assistance during the period spent in the School. I am very grateful to Dr. 0 .0 . Akinyinka of the Department of Pnediatrics, U.C.H., Ibadan for the collection of human blood samples used for one of the experiments reported in this thesis. I appreciate tha moral support and the encouragement of my cr. lleagues at Ogun State UNIVERSITY OF IBADAN LIBRARY T University. I am pariicularly grateful to my mentor and benefactor - Professor F.O. Osiyemi who introduced me to Professor Bababunmi. I want to thank Dr. J.O. Olowookere, my Senior colleague, and collaborator, for his advice encouragement and undei Standing. I appreciate the love, support and encouragement given to me by my friends - Messrs K.B. Olurin and O.A. Sodeinde. I also want to thank the entire staff of the Departments of Biological Sciences and Biochemistry, Opun State University, for their support. I want to thank Dr. Taiwo Ajayi and his brother Mr. Tosin Oladipo as well as Mr. Kunle Sonibare for their support and encouragement particularly when things oecame “ rough” during my stay in Britain. I am very gratefui to my friends and colleagues - Messrs A I. Elegbade and E.O. Okegbile for their multifacet support, encouragement and understanding during vhe period of this inves- tigation; particularly during my study leave to the University of Liverpool. The exciting and sometimes difficult moments at the Department of Biochemistry were shared with a number of colleagues: Dr. W.G. Okunade, Dr. Ronke Osowole, Ronke Adebayo, Mrs. Nike Ibitayo (Noa Fasogbon), Sunday Atawodi, Levi Mgbojikwe, Tunde Farounbi, Wole Owojuyigbe and a h >st of others too numerous to list here. I remember with gratitude, my late maternal grandfather, Pa W.O. Okeowo and my late mother, Mrs. Deborali Adenuga (Nee Okeowo) for their contributions to my academic and overali development while they were alive. I also appreciate the kindness and genuine love of my great uncle Chief A. T. G. Okeowo. I appreciate the support of my extended family, the ATUNLUTE FAMILY, and thank them for bearing my absence from home. Finally, I wish to thank Miss Tosin Ogunsanwo for wordprocessing the thesis. This work was yupported by (i) The British Council Fellowship (ii) Children’s Research Fund (U.K.) and (iii) Ogun State University Study leave grant for which I am very grateful. UNIVERSITY OF IBADAN LIBRARY 5 D EDI CATION This work is dedicated to my father Mr. Emmanuel ADENUGA Senboyejo and the entire ADENUGA FAMILY. UNIVERSITY OF IBADAN LIBRARY z CERTIFICATION I certify that this work was carried out by Mr. Gbenga Adebola ADENUGA at the Department of Biochemistry, University of Ibadan, Nigeria. Professor E.A. Bababunmi Ph D., d.Sc., FRCPath. SUPERVISOR. UNIVERSITY OF IBADAN LIBRARY LIST OF PLATES Plate 1. SDS-PAGE and densitometric scan of the microsomal proteins of normal r a t ....................................................................................................... 95 Plate 2. SDS-PAGE and densitometric scan of the microsomal proteins of normal, DDT- and PB-treated rats............................................................... 102 Plate 3. SDS-PAGE and densitometric scan of the microsomal proteins of normal and protein-malnourished animals................................................... 107 Plate 4. SDS-PAGE and densitometric scan at the microsomal pro.ein of animal on low protein diet with or without aflatoxin B treatment................. 117 UNIVERSITY OF IBADAN LIBRARY 8 LIST OF FIG URES Figure 1. Stages in tumour growth and metastasis................................................. I5 Figure 2. Structures of selected Chemical carcinogens............................................ 18 Figure 3. Principal aflatoxin-B^ toxifying and detoxifyingp athways......................... 19 Figure 4. Multistep carcinogenesis: Mechanistic model............................................ 21 Figure 5. Structures of some Chemicals relevant to tumour growth and development....................................................................................... 30 Figure 6. Simplified reaction pathway for P-type ATPases................................. 38 2+ Figure 7. Ca -transporting Systems in eukaryoticc e lls ............................................40 2+ Figure 8. A scheme of the molecular Organization of the Ca -pump in the plasma membrane............................................................................. 43 2+ Figure 9. Schematic summary of some of the Ca -mediated processes relevant to toxicology................................................................................ 48 Figure 10. A schematic summary of the two temporally distinct phases of prolonged cellular response................................................................ 49 Figure 11. Mechanisms of oxidant carcinogenesis................................................. 57 Figure 12. Protein calibrated p lo t.............................................................................. 67 Figure 13. Phosphate calibrated plot ..................................................................... 73 Figure 14. AMC calibrated p lo t................................................................................ 80 Figure 15. pH-dependence2gf the activity of rat liver membrane-bound microsomal Ca -ATPase......................................................................... 91 2f Figure 16. Ca -dependenc^ of the activity of rat liver membrane-bcund microsomal Ca '-ATPase......................................................................... 92 Figure 17. ATP-dependenc^ of the activity of rat liver membrane-bound microsomal Ca +-ATPase......................................................................... 93 2+ Figure 18. Ca -dependenc^ of the activity of membrane-bound microsomal Ca -ATPase of Normal, DDT- and PB-treated rats . . . . 101 Figure 19. Schematic diagram of the carcinogenic treatment protocol for animals on normal diet....................................... 111 Figure 20. Schematic diagram of the carcinogenic treatment protocol tor animals cn low protein d ie t...................................................................... 112 Figure 21. The2j;espcnsiveness of the erythrocyte ghost membrane Ca -ATPase of control kwashiorkor and liver cancer patients to calmodulin.................................................................................. 122 UNIVERSI Y OF IBADAN LIBRARY 9 LIST OF TABLES Table 1. Classification of carcinogenic Chemicals..........................................................17 Table 2. Protein products of some known oncogenes..................................................25 Table 3. Some p.operties of enzyme-altered foci and hyperplastic nodules in the rat liver by Chemical carcinogens....................................................... 26 Table 4. Examples of some ion motive ATPases discovered to date..................... 36 Table 5. Protocol for protein calibration and estimation................................................66 2+ Table 6. Protocol for assay for microsomal Ca -ATPase a c tiv ity .............................. 70 Table 7. Protocol for inorganic phosphate calibration and estima’io n ......................... 72 2+ Table 8. Protocol for assay for plasma membrane (EGM) Ca -ATPase activity . . 76 Table 9. Protocol for AMC calibration and estimation.....................................................79 Table 10. Protoccl for the preparation of running g e l................................................. 86 Table 11. Protocol for the preparation of Stacking g e l............................................... 87 Table 12. Effects of calmodulip and Vanadate on the membrane-bound rat liver miciosomal Ca ^-ATPase...........................................................................94 Table 13. The percentage composition of the microsomal proteinsof normal rat as resolved by SDS-PAGE and the densitometric scanning......................96 Table 14. Effects of dicophane, Phenobarbital and aflatoxin B on the membrane-bound rat liver microsomal Ca +-ATPas^ and other biochemical parameters..................................................................................100 Table 15. The percentage composition of the microsomal proteins of normal, DDT- and PB-treated rats as resolved by SDS-PAGE and the densitometric scanning...................................................................................103 ?+ Table 16. The Ca -ATPase activity and other biochemical parameters of normal and protein-malnourished rats........................................................... 106 Table 17. The percentage composition of the microsomal proteins of normal and protein-malnourished rats as resolved by SDS-PAGE and the densitometric scanning...................................................................................108 Table 18. Long-term study on the effect of dicophane administration after carcinogenic initiatior^with aflatoxin B on the rat liver membrane-bound Ca +-ATPase and cfther biochemical parameters..........114 Table 19. The percentage composition of the microsomal proteins of protein malnourished rats treated with or without aflatoxin B as resolved by SDS-PAGE and the densitometric scanning . . -1................................ 118 2+ Table 20. The erythrocyte ghost membrane Ca -ATPase activity of kwashiorkor and liver cancer patients..........................................................121 UNIVERSITY OF IBADAN LIBRARY 10 TABLE OF C O N TEN TS ABSTRACT.......... ............................................................................................................ 2 ACKNOWLEDGEMENTS.............................................................................................. 3 DEDICATION.................................................................................................................. 5 CERTIFICATION............................................................................................................. 6 LIST OF PLATES................................................................................... 7 LIST OF FIGURES........................................................................................................ 8 LIST OF TABLES ........................................................................................................ 9 TABLE OF CONTENTS.................................................................................................. 10 ABBREVIATIONS...............................................................................................................11 CHAPTER ONE: INTRODUCTION.............................. 14 1.1 Definitions and concepts of the multi-stage process of carcinoyenesis................................................................. 14 1.2 Social and environmental factors in the development of cancer: Roles of food contaminants environmental pollurants, naturaüy occurring flavonoids, malnutrition and d ru g s ............................... 27 1.3 Calcium] ion, Calmodulin and Oncomodulin and the cont.ol of prolifera'iion in normal and cancer c e lls ................................................... 32 1.4 The regulation of intracellular calcium ....................................................... 37 2+ 1.5 lntrace!!ular Ca signalling for transient and sustained cellular respor.ses......................................................................................................46 2+ 2+ 1.6 Cellular Ca homeostasis and Ca -mediated cellular processes as critical targets for toxicant a c tio n ........................................................ 50 2 f 1.7 Ca homeostasis with particular reference to the role cf the endoplosmic reticulum int umourigenesis by Chemical agents................... 53 1.8 Tumour promoter and biochemical mechanism of action.......................... 55 1.9 Aims of the s tu d y .......................................................................................... 58 CHAPTER TWO: MATERIAL AND METHODS........................................................ 60 2.1 Materials....................................................................................................... 60 2.2 Preparation of membranes..........................................................................60 2.2.1 Preparation of the light microsomal fraction. ..............................60 UNIVERSITY OF IBADAN LIBRARY 11 2.2.2 Preparation of erythrocyte ghost membranes ................................62 2.3 Protein determination....................................................................................... 64 2.4 Enzyme assay and microsomal haemoprotein content determination . . . . 68 2.4.1 Determination ĉf+the activity of membrane-bound rat liver microsomal Ca +-ATPase................................................................... 68 2.4.2 Determination of the activity of th^membrane-bound erythrocyte ghost membrane Ca +-ATPase................................... 74 2.4.3 Determination of the activity of gamma glutamyl transpeptidase (G G T)...................................................................... 77 2.4.4 Determination of microsomal CytochromeP 450 ............................ 81 2.5 Separation of membrane proteins by SDS-PAGE...................................... 82 CHAPTER THREE: EXPERIMENTS AND RESULTS................................................. 89 Experiment 1: Characterization of rat liver microsomal membrane-bound Ca +-ATPase.................................................................................... 89 Experiment 2; Short-term in-vivo studies of the effect of dicophane on the activity of rat liver microsomal membrane-bound Ca -ATPase........................................................................................97 Experiment 3: Comparative Studies on the action of low protein intake and the effect of dicophane administration wil,h respect to the depression of rat liver microsomal Ca'+-ATPase................ 104 Experiment 4: Effect of carcinogenic promotion (by dicophane administration) on liver microsomal membrane-bound Ca -ATPase after carcinogenic initiation (by aflatoxin treatment) in ra ts ...........................................................................109 Experiment 5: An asses^irient of the activity of erythrocyte membrane- bound Ca -ATPase in humans suffering from protein- energy-malnutrition and liver cancer..................................................119 CHAPTER FOUR: DISCUSSION AND CONCLUSION................................................... 123 4.1 Discussion . ......................................................................................................... 123 4.2 Summary of results............................................................................................... 134 4.3 Contributions to knowledge...................................................................................135 REFERENCES.....................................................................................................................136 APPENDIX............................................................................................................................. 153 UNIVERSITY OF IBADAN LIBRARY 12 ABBREVIATIONS DDT - Dicop'iane or Dicholorodiphenyltrichloroethane or 1,1,1, trichloro 2,2-bis (p-ch!crophenyl) ethane PB - Phenobarbital EGM - Erythrocyte Ghost Membrane LPI - Low Protein Intake AFB - Aflatoxin B 1 1 PLC - Primary Liver Cancer 2+ 2+ 2+ 2+ Ca -ATPase or (Ca + Mg ) - ATPase - Mg -stimulated calcium-dependent Adenosine triphosphatase or ATP phosphohydrolase GGT - Gamma Glutamyl Transpeptidase SDS-PAGE - Sodium Dodecyl Sulphate Polyacrylamide Gei Electrophoresis DNA - Deoxyribonucleic acid PI - Isoelectric point cAMP - Cyclic Adenosinetriphosphate KDa - Kilodalton FITC - Fluorescein Isothiocyanate CaM - Calmodulin 2+ hPMCA - Human Plasma Membrane Ca -ATPase 2+ rPMCA - Rat Plasma Membrane Ca -ATPase ATP - Adenosine T riphosphate TFP - Trifluoperazine [Ca~ ]- Submembrane Concentration of Calcium sm | PKC - Protein Kinase C Bis-acrylamide - N, N’methylene-bis-acrylamide PIP - Phosphatidylinositol-4,5-bisphosphate 2 UNIVERSITY OF IBADAN LIBRARY 13 IP - Inositoitriaphosphate 3 DAG - Diacylglycerol GTP - Guanosinetriphosphate [O] - Activated Oxygen (AO) TPA or PMA - 12-0-Tetradecanoylphorbol-13-acetate orPhorbol-12-Myristate-13-acetate PLC - Phospholipase C PDGF - Platelet Derived Growth Factor EGF - Epidermal Growth Factor S6 - Small Flibosomal Subunit protein 6. AP - Transciiption factor AP 1 1 ADPR - ADP-ribose or Adenosinediphosphate ribose ER - Endoplasmic reticulum Tris - Tris (hydroxymethyl) mothylamino or 2-amino-2-(hydroxymethyl) propane-1,3-diol EGTA - Ethyleneglycol-bis-( -aminoethyl ether) NNN’N’-tetraacetic acid PMSF - PhenylmethylSulfonylfluoride EDTA - Ethylenediaminetetraacetic acid HEPES - N-2-hydroxyethylpiperazine-N’-2-ethane-sulfonicacid BSA - Bovine Serum albumen AMC - 7-Amino-4-methyl-Coumarin Ammediol-2-amino 2-methyl-1,3, propanediol TEMED - N,N,N’,N’-tetramethylethylenediamine SR - Sarcoplasmic reticulum HBV - Hepatitis B Virus DMSO - DimethylSulfonyloxide 4 UNIVERSITY OF IBADAN LIBRARY 14 CHAPTER ONE 1NTRODUCTION 1.1 Definitions and concepts of the multi-staqe process of carcinoqenesis A tumour is a large aggregation of cancer cells, derived from a s>ngle “founder” cell. The single ancestor was once a normal cell, with a normal function in a particular tissue but somehow underwent a fundamental change. As a result of that change it began to divide and proliferate in response to some imperative of its own rather than in response to the external Stimuli ordinarily required for cellular growth. (Weinberg, 1983). Tumours arise with great frequency especially in older animals and humans, but most pose little risk to their host because they are localized. On rare occasions, they become life-threatening because they spread throughout the body and become malignant (Fig. 1). Benign tumours contain cells that closely resemble normal ce'ls and may function as such. The forces that keep benign tumour cells (and normal cells) localized to appropriate tissues are not clearly understood. A fibrous capsule usually delineates the extent of a benign tumour and makes it an easy target for the surgeon. They become serious medical problems only if their sheer bulk interferes with normal functions or if they seerete excess amounts of biologically active substances like hormones. The major characteristics that differentiate malignant tumours from benign ones are their properties of invasiver,ess and spread. Malig­ nant tumours do not remain localized and encapsulated: they invade surrounding tissues, get into the body’s circulatory System, and set up areas of proliferation a vay from the site of their original nppenrnnco In a proces3 known 03 metnstasis (Darnell et n|., 1906). The abnormal behaviour of cancer cells is characterized by many distinctive traits the most obvious of wh'ch is uncontrolled growth. Cancer cells often exhibit a shape that is very different from that of their normal counterparts. They fail to respect the territorial rules that confine normal cells to particular tissues. Many of them import sugar molecules at an unusu- ally high rate. They also rely to an unusual extent on anaerobic metabolism. The outer membrane of cancer cells is different from that of a normal cell and displays special tumour UNIVERSITY OF IBADAN LIBRARY 15 Mass of tum or cells Ui) (locali/cd, benign tum or) Tumor cells ^ invade blood vessel5 \^ Figure 1. Stages in tumour growth and metastasis (Darneil et al.. 1986) UNIVERSITY OF IBADAN LIBRARY 16 antigens giving the cells distinctive immunoiogical properties (Weinberg, 1983). Malignant cells usually have enough of the hallmarks of the normal cell type from which they were derived, that it is possible to classify them by their relationship to normal tissue. Normal cells arise from one of three embryonic cell layers-endoderm, ectoderm or mesoderm. Malignant tumours derived from epithelial tissues such as the skin and the lining of the digestive or respiratory tract, are called carcinomas. Those derived from tissues of meso­ dermal origin, such as connective tissues, bones, or muscles are referred to as sarcomas. The leukemias are a subdivision of sarcoma (Klein, 1982; Darneil et al. 1986). Neoplasm that arise in animals that have not been treated in anyway to cause the development of cancer are known as spontaneous while those that are produced purposely by a cancer-producing agent or carcinogen are termed induced neoplasm. Carcinogens can be classified into three major groups viz physical, Chemical and biologi- cal. Radiation (e.g UV radiation) Chemical compounds (e.g polycyclic hydrocarbons and aromatic amines) biological factors (e.g hormones and the oncogenic viruses) are examples of physical, Chemical and biological carcinogens respectively. The most widely studied and better understood of the three is Chemical carcinogens. While some Chemical carcinogens are genotoxic, others are not, and are referred to as epigenetic carcinogens (Table 1). Tumour Promoters belong to the latter group (Weisburger and Horn, 1982). There are two broad categories of genotoxic Chemical carcinogens, direct-acting and indirect-acting, with the latter requiring metabolic activation to become carcinogens (Fig. 2). The direct-acting carcino­ gens, of which there are only a few, are reactive electrophiles (compounds that seek out and react with negatively charged centers in other compounds). The metabolic activation process of indirect carcinogens makes ultimate carcinogens from the preeursors by giving them electrophilic centers The metabolic activation of carcinogens is carried out by drug metabo- lizing enzymes which are bound to the endoplasmic reticulum. The pathway involved in the metabolic activation of aflatoxin - an indirect-acting carcinogens - is illustrated in Fig. 3. Chemical carcir;ogenesis is a multistep process. Rous and Kidd (1941) were among the first to provide experimental evidence suggesting a two-stage mechanism for carcinogenesis in skin. Within the last two decades, however, it has become apparent that the two-stage phenomenon in the development of neoplasm is not unique to the skin. According to this concept, there is a stage of initiation when a cell is affected by a carcinogen and the promotion stage when tumour growth occurs. UNIVERSITY OF IBADAN LIBRARY 17 Table 1: CLASSIFICATION OF CARCINOGENIC CHEMICALS Type Mode of action Example Genotoxic 1. Direct-acting Electrophile, organic compound Ethylene imine, bis genotoxic, interacts with DNA (chloromethyl) ether 2. Procarcinogen Requires conversion through Vinyl Chloride, metabolic activation by host benzo (a) pyrene or in vitro to type 1 2-naphthylamine, dimethylnitrosamine 3. Inorganic Not directly genotoxic, leads Nickel, chromium carcinogen to changes in DNA by selective alteration in fidelity of DNA replication EDiaenetic Solid-state Exact mechanism unknown: Polymer or metal carcinogen usually affects only mesenchy­ foils, asbestos mal cells and tissues: physical form vital Hormone Usually not genotoxic: mainly alters Estradiol, diethyl- endocrine System balance and stilbestrol differentiation: often acts as Promoter Immuno- Usually not genotoxic: mainly stimulates Azathioprine, antilym- suppressor “virally induced” , transplanted or phocytic serum metastatic neoplasms Cocarcinogen Not genotoxic or carcinogenic Phorbol esters, catechol, but enhances effect of type ethanol n-dodecane, 1 or type 2 agent when given S 02. at the same time. May modify conversion of type 3 to type 1 Promoter Not genotoxic or carcinogenic, Phorbol esters, phenol but enhances effect of type 1 anthralin, bile or type 2 agent when given acids,tryptophan subsequently metabolites, Saccharin (Weisburger and Horn, 1982) UNIVERSITY OF IBADAN LIBRARY 18 D llll CT AC [ING CAHCINOGENS INOIHECT ACTING CARCINOGENS 0 CO 1 I li/C CH.. K l ) l /I l ’ ropinlactonu IC 1 1 ) 1 0 Benzolalpyrono (3,4-bonzpyrunn) H,C S O CH. CH, ' 0I Ethyl m utlum osulfonutu (EMS) . _ O O II jö r o O r II,C O S O CH, ( Q l " ' " 0II Dibenz(a,/;)anthracene D im e'.iy l sulfato (DMS) CI O H j— C\H2 N - C H 3 CI CH2— CH2 2-Naphthylamino Nitrogen m ustard H,C N C NH, I I N O OI H,C D im nlhyln itronnm inn M ethyl n ltrosouiua (MNU) H2C— CH— CI Vinyl Chloride H2 4 3 2-Acetylam inofluorene h 2c — c h ~ c h 2 Safrole (sassafras) A f la to x in Bi (Aspergillus flu vus) Fig. 2 Structures of selected Chemical carcinogens (Darnell et aj., 1986) UNIVERSITY OF IBADAN LIBRARY 19 I' c l'Uft 1 Vj<) lO»» tOKIC Fig. 3 Principal aflatoxin-B1 toxifying and detoxifying pathways (Mandel et al., 1987) UNIVERSITY OF IBADAN LIBRARY 20 A more general concept which extends the two stage mechanism is that of tumour Progres­ sion proposed by Foulds (1969). The importance of the progression stage was recently emphasized by Cerutti (1987) (Fig. 4). First, a carcinogenic initiator causes genetic damage out no visible tumour. There is then a promotion stage during which the Controls of cell growth and differentiation are disturbed. The affected cells expand to a visible tumour at the expense of its neighbours. Finally, there can be a progression stage during which the tumour becomes malignant - it infiltrates surrounding tissue and metastasizes. An initiating agent or initiator is a Chemical, physical or biological agent which is capable of directly altering in an irreversible manner the native molecular structure of the genetic component (DNA) of the cell. Such alteration(s) maybe the result of a covalent reaction of DNA with the initiating agent itself or with one of its metabolites, but this alteration may also include a distortion of the structure of DNA without covalent binding to its components. Initiation is irreversible, and a cell once initiated, does not lose this induced property with time. The initiated cells and immediate progeny are not usually identifiable. Pure initiator (incom- plete carcinogen) causes irreversible change but not neoplasm unless a promoter is applied. This process is dependent on cell cycle and for many Chemicals on the metabolism of the cell (Pitot and Sirica, 1980). A promoting agent or promoter is an agent that alters the expression of genetic Informa­ tion of the cell. Example of such agents include hormones, drugs, plant products etc., which in themselves do not directly react with the genetic material but rather affect its expression by a variety of meehanisms including their interaction with cell surface receptors or with cytoplasmic and nuclear protein receptors, or by an alteration of other cellular components and functions. The promoting stage is reversible, at least in early stages. Promoted neo­ plasm can be seen grossly. Promoting agents are not carcinogenic but may promote fortu- itously initiated cells. Promotion is modulated by dietary, hormonal, environmental, and related factors (Pitot and Sirica, 1980). Carcinogens may gain access to the body by several routes (a) through the skin e.g. polycyclic hydrocarbons and products containing them such as tars and soot (b) through the respiratory tract (by inhalation) e.g. soot, poiycyclic hydrocarbons, asbestos, nickel, vinyl Chlorides and naphthyl amine, smoke of Cigarette and UNIVERSITY OF IBADAN LIBRARY Phenotypically Benign Tumour Malignant Tumour unaltered cell V INTIATION PROMOTION PROGRESSION conversion l clonal expansion F'9- 4 Multistep carcinogenesis: Mechanistic model (Cerutti, 1987) UNIVERSITY OF IBADAN LIBRARY 22 (c) through the digestive tract. Carcinogens can originate in the digestive tract from several sources: 1. They can be natural constituents of the food or metabo!;tes of fungal organisms contaminating it e.g. mycotoxins such as aflatoxin B . 2. They can be formed by interactions of foodstuffs e.g. formation of nitrosamines from secondary amines which can be present as components of fish products and nitrites which can be ingested with plant products or added to food products as preservative. 3. They can be formed during cooking e.g. imidazoquino lire may be generated by the heating of a mixture of Creatinine, glycine and glucose, and nitropyrenes are formed when meat is cooked over a smoky flame i.e. barbecued meat. 4. They can be formed by bacterial action on partially digested foods or on the bile acids (Thomas and Gillham, 1985). Dose and frequency of application, synergism and Promoters are some of the factors affecting the carcincgenicity of Chemicals. The incidence of tumours produced by any Chemi­ cal is dependent on the total dose and the frequency of application, although simple quanti­ tative predictions are seldom possible. Two Chemicals may frequently exhibit synergistic effects. Although it is possible that one of the Chemicals may be acting as a promoter, this is by no means dennite. Addition of promoter after initiator will aiways induce tumours, whereas, when promoter precedes initiator no tumours develop (Thomas and Gillham, 1985). In a specific tissue, cancer cells are usually recognized by the characteristics of rapidly growing cells : a high nucleus-to-cytoplasm ratio, prominent nucleoli, many mitoses, and relatively little specialized structure. The presence of invading cells in an otherwise normal tissue section is the most diagnostic indication of a malignancy (Darnell et al„ 1986). Cancer cells have abnormal and unstable numbers of chromosomes as weis as many chromosomal abnormalities. The study of cells in an animal is impractical because of the difficulty of identifying the relevant cells, manipulating their behaviour in a controlled manner, and separating the effects due to the intrinsic properties of the cells from the effects due to the interactions among the many cell types present in the organism. When cells growing in culture are exposed to a UNIVERSITY OF IBADAN LIBRARY 23 carcinogen, these problems can be controlled. The environment of the cell can be controlled. The environment of the cell can be manipulated by the investigator, the target cell can be well defined, the changes in the cell following treatment can be examined, and the fate of the carcinogen can be determined. Furthermore, in culture, the cells can be quiescent to growing; they can have, in fact, precisely defined growth parameters. They can also be manipulated genetically. For these reasons, studies of normal cell growth as well as of cancer induction depend heavily on the use of cultured cells. Exposure of cells to carcinogens can dramati- cally change their growth properties in culture. Furthermore, these treatments can cause the cells to form tumours after they are injected into susceptible animals. Such changes in the growth properties of cells and their subsequent development of tumour forming capacity are collectively referred to as malignant transformation, or just transformation. Because transfor- mation can be carri-sd out entirely in culture, it is widely studied as an analog of cancer induction in animals, although the relationship between the two processes has not been established. The transformation of adherent cells involves changes in a constellation of cellular prop­ erties. These include: Increased Saturation density, decreased growth factor requirements, loss of capacity for growth arrest, loss of dependence on anchorage for growth, changed cell morphology and grov/th habits, loss of contact inhibition of movement, cell surface alterations, easier agglutination by lectins, increased glucose transport, reduoed or absent surface fibronectin, loss of actin microfilaments, release of transforming growth factors, protease secretion, altered gone transcription and immortalization (Darneil et al.. 1986). An important Step forward in the understanding of one way in which Chemical carcino­ gens might cause transformation of cells was made when it was app; eciated that some cells transformed in this way possess activated oncogenes i.e. genes that cause cancer. It is now recognized that two classes of oncogenes exist in nature: endogenous or cellular oncogenes, which are present in the germ line, and exogenous or viral oncogenes which may be found in several types of retrovirus or in the genomes of tumour cells. In fact, the term oncogene is not quite accurate as far as the endogenous genes are concerned since they are not normally oncogenic (although they may well be expressed as normal eenes). The term proto- oncogene has, therefore, been introduced to indicate that they require some sort of alteration UNIVERSITY OF IBADAN LIBRARY 24 or mutation (which may be induced by Chemical carcinogens) for their oncogenic potential to be expressed (Bishop, 1982). A number of molecular mechanisms have been implicated in the conversion of proto-oncogenes to oncogenes. In the case of mammalian tumour genes they include point mutation, chromosomal rearrangement and gene amplification. In the case of retro-virus associated genes, the transduced gene may undergo mutation or may end up adjacent to a viral '•egulator that increases its level of expression (Weinberg, 1983). It is now clear, from an evolutionary point of view, that proto-oncogenes have been greatly conserved. This Observation has led to the conclusion that thoy play an important role in normal cell biology and that this in some way becomes perverted in transformed cells. A possible biological role for the proto-oncogenes being actively considered at present, and for which there is some evidence is that they are involved in development (Thomas and Gillham, 1985). Like other genes, oncogenes encode proteins. However, the proteins encoded by oncogenes function abnormally, and somehow induce the transformation of a normal cell into a cancer cell. Some known oncogenes, their protein products and their functions are shown in Table 2. Some are protein kinases, others are growth factors, GTP-binding proteins or DNA-binding proteins (Hunter, 1984). Hepatocellular carcinoma, is one of the ten most frequent cancers worldwide accounting for 4% of the total. While relatively rare in Europe and the Americas, it is frequent in The People’s Republic c» China, where almost one-half of the new casos in the world (251, 200 cases) occur, and in Africa. The aetiology of this cancer has been associated with two major risk factors, persistent hepatitis B virus (HBV) infection and exposure to dietary aflatoxins, although other aetiological agents like smoking, and some occupational exposures, have also been implicated (Cova et al.. 1990). A number of studies have demonstrated many similarities between the pathogenesis and morphological changes of experimental and human liver can­ cer. Evidence tha: specific environmental Chemicals from industiial, medical and dietary sources are carcinogenic to the human has now become clear (Phot and Sirica, 1980). It is well established that during the administration of hepatocarcinogens to the rodent, several distinct focal and nodular hyperplastic lesions develop in the liver before the appearance of hepatocarcinoma. Some of the specific biochemical markers used to distinguish these preneoplastic lesions from normal liver parenchyma are shown in Table 3. UNIVERSITY OF IBADAN LIBRARY 25 Table 2: Protein products of known Oncogenes ------------------- - v —,. ■ ÜNCOUKNIC PROTEIN ' .«A/uS 0 }? ULT HO VI aus TUMOR ZELLULAR LOCATION FUNCTION CLASijOHCOCCNL orc CHICKEN SAUCOMA - ’LASMA MEMBRANE yet; CHICKEN SAHCOMA PLASMA MEMBRANE(?) CLASS 1 fg r CAT SAUCOMA - (7 ) TYROSINE-SPECIFIC (CYTOPLASMIC abl MOUSE LEUKEMIA HUMAN LEUKEMIA PLASMA MEMBRANE PROTEIN KINASE TYR0S1NE PROTEIN KINASE fp s CHICKEN SAUCOMA CYTOPLASM (PLASMA MEMBRANE?) CYTÜPLASM l'ea CAT SARCOMK - (CYTOSKELKTON?) Cll ICK KN SAUCOMA - (7 )______________ EGF RECEPTOR'S PLASMA AND ( CYTOPLASMIC TYRO-. erb-B CHICKEN LEUKEMIA . "EC.IFIC !«« . CYTOPLASMIC1 1 MEMBRANES KINASE u* ,.W CLASS 1-RELATE PLASMA AND CYTOPLASMIC DÜMAINl (POTENTIAL fms CYTOPLASMIC OF a 'crowth- factoh PROTEIN MEMBRANES HECEPTOK ( ? ) K1HASES) ml 1 CI! ICK EN CARCINOMA ... CYTOPLASM ( I L ____ ral’ MUUSE SAIICOME - CYTOPLASM _______ h . . . fuo il MOUSE SAHCOMA MOUJE LEUKEMIA CYTOPLASM i ? r aia MONKEY SAUCOMA ■- SECHETED PDGF-L1KE CLASS 2CHOWTII FACTOR (OHOWl'H FACTOR 11 a -rau HAT SAHCOMA HUMAN CARCINOMA HAT UAHCINOMA PLASMA MEMBRANE HUMAN CALCI NOMA , K i-raa HAT SAHCOMA LEUKEMIA ANH PLASMA MEMBRANE CLASS 3 SAUCOMA GTP-BINDINC (CYTOPLASMIC, OTP-BIND1NC) N -ras - HUMAN LEUKEMIA ANU CARCINOMA PLASMA MEMBRANE lOS KOUSK SAHCOMA - NUCLEUS (7 ) myc CHICKEN LEUKEMIA. HUMAN LYMX1I0MA NUCLEUS DNA-BlND1NC myb CHICKEN LEUKKKA HUMAN LEUKEMIA NUCLEUS (7 ) claoc I4 (NUCLEAR) B-lym - CHICKEN LYMl'HOMA, HUMAN LYMl'HOMA NUCLEUS ( ? ) (7 ) yki CHICKEN SAHCOMA - NUCLEUS ( ? ) (7 ) ru l TURKEY LEUKEMIA - (7 ) (7 ) erb-A CHICKEN LEUKEMIA - (7 ) (7 ) UNCLASSI^IED u tu CHICKEN LEUKEMIA - (7) ( ? ) (lluntpr. loHM UNIVERSITY OF IBADAN LIBRARY 26 ^able 3: Some properties of enzyme-altered foci and hyperplastic nodules induced in the rat liver by Chemical carcinogens. 1. Show altered metabolism of glycogen 2. Deficient in the enzyme activities of: glucose-6-phosphatase adenosine triphosphatase (canalicular) ß-glucuronidase Serine dehydratase acid phosphatase 3. Exhibit increased activity of the fetal hepatocyte enzyme ft-glutamyl transpeptidase. 4. Express preneoplastic antigen. 5. Show a deficiency to störe iron. 6. Resistant to the cytotoxic action of hepatotoxins and carcinogens. 7. Exhibit an elevated DNA synthesis and mitosis. 8. Possess phenotypic heterogeneity with respect to above characteristics. (Pitot and Sirica, 1980). UNIVERSITY OF IBADAN LIBRARY 27 1.2 Social and environmental factors in the development of cancer. Since the energy and substance for the development of the first cancer cells are derived principally from the animal and since the new cell type increases in number by assimilating nutrients from the hcst; it might be expected that the social and the environmental conditions of the host influence the formation and growth of tumours. These factors include food con- taminants (e.g. the mycotoxin aflatoxin B^), environmental pollutants (e.g. the insecticide dicophane), naturaliy occurring flavonoids (e.g. the antioxidant quercetin), malnutrition (e.g. protein malnutrition) and drugs (e.g. phenobarbital). Food contaminants Perhaps one of the rnost widely distributed food contaminants in Tropical Africa is the myco­ toxin aflatoxin (See Fig. 5 for structure). Aflatoxins are metabolic products of a few strains of Aspergillus flavus and A.parasiticus. which grow on virtually all types of food in warm humid environments. It is therefore not surprising that aflatoxins contaminate most foodstuffs in tropical and subtropical regions of the world where the warm and humid conditions exist that are necessary for the growth of the fungi which produce the toxins (Bababunmi et al.. 1978; Wogan, 1966, 1969). This explains the widespread ingestion of these toxins with food in these areas by both old (Bababunmi et al„ 1976) and young (Coulteret al.. 1986; Hendrickse et al.. 1982; De-Vries et al.. 1987,1989). Aflatoxin B̂ appears to be the most potent member of the aflatoxin series as regards its toxicity to animals (Wogan, 1969) and in naturaliy contaminated materials, the B̂ compound occurs predominantly (Feuell, 1969). The liver is the principal target for toxicity and pathological changes include fatty Infiltration, biliary prolif- eration, acute toxic necrosis and portal fibrosis (Busby and Wogan, 1981). Series of investigations have shown aflatoxin B̂ to have a potent hepatocarcinogenic, teratogenic and mutagenic activities in many species (Busby and Wogan, 1981). Moreover, it has been observed that young animals are more susceptible to the toxic effects of aflatoxin than older one (Patterson, 1973; Butler, 1964). Furthermore, female rats are less prone to the carcinogenic effect of aflatoxin than male rats (Ward et al.. 1975; Butler, 1964). Epidemiological studies from some developing countries suggest a relationship between ingestion of aflatoxin B̂ (AFB^) - contaminated food and increased frequency of human liver cancer (Bababunmi, 1976; Alpert et al.. 1971; Shank et al.. 1972; Peers and Linsell, 1973). UNIVERSITY OF IBADAN LIBRARY 28 Environmental pollutants The problem of pesticides as environmental pollutants particularly as carcinogens for man has become a matter of special concern, especially because sonne of them (e.g. the chlorine-containing DDT) tend to accumulate in nature, as they are not destroyed by bacteria in the soil and water, or by plant or animal tissue. This raises othe; toxicological problems apart from carcinogenicity (Berenblum, 1974). DDT is a Chemical widely spread in the environment, particuiarly in the developing countries, to which man is known to be exposed and for which experimental evidence for carcinogenicity exists (Shabad et al„. 1972; Tarjan, 1969; Turusov etal., 1973; Berenblum, 1974). It has been proven that the carcinogenic effect of DDT on the liver ss due to a promotion action since DDT is not genotoxic (Williams, 1980; 1983). DDT (See Fig.5 for structure) has therefore been classified as an epigenetic carcino- gen functioning as l;ver tumour promoter (Williams, 1983). DDT has been shown to enhance the effects of 2-acetyl-amino luorene (Peraino et al„ 1971), diethylnitrosamine (Nishizumi, 1979; Kitagawa and Sugano, 1978), 3-methyl-4- (dimethylamino)-azobenzene (Kitagawa and Sugano, 1978) and aflaioxin B̂ (Rojanapo et al.. 1988) in inducing hepatocellular carcinoma in rats and mice. In addition, DDT has been shown to enhance the development of mammary gland tumours in male rats treated with 2- aminophenanthrene (Scribner and Mottet, 1981). Naturallv occurring flavonoids Worldwide, quercetin - a flavonoid-occurs in conjugated or as free forms in many edible plant foods (Herrmann, 1976; Harborne and Williams, 1975; Wollenweber and Dietz, 1981; Mabry and Ulubelen, 1980). Numerous observations both in-vivo and in-vitro have demon- strated a wide variety of physiological and pharmacological effects fc( a number of flavonoids, many of which could be beneficial to health (Bohm, 1968; Brown, 1980; Kuhnau, 1970). Quercetin (See Fig.5 for structure) has been reported very widely as a non-carcinogenic naturally occurring flavone (De Eds 1968; Sugimura, 1979; Wang et al., 1976). Some work- ers, however, have demonstrated the mutagenicity or carcinogenicity of Bracken Fern (Pteridium aquilinum) a component of which is quercetin. (Bryan and Pamukci*. 1979; Pamukcu et al.. 1980). Quercetin has been shown to affect the growth of transplnnted sarcoma (Bohm, 1968). Yoshida et al. (1990) have reported recently that quercetin markedly inhibits the UNIVERSITY OF IBADAN LIBRARY 29 gnowth of human gastric cancer cells and biocks cells progression from the G to the S chase. In addition, quercetin inhibits the activities of miscellaneous enzymes including the on-motive ATPases (Lang and Racker, 1974; Suolinna et al.. 1974; Graziani, 1977; Apps et al., 1982) and glutathione-s-transferase (Fröhlich et al.. 1989). It has been recognized that nhibitors of glutathione-s-transferase can be effective as cell antiproliferating agents (Sato et al., 1990). At very low concentrations, quercetin inhibits microsome-catalyzed adduct forma- üon between the Chemical carcinogen benzpyrene and DNA (Shah and Bhattacharya, 1986). Quercetin has also been shown to enhance the antiproliferative activity of Cis- diaminedichloroplatinum (II) both in vitro and in-vivo (Hofmann et ai.. 1988; Hofmann et al.. 1990). Quercetin is also an antioxidant. Its antioxidant property is due primarily to the presence of phenolic hydroxyl groups; the antioxidant effect increasing with the degree of hydroxylation of the rings (Brown, 1980). In carcinogenesis activated form of oxygen appears to play a role mostly in the promotion phase during which gene expression of initiated cells is modu- lated by affecting genes that regulate cell differentiation and growth (Freeman and Gapo, 1982). Many tumour p-omoters are recognized as oxidant carcinogens which induce cellular pro-oxidant state (Cerutti, 1987). Furthermore, Cerutti (1985) has outlined evidence in Sup­ port of the hypothesis that many anti-oxidants are anticarcinogens. Malnutrition. Malnutrition, particularly protein malnutrition is widespread and endemic in developing countries and is universally recognized as the single most important contributor to the high sickness and death rates in childhood in these countries (Hendrickse et al, 1982). It is probable, therefore, that protein-malnutrition acting in concert with environmental carcinogens which are also widespread in the developing countries might influence the incidence of hepa- tocellular carcinoma in Africa and Asia. A number of studies have shown that the development of cancer can be modified either by changing the quality or the quantity of dietary protein. This has been demonstrated for tumours of the liver (Tannenbaum and Silverstone, 1953). A reduced incidence of spontane- ously occurring benign hepatoma in mice fed 9 per cent casein diets ad libitum or isocalorically 4 and also in experiments in which caloric intakes were controlled to maintain equivalent body weights has been reported (White et al.. 1947; Silverstone and Tannenbaum, 1951). UNIVERSITY OF IBADAN LIBRARY 30 © 0 (DDT) PHENQBARBITAL OH QUERCETIN Fig.5 Structures of some Chemicals relevant to tumour grov/th and development UNIVERSITY OF IBADAN LIBRARY 31 In laboratory rats treated with aflatoxin B , low protein intake has been shown to inhibit the development of both liver tumours and putatively preneoplastic foci (Madhavan and Gopalan, 1968, Appleton and Campbell, 1982). Foci developments have been found to be associated with casein levels above 10-12% in the diet (Dunaif and Campbell, 1987). The effect of low protein intake it has been suggested is to decrease the enzymatic activation of aflatoxin B to form covalent adducts with DNA, RNA and protein (Preston etaj., 1976) thereby suppress- ing the formation of initiated cells. However, low protein intake has a more significant effect on development of foci after aflatoxin B̂ initiation is completed (Appleton and Campbell, 1983. Dunaif and Campbell, 1987). In addition, it has been shown that diets deficient in essential amino acids e.g. cystine (White et al.. 1947) and lysine (Voegtlin and Thompson, 1936) suppressed the genesis of certain tumours. The inhibition of the development of foci either by decreasing the quantity of protein intake and holding the quality of the protein constant or by decreasing the quality and holding the quantity constant as reported recently by Schulsinger et al. (1989), has again demonstrated the important role of dietary protein in the development of hepatocellular carcinoma. Further- more, it has been shown that supplementing the diet of rats with dietary protein protects against the acute toxicity of aflatoxins but enhances the risk of carcinoma (Madhavan and Gopalan, 1968; Mirmomeni et al. (1979). Drug The drug phenobarbital has long been recognized as one of the agents that stimulate the proliferation of the smooth portion of the endoplasmic reticulum of the liver. Subsequently, it was demonstrated that phenobarbital, like DDT is a tumour promoier of the epigenetic dass (Williams, 1983; Wantanabe and Williams, 1978; Shimada et al.. 1981). It is recognized that this compound affects the hepatic activity of several membrane-associated enzymes (Ratanasavanh et ad-. 1979; Williams et al.. 1980). Phenobarbital (See Fig.5 for structure) has been shown to enhance the hepatocarcinogenidty of 2-acetylaminofluorene (Peraino et al., 1973 & 1975), diethylnitrosamine (Ito et al.. 1980; Kitagawa and Sugano, 1978; Nishizumi, 1979) and 3 methyl-4-(dimethylamino) azobenzene (Kitagawa and Sugano, 1978) in animals. UNIVERSITY OF IBADAN LIBRARY 32 1.3 Calcium ion. Calmodulin and Oncomodulin and the control of proliferation in normal and cancer cells. 2+ A significant body of circumstantial evidence suggests that Ca and Calmodulin are important regulators of cell growth and malignant transformation (Veigl et al., 1984). The divalent ion calcium, like the cyclic nucleotides, is a major second messenger for regulation of numerous physiological processes, among them cell motility, egg activation, muscle con- traction, neurotransmission, secretion and cell proliferation (Whitfieid et al.. 1979). It has been established as a general phenomenon, in both fibroblast and epithelial cell types, that the proliferation of normal, non-neoplastic cells is tightly controlled by extracellular 2+ 2+ Ca concentration: proliferative quiescence sets in as the Ca level is reduced below 1 mM (Durham and Walton, 1982). Neoplastic cells transformed either naturally in-vivo; or in-vitro 2+ by viruses, Chemicals etc., can proliferate at much lower Ca concentrations (1 to 10 j jM). 2+ These data suggest that Ca regulates some aspects of normal ccil division that is aborted upon cell transformation. This inability of calcium to control tumour cell proliferation is one of the few (maybe the only) qualitative differences between normal and cancer cells (Whitfield et al.. 1979). 2f There is a good correlation between loss of Ca control and progression towards tumourigenicity. As rodent cells become preneoplastic by repeated passage in culture, and then neoplastic by treatment with Chemical carcinogens or transforming viruses, the level of 2+ Ca ions required for cell replication progressively decreases (Boynton et al., 1977; Boynton and Whitfield, 1973). In addition, Rixon and Whitfield (1976) observed that the cellular pro­ liferation in rat liver following partial hepatectomy is prevented in the presence of hypocalcemia induced by prior parathyroidectomy. Furthermore, it has been shown that growth of tumours in-vivo in young animals can be inhibited by calcitonin treatment (Anghileri et al., 1980). Numerous studlea have shown that calcium can exert its oocond messongor function via interaction with a family of small acidic, structurally similar proteins, which bind calcium through helix-loop-helix structures. This family includes calmodulin, troponin C, parvalbumins and a new protein that has thus far only been detected in tumour tissue-oncomodulin. The most multifaceted member of this family is the protein calmodulin, which is present in all eukaryotes and activates a wide variety of target proteins (Veigl et al.. 1984). Calmodulin is a single UNIVERSITY OF IBADAN LIBRARY 33 solypeptide chain with a molecular weight of 16,700 dalton and 148 amino acid residues. Some 30 percent of its amino acids consists of aspartate and glutamate - accounting for the 2+ soelectric point oi 4.3. Calmodulin contains four Ca - binding s4es. This protein itself is not active; the active form is the calmodulin - calcium complex (Cheung, 1980). A number of iaboratories over the past several years have reported that calmodulin levels are higher in trans'ormed and rapidly dividing normal cells and tissues than in their quiescent normal counterparts (Watterson et al.. 1976; LaPorte et al.. 1979; Uenishi et al, 1980; MacManus et al., 1981; Veigl et al., 1982; Van Eldik and Burgess, 1983; and Nakajo et al.. 1983). This Observation suggests that calmodulin levels regulate cell division. Furthermore, investigation of synchronized cells in culture indicates that calmodulin content of cells in- crease at a specific time in the cell cycle. Indeed, it has been shown that calmodulin is synthesized at the G JS interface (Veigl et al., 1982; Chafouleas et al; 1982). The best correlative in-vitro change in cells with in-vivo tumour formation appears to be acquisition of anchorage-indepondence-reflected in the ability of transformed cells to grow in a semi-solid medium. The ability of transformed cells to grow without attachment to a substratum con- trasts with normal cells, which can grow only on a surface that will support a flattened cell morphology (Folkman and Moscona, 1978). Calmodulin has been shown to regulate a wide variety of Cellular activities including cell morphology, motility, communication with other cells and interaction with the growth environment - all processes that change upon cell transforma- tion (Veigl et al.. 1984). Recent work :n a number of Iaboratories suggests that calmodulin is a major regulator of cyto-skeletal function through a variety of mechanisms, including regulation of filament-based motility Systems in both muscle and non-muscle tissues of eukaryotes. The role of calmodulin 2+ as a Ca dependent activator of myosin light-chain kinase in these tissues is now well established. Calmodulin and specific phosphatases provides for reversible, specific activa- tion of the actomyosin ATPase activity responsible for contraction (Adelstein, et al., 1980; Gorecka et al.. 1976). Interestingly, cAMP - dependent phosphorylation of myosin light chain 2+ kinase appears to inhibit the activation by Ca calmodulin. This may allow coupling between regulatory Signals, thus providing a rational regulatory cycle in smooth and non-muscle tis­ sues (Veigl et al., 1984). In addition to the regulation of ATP hydrolysis in these motility UNIVERSITY OF IBADAN LIBRARY 34 Systems, calmodulin may play an important role in regulating the polymerization of the asso- ciated filament Systems. Calmodulin has been shown to become associated during mitosis with the polar regions of the spindle fiber apparatus believed to con'.ain micro-tubule organiz- ng centers (Willingham et al., 1983; Welsh et al., 1978,1979) and mcves together with tubulin, actin and myosin during axonal transport (Erickson et al.. 1980). In 1979, a novel calcium-binding protein was detected in lysates of Morris hepatoma 5123tc. The protein was subsequently named “oncomodulin” in lecognition of its frequent expression in neoplastic tissues and its ability to stimulate cyclic nucleotide phosphodiester- ase in a calmodulin-like manner (Henzl and Birnhaum, 1988). Oncomodulin is an oncodevelopmental calcium-binding protein. Its expression is restricted to tumour cells and the placental cytotrophoblasts of rodents and man (MacManus et al., 1983; Gillen et al.. 1987). Oncomodulin strongly resembles another calcium-binding protein called parvalbumin, and shows a very high degree of sequence homology. Oncomodulin isolated from rat tumour contains 108 amiro acid residues; parvalbumin from skeletal muscle from the same animal contains 109 residues. The reported sequences are identical at 55 positions. Both proteins possess two high affinity calcium-binding domains which are referred to as the CD (residues 51-63) and EF (residues 90-102) sites. In the rat, the EF sites of oncomodulin and parvalbumin are nearly identical (11 of 12), while the CD site exhibits somewhat less homology (7 of 12 residues). However, despite the pronounced similarity between the two proteins, it is becom- ing increasingly clearthat oncomodulin is not merely another parvalbumin (Henzl and Birnbaum, 1988). Although it has been recognized that chemically transformed rat fibroblast cell line ex- presses high levels of oncomodulin (Sommer et al.. 1989), the role of oncomodulin as well as its possible function within the tumour cell remains unclear. Furthermore, it has been shown that the expression of oncomodulin does not lead to the transformation or immortalization of mammalian cells in-vitro (Mes-Masson et al.. 1989). It has been proposed that the active molecule could be a cysteine linked oncomodulin dimer (Mutus et_ai., 1988). The disulfide- linked dimer of oncomodulin appears to be more similar to calmodulin than oncomodulin since the dimer displayed “calmodulin-like” affinity for the amphiphilic peptide melittin. In addition, Oncomodulin dimer was shown to activate two calmodulin-dependerit enzymes, cyclic nucle- UNIVERSITY OF IBADAN LIBRARY 35 otide phosphodiesterase and calcineurin phosphatase. Nevertheless, recent studies indicate that oncomodulin can regulate the activity of glutathione reductase (Palmer et al.. 1990). Furthermore, glutathione reductase was able to catalyze the reduction of the disulfide-linked dimer of oncomodulin. While elevated levels of calcium, calmodulin and oncomodulin in tumour cells have been established, neither the underlying factors nor the significance of the elevation is fully understood at present. UNIVERSITY OF IBADAN LIBRARY able 4 Examples of some ion motive ATPases discovered to date ATPase Source Membrane ATPase dass h f Lower eukaryotes (yeast, fungi) Plasma P H+ Higher eukaryotes plants Plasma P animals Plasma (bladder,tumor) P K+ E. coli, S. faecalis Inner P H+/K+ Higher eukaryotes (animals) Plasma (intestine) P Na+/K+ Higher eukaryotes (animals) Plasma P Ca2+ Higher eukaryotes (animals) Plasma P Ca2+ Higher eukaryotes (animals) Sarcoplasmic reticulum P Ca2+ Higher eukaryotes (animals) Lysosomes, Go'gi P H+ Lower eukaryotes (yeast, fungi) Vacuoles V H+ Higher eukaryotes (plants) Tonoplasts V H+ Higher eukaryotes (animals) Lysosomes V H+ Higher eukaryotes (animals) Endosomes V H+ Higher eukaryotes (animals) Secretory granules V H+ Higher eukaryotes (animals) Storage granules V H+ Higher eukaryotes (animals) Clathrin coated vesicles V H+ Most bacteria Inner F H+ Eukaryotes animals, plants Mitochondrial inner F H+ plants Chloroplast thylakoid F (Pedorsen and Carafoli, 1987) UNIVERSITY OF IBADAN LIBRARY 37 1.4 The requlation of intracellular calcium ATPases invoived in ion translocation are present in a diverse variety of biological membranes. The ion-motive ATPases discovered to date can be grouped into three major categories desigriated as “P” (Phosphorylated), “V” (Vacuolar) and “F” (F -F ) type (Pedersen o 1 and Carafoli, 1987) (Table 4). Ion-motive ATPases of the “V” type are defined as those associated with membrane bound organelles other than the mitochondria and the endoplasmic or sarcoplasmic reticulums. They are found in the Vacuoles (hence the Symbol “V”) of Neurospora and yeasts and in tonoplasts of plants, as well as in lysosomes, endosomes, clathrin coated vesicles, hormone storage granules, secretory granules and Golgi vesicles. This may be the largest of the three major classes of ion-motive ATPases. Ion-motive ATPases of the “ F” category are defined as those of the F F type found in O 1 bacteria, chloropiasts and mitochondria. These are the most complicated of the ion-motive ATPases, consisting of a water soluble F moiety invoived in catalytic activity (i.e either ATP synthesis or ATP hydrolysis) and an F moiety invoived in H+ translocation (Pedersen and 0 Carafoli, 1987). ATPases of the “ P” dass are defined as those which form a covalent phosphorylated intermediate (see Fig.6) as part of their reaction cycle (hence the symbol “P”). Examples are the Na+/K+, Ca2+, and H+ transporting ATPases of the plasma membranes of eukaryotic cells and the Ca2f transporting ATPase of the plasma membranes of eukaryotic cells and the Ca2f transporting ATPase of the sarcoplasmic and Endoplasmic reticulum. The K+ transporting ATPases of E. coli and Streptococcus faecalis are also of the ‘p' type. The common catego- rization of this dass of ATPases as E^-E^ is inappropriate (Pedersen and Carafoli, 1987). Such ATPases are inhibited by vanadate, a transition state analog of phosphate and an inhibitor which aiso distinguishes many of the ATPases of the P-type from those of the V and F types. Structurally, these ATPases all consists of a peptide ( a ) of 70-100 KDa which contains the phosphorylation and ATP binding sites. The Na%K+ - ATPase, unlike other known P-type ATPases, contains in addition, fl peptide of approximately 55 KDa; its function is, however, unknown. Several of the P-type ATPases have now been sequenced, and it has been observed that significant sequence homology exists among them suggesting that they may have been derived from a common ancestor. Further more, comparison of the a-subunit UNIVERSITY OF IBADAN LIBRARY 38 nl out nl in Fig.6 Simplified reaction pathway for P-type ATPases (Pedersen and Carafoli, 1987) UNIVERSITY OF IBADAN LIBRARY 39 of the Kidney Na+/K -ATPase with that of the Ca2+-ATPase of carctiac muscle shows that the two proteins are very similar in size and virtually identical in the location of their phospho- rylation and ATP binding sites. Thus it seems likely that all ATPases of the P-type will be found to have many reaction mechanisms in common. Despite the striking similarities among the P-type ATPases, there are critically important differences which reflect their specific "ion-motive” properties; these include (1) the amino acid sequence of the ion binding domains (or channels) (2) inhibitor sensitivities and (3) effect of some natural protein activators. Finally, it seems likely that some ATPases may exhibit some sublle but very important differences even when they translocate the same ion (Pedersen and Carafoli, 1987). 2+ The role of Ca -ATPase in the regulation of intracellular calcium 2+ The messenger role of Ca' requires its maintenance withiri cells at a very low (submicromolar) ionic concentration, and Systems to modulate it in the different cell compart- ments in keeping witn the requirements of the messenger function. The control of cellular Ca2+ is based on the reversible complexation to specific proteins that are either soluble in the cytoplasm (e.g. calmodulin), intrinsic to membranes (e.g. ATPases, Ca exchangers), or 2-» organized in nonmembranous structures (e.g. troponin C). Two Ca pumps (i.e. ATPases) are responsible for a large portion of the Ca?f movements across me nbrane barriers. One is located in Sarco (Endo)-plasmic reticulum. The other is located in the plasma-membrane (Carafoli, 1988a). (Fig. 7). Plasma membrane Ca2"-ATPase 2+ The Ca -ATPase of the plasma membrane interacts with Ca with high affinity (Km, about 0.5 pM), but has low total transport capacity: In the heart, it corresponds to about 0.5 nmol/ mg of membrane prctein/s. Given its high Ca2+ affinity, the enzyme is qualified to export Ca2+ from cells continuousiy, not only when its concentration in the cytosol has increased. Thus, the Ca2+-ATPase plays the most important role in maintaining the 104 fold gradient of Ca2+ between cells and medium. The enzyme is an ATPase of the P-class, i.e it forms an aspartyl Phosphate during the reaction mechanism and is inhibited by vanadate. It is a target of calmodulin Stimulation The purified enzyme is a single polypeptide of about 138 KDa, and has been reconstitutea in liposomes with optimal transporting efficieney (Carafoli, 1988b). UNIVERSITY OF IBADAN LIBRARY 40 2+ Fig.7 Ca -transporting Systems in eukaryotic cells (Ca-afoli, 1988b) UNIVERSITY OF IBADAN LIBRARY 41 2* The liposomal System shows that the ATPase transports Ca with a 1:1 stoichiometry to ATP hydrolysis, i.e it is less “efficient” than the analogous enzyme of sarcoplasmic reticulum. The enzyme is also stimulated by a cAMP-linked phosphorylation process. Work on the 2+ purified Ca -ATPase has established that the enzyme can be stimulated by several treat- ments alternative to calmodulin, among them the exposure to acidic phospholipids and poly- unsaturated fatty acids, and by a controlled proteolytic treatment with a number of proteases. Among the proteases that activate the purified ATPase, trypsin has been particularly useful, since it has permitted one to establish that the enzyme is degraded in sequence to products 2+ of 90-85-81-76 KDa, all of them acting as Ca stimulated ATPases. The first three have been reconstituted in liposomes and shown to be able to pump Ca. Akhough the 90 KDa compo- nent is fully reactive to calmodulin, the 85 KDa component binds calmodulin but does not respond to it (or responds negligibly), and the 81 and 76 KDa components do not bind calmodulin. The results have led to the Suggestion that the calmodulin binding domain is contained in a 9 KDa fragment of the ATPase and contains a 4 KDa domain that binds calmodulin, and a 5 KDa domain that is required for the expression of calmodulin Stimulation (Penniston, 1983, Carafoli 1984, Carafoli, 1987, Carafoli, 1988a). It has been shown that protein Kinase C stimulates the activiV/ of the plasma membrane 2+ Ca -pump by a direct effect on the pump protein (Smallwood et aL, 1988). The effect was a 5-7-fold increase of Vmax without a significant change in the apparent Km for Ca?+. Fur- thermore, the effect of protein Kinase C and Calmodulin on Ca2+ uptake were nearly additive and the Stimulation by protein kinase C is reversible by treatment with alkaline phosphatase (Smallwood, et a!., 1988). 2+ The amino acid sequence of the plasma membrane Ca pump has been established by protein Chemistry and DNA cloning techniques using a human teratoma Agt 10 library (Verma ei al., 1988). The isoform of the enzyme present in human teratoma cells contain 1220 amino acids, corresponding to a M of 134,683. Asp 475 forms the acyl phosphate during the r reaction cycle, and Lys 601 binds the ATP antagonist FITC. Asp 475 and Lys 601 are separated by a domain which is relatively conserved in the ion molive ATPases of the P-class and which may function as a hinge that permits the two residues to come close to each other in space during the reaction cycle. The calmodulin (CaM) binding domain has been identified next to the C-tei minus (residues 1100-1127). Distinctive properties of this domain are the UNIVERSITY OF IBADAN LIBRARY 42 Dredominance of basic residues, the propensity to form an amphiphilic helix and a tryptophan in the N-termina! portion of the domain (position 1107). It has been observed that if tryptophan 1107 is substituted with an alanine the affinity for calmodulin decreases several fold. The calmodulin binding domain interacts with a sequence adjacent to it on the N-terminal side which is very rieh in Asp and Glu and which may play a role in the binding of Ca?t and in reguiating the interaction of CaM with the pump. Aithough the Asp- and Glu- rieh domain does not interact directly with calmodulin it apparently changes conformation when calmodulin interacts with the ATPase. The pump also contains, next to the N-terminus, two eleven amino acid Stretches which resemble EF-hands (residues 22-33 and 310-321), and could thus also 2f form Ca -binding sites. Fig.8 shows a scheme of the architecture of the pump in the plasma membrane. Up to ten hydrophobic domains, presumably spanning the membrane, have been identified: 4 are located in the N-terminal portion of the pump, 6 in the C-terminal portion. The exact number of the membrane-spanning domains, however, is still uncertain. The mid portion of the pump (about 500 residues) contains no hydrophobic domains. Ser 1178, located on the C-terminal side of the CaM binding domain, is phosphorylated by the cAMP- dependent kinase, increasing the Ca-affinity of the pump. Studies on several isoforms of the pump from different tissues (e.g. muscle, brain, intestinal mucosa) show that they differ essentially in the calmodulin and cAMP-regulated domains. 2+ The liver plasma membranes contain a high-affinity Ca -ATPase (Kessler, et al.. 1990) that is not sensitive to calmodulin but can be activated by another activator present in the cytosol (Lotersztajn et al„ 1981). Furthermore, this ATPase is inaotive in the absence of the protein activator. Lotersztajn and Pecker (1982) have shown the existence of a protein 2+ inhibitor of the purified Ca -ATPase. The effect of this inhibitor is reversed by the addition of their activator, and the inhibitor's activity is dependent upon the presence of Mg?+. The liver 2+ Ca -ATPase in intact plasma membranes is not sensitive to concentrations of orthovanadate as high as 2mM (Iwasa et al., 1982). The Ca2+-ATPase has been shown to be a multigene family. Ssoforms of the pump have been identified at least in humans. Four different products of this multigene family have been identified in rats and humans; and classified as h (for humans) anu r (for rat) isoforms PMCA 1-4 (Carafoli, 1991; Greeb and Shull, 1989; Shull and Greeb, 1988; Strehler et al.. 1989; UNIVERSITY OF IBADAN LIBRARY 20 AA 10 AA 8 AA 23 AA 8 AA HINGE REGION u4̂> Active Putative Site (~P) Ca-binding— * domain CaM-binding domain (amphiphilic helix) cAMP-dependent phosphorylation site Fi3'8 membrane ^ f o " b S ° rg“ n ° ' 'hS in the UNIVERSITY OF IBADAN LIBRARY 44 Strehler et al., 1990). Each corresponds to a family of alternative^' spliced isoforms. Fur- thermore, Isoform specific functional differences has been observed; for instance hPMCA 4 isoform has been shown to lack the cAMP-sensitive phosphorylation site (Khan and Grover, 1991). The Ca2 t -ATPase of the Endo (sarco) plasmic reticulum: The endo(sarco) plasmic reticulum is the membrane System responsible for the fine 2+ 'egulation of Ca in the cytosol. Most of the work on this membrane System has been oerformed on sarcoplasmic reticulum, essentially due to the easier availability of its key enzyme, the Ca-ATPase, with respect to endoplasmic reticulum. The latter organeile, how- ever, has recently become prominent as a result of the discovery that its Ca pool is sensitive to inositol tris-phosphate(Carafoli 1988b). The sarcoplasmic reticulum Ca2+-ATPase is of the same dass as that of the plasma membrane, has a Km below 0.5 p.M, and is present in very 2+ large amounts. As a result, the total Ca transporting capacity of the organelle is very high, particularly in fast skeletal muscles where it may reach 70 nmol/my of membrane protein/s. The ATPase has been purified as a single polypeptide of about 100kDa. It forms an aspartyl Phosphate, is inhibded by vanadate, and can be reconstituted into ’.posomes, where is trans- ports Ca2* with a .2:1 stoichiometry to the hydrolyzed ATP. The primary structure of this ATPase has also been determined (MacLennan et al.. 1985). It appears that the enzyme contains 10 transmembrane Stretches, and a very large portion prolruding from the cytosolic side of the membrane. The latter portion is postulated to consist of several domains, contain- ing the ATP-binding sequence, the aspartic acid residue which beccmes phosphorylated, the portion of the molecule that transduces the ATP energy to the region that translocates Ca2+, 2+ and the Ca -bindirig domain proper. The latter has been ascribed to a region of the molecule next to the hydropnobic intra-membrane a -helixes that contains an unusually high concen- 2 t tration of glutamic acid residues. Thus, the primary structure of the Ca -ATPase of the fast skeletal muscles repeat the essential features of the P-type ATPases. The ATPase in heart, smooth, and slow (but not fast) skeletal muscles is modulated by an acidic proteolipid termed phospholamban. fhis proteolipid, which is a pentamer of five identical subunits of about 6 KDa, is phosphorylated by two kinases, one of which is cAMP depandent while the other is calmodulin dependent (Carafoli, 1988b). Phosphorylated phospholamban activates the UNIVERSITY OF IBADAN LIBRARY 45 ATPase-linked transport of Ca2+, thus transmitting to sarcoplasmic reticulum hormonal mes- sages from the plasma membrane (Carafoli, 1988a). While the (Ca2' + Mg2')-ATPase of heart sarcoplasmic reticulum is under the control of a phosphoryiation process mediated by cAMP and CaM, in skeletal muscle these regulatory Systems are apparently absent. Much 2-f iess, however, is known of the Ca transport Systems in liver (Famulski and Carafoli, 1984). It has been demonstrated that the mycotoxin cyclopiazonic acid is a highly specific and potent inhibitor of the Ca -ATPase of sarcoplasmic reticulum (Seidler, et al., 1989). Furthermore, it has been argued that there are many more cor.formational changes in the cycle for calcium pumping than the single conformational change from to E and back from E - P.Ca to E -p.Ca , that is postulated in the E -E model (Jericks, 1989). 1 1 2 ? 1 ? 2f ln liver, the endoplasmic reticulum seems to have a high affinity for Ca , but a very small capacity for storage of Ca?+. (Penniston, 1983). The hepatic Gz "-ATPase contains tightly bound calmodulin. Partial removal of this calmodulin by EGTA treatment results in an in- creased 4’Ca2+ uptake by added exogenous calmodulin and inhibition by trifluoperazine (TFP). The native microsomal enzyme is not activated by added calmodulin (Moore and Kraus-Friedmann 1983). The Stimulation of Ca2' transport is associated with the Operation of a protein phospho- 2 F rylation System dopendem on Ca and calmodulin. Transport is inhibited by a protein phos- 24 phatase which is stimulated by Ca and calmodulin as well. This finding strongly supports the Suggestion that the protein phosphorylation-dephosphorylation cycle is a regulatory device 2+ 2+ which Controls the (Ca + Mg -) ATPase in liver microsomes (Famulski and Carafoli, 1984). Recently, it was demonstrated that acyl phosphatase is a scluble non-calmodulin activa- 2+ tor of erythrocyte membrane Ca -ATPase; and that Stimulation by acyl phosphatase was additive to that änduced by calmodulin (Nassi et al, 1990). UNIVERSITY OF IBADAN LIBRARY 46 1.5 Intracellular Ca~ siqnallinq for transient and sustained cellular responses. A common trigger precipitates biological events as diverse as the contraction of a muscle and the secretion of a hormone. The trigger is a minute flux of caic'um ions. Calcium is one of the body’s “second messengers” : it relays electrical and Chemical messages that arrive at a cell’s surface membrane to the biochemical machinery within the cell. To control cellular processes effectively, calcium itself must be regulated. Thus cells have evolved an elaborate System of proteins that interact with the calcium ion, governing the transmission and reception of the intracellular message (See section 1.4). The mechanisms that regulate calcium in the cell do not operat.s In isolation. Networks rather than simple pathways typify cell physiology, and intracellular signalling Systems are no exception. The modification by cyclic AMP of calcium channels and the calcium-pumping ATPase in the plasma membrane is one example of the interactions among messenger Systems (Carafoli and Penniston, 1985). Thus, the old belief that Ca2' and cAMP are separate intracellular messengers and that these two messen­ ger Systems functioned separately - Ca2' in excitable cells and cAMP in non-excitable cells is no longer tenable. It is now recognized that intracellular Ca?' signalling is more complex than previously thought for the foilowing reasons: 2+ (i) There is a synarchic relationship between Ca and cAMP (Rasmussen, 1970). 2 f 2 \ (ii) Intracellular free Ca ion concentration [Ca ]i was transient even though the response was sustained (Grynkiewicz et al„ 1985; Cobbold and Rink, 1987; Morgan and Morgan, 1984). (iii) Ca2' cycling (i.e sustained increase in Ca?' influx without an increase in [Ca2t]i or in total ceü calcium) alone without a Ca?' - sensitive transducer e.g. Protein Kinase C was noi sufficient to induce a sustained cellular response (Rasmussen, 1989; 1990). In several transient cellular responses, including the secretion of neurotransmitters by nerve cells and the contraction of skeletal and cardiac muscle cells, calcium serves as a simple on-off switch that conveys information from the cell surface *o the cell interior. When a cell is stimulated by an extracellular Signal, channels in its plasma membrane open and allow calcium ions to enter at from two to four times the normal rate, When the concentration UNIVERSITY OF IBADAN LIBRARY 47 rises, calcium-binding proteins in the cytosol, such as the specific receptor calmodulin, attach to calcium ions; the calcium-protein complexes then interact with other proteins in the cell to alter their functions. When the calcium concentration in the cy'osol falls again, the ions dissociate from the receptor proteins and the system turns off. In each case, the rise in calcium ion concentration in tho cytoeol initiatoc tho response, and tho fall in calcium ion concentration terminates it (Rasmussen, 1989). The role of calcium in sustained cellular responses such as the secretion of insulin or the contraction of the smooth muscle surrounding the blood vessels, has historically been more elusive than its part in transient responses. It is now recognized that prolonged or sustained cellular response can be divided into two temporally distinct phases - a calmodulin branch which is active during the initial phase of the response and a protein Kinase C branch that operates in the second sustained phase (Rasmussen, 1989, 1990). Fig. 9 is a schematic 2+ summary of sonne of the Ca -modiated proeessos relevant to toxicology. In the initial phase, the binding of an txtracellular Signal to its receptor prompts the break-down of the membrane component, PIP into IP and DAG. IP causes the release of calcium ions from intracellular 2 3 3 compartments called calcisomes or endoplasmic reticulum, resulting in a transient rise in 2+ cytosolic calcium (Ca ). The ions bind to calmodulin, and the calcium-calmodulin complex activates protein kinases i.e calmodulin dependent protein Kinases (enzymes that transfer Phosphate groups to proteins). The phosphorylated proteins initiate the cellular response. The calcium ions released from the calcisomes and the increaso in DAG also prompt the enzyme PKC to associate with the membrane. In the sustained phase, the Signal increases calcium cycling across the membrane, and the resulting rise in the submembrane concentra- 2+ tion of calcium [Ca sm] activates the membrane-associated PKC. This activation brings about the phosphorylation of a different set of proteins that sustain the response (Rasmussen, 1989). In these two phases, however, the Operation of the calcium-messenger System de- pends heavily on the activity of the cAMP-messenger system. Furthermore, one or more protein Kinase cascades function to convey information from cell surface to cell interior when 2+ the protein Kinase C branch of the Ca -messenger system is activated (Rasmussen, 1990). Fig. 10 is a schematic expression of the initial and sustained phases of a prolonged cellular response. UNIVERSITY OF IBADAN LIBRARY 48 Hormonal and electrica! Signals Phosphatidylinositol cycle Arachidonic acid Cascade cAMP messenger System t [Ca++] i Calmodulin Branch C-Kinaso Branch (brief response or initial (sustainea response) phase of sustained response) neurotransmitter release cell division skeletal muscle contraction cell prolifsration cardiac nvjscle contraction cell-cell communication platelet-release reaction Organization cf cytoskeleton endocrine secretion smooth muscle contraction exocrine recretion intermediary metabolism aldosterone secretion endocrine secetion exocrine secretion insulin secretion 2+ Fig. 9 Schematic summary of some of the Ca -mediated processes relevant to toxicology (Pounds and Rosen, 1988) UNIVERSITY OF IBADAN LIBRARY 49 L ^ - m u P t\A S E i Fig. 10 A sechematic summary of the two temporally distinct phases of prolonged cellular response (Rasmussen, 1989). UNIVERSITY OF IBADAN LIBRARY 50 1.6 Cellular Ca homeostasis and Ca -mediated cellular processes as critical targets for toxicaat action. ?\ On the basis of the central role of the Ca -messenger System in several aspects of cell 2+ 2+ function, it is logical to examine possible disturbances in Ca homeostasis and Ca -medi­ ated functions as underlying mechanisms of toxicant effects at different levels of biological 2+ Organization (Pounds and Rosen, 1988). Perturbations in the Ca messenger System by toxicants may place the regulation of Cellular processes out of the normal ränge of physiologi- 2+ 2+ cal control. Hence, disturbances in intracellular Ca homeostasis and Ca -mediated func­ tions have becomu attractive and frequently postulated targets for numerous pathophysiologi- cal processes including neurosecretion, ischemic injury and toxicant-induced cell death, cell growth and transformation, tumour promotion, and hypertension (Dubovsky and Franks, 1983; Lock-Caruso and Trosko, 1985; Metcalfe et al.. 1985; Starke et al.. 1986; Trump and Berezesky, 2+ 2+ 1985; Viegl et al., 1984). Perturbations of Ca homeostasis and Ca -mediated cell functions following toxicant exposure may each result from at least three general types of interactions : direct, indirect and secondary interactions (Pounds and Rosen, 1988). Toxicant Effects on Ca 2i Cellular Homeostasis 2s 2 t Toxicants may directly alter Ca homeostasis by substituting for Ca at specific sites of Ca2+ transport or storage. In this sense these interactions are competitive with Ca2+ and generally reversible at the molecular level. Direct interactions are most commonly observed with other divalent metals, including lead, ruthenium, cadmium, and others, which directly 2+ compete with or displace Ca at transport sites in the plasma membrane (Atchison and Narahashi, 1984; Cooper et al.. 1984; Simons, 1986). Other examples include the direct actions of the calcium channel blockers, nifedipine or verapamil which block calcium mobili- zation through membrane channels in myocardial and smooth muscle cells. It must be recognized that a direct interaction does not necessarily result in an inhibition of a Ca2+- mediated function, but may also produce an exaggerated Ca2f-mediated response if the 2 + clearance of Ca from the cytosol and other cellular compartments is impaired (Pounds, 1984). Toxicants may indirectly act on specific Ca2+-homeostatic processes, such as Ca2+ 2+ pumps or gates, but at molecular sites that are independent of Ca , i.e., presumably not at UNIVERSITY OF IBADAN LIBRARY 51 2+ the Ca transport or binding site. Thus the interaction is noncompetitive in that adding more calcium will not alter the biochemical lesion. This type of interaction is observed with many organic toxicants. The most fully characterized example of an ind;rect action is the inactiva- 2+ tion of the Ca transporter in smooth endoplasmic reticulum by carbon tetrachloride leading eventually to cel! death (Long and Moore, 1986, Recknagel, 1983). An indirect interaction may be reversible at the molecular level, but if the injury is sublethal, the cell and organism may be able to repair and recover, within this instance, the biosynthesis of new endoplasmic reticulum and the associated Ca -pump. 2+ Toxicants may secondarily alter Ca homeostasis through n.onspecific effects on cell function that are biochemically and functionally remote from the piocesses of Ca2+ transport and storage. For example, ethanol and other aliphatic alcohols and local anesthetics, includ- ing dibucaine or tetracaine, produce changes in nerve cell excitability and neurotransmitter release which are the result of physical-chemical changes in the cell membrane, including increased fatty chain motion within the membrane bilayer, expar.sion of membrane volume, 2+ and increased membrane fluidity resulting in altered Ca permeability and transport properties of the membrane (Michaelis and Michaelis, 1983). Furthermore, toxicants which dissipate Na' gradient will secondarily alter Ca2' homeostasis in cells where Ca‘ '/Na' exchange is a 2+ component of Ca homeostasis. 2+- Toxicant Effects on Ca -mediated cell Functions. 2+ 2+ Toxicants may directly act on Ca -mediated cell functions, including Ca receptor pro- teins or Substrates, without necessarily altering Ca2' homeostasis. A well characterized example is the inhibition of Ca-calmodulin-mediated processes by the phenothiazines which bind to the hydrophobic region of calmodulin and prevent the formation of a Ca2'-calmodulin 2< receptor enzyme complex, thereby inhibiting Ca -mediated functions. It must be noted, however, that some calmodulin antagonists also alter membrane currents of potassium and sodium (Klockner and Isenber, 1987). Lead, aluminum, cadmium, and other divalent metals 2+ 2+ effectively compete with Ca for binding sites on a variety of Ca -binding proteins including calmodulin (Fullmer et al.. 1985). Replacement of Ca in Ca receptor proteins by other metals may alter Ca2+-mediated function in unpredictable ways. However, due to autoregulation 2+ of Ca homeostasis by calmodulin, direct actions of toxicants on calmodulin and related UNIVERSITY OF IBADAN LIBRARY 52 2+ 2+ "oteins may likely result in alterations in Ca homeostasis (an indirect effect on Ca ho- '-eostasis). 2+ Toxicants may indirectly alter Ca mediated cell functions, incependently of changes in Ca2+ homeostasis and independently of direct interaction with the Ca2+ receptor or effector croteins. The response to a Ca2" Signal is a cascade of biochemical events which often mclude phosphorylation, and is subject to feed-back control by oyclic nucleotides, and to sensitivity and response modulation (Rasmussen, 1986). Because many cAMP-mediated 2+ responses are antagonistic to the Ca -mediated response, toxicants which affect adenylate cyclase, phosphodiesterase, or other parts of the cAMP messenger System will likely alter 2+ Ca -mediated cell functions. Examples of indirect effects inclurie the inhibitory effects of Xanthines on phosphodiesterase leading to pharmacologic and toxic changes in muscle con- traction. 2+ Toxicants may secondarily perturb Ca -dependent functions as a result of toxic events remote from Ca-dependent processes. Toxicants which grossiy alter cell and organeile 2+ function may imp lir the ability of a cell to respond to a Ca Signal, but as a result of toxic 2+ action which has no relationship to the Ca messenger system. r or example, methyl mer- cury impairs neuronal migration and cell division, but the cellular taryet forthese actions is the 2-\ neurotubules and microtubules of the cells rather than the Ca -mediated aspects of cell movement and division. Other toxicants which alter cellular energetics, protein synthesis, 2+ etc., will alter the ability of cells and tissues to maintain proper Ca homeostasis and function. 2+ However, these secondary effects on Ca -mediated function aro more an expression of toxicity than the cause of toxicity. (Pounds and Rosen, 1988). UNIVERSITY OF IBADAN LIBRARY 53 2+ - ‘ .7 Ca~ homeostasis with particular reference to the role of the endoplasmic reticulum in tumoungenesis by Chemical agents. The oncogenic transformation of cells from a normal to malignant phenotype is associ- ated with a variety of experimentally discernible changes in the pattern of metabolism. Many of these alterations involve processes modulated by the intracellular level and distribution of calcium (Murphy and Fiskum, 1987). Pathological excess of intracellular calcium has been implicated as a final common pathway of cell death produced by a wide ränge of toxins including the well studied hepatotoxin carbon tetrachloride (Lowrey et al.. 1981a). Coinciden- tally, many of the Chemicals that induce hepatocellular carcinoma in experimental animals are also acutely hepatotoxic (Manson, 1983). ln terms of general cellular metabolism, mitochondrial Ca transport appears to be in- 2+ volved primarily in the physiological regulation of intramitochondr.al levels of Ca , whereas transport activities at the plasma membrane and endoplasmic reticulum are thought to play primary roles respectively in regulation of basal levels and transient changes in the cytosolic Ca?+ concentratidri. However, mitochondrial Ca2+ uptake and reiease may assume a more 2\ critical role in regulating cytosolic Ca under metabolically stmssful conditions, such as during ischemia and reperfusion (Murphy and Fiskum, 1987). The endoplasmic reticulum of the liver cell is the first celluiar organelle disrupted by carbon tetrachloride poisoning. The disruption is accompanied by a destruction of Calcium pump activity in the organeile (Moore et al., 1976). The destruction of liver microsomal CaU-H pump activity by carbon tetrachloride and other hepatotoxins has been confirmed by other workers (Lowrey et al.. 1981 a, b). Furthermore, carbon tetrachloride toxicity in the rat can be potentiated by pretreatment of animals with agents which induce the mixed function oxi- dase complex of the liver endoplasmic reticulum. These agents which include phenobarbital (Garner and Mclean, 1969) and 1,1,1,-trichloro-2,2,-bis (p-chlorophenyl) ethane (McLean and McLean, 1966) are well known liver tumour Promoters. It is now known, however, that the trichloromethyl radical of carbon tetrachloride initiates lipid peroxidation. It is a potent hepato­ toxin, hepatopromoter and a complete carcinogen (Cerutti, 1985), Thapsigargin, a naturally occurring sesquiterpene lactone and tumour promoter has been found to be a higfily selective and potent inhibitor of the endoplasmic reticulum Ca2+-ATPase. UNIVERSITY OF IBADAN LIBRARY 54 2+ 24 Thapsigargin induced ER-Ca -ATPase inhibition abolishes the Ca -releasing ability of both GTP and lns-1,4,5~P3 (Thastrup, 1990, Thastrup et al.. 1990). It has been observed that the rate of Ca2+ uptake by AS-30D hepatoma microsomes is approximately twice as fast as that observed with rat liver microsomes. In addition, the AS- 2+ 30D hepatoma microsomes is more sensitive to the Ca -releasing action of inositol 1,4,5 trisphosphate (IP^) than the rat liver microsomes (Murphy and Fiskum, 1987). Whether alterations in the transport or effects of calcium are the primary mechanism by which malignant cells dictate their high growth rate or whether they are the manifestation of some other transfomning characteristic is unknown at this time. UNIVERSITY OF IBADAN LIBRARY 55 \S Tumour prompter and biochemical mechanism of action Tumour promotion encompasses the modulation of the expression of genes related to growth and differentiation of initiated cells (Cerutti, 1985). A tumour promoter is an agent that ‘acilitates formation of neoplasms from altered cells that otherwise would remain dormant. Tumour promoters are usually identified by their enhancement of the tumour yield resulting ‘rom a previously adrninistered carcinogen, called an initiating agent in the terminology adopted for multi-stage carcinogenesis. They can also be conceived to promcte tumour formation by cells altered through effects other than experimentally induced initiation, such as cells with an inherited genetic abnormality or a spontaneous mutation (Williams, 1983). While there is a wealth of information on genotoxic carcinogens, the process of tumour promotion is still poorly understood. Nevertheless, some progress have been made in this regard. Indeed, several biochemical effects of tumour promoters have been identified and these include: (i) Generation of free radicals (Troll and Weisner, 1985; Albano et al.. 1988; Connors, 1988; Das et al., 1990; Kozumbo et al., 1985; Nakamura et al., 1985; and O’Connel et a[M 1986. (ii) Impairment of antioxidant System (Perchellet et al., 1986; Perchellet et al., 1987; Kaplan and Groses, 1972; Peskin et al.. 1978; Corrocher et aj., 1986). (iii) Inhibition of intercellular communication (Williams et al.. 1S8 ;; Klaunig et al.. 1990). (iv) Stimulation of arachidonic acid metabolism (Levine, 1984 and 1988) and (v) Modulation of the activities of several enzymes including (1) Ornithine decarboxylase (Raunio and Pelkonen, 1983; O’Brien, 1976; Yanagi et al.. 1981; and Kitchin and Brown, 1987) (2) Protein Kinase C (Castagna et al.. 1982) (3) Protein phosphatases (Bialojan and Takai, 1988) and 2+ (4) Ca -ATPases (Parola et al., 1990; Lowrey et al., 1981 a; Lowrey et al., 1981b; Thastrup et al.. 1990). One major obstacle in synthesizing the multifarious effects of tumour promoters to give a comprehensive picture is the difficulty in differentiating a primary effect from a secondary effect. To date, the most acceptable and comprehensive hypothesis proposed to explain the UNIVERSITY OF IBADAN LIBRARY 56 mechanism of action of tumour Promoters appears to be that of oxiciant carcinogenesis pro- posed by Cerutti and co-workers (1989). According to this hypothesis, Promoters and progressors are oxidants and agents which induce cellular pro-oxidant States. The authors proposed that oxidants induce series of biochemical reactions that result in carcinogenic tumour promotion (Fig.11). According to the authors, many mitcgens including tumour Pro­ moters act either indirectly by producing free radicals like activated oxygen or directly by binding to an agonist-specific receptor or both. The activated oxygen induces the mitochon- 2+ drion to release :ts calcium pool, inhibits the plasma membrane Ca -ATPase and induces the release of the membrane-bound protein Kinase C. The binding to the agonist-specific recep­ tor is followed by hydrolysis of polyphosphoinositides, resulting in formation of inositol phos- phates (IPg) and diacylglycerols. These respectiveiy increase intracellular calcium concen- trations (via IP -induced calcium release from the endoplasmic reticulum) and activate protein 3 kinase C with concomitant Stimulation of protein phosphorylaticn and calcium/calmodulin- dependent processes (Nishizuka, 1984; Berridge, 1984; Graff et al. 1989; Cerutti et al„ 1989). Furthermore, the multistage process of cancer development is known to involve both muta- genic and non-mutagenic mechanisms. These result in the induction of multiple direct and indirect genetic changes at target oncogenes or tumour suppressor genes as well as alter- ations in Signal transduction pathways involved in growth controS (Perera, 1990). UNIVERSITY OF IBADAN LIBRARY OXIDANT CARCINOGENS Fig. 11 Mechanisms of oxidant carcinogenesis (Cerutti et a|., 1989) UNIVERSITY OF IBADAN LIBRARY 58 1.9 Aims of the study 2+ Protein Kinase C, the Ca and phospholipid dependent kinase, has been identified as the target of tumour Promoters (Castagna et al., 1982; Ashendei et al., 1983 a,b). This enzyme has been well characterized (Inoue, 1977). It has been shown that diacylglycerol (a product of phosphatidylinositolbisphosphate breakdown) is the endogenous activator of protein Kinase C under physiological conditions (Kikkawa et al., 1982). The addition of diacylglycerol (pM 2+ ränge) increased the enzyme’s affinity to both Ca and phospholipid (Takai et al., 1979). A number of agents, including ff - adrenergic Stimulators, growth factors (e.g. EGF), and other tumour Promoters, mitogens, peptide hormones, and neurotransmitters are able to stimulate protein kinase C activity and to accelerate phosphatidyl inositol turnover (Castagna et al.. 1982; Michell, 1979). 2+ Usually, intracellular events are signalled by a transient rise in the concentration of Ca' in the cytosol (Ca afoli, 1987). Indeed, Berridge (1987) has conduded that a rapid and 2+ transient increase in cytosolic free Ca is part of the first response of cells to growth factors and that the release of Ca?‘ from the endoplasmic reticulum (ER) is often stimulated by agents that cause cell proliferation. Inositol 1,4,5-trisphosphate (IP ) another product of phosphatidylinositolbisphosphate breakdown is the mediator in the release of intracellular Stores of Ca2+ from the ER into the cytosol (Suresh et al., 1984; Streb et al.. 1983). The mechanism by which liver tumour Promoters exert their physiological effects is poorly understood. The well-studied liver tumour promoter and complete carcincgen carbon tetrachloride (C C I) has been shown to: 4 2+ (a) induce an inhibition of the plasma membrane Ca -ATFase (Parola et al.. 1990), (b) induce the inhibition of the microsomal Ca?f-pump (Rec cnagel et ab, 1982; Long and Moore, 1986) and (c) decrease the Ca?' and phospholipid dependent protein Kinase C content (Poli et al.. 1988). 2f It has been demonstrated that the activity of the plasma membrane Ca -ATPase is regulated by protein Kinase C (Smallwood et al., 1988). However, it is recognized that tumour Promot­ ers induce cellular prooxidant state (Cerutti, 1985) and that activated oxygen inhibits plasma UNIVERSITY OF IBADAN LIBRARY 59 membrane Ca2+-A TPase activity (Hebbel et al.. 1986). However, the Inhibition of the plasma membrane Ca2+-ATPase even with the IP -induced Ca2+ release from the ER, seems not 3 2+ sufficient to account for the very high level of Ca in the cancer ceils; assuming that the ER 2+ Ca -ATPase functions normally. It might be speculated therefore. that the effects of tumour 2+ Promoters on the ER involve not only the IPg-induced Ca release from the ER, but also an inhibition of the ER Ca2+-ATPase. Since carbon tetrachloride is a complete carcinogen, results obtained by the use of the Chemical can not be taken as a true reflection of tumour promotion. 2+ This thesis was designed to evaluate the role of the endoplasmic reticulum Ca -ATPase in the process of liver tumour promotion. The specific liver tumour promoter and environmen­ tal contaminant, DDT as well as protein-malnutrition which is widely used and common re- spectively in Africa and Asia were used as models. It has been suggested that these factors might be associatsd with the preponderance of PLC in Africa and Asia (Rizvi et aj., 1987; Rojanapo e( al., 1980). The aim3 of this study are: (1) To find out the effects of the tumour promoter dicophane on the membrane-bound 2+ rat liver microsomal Ca -ATPase with the ultimate goal of using the enzyme as a marker of tumour promotion. 2+ (2) To determine the effect of low protein intake on the liver ER Ca -ATPase and 2 h (3) To assess the possibility of using the erythrocyte ghost membrane Ca -ATPase for the ciiagnosis of cancer in humans. UNIVERSITY OF IBADAN LIBRARY 60 CHAPTER TWO M A TER IALS AND M ETH O D S 2.1 Materials (i) Chemicals: All Chemicals were purchased either from Signa Chemical Co. (U.S.A.) or from BDH Chemicals Ltd., (U.K.) Poole. (ii) Animais: Male Wistar rats (150-200 g) or male Fisher F344 rats (weanlings or 8 weeks old) obtained from Bantin and Kingman Ltd., Aldbrough, Hüll, England were used for the study. (iii) Animal feeds: All animals except those on low protein diet were fed MRC 41B diet obtained from Pilsbury’s Ltd., Birmingham, England (See appendix I for composition). Low protein diet (5 % protein) was obtained from Special Diets Services Ltd., Essex, England (See appendix II for composition). 2.2 Preparation of membranes 2.2.1 Preparation of the light microsomal fraction. The eonventional microsomal fraction obtainable from the post-mitochondrial supernatant is known to originate from the endoplasmic reticulum. However, recent studies show that the post-mitochondrial supernatant of rat liver contains two vesicu- 2+ lar fractions which transport Ca actively: a heavier fraction, enriched in plasma membrane markers and a lighter fraction, enriched in endoplasmic reticulum markers (Famulski and Carafoli, 1982, 1984, Gill ef al, 1989). The neavy fraction is probably a contarninant. In the present study, the light microsomal fraction was used as microsome (endoplasmic reticulum) preparation. Reagents (A) Stock Solutions (i) 100 mM Tris; 12.114 g Tris (hydroxymethyl) methylam;r;e (BDH Chemicals Ltd., England) was dissolved in 1 litre Volumetrie flask and the volume made up to the UNIVERSITY OF IBADAN LIBRARY 61 1 litre mark with distilled water. (ü) 40 mM EGTA: 1.5216 g of ethylene glycolbis-(p-aminoethyleiher) NN’N’N’-tetraacetic acid (Sigma Chemicals Co. USA) was suspended in about 80 ml distilled water and 1M NaOH added in drops until the Suspension became clear. The pH was adjusted to 7.4 and the volume made up to 100 ml in a Volumetrie flask. (iii) 500 mM PMSF: 0.0871 g of phenylmethyl sulfonyl fluoride (PMSF)(Sigma Chemicals Co. USA) was dissolved in 1ml dimethyl formamide (BDH Chemicals Ltd., England). (iv) ß -Mercaptoethanol: ß-mercaptoethanol (BDH Chemicals Ltd.,England) was obtained in commercial form. (B) Working Solutions (i) Isolation buffer: 250 mM Sucrose, 5 mM Tris,1 mM mercaptoethanol. 0,5 mM PMSF, pH 7,4 The buffer was prepared by dissolving 86.58 g of Sucrose (BDH Chemicals Ltd., U.K.) in about 500 ml of distilled water. To the solution was added 50 ml of 100 mM Tris, 70.5 gl of ß -mercaptoethanol and 1 ml of 500 mM PMSF in 1 litre beaker and distilled water added to 900 ml mark. The pH was adjusted to 7.4 and the solution transferred into a litre Volumetrie flask. The buffer solution was made up to the 1 litre mark with distilled water. (ii) Storage buffer: 250 mM Sucrose, 5 mM Tris, 1 mM mercaptoethanol, 0.5 mM PMSF. 1 mM EGTA. pH 7.4 21.394 g of Sucrose was dissolved in about 100 ml distilled water, and to the solution was added 12.5 ml of 100 mM Tris, 17.6 gl of ß-mercaptoethanol, 250 gl of 500 mM PMSF, 6.25 ml of 40 mM EGTA in 250 ml beaker and water adderto the 200 ml mark. The pH was adjusted to 7.4 and the volume made up to 250 ml in a Volumetrie flask with distilled water. Procedure Rats were CO? exsanguinated and the liver quickly removed. Rat liver light microsomal fraction (endoplasmic reticulum) was prepared as described by Famulski and Carafoli (1982). Rat liver (maintained on ice-cold isolation buffer) was carefully weighed and homogenized in 4 times ice-cold isolation buffer using a Potter-Elvehjem homogenizor. The homogenate was UNIVERSITY OF IBADAN LIBRARY 62 filtered through 4 layers of cheese cloth and centrifuged at 3 ,000 x g for 6 min. The supernatant was centrifuged at 10000 x g for 10 min. and the post-mitochondrial supernatant was centrifuged at 17,500 x g for 20 min. to remove the heavy microsomal fraction. The obtained supernatant was centrifuged at 100,000 x g for 1 hr. The pellet (light microsomal fraction) was suspended in a small volume of storage buffer and kept frozen in aliquots at -20°c until required for use. 2.2.2 Preparation of ervthrocvte qhost membranes Erythrocyte ghost membrane (EGM) is the most studied plasma membrane chiefly be- cause it is devoid of any contaminating intracellular membranes and also because it is rela- tively easier to prepare. Furthermore it can be obtained in large quantities. The red blood cell like most cell types can be lysed by using hypotonic buffer Solutions. This is the principle on which the EGM preparation is based. Reaqents (A) Stock Solution: (i) 100 mM Tris: 12.114 g Tris (hydroxymethyl) methylamine (BDH Chemicals Ltd., England) was dissolved in 1 litre Volumetrie flask and the volume made up to the 1 litre mark with distilled water. (ii) 3.25 M KCl: 121.23 g of potassium Chloride (BDH Chemicals Ltd., England) was dissolved in 1 litre distilled water using a Volumetrie flask. (iii) 100 mM EDTA : 0.093 g of disodium salt of ethylenediaminetetraacetic acid (EDTA) (BDH Chemicals Ltd., England) was dissolved in 250 ml distilled water using a Volu­ metrie flask. (iv) 1 M HEPES: 23.830 g of HEPES (N-2-hydroxy ethylpiperazine-N1-2 ethanesulfonic acid) (Sigma Chemicals Co., USA) was dissolved in 100 ml distilled water using a Volumetrie flask. (v) 100 mM MgCh 2.3805 g of anhydrous magnesium Chloride (BDH Chemicals Ltd., England) was dissolved in 250 ml distilled water using a Volumetrie flask. (vi) 40 mM CaCk 1.4792 g of calcium Chloride 2-hydrate (BDH Chemicals Ltd.,England) was dissolved in 250 ml distilled water using a Volumetrie flask. (vii) 500 mM PMSF: 0.0871 g of phenylmethylsulfonyl fluoride (PMSF) (Sigma Chemicals UNIVERSITY OF IBADAN LIBRARY Co., USA) was dissolved in 1 ml dimethylformamide (BDH Chemicals Ltd..England). 3) Working Solutions (i) Isotonic buffer : 130 mM KCl. 20 mM Tris. pH 7.4 The buffer was prepared by mixing 80 ml of 3.25 M KCl with 400 ml of 100 mM Tris, and distilled water added to 1900 ml in a beaker. The pH was adjusted to 7.4 and the solution made up to 2 litres in a Volumetrie flask. (ii) Hypotonie (Lvsino) buffer: 10 mM Tris,1 mM EDTA, pH 7.4 The buffer was prepared by mixing 200 ml of 100 mM Tris with 20 ml of 100 mM EDTA and distilled water to 1900 ml in a beaker. The pH was adjusted to 7.4 and the solution made up to 2 litres in a Volumetrie flask. To every 500 ml of the buffer was added 0.2 ml of PMSF just before use. (iii) Washing buffer: 10 mM HEPES pH 7.4 About 1700 ml of distilled water was added to 20 ml of 1 M HEPES in a beaker and the pH adjusted to 7.4. The solution was made up to the 2 litres mark with distilled water in a Volumetrie flask. To every 500 ml of the buffer was added 0.2 ml of PMSF just before use. (iv) Storage (resealing) buffer: 130 mM KCl. 20 mM HEPES. 500 uM MgCI 2 and 50 uM CaCI dH 7.4 2 The buffer was prepared by mixing 80 ml of 3.25 M KCl with 40 ml of 1M HEPES 10 ml of 100 mM MgCI , 2.5 ml of 40 mM CaCI and about 1800 ml of distilled water 2 2 in a beaker. The pH was adjusted to 7.4 and the solution made up to 2 litres in a Volumetrie flask. To every 50 ml of the buffer was added 20 pl of PMSF just before use. Procedure Haemoglobin-free ghost deficient in calmodulin were prepared by the procedure of Niggli et aj (1981) which »6 based on the principles of hypotonic lysis developed by Dodge et aj (1963). Fresh but exposed human blood samples obtained from Liverpool Blood Transfusion Service, Merseyside, England were used for the preparation of erythrocyte ghost membranes (EGM). UNIVERSITY OF IBADAN LIBRARY The whole blood was centrifuged at 5800 x g for 10 min. The plasma and buffy coat were removed by aspiration to obtain packed erythrocytes. The erythrocytes were washed twice in 5 times volumes of isotonic buffer. Each time the cell Suspension was centrifuged at 6000 x g for 10 min. and the supernatant removed by aspiration. The cells were haemolyzed in 5 times volumes of hypotonic buffer and centrifuged at 20000 x g for 20 min. This Step was repeated four times. The membranes were then washed five times with the washing buffer by suspending the pellet in the buffer and centrifuging at 20000 x g for 20 min. The haemoglobin-free ghost membranes deficient in calmodulin were finally suspended in the storage buffer and stored at -20°c tili required for use. 2.3 Protein determination. The Lowry procedure of protein determination (Lowry et ab. 1951) is based on the use of Folin-Ciocalteu reagent (a solution of phosphomolybdictungstic complex) which reacts with and is reduced by tyrosine residues in protein at alkaline pH to give a blue colour. To buffer the pH around 10 and to neutralize the phosphoric acid produced by the degradation of the phosphomolybdictungstic complex at alkaline pH, a mixture of Na CO -NaOH is added to the 2 3 assay System. Furthermore, pretreatment of the protein sample with alkaline copper mark- edly increased the colour developed. Reaqents (A) Stock Solutions (i) Reagent A : 2 % Na CO in 0.1 N NaOH. 2 3 10 g of anhydrous Na CO (BDH Chemicals Ltd., England) was dissolved in 2 3 about 400 ml of 0.1 N solution of NaOH (4 g NaOH (BDH Chemicals Ltd., England) dissolved in 1 litre Volumetrie flask and made up to the 1 litre mark with distilled water). The Na CO solution was made up to the 500 ml mark in a 2 3 Standard Volumetrie flask with 0.1 N NaOH solution. (ii) Reagent B : 2 % NaTTĈ Tartrate 2.0 g of Sodium Potassium tartrate (BDH Chemicals Ltd., England) was dissolved in 100 ml of distilled water using a Volumetrie flask. UNIVERSITY OF IBADAN LIBRARY (iii) Reagent C : 1 % Copper Sulphate 1.0 g of Copper sulphate (BDH Chemicals Ltd., England) was dissolved in 100 ml distilled water using a Volumetrie flask. 3) Working Solutions (i) Reagent D : Alkaline copper Solution: This reagent was prepared by mixirig 50 m! of reagent A with 0.5 ml of reagent B and 0.5 ml of reagent C in that order just before use. (ii) Reagent E : Folin-Ciocalteu reagent This was obtained in commercial form from Sigma Chemicals Co., U.S.A. (iii) Bovine Serum albumen (BSA) : 1 mg/ml BSA This was prepared by dissolving 20 mg of BSA (Sigma Chemicals Co., U.S.A) in 20 ml of distilled water using a Volumetrie flask. Procedure Membrane protein determination was carried out as described by Lowry et aj. (1951). First, a protein calibrated curve was plotted using the procedure contained in Table 5. The obtained results were used to plot a protein calibrated curve Fig.12. Thereafter, 20 gl of the membrane sample was pipetted into a test-tube and 580 gl of distilled water added to it. Another test-tube containing 600 gl of distilled water served as control. To the two test-tubes were added 3 ml of reagent D and mixed. The mixture were allowed to stand for 10 min after which 0.3 ml of reagent E was added and rapidly mixed. After incubation at room temperature for 30 min, the absorbance at 750 nm was taken and the protein content extrapolated from the protein calibrated curve (Fig.12). UNIVERSITY OF IBADAN LIBRARY Table 5: Protoco! for Protein determination 1 mg/ml BSA (pl) - 10 20 40 60 80 100 H20 ( ul) 600 590 580 560 540 520 500 Reagent D (ml) 3 3 3 3 3 3 3 Mix rapidly and wait for 10 minutes Reagent E (ml) 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Mix rapidly and wait for 30 minutes A 750 nm Protein Content (pg) - 10 20 40 60 80 100 UNIVERSITY OF IBADAN LIBRARY 67 j jg proteins y = 2 6 6 -A3x - 1 - 6 2 Fig. 12 Protein calibrated plot A 750nm UNIVERSITY OF IBADAN LIBRARY 68 2.4 Enzyme assays and microsomal haemoprotein content determination. 2.4.1 Determination of the activity of membrane-bound rat liver microsomal Ca~-ATPase. Endoplasmic reticulum, the Ca2+ störe, is the most important intracellular structure 2 f 2+ in the fine regulation of intracellular Ca concentration. It takes up Ca through an 2+ E-E ATPase, and can release Ca when acted upon by a number of agonists via the action of inositoltrisphosphate (Carafoli, 1987). 2+ The ability of the ER to accumulate Ca requires the hydrolysis of ATP; with the 2+ release of its Jf-P. Determination of the Ca -ATPase activity is based on the i estimation of the released X -P in the reaction pathway. REAGENTS (A) Stock Solutions (i) 3.25 M KCl: 121.23 g potassium Chloride (BDH Chemicals Ltd., England) was dissolved in 1 litre distilled water using a Volumetrie flask. (ii) 100 mM Tris: 12.114 q Tris (hydroxymethyl) methylamine (BDH Chemi­ cals Ltd., England) was dissolved in 1 litre Volumetrie flask and the vol- ume made up to the 1 litre mark with distilled water. (iii) 100 mM NaN :̂ 0.6501 g of Sodium azide (BDH Chemicals Ltd., England) was dissolved in about 90 ml of distilled water and the volume adjusted to 100 ml in a Volumetrie flask. (iv) 1 mM diqitoxyqenin: 0.003745 g of digitoxygenin (Sigma Chemicals Co., USA) was dissolved in 10 ml DMSO (BDH Chemicals Ltd., England). (B) Working Solutions (i) 40 mM CaC k 1.4702 g of calcium Chloride 2-hydrate (BDH Chemicals Ltd., Eng­ land) was dissolved in 250 ml distilled water using a Volumetrie flask. (ii) 40 mM ATP: 0.44088 g of disodium Salt of ATP (Sigma Chemicals Co., U.S.A) was dissolved in 20 ml distilled water and kept frozen at -20°c. (iii) 40 mM EGTA: 1.5216 g of ethyleneglycol-bis-(p-amino ethyl ether) NN’N'N-tetraacetic UNIVERSITY OF IBADAN LIBRARY 69 acid (Sigma Chemicals Co., USA) was suspended in about 80 ml distilled water and 1 M NaOH added until the Suspension became clear. The pH was adjusted to 7.4 and the volume rnade up to 100 ml using a Volumetrie flask. (iv) 10 % S.D.S: 50 g of Sodium dodecyl sulphate (S.D.S) (BDH Chemicals Ltd..Eng­ land) was dissolved in 500 ml distilled water using a Volumetrie flask. (v) 9 % Ascorbic acid: 45 g of L-ascorbic acid (BDH Chemicals itd., England) was dis­ solved in 500 ml distilled water using a Volumetrie flask. (vi) Reagent A: 1.25 % Ammonium molybdate in 6.5 % (v/v) H SO : 2 4 6.25 g of ammonium molybdate (Sigma Chemicals Co. USA) was dissolved in dilute Sulphuric acid (32.5 ml of conc. H^SO^ (BDH Chemicals Ltd., England) in 400 ml distilled water) in a beaker. The volume was adjusted to 500 ml with distilled water in a Volumetrie flask. (vii) Microsomal Ca2±-ATPase assay buffer; 40 mM Tris, 200 mM KCl, 2 mM NaN and 40 p M djgitoxyg en i n The buffer was prepared by mixing 100 ml of 100 mM Tris with 5 ml of 100 mM NaN , 15.625 ml of 3.25 M KCl, 10 pl of 1 mM digitoxigenin and about 100 ml of distilled water in a beaker. The pH was adjusted to 8.0 and the volume made up to the 250 ml mark with distilled water. PROCEDURE 2+ Determination of the microsomal Ca -ATPase activity was carred out as described by Famulski and Carafoli (1982). The assay medium was prepared according to Table 6. The reaction was initiated with the addition of 100 pl of 40 mM ATP and terminated after 30 min of incubation (at 30°c in a water bath) with the addition of 200 pl of 10 % S.D.S. The difference in the arnount of Pi released in the presence and absence of 1.8 mM CaCI (final 2 concentration) was used for the calculation of enzyme activity i.e the difference between the Mg2+-ATPase and the Ca2+/Mg2+-ATPase activity. The ATP blank served as control. The phosphate liberated was determined by the Fiske-Subbarow method (1925). For this, 1ml of ammonium molybdate solution (reagent A) was added to each test tube followed immediately with the addition of 1 ml of 9 % ascorbic acid. The colour developed after 30 min. was read on a spectrophotometer at 660 nm. The arnount of phosphate was extrapolated from a phosphate calibrated curve (Fig. 13). UNIVERSITY OF IBADAN LIBRARY 70 2 f Tab|e_6: Protocoi for assay for Microsomal Ca -ATPase activity ATP blank Enzyme blank Mg2+-ATPase Ca2+/Mg2+- ATPase Microsomal assay Buffer pH 8.0 (Ml) 400 400 400 400 40 mM EGTA (MO 40 40 40 40 40 mM CaCI (MO 36 36 - 36 2 H 0 (MO 304 224 240 204 2 10 fjg/pl Membrane (pl) 20 - 20 20 40 mM ATP (MD - 100 100 100 UNIVERSITY OF IBADAN LIBRARY 71 Construction of phosphate calibrated curve The construction of a Standard curve for phosphate (Fig.13) was done using 10 mM Na^HPO^ as Standard; according to Table 7. Calculation of Ca -ATPase activity: 2+ The Ca -ATPase activity was calculated according to the equation below: 2+ Ca -ATPase activity = 2x/Y pmoles P/mg Protein/hr, i where x = P liberated in 30 min. (in pmoles) Y = Protein concentration of membrane (in mg) UNIVERSITY OF IBADAN LIBRARY 72 Table 7: Protocol for inorganic phosphate calibration H 0 (Hl) 1000 995 990 980 970 960 950 2 10 mM Na HPO (gl) - 5 10 20 30 40 50 2 4 Reagent A (ml) 1 1 1 1 1 1 1 Ascorbic acid (ml) 1 1 1 1 1 1 1 Mix rapidly and wait for 30 min. Ä nm 660 P (nanomoles) 50 100 200 300 400 500 UNIVERSITY OF IBADAN LIBRARY 73 y = 402 • 31x+ 0-85 Fig. 13 Phosphate calibrated plot A 660nm UNIVERSITY OF IBADAN LIBRARY 74 2.4.2 Determination of the activity of the membrane-bound erythrocyte qhost membrane Ca~-ATPase. 2+ 2+ The plasma membrane Ca -ATPase functions by pumping out Ca from the cell against a concentration gradient. This process is ATP-driven and is accompanied with the release of the & -P of ATP. Determination of the erythrocyte ghost membrane 2+ 2+ Ca -ATPase activity like the microsomal Ca -ATPase activity is therefore based on the estimation of the released $-P. i Reaqents (A) Stock Solutions (i) 3.25 M KCl: 121.23 g of potassium Chloride (BDH Chemicals Ltd., England) was dissolved in 1 litre distilled water using a Volumetrie flask. (ii) 40 mM EGTA: 1.5216 g of ethyleneglycol-bis-(p-aminoethyl ether) N N’ N’ N’ - tetraacetic acid (Sigma Chemicals Co. USA) was suspended in about 80 ml distilled water and 1M NaOH added until the Suspension became clear. The pH was adjusted to 7.4 and the volume made up to 100 ml using a Volumetrie flask. (iii) 1 M HEPES: 23.830 g of HEPES (N-2-hydroxy ethyl piperazine-N’-2 ethane sulfonic acid) (Sigma Chemicals Co., USA) was dissolved in 100 ml distilled water using a Volumetrie flask. (iv) 100 mM MqCk 2.3805 g of anhydrous magnesium Chloride (BDH Chemicals, Ltd., England) was dissolved in 250 ml distilled water using a Volumetrie flask. (v) 40 mM CaCh 1.4702 g of Calcium chloride-2-hydrate (BDH Chemicals Ltd., England) was dissolved in 250 ml distilled water using a Volumetrie flask. (vi) 40 mM ATP: 0.44088 g of disodium salt of ATP (Sigma Chemicals Co., USA) was dissolved in 20 ml distilled water and kept frozen at -20°C.. (B) Working Solutions: (i) EGM assay buffer : 260 mM KCl, 60 mM HEPES pH.74 The buffer was prepared by mixing 20ml of 3.25 M KCl and 15 ml of 1 M HEPES and distilled water to about 200 ml in a beaker. The pH was adjusted to 7.4 and the volume made up to 250 ml in a Volumetrie flask. (ii) 8 mM ATP: This was prepared by adding 20 ml of distilled water to 5 ml of 40 mM UNIVERSITY OF IBADAN LIBRARY 75 ATP. The solution was kept frozen at -20°c. (iii) 0.8 mM EGTA: The solution was prepared by pipetting 2 ml of 40 mM EGTA into 100 ml Volumetrie flask and the volume adjusted to 100 ml with distilled water. (iv) 80 mM MgCI : The solution was prepared by pipetting 80 ml of 100 mM MgCI into 100 ml Volumetrie flask and adjusting the volume to 100 ml with distilled water. (v) 800 pM CaCI : The solution was prepared by pipetting 2 ml of 40 mM CaCI into 2 2 100 ml Volumetrie flask and adjusting the volume to 100 ml with distilled water. Procedure 2+ The EGM Ca -ATPase activity was estimated essentially as reported by Bewaji and Bababunmi (1987). The assay media were prepared as presented on Table 8. The reaction was initiated with the addition of 100 pl of 8 mM ATP and terminated after 30 min of incubation (at 30°c in a water bath) with the addition of 200 pl of 10% S.D.S. The difference in the amount of Pi released in the presence and absence of 20 pM CaCI (final 2 concentration) was used for the calculation of enzyme activity i.e the difference between the 2+ 2+ 2+ Mg -ATPase and the Ca /Mg -ATPase activity. The ATP blank served as control. The liberated inorganic phosphate was estimated according to Fiske and Subbarow (1925) using a Standard phosphate calibrated curve as contained in section 2.4.2. Calculation 2+ 2+ of (Ca + Mg ) - ATPase activity was also done as contained in section 2.4.2. UNIVERSITY OF IBADAN LIBRARY 76 2+ Table 8: Proloco! for assay for plasma membrane (EGM) Ca -ATPase activity ATP blank Enzyme blank Mg2+-ATPase Ca2+/Mg2+- ATPase EGM assay Buffer pH 7.4 (Ul) 400 400 400 400 80 mM Mgcl^ (MO 20 20 20 20 800 pM Cac! (Ul) 20 20 - 202 0.8 mM EGTA (ui) - - 100 - H 0 (Ul) 340 260 160 2402 10jjg/jjl Membrane (pl) 20 - 20 20 8 mM ATP (ul) - 100 100 100 UNIVERSITY OF IBADAN LIBRARY 77 2.4.3 Determination of the activitv of qammaqlutamvl transpeptidase (GGT). Glutamyltranspeptidase (EC.2.3.2.2) catalyzes the transfer of the Jf-glutamyl moiety of peptides to a variety of amino acid and peptide receptors (Tate and Meister, 1978). The fluorimetric assay procedure for GGT activity is based on the Observation that L-glutamic acid-}f-(7-amido-4-methyl coumarin) is non-fluorescent while its cleavage product, 7-amino- 4-methyl coumarin (AMC) is highly fluorescent - when excited at 370 nm and the fluorescence emission taken at 440 nm (Smith et al„ 1979). Reaqents (A) Stock Solutions (i) Ammediol buffer: 0.1 M ammediol, 20 mM qlvcvlglycine. and 0.1 % Triton X- 100 pH 8.5 10.51 g of 2-amino-2-methyl-1,3, propanediol (ammediol) (Sigma Chemicals Co., USA), 2.642 g of glycylglycine (Sigma Chemicals Co., USA) and 1ml of 100 % Triton X-100 (BDH Chemicals Ltd., England) were dissolved in about 950 ml distilled water and the pH adjusted to 8.5. The solution was trans­ ferred into a Volumetrie flask and the volume made up to 1 litre mark with distilled water. (ii) 1 mM 7-amino-4-methvl coumarin (AMC): 0.003504 g of 7 amino-4-methyl-coumarin (SigmaChemicals Co., USA) was dissolved in 20 ml of methoxyethanol (BDH Chemicals Ltd., England). (iii) 10 mM L-glutamic acid-X- (7 amido-4-methylcoumarin (L-X-qlutamvl AMCL 0.00761 g of L-glutamic acid-X-(7-amido-4 methyl coumarin) (Sigma Chemi­ cals Co., USA) was dissolved in 2.5 ml of methoxyethanol and sonicated to get a homogenous Suspension. (B) Working Solution: (i) 0.05 M qlvcine buffer pH 10.4 1.877 g of glycine (BDH Chemicals Ltd., England) was dissolved in about 450 ml of distilled water (in a beaker) and the pH adjusted to 10.4. The Volume was made up to 500 ml mark in a Volumetrie flask with distilled water. (ii) 20 mM Serine/100mM Borate reaqent: 0.105 g of L-serine (Sigma Chemicals Co., USA) and 1.907 g of borax UNIVERSITY OF IBADAN LIBRARY 78 (disodium tetraborate) (BDH Chemicals Ltd., England) were dissolved in about 40 ml of distilled water and made up to the mark in a Volumetrie flask to 50 ml with distilled water. (iii) Buffered Substrate for GGT assav The solution was prepared by adding 0.2 ml of 10 mM L-glutamyl-AMC to 9.8 ml of ammediol buffer pH 8.5. Procedure Liver GGT activity was measured according to the procedure of Smith et al.. (1979) using L-tf-glutamy- AMC as Substrate. The assay medium contained 142.86 pM Substrate, 70mM Ammediol, 14.0 mM glycylglycine, 0.07 % Triton X-100 and liver homogenate. This was prepared by adding 35 pl of liver homogenate (0.25 g/m!) to 250 pl of buffered Substrate in a final volume of 350 pl. Sample containing serine/Borate (2 mM/10 mM) as an inhibitor of GGT as suggested by Tate and Meister (1978), was added to check for non-specific protease activity. The mixture was incubated at 37°c for 10 minutes. The reaction was stopped by adding 2 ml of ice-cold glycine buffer pH 10.4. A Standard curve was prepared by using 7- amino-4 methyl coumarin (AMC) in ammediol buffer (0.14-3.5 nmoles). The intensity of fluorescence emission was measured at 440 nm after excitation at 370 nm. Slits = 6 and sample sensitivity = 0.3. Construction of AMC Calibrated Curve The construction of the 7-amino-4-methyl coumarin (AMC) calibrated curve (Fig.14) was done according to Table 9. Calculation of GGT Activity The fluorescence emission of the sample at 440 nm after excitation at 370 nm was used to calculate the amount of AMC released from the Substrate (extrapolated from Fig.14) and the vaiue used to calculate the GGT activity as given below. GGT activity was expressed as nmoles AMC/g liver/min. GGT activity = x/10Y nmoles AMC/g liver/min. where x = nmoles AMC released after 10 min of incubation. 10 = Period of incubation in min. Y = Weight of liver (in grams) in pipetted volume of liver homogenate. UNIVERSITY OF IBADAN LIBRARY 79 Table 9: Protocol for AMC calibration/estimation 1 2 3 4 5 6 7 8 9 Ammediol buffer pH 8.5 250 250 250 250 250 250 250 250 250 7 pM* or 70 pM+ AMC (pl) - 20* 40* 50* 75* 10+ 20+ 50+ H O (pl) 100 80 60 50 25 90 80 60 50 0.05M Glycine buffer pH 2 2 2 2 2 2 2 2 2 10.4 (ml) (ml) nmoles AMC - 0.14 0.28 0.35 0.53 0.70 1.40 2.80 3.50 Fluorescence Emission ( ext = 370 ; emis = 440) UNIVERSITY OF IBADAN LIBRARY +o •'t 80 y = 0-0975» 0-0038 Fig. 14 AMC calibrated plot UNIVERSITY OF IBADAN LIBRARY 8 1 2.4.4 Determination of microsomal Cvtochrome P450 Content. Cytochrome P is a family of haemoproteins involved in the detoxification of a wide ränge of Chemical compounds. Use is made of the fact that when the haem iron is reduced and complexed with carbon monoxide, a characteristic absorption spectrum results. The reduced, carbon monoxide difference spectrum of cytochrome P absorbs maximally at around 450 nm (hence the name) and the extinction coefficient for the wavelength couple 450- 490 nm has been accurately determined to be 91 nM 1 cm 1 thus allowing quantitative deter- mination of this haemoprotein. Reaqents (i) 0.1 M Tris pH 7.4 containinq 20 % (v/v) qlycerol: 6.057 g of Tris (hydroxymethyl) methyl amine (BDH Chemicals Ltd., England) was dissolved in about 350 ml of distilled water. 100 ml of glycerol (BDH Chemicals Ltd., England) was added and the pH adjusted to 7.4. The solution was made up to the 500 ml mark with distilled water in a Volumetrie flask. (ii) Solid Sodium dithionite (BDH Chemicals Ltd., England). (iii) Carbon monoxide (British Gas, U.K.) Procedure Cytochrome P content was determined according to Omura and Sato (1964). 100 p I of microsome sample was added to 1900 pl of Tris/glycerol buffer. The solution was mixed and distributed equally into 2 cuvettes (a sample and a reference cuvette) with a path length of 1 cm. A few grains of solid sodium dithionite were added to each cuvette and stirred gently. The sample cuvette only was then gently bubbled with carbon monoxide for approximately 1 minute. The spectrum was scanned from 400-500 nm on a Cecil series 6000 double beam spectrophotometer. Caiculation The absorbance difference between 450 and 490 nm was used for the caiculation of cytochrome P content knowing (1) that the extinction coefficient (450-490) equals 91 nM’1 cm’1 and UNIVERSITY OF IBADAN LIBRARY 82 (2) the protein concentration in mg/ml. Y x 1000 Cytochrome P nmol mg 1 content 450 91 x a where Y = absorbance difference (A - A ) 450 490 a = Protein concentration in mg/ml. 2.5 Separation of membrane Proteins by SDS-PAGE. The investigation of the molecular Constitution of membranes was revolutionized by the solubilization of membranes in SDS and Separation of the polypeptides by elec trophoresis in a poly acrylamide gei matrix in the presence of sodium dodecyl sulphate (SDS). It has been observed that the electrophoretic migration rates have a fairly predict able relation to the molecular weight of proteins, if the proteins are dissociated and denatured with sodium dodecyl sulphate before and during electro- phoresis (Weber and Osborn, 1969). SDS binds to proteins and confers on them a net negative Charge. As the peptides become saturated with SDS, they fold into helical rods surrounded by an SDS-shell. The mobility of these rods through a polyacrylamide gel is inversely proportional to the logarithm of the molecular weight of the polypeptide. Thus with this technique, it is possible to separate and identify membrane proteins. In order to get complete monomerization and unfolding of proteins, it is often useful to reduce dis- ulfide bonds with mercaptoethanol followed by a brief exposure at 100°c. Reagents (i) Stock A : 30 % Acrvlamide/0.8 % Bis-acrylamide 30 g of acrylamide (Sigma Chemical Co., USA) and 0.8 g Bis-acrylamide (Sigma Chemical Co., USA) were dissolved in a little quantity of distilled water and then made up to the mark in a 100 ml Volumetrie flask. The solution was filtered and stored in a reagent bottle at 4°c for several weeks. This solution is used for low cross-links separating gels and for all stacking gels. UNIVERSITY OF IBADAN LIBRARY 83 (ii) Stock B : 30 % Acrvlamide 1,5 % Bis-acrvlamide 30 g acrylamide (Sigma Chemical Co., USA) and 1.5 g Bis-acrylamide were dis- solved in a little quantity of distilled water and then made up to the mark in a 100 ml Volumetrie flask. The solution was filtered and stored in a reagent bottle at 4°c for several weeks. This solution is used for high cross-links separating gels. (iii) Stock C : 1.5 M Tris-HCl. pH 8.8 18.17 g of Tris (hydroxymethyl)-aminomethane (Sigma Chemical Co., USA) was dissoived in about 90 ml of distilled water and the pH adjusted to 8.8 with hydro- _chloric acid, HCl. The solution was then made up to the mark in a 100 ml Volumetrie flask, filtered and stored in a reagent bottle at 4°c for several weeks. This solution is used for buffering separating gels. (iv) Stock D : 10 % SPS fwM 10 g of sodium dodecyl sulphate, SDS (Sigma Chemical Co., USA) was dis­ soived in a little quantity of distilled water and made up to the mark in a 100 ml Standard Volumetrie flask. The solution was stored in a reagent bottle at room temperature. SDS is an anionic detergent. The solution is used in sample buffer, stacking and separating gel buffers. It.denatures proteins and imparts a uniform negative Charge. (v) Stock E : 1.25 M Tris-HCl. dH 6.8 15.15 g of Tris(hydroxymethyl)-aminomethane (Sigma Chemical Co., USA) was dissoived in about 90 ml of distilled water and the pH was adjusted to 6.8 with hydrochloric acid, HCl. The solution was made up to the mark in a 100 ml Standard Volumetrie flask with distilled water and stored in a reagent bottle at 4°c for several weeks. (vi) Glvcerol This was obtained in commercial form from BDH Chemicals Ltd., England; and - stored at room temperature. (vii) TEMED. N,N,N,N-tetramethy!ethelenediamine, TEMED (Sigma Chemical Co., USA) was obtained in commercial form and stored at 4°c. UNIVERSITY OF IBADAN LIBRARY 84 (viii) Ammonium persulphate (a) Solution for stacking gels. This was prepared by dissolving 75 mg ammonium persulphate (NH ) S O (Sigma Chemical Co., USA) in a 5 ml of distilled water. The solution should always be prepared fresh betöre pouring gel. (b) Solution for separating gels. This was prepared by diluting the ammonium sulphate solution for stacking gels in the ration 1:1 with distilled water. (ix) 10X-Stock Running buffer:250 mM Tris.1.92 M Glycine. 1 % SPS, pH 8.3. 30.9 g of Tris(hydroxymethyl)-aminomethane (Sigma Chemical Co., London), 144.1 g glycine (BDH Chemicals Ltd., England), 10 g SDS were dissolved in about 900 ml of distilled water using a magnetic stirrer. The solution was made up to the mark in 1 litre Volumetrie flask and stored in a reagent bottle at room temperature for several weeks. The pH of this solution should not and was not adjusted. (x) Working running buffer This was prepared by diluting the stock running buffer in the ratio 1:9 with distilled water. To do that, 150 ml of 10X stock was added to 1350 ml of distilled water to prepare 1500 ml solution. (xi) Sample buffer Sample buffer was prepared by adding 1.0 ml of stock E, 4 ml of stock D, 2 ml of glycerol, 1 ml of ß-mercaptoethanol, 0.2 ml of 2 % Bromophenol blue, to 11.8 ml of distilled water. This solution was stored in 1 ml aliquots and kept frozen until required for use. (xii) Preparation of gel Solutions The running gel and the stacking gel Solutions were prepared according to Table 10 and Table 11 respectively. CAUTION: It should be noted that acrylamide is neurotoxic. Observe extreme caution to minimize skin contact and inhalation. Apparatus (i) Power pack: Power supply B605 D/S (OLTRONIX, USA) UNIVERSITY OF IBADAN LIBRARY 85 (ii) Electrophoretic unit: Vertical slab gel unit SE600 series (Hoefer Scientific Instru­ ments, USA). Procedure (i) Setting up the qlass Sandwich: Two glass plates (18 x 16 cm each) were thoroughly cleaned and a Sandwich of the two made by using the spacers and clamps. The Sandwich was then cammed into the casting stand with the rubber gaskets to form a mold for the gel. (ii) Pourinq the gradient gel The gradient maker was used for this purpose. A long cannula was con­ nected to the free end of the outlet of the gradient maker and serves as a pump for filling the glass Sandwich with gel. The mixing chamber of the gradient maker was filled with the heavy (20 %) monomer solution (See Tablei^iand the reser- voir chamber with the light (5 %) monomer solution (see Table 10 also). A stirrer was placed in the mixing chamber. The valves of the gradient maker were opened and a syringe used to suck the solution to bring about the flow through it of the solution. The free end of the cannula was then placed between the glass plates of the Sandwich. When all the gel has been pumped into the glass Sandwich, the gel was water-layered. After polymerization, the water was poured off and the surface of the gel rinsed with distilled water. A stacking gel monomer solution was prepared (See Table 11) and poured by using a pasteur pipette. A comb was inserted in the stacking gel to form the sample wells. (iii) Loading the sample Using a Hamilton syringe, each well was underlayered with the sample. (iv) Final assembly of the unit After the gel has polymerized and samples applied, the top of the gel Sand­ wich was attached to the underside of the upper buffer chamber using the rubber gaskets. This assembly was lifted off the casting stand and lowered into the lower buffer chamber. Buffer was added to each chamber and the electrical Circuit was completed by fitting the safety lid onto the apparatus and connecting the leads to power supply. UNIVERSITY OF IBADAN LIBRARY 86 TABLE 10. PROTOCOL FOR THE PREPARATION OF THE RUNNING GEL 20% 5% STOCK “A” 11.33 ml 2.83 ml STOCK “C” 4.35 ml 4.25 ml H 0 - 9.18 ml ? STOCK “ D” 0.17 ml 0.17 ml GLYCEROL 0.98 ml 0.23 ml TEMED 0.01 ml 0.005 ml AMMONIUM PERSULPHATE 0.26 ml 0.335 ml UNIVERSITY OF IBADAN LIBRARY 87 TABLE 11. PROTOCOL FOR THE PREPARATION OF THE STACKING GEL STOCK “A” 2.66 ml STOCK “C” 5.28 ml STOCK “D” 0.22 ml H 0 11.22 ml 2 TEMED 28 ul AMMONIUM PERSULPHATE 0.57 ml UNIVERSITY OF IBADAN LIBRARY 88 (v) Staininq and detaininq. After removing the gel, it was fixed in a solution containing 50 % methanol in 7.5 % Acetic acid for about 30 minutes and then stained with Coomasie brilliant blue solution for 45 minutes. The gel was destained with 20% methanol in 7.5 % acetic acid solution. 2.6 Analysis of data: All data were statistically analyzed using the Software package “ MINITAB” (MINITAB Inc., Ü.S.A.). UNIVERSITY OF IBADAN LIBRARY 89 CHAPTER THREE EXPERIMENTS AND RESULTS Experiment 1: Characterization of rat liver microsomal membrane-bound Ca2+-ATPase. Introduction The oncogenic transformation of cells from normal to malignant phenotype is associated with a loss of the ability of Calcium to control cell proliferation (Murphy and Fiskum, 1987). 2+ ln addition, tumour cells generally contain abnormally high levels of endogenous Ca (Fiskum, 1985); suggesting that the calcium regulatory System of tumour cells is defective. The control of calcium is essentially performed by the reversible complexation to specific 2+ proteins. Soluble proteins contribute to Ca buffering, but membrane-intrinsic proteins play 2+ the main role in buffering of cell calcium. They control Ca very precisely and with high affinity (ATPases) or with lower affinity (channels, exchangers, the electrophoretic uniporter) (Carafoli, 1987). The endo(sarco) plasmic reticulum is responsible for the fine regulation of intracellular calcium (Carafoli, 1987). Most of the reports on the sarco(endo) plasmic reticulum were 2+ performed with the sarcoplasmic reticulum (SR) mainly because the Ca -ATPase content of the SR is very abundant, representing as much as 90% of the total protein of SR, whereas 2+ the content of Ca -ATPase in the endoplasmic reticulum (ER) is relatively minor (Carafoli, 1987; Penniston, 1983). More recently, the ER has gained considerable attention because 2+ of the discovery that its Ca pool is sensitive to inositoi 1,4,5 trisphosphate (IP ) the mediator 2+ ' 3 in the release of intracellular Stores of Ca (Carafoli, 1987). 2+ The paucity of Information on the ER Ca -ATPase and the fact that the ER of the liver cell is the first cellular organelle which is disrupted by Chemical poisoning (Moore et al.. 1976) has prompted, the partial characterization of the membrane bound ER Ca2+-ATPase as a 2+ prerequisite to the investigation of the role of the ER Ca -ATPase in DDT-induced liver tumour promotion. UNIVERSITY OF IBADAN LIBRARY 90 Procedure Three male Wistar rats weighing 150-200 g were used for the experiment. Rats were housed together in a plastic cage. The animals were maintained in a 12hour light/12 hour dark regime. Temperature was kept at constant 70°F + 2°F. The relative humidity was 50 -*5%. All animals were given MRC 41B diet and water ad.lib. Animals were CO^ exsanguinated. Livers were quickly removed and placed on ice-cold isolation buffer. Each liver sample was separately weighed and used for the preparation of endoplasmic reticulum (light microsomal fraction) as described in section 2.2.1. The protein content of the membrane preparations was determined as described in section 2.3, and the activity of the membrane-bound microsomal Ca2+-ATPase determined as described in section 2.4.1. 2+ Assessment of the pH-dependence of the membrane-bound Ca -ATPase was carried out by using microsomal assay buffers with pH ranging from 6.0 to 8.5. To establish the Ca2+- dependence profile, 40 mM CaCI^ was added to the assay medium to final concentrations between 1 and 4 mM CaCK The free calcium concentration was estimated by using a Computer Programme developed by Fabiato and Fabiato (1979). The ATP-dependence of the membrane-bound enzyme was determined by adding 40 mM ATP to the assay medium to final concentrations ranging from 1 to 7 mM ATP. The S.D.S poly acrylamide gel electro- phoresis of microsomal proteins was carried out as described in section 2.5. In Order to evaluate the responsiveness of the membrane-bound enzyme to calmodulin and Vanadate, the modulators were added to the assay media to a final concentration of 2.0 gg/ml (calmodulin) and .75 pM (vanadate). Results 2+ The membrane-bound rat liver microsomal Ca -ATPase has a specific activity of 4.543 + 0.857 pmole Pi/mg Protein/hr. at pH 8.0 (Fig.15). It has a high affinity for Ca2+ (Fig. 16), but the membrane-bound enzyme did not seem to obey the Michaelis-Menten Kinetics (Fig. 17). Furthermore, the enzyme in unfractionated microsomes was not sensitive to calmodulin but siightly sensitive to vanadate (Table 12). The S.D.S. PAG electrophoresis of microsomal protein and the densitometric scan of the resolved polypeptide revealed the presence of 19 polypeptide (Plate 1,Table 13) with molecu- larweights ranging between 14,000 and 100,000 dalton. UNIVERSITY OF IBADAN LIBRARY 91 FIG. 15 pH-dependence of the activity of rat liver membrane-bound microsomal Ca^+-ATPase UNIV - A T P a s e act iv ityERSITY OF IBADAN LIBRARY 92 - I° g ,0 (?a2i ] FIG. 16 Ca2+-dependence of the activity of rat liver membrane-bound microsoma! Ca2+-ATPase UNIVERSITY OF IBADAN LIBRARY 93 FIG. 17 ATP-dependence of the activity of rat liver membrane-bound microsomal Ca2+-ATPase UNIVERSITY OF IBADAN LIBRARY Table 12: Effects of calmodulin and vanadate on the membrane-bound rat liver 2± microsomal Ca' -ATPase. Ca2+-ATPase activity (limole P/mg Protein/hr) Control (no additions) 2.502 + 0.134 2 ng/ml calmodulin 2.415 + 0.078 0.75 |iM vanadate 2.030 + 0.632 UNIVERSITY OF IBADAN LIBRARY 95 Plate 1 SDS-PAGE and densitometric scan of the microsomal proteins of normal rat. UNIVERSITY OF IBADAN LIBRARY 96 Table 13: The percentage composition of the microsomal polypeptides of a normal rat as resolved by SDS-Page and the densitometric scanning (Platel) Bands % Composition 1 0.59 2 0.58 3 4.79 4 1.99 5 14.82 6 25.10 7 14.82 8 8.72 9 3.78 10 1.56 11 7.46 12 1.22 13 1.51 14 1.16 15 2.00 16 1.24 17 3.71 18 2.43 19 2.41 UNIVERSITY OF IBADAN LIBRARY 97 Experiment 2: Short-term in-vivo studies of the effect of dicophane on the activity of rat liver microsomal membrane-bound Ca2+-ATPase. Introduction Dicophane (DDT) is a well known liver tumour promoter (Klaunig et al, 1990; Williams, 1983; Williams et al.. 1981; Kitchin and Brown, 1987). It has been shown to enhance the effect of 2-acetylaminofluorene, diethylnitrosamine, and 3’-methyl-4-(dimethylamino)-azobenzene in inducing liver tumours in rats and mice when given after these carcinogens (Kitagawa et aj., 1984; Nishizumi, 1979; Peraino et al.. 1975; Williams and Numoto, 1984). Although the use of this Chemical is curtailed or banned in the developed countries, they are still widely used in the developing countries of Africa and Asia (Corbett,1974; WHO/UNEP, 1988; Rojanapo et al.. 1988). This is a matter of concern as primary liver cancer (PLC) is one of the most prevalent forms of cancer in Africa and Asia (Parkin et al.. 1988). The mechanism of action of liver tumour Promoters is poorly understood. Nevertheless, an inhibition of intercellular communication has been advocated as a mechanism of the action of liver tumour Promoters (Klaunig et al.. 1990; Williams, 1983; Williams et al.. 1981); but it has long been recognized that gap junctional intercellular commu­ nication is a function of cytoplasmic calcium concentration. Gap junctional intercellular communi­ cation decreases as cytoplasmic calcium concentration increases (Loewenstein, 1979). This Observation suggests that the process of intercellular communication is perhaps a secondary event in the action of liver tumour Promoters. A rapid and transient increase in cytosolic free Ca is part of the first response of cells to mitogens including growth factors and tumour Promoters (Berridge, 1987). The endo(sarco) plasmic reticulum is the most important structure in the fine regulation 2+ of intracellular Ca concentration (Carafoli, 1987). Endoplasmic retiqulum (ER), the intracel- 2+ 2+ 2+ lular Ca störe, accumulates Ca from the ambient through the ER Ca -ATPase. Recently, it was reported that thapsigargin-a skin tumour promoter-inhibits the ER-Ca2+-ATPase 2+ (Thastrup, et al, 1990) and that the inhibition is accompanied with Ca mobilization. Since the tumour promoter-induced inhibition of ER Ca -ATPase is accompanied with 2+ Ca mobilization, it appears that the enzyme might play a crucial role in the process of intercellular communication and the mechanism of action of tumour Promoters. The aim of the UNIVERSITY OF IBADAN LIBRARY 98 experiment was to determine the effect of the specific liver tumour promoter,dicophane on the liver ER Ca2+-ATPase. Procedure Male Wistar rats weighing 150-200 g were used for the experiment. Rats were housed 3 per cage in plastic cages. The animals were maintained in a 12 hour light/12 hour dark regime. Temperature was kept at constant 70°F + 2°F. The relative humidity was 50 + 5%. All animals were given MRC 41B diet and water ad.libitum. The experiment was performed in three parts: Part one was designed to investigate the effect of the liver tumour promoter, dicophane (DDT). To accomplish this, six animals were divided into 2 groups A and B of 3 animals each. Group A (control) were given a single dose of subcutaneous injection of Olive oil and group B (DDT-treated) were given a single dose of subcutaneous injection of 2 pl/g.body wt. of 37.5 mg/ml of DDT in olive oil, corresponding to 75 mg/kg.body wt. of DDT. Animals were fasted overnight on day seven and CO? exsanguinated on day eight. The dosage is as reported by McLean and McLean (1966). Part two investigated the effect of another liver tumour promoter, phenobarbital (PB). For this investigation, six animals were also divided into 2 groups A and B of 3 animals each. Group A (control) were injected with 0.1 M NaOH (pH 9.0) intraperitoneally and group B (PB-treated) were injected with 4 pl/g.body wt. of 20 mg/ml of phenobarbital dissolved in 0.1 M NaOH pH 9.0, corresponding to 80 mg/kg.body wt. of phenobarbital once everyday for 4 days. Animals were fasted overnight on day four and CO 2 exsanguinated on day five. Part three was set up to determine the effect of the carcinogenic initiator aflatoxin B .̂ To do this, six animals were also divided into 2 groups A and B of 3 animals each. Group A (control) were injected with a single dose of 2 pl/g.body wt. of 1 %(v/v) of DMSO intraperi­ toneally and group B (aflatoxin B^-treated) were injected with a single dose of 2 pl/g.body wt. of 0.36 mg/ml of aflatoxin B (dissolved in DMSO) intraperitoneally. Animals were fasted overnight on day six and CO? exsanguinated on day seven. Livers of animals were quickly removed after they were socrificed and placed on ice-cold isolation buffer. Each liver was separately weighed and used for the preparation of endoplas- UNIVERSITY OF IBADAN LIBRARY 99 mic reticulum (light microsomal fraction) as described in section 2.2.1. The protein content of the microsome preparations was determined as described in section 2.3, and the activity of the membrane-bound microsomal Ca2+-ATPase determined as described in section 2.4.1 The S.D.S-PAG electrophoresis and the densitometric scan of the resolved microsomal pro- teins were carried out as described in section 2.5. Results After eight days of the administration of DDT to rats, significant increases (P < 0.05) in the relative liver weight (liver weight/body weight) and in the cytochrome P content were 450 observed for DDT-treated rats compared with control animals (Table 14a). However, the microsomal Ca2+-ATPase activity was significantly lower (P < 0.01) while an insignificant decrease (P < 0.05) in liver GGT activity was observed. Also, after four days of the administration of phenobarbital to rats, significant increases (P < 0.05) were observed in the relative liver weight (liver wt./body wt.) and in the cytochrome 2+ P content (Table 14b). A significant decrease (P < 0.05) in the Ca -ATPase activity was 450 also observed; and the liver GGT activity was significantly increased (P < 0.05). The values obtained for aflatoxin B^-treated rats including the relative liver weight (liver wt./body wt.) cytochrome P content, and microsomal Ca2+-ATPase activity were not sig- 450 nificantly different (P > 0.05) from the values obtained for control animals (Table 14c). 2+ Although there were changes in the specific activity of the microsomal Ca -ATPase of promoter-treated rats, the affinity of the enzyme for calcium remained unchanged (Fig. 18). Comparison of the S.D.S. PAG electrophoretic pattem and the densitometric scan of the resolved polypeptide (Plate 2, Table 15) of microsomal proteins of normal, DDT-treated and PB-treated rats showed changes in the relative content of some of the microsomal proteins UNIVERSITY OF IBADAN LIBRARY 100 Table 14(a-c): Effects of (a) dicophane (DDT), (b) phenobarbital (PB) and (c)^flatoxin-B (AFB ) on the membrane-bound rat liver microsomal Ca +-ATPase kctivit^ and other biochemical parameters. (a) Relative Cytochrome+ Liver GGT$ Ca2+-ATPaseN= Liver wt. P content activity activity 450 control 0.0350 1.256 25.160 3.290 (n =3) ±0.0013 4p.162 +5.550 +0.754 DDT-treated 0.0451' 1.947 8.747 1.699” +0.0007 +0.281 +0.552* +0.189 (b) Relative Cytochrome+ Liver GGT$ Ca2+-ATPaseN= Liver wt. P content activity activity aro control 0.0388 0 .9 37 13.200 3.214 (n =3) +0.0030 453.090 +2.750 H53.470 PB-treated 0.0489* 2 .7 4 5 ” 25.015* 1.261 (n =3) j5).0013 +0.214 j53.340 +0.071 (c) Relative Cytochrome* Liver GGT$ Ca2+-ATPaseN= Liver wt. P content activity activity a r n control 0.0387 0.414 - 3.260 (n =3) -rO.OOl 8 +0.017 - +0.913 AFB -treated 0.0356 0.416 - 3.492 (n=J) +0.0061 +0.077 +0.644 The value in parenthesis represents the number of animals and microsome preparations. *p < 0.05 ** P < 0.01 n - no of membrane preparations or animals. + - nmole Cyt. P /mg protein 450 $ - nmole AMC/g liver/min. N =-ji mole Pi/mg protein/hr. Relative Liver wt. is the ratio of Liver weight to body weight. UNIVERSITY OF IBADAN LIBRARY 00 ii 4 > £ Q) O < 2 N orm al D D T -T reated c/> _ P B -T reated CO □- E J— < ^ . Q. O+l 0) CO o u E =1 0 — j---------------------------- »--------------------------1+ 9 8 7 pCa Ca -dependence of the activity of membrane-bound microsomal Ca2+-ATPase. of Normal, DDT- and PB-treated rats UNIVERSITY OF IBADAN LIBRARY 102 Plate 2. SDS-PAGE and densitometric scan of the microsomal proteins of (1) normal, (2) DDT- and (3) PB-treated rats UNIVERSITY OF IBADAN LIBRARY 103 Table 15: The percentage composition of the microsomal polypeptides of normal (1), DDT- (2) and PB(3)- treated rats as resolved by SDS-PAGE and the densitometric scanning (Plate 2) % Composition Bands 1 2 3 1 0.05 2 1.41 3 0.67 1.09 1.17 4 1.52 1.37 0.96 5 1.95 2.25 1.77 6 2.30 2.01 1.83 7 4.62 3.95 4.64 8 1.94 0.91 ) 2.21 9 0.99 0.93 10 4.11 3.00 2.57 11 7.57 7.56 8.37 12 32.00 33.12 41.18 13 8.47 10.21 13.82 14 8.44 4.63 ) 1.95 15 3.65 2.14 16 0.46 0.39 0.56 17 1.18 0.68 ) 1.38 18 0.55 0.32 19 4.97 4.67 2.91 0.78 20 1.94 1.91 ( 1.01 21 3.06 3.00 2.34 22 0.82 1.07 0.73 23 4.78 6.05 4.17 24 2.30 3.47 1.47 25 0.96 4.79 4.48 UNIVERSITY OF IBADAN LIBRARY 104 Experiment 3: Investigation of the action of low protein intake on the activity of rat liver microsomal membrane-bound Ca2+-ATPase. Introduction A number of studies have shown that the development of cancer can be modified either by changing the quality or the quantity of dietary protein. This has been unequivocally demonstrated for tumours of the liver (Tannenbaum and Silverstone, 1953; Rizvi, et al.. 1987; Schulsinger, et al.. 1989; Mathur and Nayak, 1989). However, the reports of actual experi­ mental studies are conflicting. While some scientists (Rizvi et al.. 1987; Mathur and Nayak, 1989) suggest that protein malnutrition could enhance or promote tumour development, some others (Madhavan and Gopalan, 1968; Schulsinqer et al.. 1989) have reported an inhibitory effect (Rizvi et al.. 1987). Many tumour Promoters are almost exclusively membrane active agents (Troll and Weisner, 1985), and low protein intake has been shown to have a pronounced effect on the structural integrity of biomembranes (Coward, 1971). This similarity suggests that low protein diet might act as a tumour promoter. This experiment was designed to test the hypothesis that low protein intake acts as a liver tumour promoter using the inhibition of liver ER Ca?'-ATPase as a mechanistic model. Procedure Male weanling Fisher F344 rats were used for the experiment. Animais were divided into two groups A and B of four and seven animals. Group A were given normal (MRC 41B) diet and water ad. libitum while group B were given low protein diet (5 % protein) and water ad. libitum for twelve weeks. All animals were housed in plastic cages and were maintained in a 12 hour light/12 hour dark regime. Temperature was kept at constant 70°F + 2°F. The relative humidity was 50 +.5 %. Animals were fasted overnight and CO^ exsanguinated after twelve weeks of feeding. Liver of animals were quickly removed after they were sacrificed and placed on ice-cold isolation buffer. Each liver was separately weighed and used for the preparation of endoplas- mic reticulum (light microsomal fraction) as described in Section 2.2.1. The protein content of the microsome preparations was estimated as described in Section 2.3, and the activity of the UNIVERSITY OF IBADAN LIBRARY 105 microsomal Ca2 -ATPase determined as contained in Section 2.4.1. The S.D.S, PAG elec- trophoresis was carried out as described in Section 2.5. Results There was no significant difference (P > 0.05) in the relative liver weight (liver weight/ body weight) for protein-malnourished animals compared with well-fed Controls (Table 16). The cytochrome P content and the liver GGT activity also remained normal. A significant depression (P < 0.05) in the microsomal Ca2+-ATPase activity was, however, observed. A comparison of the S.D.S. PAG electrophoretic pattem of normal and protein-malnour­ ished animals showed differences in the relative content of some of the polypeptides (Plate 3, Table 17). UNIVERSITY OF IBADAN LIBRARY 106 Table_16: The Ca^-ATPase activity and other biochemical parameters of normal and protein-malnourished rats. Relative Cytochrome' Liver GGT' Caa+-ATPaseN= Liver wt. P content activity activity 450 Controls 0.0306 0.621 22.871 3.907 (well-fed) (n=4) +0.0009 +0.126 +17.090 +1.552 Protein-mal 0.0292 0.729 33.015 1.226 nourished (Low protein diet) (n = 7) +0.0012 +0.093 +29.201 +0.268 * P < 0.05 ‘P < 0.01 + nmole cyt. P /mg protein 450 $ nmole AMC/g liver/min. N jimole P/mg protein/hr. n = no of animals or membrane preparations. Relative Liver weight is the ratio of Liver weight to Body weight. UNIVERSITY OF IBADAN LIBRARY 107 Plate 3. SDS-PAGE and densitometric scan of the microsomal proteins of (1) normal, (2) protein-malnourished animals UNIVERSITY OF IBADAN LIBRARY 108 Table 17: The percentage composition of the microsomal polypeptides of normal (1) and protein-malnourished (2) rats as resolved by SDS-PAGE and the densitometric scanning (Plate 3). % Composition Bands 1 2 1 0.59 0.70 2 0.58 0.91 3 4.79 3.85 4 1.99 1.64 5 14.82 14.20 6 25.10 22.17 7 14.82 10.36 8 8.72 11.11 9 3.78 9.65 10 1.56 1.64 11 7.46 7.47 12 1.22 ) 2.29 13 1.51 14 1.16 1.09 15 2.00 1.05 16 1.24 1.18 17 3.71 3.98 18 2.43 2.74 19 2.41 3.92 UNIVERSITY OF IBADAN LIBRARY 109 Experiment 4: Effect of carcinogenic promotion (by dicophane administration) on liver 2+ microsomal membrane-bound Ca -ATPase after carcinogenic initiation (by aflatoxin B treatment) in rats. Introduction Hepatocellular carcinoma is one of the ten most frequent cancers encountered world- wide, accounting for 4 % of the total. While relatively rare in Europe and the Americas, it is frequent in the People’s Republic of China and in Africa (Parkin et aL, 1988). The aetiology of this cancer has been associated with two major risk factors, persistent hepatitis B virus (HBV) infection and exposure to dietary aflatoxins, although other aetiological agents, like smoking and some occupational exposures, have also been implicated (Cova et al.. 1990). DDT, the persistent and lipophilic pesticide is widely used in the control of insect vectors of disease in several parts of the world including Africa where the incidence of human primary liver Cancer (PLC) is highest (Bababunmi, 1976; Rojanopo et al... 1988; Rizvi et al., 1987). Likewise, malnutrition, particularly protein malnutrition is widespread and endemic in develop- ing countries and is universally recognized as the single most important contributor to the high sickness and death rates in childhood in these countries (Hendrickse et aj., 1982). It is probable therefore, that dicophane (DDT) and protein malnutrition acting in concert with en­ vironmental carcinogens which are also widespread in the developing countries might influ- ence the incidence of hepatocellular carcinoma in Africa and Asia. Aflatoxin B̂ (AFB^), a metabolite of the fungi Aspergillus flavus and Aspergillus paraciticus. is a widespread food contaminant and a potent hepatocarcinogen for several species of animals including primates (Busby and Wogan, 1981). Epidemiological studies from some developing countries suggest a relationship between ingestion of AFB -contaminated food and increased frequency of human liver cancer (Bababunmi, 1976; Alpert et al; 1971; Shank et al.. 1972; Peers and Linsell, 1973). This experiment examined the effect of carcinogenic promotion on liver microsomal membrane-bound Ca2+-ATPase. Procedure This experiment was performed in two parts. Part one investigated the long-term effect of the administration of promoter only to rats and also the effect of the treatment after carci­ nogenic initiation with aflatoxin B on the microsomal Ca2+-ATPase. Seventeen eight weeks UNIVERSITY OF IBADAN LIBRARY 110 old Fisher F344 rats divided into four groups A, B, C and D of four, four, five and four animals respectively were used for the investigation. Animals were treated as described in Fig. 19. Groups A and B were given 2 pl/g.body wt. of 6.25 % (v/v) of DMSO in oil and groups C and D were given 2 pl/g. body wt. of 0.125 mg/ml of aflatoxin B̂ (dissolved in DMSO) in oil corresponding to 0.250 mg/kg. body wt. orally for 10 days (5 days per week for 2 weeks). Düring promotion, group A and D were given a single subcutaneous injection of 2 pl/g. body wt. of oil and groups B and C were given a single subcutaneous injection of 2 pl/g. body wt. of 37.5 mg/ml of DDT in oil corresponding to 75 mg/kg.body wt. of DDT. All animals were given normal (MRC 41B) diet and water ad.libitum for twelve weeks. Part two investigated the effect of aflatoxin B ingestion on the microsomal Ca2+-ATPase of animals on low protein diet i.e. protein-malnourished animals. This procedure was adopted because it is more physiological than the pretreatment of animals with aflatoxin B followed 1 by placement on low protein diet. Weanling Fisher F344 rats divided into 2 group A and B of seven and nine animals were used for the investigation. All animals were given low protein diet (5 % protein) and water ad. libitum for twelve weeks. After one week of feeding on low protein diet, group A were given 2 pl/g.body wt. of 6.25 % (v/v) of DMSO in oil and group B were given 2 pl/g.body wt. of 0.125 mg/ml of aflatoxin (dissolved in DMSO) in oil orally for 10 days (5 days per week for 2 weeks); corresponding to 0.250 mg/kg.body wt. of aflatoxin B . The procedure is depicted in Fig.20. After twelve weeks animals were CO^ exsanguinated. Livers were quickly removed after animals were sacrificed and placed on ice-cold isolation buffer. Each liver was separately weighed and used for the preparation of endoplasmic reticulum (light microsomal fraction) as described in section 2.2.1. The protein content of the microsome preparations was deter- mined as described in Section 2.3, and the activity of the membrane-bound microsomal Ca2*- ATPase determined as contained in section 2.4.1. The S.D.S. PAG electrophoresis was carried out as described in Section 2.5. Results After four weeks of the administration of DDT to rats, there were no differences in the Cytochrome P content and the liver GGT activity; but the relative liver weight (liver wt./ 4t>ü body wt.) was significantly differerii (P < 0.05) frorn ine vaiue obiaineu for coniroi ariimais. 2+ There was also an insinnificant decrease in the microsomal Ga -ATPase acti'/‘tv rTshio UNIVERSITY OF IBADAN LIBRARY 111 initiation Promotion '''//tfwf'///'. End 1 2 3 4 5 6 7 8 9 10 11 12 weeks > normal diet < Fig.19 Schematic diagram of the carcinogenic treatment protocol for animals on normal diet UNIVERSITY OF IBADAN LIBRARY Initiation ■ n p i l § End8 1 1 2 3 4 5 6 7 8 9 10 11 12 We.e,KS > Low protein diet < Fig. 20 Schematic diagram of the carcinogenic treatment protocol for animals on low protein diet UNIVERSITY OF IBADAN LIBRARY 113 2+ The relative liver weight, Ca -ATPase activity and Cytochrome P content of DDT- 450 treated animals were not different from those of DDT-treated animals after carcinogenic ini- tiation with aflatoxin (Table 18b). The liver GGT activity was, however, significantly increased (P < 0.05). There were observed increases (P < 0.05) in the relative liver weight as weil as the 2+ 1 Ca -ATPase activity of protein-malnourished animals treated with aflatoxin B compared with protein-malnourished Controls; but the cytochrome P content and the liver GGT activity were not different (P > 0.05) from the values obtained for control animals (Table 18c). Aflatoxin B1 treatment alone (carcinogenic initiation) had no effect on all measured bio- chemical parameters after nine weeks (Table 18d). UNIVERSITY OF IBADAN LIBRARY 114 Table 18 (a-d): Long-term study on the effect of dicophane (DDT) administration after carcinogenic initiation with aflatoxin B on the rat liver membrane-bound Ca2+-ATPase. (al Relative Cytochrome+ Liver GGT$ Ca2+-ATPaseN= Liver wt. P content activity activity 450 per g liver/ Der mg protein/liver control 0.0302 0.794 7.900 4.560 (n=4) +0.0009 +0.134 +3.030 +1.540 DDT-treated 0.0327 1.107 7.080 2.654 (n=4) +0.0009 +0.219 _+2.830 +0.677 (b) Relative Cytochrome+ Liver GGT$ Ca2+-ATPaseN= Liver wt. P content activity activity 450 , DDT-treated 0.0327 1.107 7.080 2.654 (n=4) +0.0009 +0.219 +2.830 +0.677 AFB +DDT 0.0340 1.245 13.550* 2.930 (n=5) +0.0026 +0.089 +4.180 +1.470 (c) Relative Cytochrome+ Liver GGT$ Ca2+-ATPaseN= Liver wt. P content activity activity 450 Protein- malnourished 0.0292 0.729 33.015 1.226 (n=7) +0.0013 +0.093 +29.201 +0.268 Protein-mal nourished 0.0333* 0.653 19.772 1.886* and AFB -treated (n = 9) 1 +0.0021 +0.094 +13.652 +0.677 UNIVERSITY OF IBADAN LIBRARY 115 (d ) Relative Cytochrome+ Liver GGT$ Ca2+-ATPaseN= Liver wt. P content activity activity 450 Control 0.0305 0.735 6.630 3.519 (n=4) +0.0015 +0.121 +2.410 +0.564 AFB -treated 0.0314 0.798 10.590 3.214 (n=4) +0.0009 +0.076 +^2.730 +0.470 + nmole Cyt. P /mg protein 450 $ nmole AMC/g liver/min. N mole P/mg protein/hr. * P < 0.05 n = no of animals or microsome preparations. Relative Liver weight is the ratio of Liver weight to body weight. UNIVERSITY OF IBADAN LIBRARY 116 The S.D.S. PAG electrophoresis of microsomal proteins and the densitometric Scan of the resolved polypeptides (Plate 4, Table 19) of protein malnourished animals and theircoun- terparts (protein-malnourished animals after carcinogenic initiation with aflatoxin B ) showed r differences in the relative content of some of the proteins. UNIVERSITY OF IBADAN LIBRARY 1 17 Plate 4 SDS-PAGE and densitometric Scan of the microsomal protein of animals on low protein diet (1) without and (2) with aflatoxin Bt treatment UNIVERSITY OF IBADAN LIBRARY 1 IX Table 19: The percentage composition of the microsomal polypeptides of protein- malnourished (1) and protein-malnourished rats treated with aflatoxin B (2) as resolved by SDS-PAGE and the densitometric scanning (Plate 4) % Composition Bands 1 2 1 0.70 0.44 2 0.91 1.20 3 3.85 4.04 4 1.64 0.90 5 14.20 12.56 6 22.17 21.23 7 10.36 8.48 8 11.11 9.38 9 9.65 10.31 10 1.64 1.89 11 7.47 9.98 12 2.29 2.49 13 1.09 1.24 14 1.05 0.89 15 1.18 1.34 16 3.98 5.06 17 2.74 3.57 18 3.92 4.91 UNIVERSITY OF IBADAN LIBRARY 119 2+ Experiment 5: An assessment of the activity of erythrocyte membrane-bound Ca -ATPase in humans suffering from protein-energy-malnutrition and liver cancer. Introduction There is abundant evidence to suggest that an inhibition of the liver endoplasmic reticu- lum (ER) Ca2+-ATPase could be a useful marker of the action of liver tumour Promoters (Lowrey et al.. 1981; Recknagel et al.. 1982; Long and Moore, 1986; Thastrup et al.. 1990; Adenuga et al: 1992). However, the limitation of using human liver for diagnostic purposes might hamper the extrapolation of the result to man. One way to circumvent this problem, is to examine the erythrocyte ghost membrane (EGM) plasma membrane Ca2+-ATPase, since blood is more readily available. Furthermore, protein kinase C - the well known receptor of tumour Promoters has been shown to interact with and modulate the activity of the EGM Ca2+-ATPase (Smallwood et al, 1988). This preliminary investigation was conducted to compare the erythrocyte ghost membrane (EGM) Ca2+-ATPase activity of normal humans with those suffering from liver cancer. Furthermore, the Ca2+-ATPase activity of kwashiorkor humans was compared with that of their normal counterparts. Kwashiorkor (protein-energy-malnutrition) has been shown to be a positive modulator of hepatocarcinogenesis (Rizvi et al., 1987; Mathur and Nayak, 1989). It has been shown that the action of tumour Promoters is accompanied with an inhibition of the plasma membrane Ca2+-ATPase (Cerutti et al.. 1989; Parola et a}., 1990; Lowrey et aL, 1981a, 1981b). Procedure Blood samples were obtained from patients who were newly identified as having Kwashiorkor (by the paediatric Department) or liver cancer (by the Department of Medicine) of the University College Hospital, Ibadan. The patients were not receiving any dietary therapy or medication at the time of collection of blood. Normal human blood samples were collected from healthy donors in the same age group as the kwashiorkor and liver cancer patients. All samples were collected in acid-citrate-dextrose buffer and stored at 4°c before they were used for membrane preparation. Erythrocyte ghost membranes (EGM) were prepared as described in section 2.2.2, and the membrane protein determined as described under Section 2.3. The membrane-bound UNIVERSITY OF IBADAN LIBRARY 120 Ca?+-ATPase activity was measured as described in Section 2.4.2. Results 2+ The specific activity of the erythrocyte ghost membrane (EGM) Ca -ATPase of liver cancer patients is not significantly different (P > 0.05) from values obtained for adult Controls (Table 20). Also, the specific activity of the enzyme in the EGM of kwashiorkor patients although higher, is not significantly different (P > 0.05) from values obtained for paediatric Controls. However, the enzyme in the EGM of both liver and kwashiorkor patients were less sensitive to the stimulatory effect of calmodulin compared to Controls (Fig.21) but these values also did not reach Statistical significance (P > 0.05). UNIVERSITY OF IBADAN LIBRARY 121 Table 20: The erythrocyte ghost membrane Ca2+-ATPase activity of kwashiorkor and liver cancer patients. 2+ Ca -ATPase activity - Calmodulin + Calmodulin Adult Control (5) 0.189 + 0.067 0.370+^0.169 Liver Cancer (5) 0.202 + 0.055 0.365 + 0.112 Paediatric Control (6) 0.092 + 0.047 0.177 + 0.098 Kwashiorkor (8) 0.120 + 0.037 0.180 + 0.064 The number in parenthesis represents the number of membrane preparations using dif­ ferent blood samples. UNIVERSITY OF IBADAN LIBRARY 122 4* o o '00 - 80 - 160- 40 - 20- 100- 80 - 60 - 40- 20 FIG. 21 The responsiveness of the erythrocyte ghost membrane 2+ Ca -ATPase of control, kwashiorkor and liver cancer patients to calmodulin PC - Paediatric Control KW - Kwashiorkor AC - Adult Control LC - Liver Cancer UNIVERSITY OF IBADAN LIBRARY 123 CHAPTER FOUR DISCUSSION The majority (70-90%) of human cancers have been associated with environmental causes. Actually, a number of food additives, pesticides, insecticides, and industrial Chemicals introduced commercially during the last 50 years have exhibited carcinogenic properties in animal models. Historically, Chemical exposure due to occupation or to drugs has led to human cancers (Weisburger and Hom, 1982). The liver has received considerable attention as a target for Chemical carcinogenesis since the discovery of liver cancer induction with o-amino-azotoluene (Färber, 1980). A variety of Chemicals of diverse structure, radiations and two viruses have been implicated in the genesis of experimental liver cancer in several species and various Chemicals including hormones and possibly one virus, hepatitis B, in humans (Färber, 1980). Be- cause of its susceptibility to cancer induction under a variety of conditions and because of a large and growing body of knowledge about its cellular and molecular biology andpathology, the liver has been examined many times with many carcinogens for bio- chemical, physiological and morphological alterations as a function of time during develop­ ment of cancer. Such studies have consistently found many cellular and tissue changes involving hepatocytes and other types of cells in the liver before the development of unequivocal hepatocellular carcinoma, the commonest form of liver cancer (Färber, 1980). Indeed, primary liver cancer (PLC) is one of the most prevalent forms of cancer in Africa and Asia (Parkin et aL, 1988). Currently, attention is being focused on the mechanisms of action of tumour Promot­ ers. Skin is the classical target organ of experimental multi-stage tumourigenesis and the tumour promoter TPA(12-0-tetradecanoyl-phorbol-13-acetate) is the classical skin tumour UNIVERSITY OF IBADAN LIBRARY 124 Promoter. Three biochemical mechanisms have been proposed to explain the action of some skin tumour Promoters. (i) Stimulation of the signal-transduction enzyme protein kinase C ( Castagna et ab, 1982). (ii) Inhibition of several protein phosphatases (Bialojan and Takai, 1988). (iii) Inhibition of the endoplasmic reticulum Ca?+-ATPase (Thastrup ef ab, 1990). The Identification of these biochemical mechanisms for the action of skin tumour Promoters calls attention to the importance of understanding the cellular mechanisms of action of other groups of tumour Promoters; particularly liver tumour Promoters. It has been well documented though, that a rapid and transient increase in cytosolic free calcium is part of the first response of cells to mitogens including growth factors and tumour Promoters (Berridge, 1987). This Observation suggests that the calcium regulatory System plays a crucial role in the process of tumour promotion. The endo (sarco) plasmic reticulum is the membrane System responsible for the fine regulation of calcium in the cytosol. It contains an ATPase which has high affinity for calcium. Although there is a wealth of information on this membrane System and its enzyme, most of the work on the membrane System has been traditionally carried out on the microsomal fraction of muscle cells (sarcoplasmic reticulum); and only very little information is available on the ATPase of the microsomal fraction of non-muscle cells (endoplasmic reticulum) (Carafoli, 1987; 1988 a,b; Penniston, 1983). As a prerequisite to this investigation on the role of the 2+ ER (microsomal) Ca -ATPase in carcinogenic promotion therefore, a partial characterization of the membrane-bound (native) enzyme was first conducted. Most of the results obtained in this study on the characterization of the native micro- 2+ somal Ca -pump are in agreement with earlier reports. It was observed that the native 2+ microsomal Ca -ATPase is a high affinity enzyme (Fig.16) as has been reported by other UNIVERSITY OF IBADAN LIBRARY 125 workers (Moore and Kraus-Friedmann, 1983; Famulski and Carafoli, 1982). This result 2+ confirms the assumption that most of the properties of the ER Ca -ATPase repeat those of the Sarcoplasmic reticulum (SR) ATPase (Carafoli, 1987). Also, the native enzyme is not activated by added calmodulin (Table 12). This result is in agreement with the report of 2+ Moore and KrausFriedmann (1983) that the native microsomal Ca -ATPase contains tightly bound calmodulin; but that the partial removal of the calmodulin by EGTA treatment results 45 2+ in an increased Ca uptake by added exogenous calmodulin and inhibition by trifluoperazine (TFP). The native (membrane-bound) microsomal enzyme was also found to be just slightly sensitive to vanadate (Table 12), the phosphate analog that is now considered as the classic inhibitor of P-type ion-motive ATPases (Carafoli, 1991). This low sensitivity to vanadate observed for the native microsomal enzyme could be due to two factors. 2+ (i) It might be due to the very low level of mg in the assay medium. It has been observed that the potency of vanadate as an inhibitor greatly depends on the ionic composition of the medium and on the concentration of ATP (Barrabin et aj., 1980). The ions Mg2\ Kf and Na+ enhance the inhibition by increasing the affinity of the pump 2+ for Ca . In fact, vanadate has been shown to be almost ineffective in the absence of Mg2+. (ii) It might also be due to the tightly bound calmodulin, as calmodulin has been shown to protect the pump against vanadate. It is also probable that the two factors act syn- ergistically to bring about the low sensitivity of the pump to vanadate. However, the Observation in this study that the enzyme has a maximum activity (4.543 + 0.857 pmole P/mg Protein/hr) at pH 8.0 (Fig. 15) contrasts with earlier reports of pH 6.4 - 6.8 usually used for the enzyme assay (Famulski and Carafoli, 1982; Moore and Kraus­ Friedmann, 1983). In addition, the enzyme like the liver plasma membrane Ca2+-ATPase (Lotersztajn et aj., 1981) did not obey the Michaelis-Menten kinetics (Fig.17). This may be 2+ a peculiarity of liver Ca -ATPases (both plasma membrane and microsomal). UNIVERSITY OF IBADAN LIBRARY 126 Equipped with the kinetic and the physico-chemical properties of the membrane- 2+ bound ER CA -ATPase, the question of the effect of the environmental pollutant and tumour Promoter, DDT on the activity of the enzyme wasaddressed. Liver tumour Promoters are known to induce cell proliferation and microsomal enzymes and to increase cytochrome P 450 content (Orrenius, 1965; Orrenius and Ericsson, 1966; McLean and McLean, 1966; Mannering, 1971). In this study, increases in the relative liver weight (liver weight/body weight ratio) and cytochrome P content were used as criteria of liver tumour promotion. Using these 450 criteria, it was confirmed that DDT is a liver tumour promoter (Table 14a). Surprisingly, the GGT activity of DDT-treated rats were found to be lower than those of normal (control) rats. Elevated GGT levels is widely used as a marker of hepatocellular carcinoma but the role of this enzyme in the carcinogenic process is not yet clear. The cause and the significance of the DDT-induced inhibition of GGT activity is presently not known. However, since GGT is a membrane-bound enzyme and DDT a lipophilic compound, it might be suggested that the inhibition is due to the membrane-active effect of DDT. The drastic depression in the micro- 2+ somal Ca -ATPase activity, also, requires comment. The result supports our earlier premise 2+ thatmitogen-induced Ca release from the microsome could be meaningful only if it is ac- companied with an inhibition of the microsomal Ca2+-ATPase. The DDT-induced inhibition of 2+ the ER Ca -ATPase could be (i) the result of a direct effect of DDT on the enzyme as DDT has been shown to activate the SR Ca2f-ATPase jn vitro (Antunes-Madera and Madera, 1982). (ii) It could be due to an indirect effect on the enzyme which could be via (a) an effect on the membrane or (b) the effect of a molecule generated as a result of the metabolism of DDT on the enzyme itself or the membrane. In addition, tumour Promoters are known to induce the formation of free radicals 2+ (Cerutti, 1985) and these have been shown to be capable of inhibiting the Ca -pumping UNIVERSITY OF IBADAN LIBRARY 127 ATPase (Hebbel et aL, 1986). 2+ To confirm whether or not the DDT-induced Inhibition of microsomal Ca -ATPase is peculiarto DDT, the experiment was repeated with another livertumour promoterphenobarbital (PB). The obtained results (Table 14b) are identical to those obtained with DDT. It was confirmed that PB is also a liver tumour promoter, using the relative liver weight and the Stimulation of cytochrome P as criteria. Here, however, unlike DDT-treated animals, a 450 decrease in body weight was observed; this result could be interpreted to mean that the PB treatment showed some toxicity whereas DDT treatment did not. Also, in contrast with DDT- treated rats, PB-treated rats showed increased GGT activity. It is well documented that phenobarbital treatment leads to increased GGT activity in rats (Ratanasavanh et aL, 1979). The differences in the results obtained for DDT and PB-treated animals on changes in body weight and GGT activity suggest that the two Promoters might be different in their toxicities to the animals at concentrations used for this investigations. The differences mentioned above for DDT-treated and PB-treated rats notwithstand- ing, phenobarbital treatment like DDT treatment also resulted in a significant decrease in the 2+ microsomal Ca -ATPase activity. This result confirms thatthe promoter-induced inhibition of the enzyme is probably a characteristic of tumour Promoters. It is worth noting that the 2+ promoter-induced depression of the microsomal Ca -ATPase activity is not due to differ­ ences in total microsomal protein content; as there were no significant changes in the total microsomal protein for DDT-or PB-treated rats compared to the control rats. Also, the tumour 2+ promoter-induced inhibition of the ER Ca -ATPase is not accompanied by changes in the affinity of the enzyme for calcium (Fig.18). Lowrey et aL, (1981) had observed that decreased microsomal calcium pumping is one of the earliest signs of Chemical hepatotoxicity (carcinogenicity) using haloalkane as their study tools. Recknagel et aL (1982) and more recently Long and Moore (1986) observed that carbon tetrachloride elevates cytosolic calcium in rat hepatocytes and that it is the free UNIVERSITY OF IBADAN LIBRARY 128 radicals generated from the Chemical solvent by the liver ER which inactivate the calcium pump activity of the liver ER. In addition, the consensus of opinion is that the ER of the liver cell is the firstcellular organeile which is disrupted by the Chemical poisoning. It has been observed that a naturally occurring tumour promoter, thapsigargin, is a potent inhibitor of the 2+ Ca -pumping ATPase of the ER (Thastrup, et aL, 1990). The authors also suggested that 2+ the modulation of Ca -pump activity could provide the means of generating ionic Signal 2+ 2+ based on the Observation that thapsigargin induces Ca -mobilization by ER Ca -pump inhibition. Pounds and Rosen (1988) considered the three ways in which a toxicant may alter 2+ Ca cellular homeostasis: 2+ 2+ (a) by substituting for Ca at specific sites of Ca transport or storage such as a competitive interaction observed with other divalent metals such as lead and cadmium (b) through non-specific effects on cell function that are biochemically and function- ally remote from the processes of Ca2+ transport and storage, such as physical- chemical changes in the membrane which are induced by ethanol and other aliphatic alcohols and local anesthetics (includingdibucaine and tetracaine) and (c) through non-specific effects such as an indirect action of the inactivation of the 2+ Ca -transporter of the smooth ER. It is this third hypothesis that is curreritly receiving much attention in studies which concem the modulation of Ca2+ cellular homeostasis in the process of liver tumour promotion. To date, the only acceptable biochemical mechanism that has been proposed to explain the action of some liver tumour Promoters is the inhibition of gap junctional intercel­ lular communication. However, as earlier suggested and as supported by the results of this investigation, it seems that the inhibition of intercellular communication might be secondary 2+ to the inhibition of the microsomal Ca -pumping ATPase as the former is dependent on UNIVERSITY OF IBADAN LIBRARY 129 2+ 2+ intracellular Ca concentration. The present Observation on the marked depression of Ca - ATPase activity in rat liver microsomes may be a useful indicator of the physiological and/ or biochemical function of specific liver tumour Promoters. It is well recognized that mitogenesis is an important factor in carcinogenesis particu- larly at the stage of tumour promotion (Färber, 1980). This notion is supported by the finding that both PB and DDT induced the proliferation of the endoplasmic reticulum as stated earlier on. Three mechanisms have been suggested for the promoter-induced differential rapid growth of initiated cells; these include: (a) differential Inhibition (b) differential Stimulation and (c) differential recovery (Färber, 1980). Differential inhibition proposes that the initiator is an inhibitor of cell proliferation except for initiated (resistant) cells. This differential enables the rapid growth of resistant (initiated) cells when a Stimulus for proliferation (tumour promoter) is applied. Differential Stimulation proposes that promoter selectively stimulates initiated cell to grow. It also suggests that some promoters might stimulate both initiated and uninitiated cells but that the later loses their abilityto respond while this capability continues in initiated cells. This differential Stimulation creates a progressive enlargement or proliferation of initiated cells. Differential recovery proposes that most initiated cells may undergo reversion to normal - appearing liver - a process called regression. While some cells show rapid rever­ sion, others show no reversion; and that the process of reversion may be similar in principle to the retum of the liver to a resting phase (G ) after one or more cycles of cell proliferation. O The failure to retum to a G state could conceivably be part of an altered biochemical O Programme that prevents normal recovery. Phenobarbital has been shown to act through the differential Stimulation mechanism UNIVERSITY OF IBADAN LIBRARY 130 (Färber, 1980). It is very probable that DDT acts through same mechanism. In Order to determine whether genotoxic carcinogens such as aflatoxin B could also 2+ induce an Inhibition of the microsomal Ca -ATPase as observed for the non-genotoxic carcinogens (tumour Promoters), the short-term effect of aflatoxin administration on the 2+ liver microsomal Ca -ATPase was investigated. The obtained results (Table 14c) show that 2+ genotoxic carcinogens (in this instance aflatoxin B )̂ do not have any effect on the ER Ca - ATPase. Aflatoxin B̂ is a well known hepatotoxin, hepatocarcinogen and membrane active agent (Cerutti, 1985). Thus, the result shows that the promoter-induced depression of mi- 2+ crosomal Ca -ATPase is probably not due to the membrane-active properties of the tumour Promoters. Having established that liver tumour Promoters are inhibitors of membrane-bound 2+ microsomal Ca -ATPase, the effect of another modulator of hepatocarcinogenesis, that is, low protein intake (or protein malnutrition) on the activity of the enzyme was investigated and compared with obtained results for tumour Promoters. The experiment was based on the premise that since both liver tumour Promoters and low protein intake modify the structural integrity of biomembranes (Troll and Weisner, 1985; Coward, 1971), they might affect the activity of membrane-bound enzymes such as the Ca2+-ATPase. The results obtained using the two adopted markers of tumour promotion in this study i.e. cytochromeP content and 450 the relative liver weight (liver weight/body weight ration) show that low protein diet does not belong to the same dass of modulators of hepatocarcinogenesis as DDT and PB. Despite these differences, low protein diet like liver tumour Promoters induced a significant decrease 2+ in microsomal Ca -ATPase activity (Table 16). Suggesting that positive modulators of liver 2+ cancer are probably inhibitors of microsomal Ca -ATPase. These results suggest that there is a common mechanism for the inhibition of the microsomal Ca2+-ATPase by positive modu­ lators of hepatocarcinogenesis. Tumour Promoters have been shown to be oxidants or to induce cellular prooxidant UNIVERSITY OF IBADAN LIBRARY 131 state (Cerutti, 1987). Also, certain antioxidant defence Systems of protein-energy-malnour- ished animals and humans have been shown to be depressed (Golden, 1987; 1988; Read, 1990). This condition might result in increased free radical level or an induction of cellular prooxidant state in the affected animal or individual. This is important because the Ca2+- ATPase has been shown to be inhibited by free radicals particulariyactivated oxygen (Hebbel et al.. 1986). It seems very likely therefore that the inhibition of the microsomal Ca2+-ATPase in promoter-treated rats and protein-malnourished animals might be due to the inhibitory effect of free radicals (probably generated during the period) on the enzyme. As earlier mentioned, the short-term effect of aflatoxin B administration does not i 2+ include an effect on the microsomal Ca -ATPase. However, it is not known what the effect on the enzyme would be on a long-term basis when preneoplastic foci might (presumably) have been formed. As presented on Table 18(d), aflatoxin B administration (cancer initia- 2+ tion) has no effect on the Ca -ATPase activity after 9 weeks of its administration. It must be noted, however, that the activity of the liver cancer marker enzyme GGT, does not confirm the development of a preneoplatic foci during same period. Although this result cannot be taken as conclusive, it seems that the initiation stage of cancer development and growth is characterized by cells with normal and functional ER Ca2+-ATPase activity. On the other hand, depressed ER Ca2+-ATPase appears to be a characteristic of the promotion stage. Since the process of tumour growth and development is a long-term process, some experiments were set up to determine whether the promoter-induced inhibition of Ca2+ -ATPase is a short-lived or a long-lasting response. As shown on Table 18(a) after 4 weeks 2+ of DDT treatment, there were no significant changes in microsomal Ca -ATPase activity compared to control animals. The results contrast with our earlier Observation on the de- 2+ creased microsomal Ca -ATPase activity after eight days (DDT) or four days (PB) of the administration of the liver tumour Promoters. These results could be interpreted to mean that UNIVERSITY OF IBADAN LIBRARY 132 the effects of both DDT and PB are short-lived and probably reversible. It is known that the effect of tumour Promoters is reversible at least in early stages (Pitot and Sirica, 1980) and that the process of carcinogenic promotion requires the prolonged exposure to the tumour Promoter (Faber, 1980; Pitot and Sirica, 1980). It is presently not known what the effect of 2+ prolonged exposure to the tumour promoter would be on the microsomal Ca -ATPase activ- ity. The requirement for a prolonged exposure to tumour Promoters is satisfied in the case of chronic dietary protein malnutrition (low protein intake (LPI) for twelve weeks) as earlier reported. The effect of a short-term LPI was not investigated in this study. Paradoxical enough, aflatoxin ingestion affords some protection against the low-pro- 2+ tein intake-induced inhibition of microsomal Ca -ATPase activity (Table 18c). The mecha- nism of such protection is not clear. It could be an indirect effect linked to second messenger production or to effects on membrane phospholipids particulariy the lipid ambient surrounding the pump. Also, if the free radical theory employed to explain the promoter - and low protein 2+ intake-induced inhibition of microsomal Ca -ATPase is correct, then, it might be possible that aflatoxin (AFB^ - particulariy its epoxide-acts as scavenger of free radicals. This last Suggestion is interesting; but there are no indications to-date that aflatoxin B , or its epoxides can act as antioxidants. However, the ingestion of AFB^ does not have a significant effect on the microsomal Ca2+-ATPase of DDT-treated rats (Table 18b). This might be due to the fact that the effect of DDT is short-lived as has been discussed earlier. 2+ To determ ine the Status of the plasma membrane Ca -ATPase in hepatocarcinogenesis, a preliminary study on the activities of the erythrocyte ghost mem- 2+ brane Ca pump of liver cancer and kwashiorkor-subjects was carried out. The preliminary 2+ results show that the activity of the EGM Ca -ATPase of cancer patients is comparable to that of normal individuals (Table 20). However, a reduced responsiveness of the enzyme to calmodulin was observed even though the value is not significantiy different (P > 0.05) from control value (Table 20). These results do not support the weli-documented Observation on UNIVERSITY OF IBADAN LIBRARY 133 the increased calcium concentration in cancer cells in culture. It however, suggest the tendency of an increased affinity of the enzyme for calmodulin in cancer cells. The results 2+ obtained with the EGM Ca -ATPase of kwashiorkor humans is similar to that obtained for cancer patients (Table 20). This resulfupports the result on decreased responsiveness to 2+ calmodulin reported for the EGM Ca -ATPase of kwashiorkor patients (Olorunsogo, 1989). As earlier reviewed (Section 1.8), in all eukaryotic cells so far investigated, and in several transformed cells, the biological message of ß -adrenergic agonists, several hor- mones, tumour Promoters,growth factors and oncogene products is intracellularly propagated by receptor-mediated generation of the second messengers diacylglycerol (DG) and inositol 2+ 1,4,5 trisphosphate (IP ). IP initiates reversible release of Ca from the endoplasmic 3 3 ^ reticulum (ER), or a specialized part of it. The liberated Ca + now acts as a “third messen­ ger” by activating Ca2+-dependent reactions (Heilmann et aj., 1989). The Ca2+-pool in the 2+ ER is refilled by an ATP-energized Ca -pump. The question on whether the action of IP 2+ 3 is accompanied by the Ca -pump activity or inhibition has not been well studied. One of the major results of this study is that the Ca2+-pump is inhibited during the action of the above-mentioned agonists (particularly liver tumour promoters andlow protein intake). This appears reasonable as the existence of a normal pump would have made the action of IP a futile one and without effect. Despite the fact that there have been reports on the effects of some hepatotoxins (Lowrey et a|., 1981, Recknagel et a}., 1982, Long and Moore, 1986) and other non-hepatic tumour promoters (Thastrup et aL, 1990) on the activity 2+ of the microsomal Ca -ATPase, it has been difficult to make this generalization since the 2+ microsomal fraction is known to contain two distinct classes of Ca -pumping organeile (Famulski and Carafoli, 1982; Gill et ak, 1989). The enzyme described in this study, obtained as the light microsomal fraction, has been shown to originate from the endoplasmic reticulum (Famulski and Carafoli, 1982). It seems reasonable therefore to conclude that the inhibition of the liver ER Ca2+-ATPase is a characteristic of liver tumour promoters. UNIVERSITY OF IBADAN LIBRARY 134 4.2 Summary of results 2+ The specific activity of membrane - bound microsomal Ca -ATPase of the livers of untreated rats was 4.543 + 0.857 pmole P/mg protein/hr. at pH 8.0 and was insensitive to ~~ i calmodulin. The specific activity of the enzyme was significantly decreased (P < 0.01) following subcutaneous administration of a single dose of 75 mg dicophane/kg body wt.; the affinity of the enzyme for Ca2+ was however unaffected. Similarly, liver microsomal Ca2+- ATPase activity was significantly diminished following the ingestion of low protein diet by rats . for 12 weeks. The mean Ca2+-ATPase activity of AFB^-treated animals (in the absence of dicophane) was not statistically different (P > 0.05) from that of AFB-treated rats which 2+ subsequently received dicophane. In contrast, liver microsomal Ca -ATPase activity of animals fed low protein diet prior to and after AFB ingestion was higher ( P < 0.05) than that of animals which were fed low protein diet only. Basal activity of erythrocyte Ca2+-ATPase of paediatric Controls and those having kwashiorkor (protein-energy-malnutrition) were similar (P > 0.05); similar observations were made between normal adults and those suffering from 2+ PLC. Erythrocyte Ca -ATPase of either PLC or kwashiorkor patients was however, some- what, less sensitive (15-40 %) to the stimulatory effect of calmodulin, an endogenous acti- 2+ vator of the Ca -pump. UNIVERSITY OF IBADAN LIBRARY 135 4.3 Contributions to knowledqe The highlights of the contributions of this thesis to knowiedge include the observations: 2+ (1) That the inhibition of microsomal Ca -ATPase appears to be a characteristic of liver tumour Promoters. (2) That low protein diet mimics the effect of liver tumour Promoters with respect to the 2+ inhibition of the rat liver microsomal Ca -ATPase. (3) That the genotoxic carcinogen aflatoxin despite its membrane-active property 2+ does not affect the membrane-bound microsomal Ca -ATPase. (4) That aflatoxin B protected the membrane-bound Ca -ATPase against LPI-in-1 + 2+ duced inhibition of microsomal Ca -ATPase in a way that is not yet understood and finally 2+ (5) That the erythrocyte ghost membrane-bound Ca -ATPase of both kwashiorkor and primary liver cancer patients are less sensitive to the stimulatory effect of calmodulin. UNIVERSITY OF IBADAN LIBRARY 136 REFERENCES Adelstein, R. S., Conti, M. A. and Pato, M.D. (1980) Regulation of myosin light Chain kinase by reversible phosphorylation and calcium-calmodulin. Ann. N.Y. Acad. Sei.. 356. 142-150. Adenuga,+ G. A., Bababunmi, E. A. and Hendrickse, R. G. (1992) Depression of the Ca +-ATPase activity of the rat liver endoplasmic reticulum by the liver tumour Promo­ ters 1,1,1-trichloro-2,2,-bis (p-chlorophenyl) ethane and phenobarbital. Toxicoloav. 71, 1- 6. Albano, E., Tomasi, A., lannone, A., Goria-Gatti, Vannini, V. and Dianzani, M.U. (1988) Free radical intermediates of carcinogenic hydrazines. In: Eicosanoids, Lipid peroxidation and Cancer. Nigam, E. (Ed). Springer-Verlag Berlin Heidelberg, pp. 273-280. Alpert, M.E., Hutt, M.S.R., Wogan, G.N., Davidson, C.S. (1971) Association between afla- toxin content of food and hepatoma frequency in Uganda. Cancer. 28, 253-260. Anghileri, L.J., Delbrück, H., Cressent, M. and Pidoux, E. (1980) Tumour growth inhibition by calcitonin-role of the age of the animal and the hypocalcemic response. Arch. Geschwulstforsch. 50, 635-640. Autunes-JVIadeira, M.C. and Madeira, V.M.C. (1982) Interaction of insecticides with the Ca +-pump activity of Sarcoplasmic reticulum. Pesticide Biochem. Physiol.. 17, 185- 190. Appleton, B. S. and Campbell, T. C. (1982) Low dietary casein inhibits the development of aflatoxin B̂ initiated preneoplastic liver lesions. Nutr. Cancer. 3, 200-206. Appleton, B. S. and Campbell, T. C. (1983) Effect of high and low dietary protein on the dosing and post-dosing periods of aflatoxin B -induced hepatic preneoplastic lesion development in the rat. Cancer Res., 43, 215052154. Apps, D. K., Pryde, J. G. and Sutton, R. (1982) The H+-translocating ATPase of chromaffin granule membranes. Ann. N. Y, Acad. Sei.. 402, 134-145. Ashendel, C. L., Staller, J. M. and Boutwell, R. K. (1983a) Identification of a calcium and phospholipid dependent phorbol ester binding activity in the soluble fraction of mouse tissue. Biochem. Biophvs. Res. Comm., 111. 340-345. Ashendel, C. L., Staller, J.M. and Boutwell, R. K. (1983b) Solubilization, purification and reconstitution of a phorbol ester receptor from the particulate protein fraction of mouse brain. Cancer Res.. 43, 4327-4332. Atchison, W. D. and Narahashi, T. (1984) Mechanism of action of lead on neuromuscular junctions. Neurotoxicoloav. 5, 267-282. Bababunmi, E. A. (1976) Excretion of aflatoxin in the urine of normal individuals and patients with liver disease in Ibadan (Nigeria). ln:Proc. 3rd Int. Symp. Depca, New York, pp 1729-1736. UNIVERSITY OF IBADAN LIBRARY 137 Bababunmi, E. A. Uwaifo, A. O. and Bassir, O. (1978) Hepatocarcinogens in Nigerian Foodstuffs. Wld. Rev. Nutr. Diet.. 28, 188-209. Barrabin, H., Garrahan, P. J. and Rega, A. F. (1980) Vanadate inhibition of the Ca2+-ATPase from human red cell membranes. Biochim. Biophys. Acta.. 600. 796-804. Berenblum, I. (1974) Pesticides, detergents and related products. ln:carcinogenesis as a biological problem. Frontiers of biology vol.34. North-Holland Publishing Co. Amsterdam Oxford. Neuberger, A. and Tatum, E.L.(Eds.) pp 105-106. Berridge, M. J. (1984) Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J.. 220. 345-360. Berridge, M. J. (1987) Inositol lipids and cell proliferation. Biochim. Biophys. Acta. 907. 33-45. Bewaji, 0 ,0 . and2Bababunmi, E.A. (1987) Further characterization of the membrane-bound (Ca + + Mg +)-ATPase from porcine erythrocytes. Int. J. Biochem.. 19. 721-724. Bialojan, C. and Takai, A. (1988) Inhibitory effect of a marine-sponge toxin, okadaic acid on protein phosphatases Biochem. J.. 256. 283-290. Bishop, J. M. (1982) Oncogenes. Scientific American. 246. 68-78. Bohm, K. (1968) The flavonoids: A review of their physiology, pharmacodynamics and therapeutic uses. Editio Cantor KG-Aulendorf i. Wurtt. pp. 14-74. Boynton, A. L. and Whitfield, J. F. (1978) Calcium requirements for the proliferation of cells infected with a temperature-sensitive mutant of Rous sarcoma virus. Cancer Res.. 38, 1237-1240. Boynton, A. L., Whitfield, J. F., Isaacs, R. J. and Tremblay, R. G. (1977). Different ex­ tracellular calcium requirements for proliferation of nonneoplastic preneoplastic and neoplastic mouse cells. Cancer Res.. 37, 2657-2661. Brown, J. P. (1980) A review of the genetic effects of naturally occuring flavonoids, anthra- quinones and related compounds. Mutation Res.. 75, 243-277. Bryan, G. T. and Pamukcu, A. M. (1979) Bracken fern (BF), a naturally urinary bladder carcinogen. ln:Proceedings of the tenth Inter-American Conference on Toxicology and occupational Medicine, Amsterdam: Elsevier/North Holland. Deichmann, W.B. (Ed.) pp. 229-232. Busby, W. F. and Wogan.G.N. (1981) AFlatoxins. ImMycotoxins and N-nitroso compounds: Environmental risks. Shank, R.C. (Ed.), Vol. II, Boca Raton, Florida CRC Press Inc., pp. 3-27. Butler, W.H. (1964) Acute toxicity of aflatoxin B̂ in rats. Br. J. Cancer. 18, 756-762. Carafoli, E. (1987) Intracellular calcium homeostasis. Ann. Rev. Biochem.. 56, 395-433. UNIVERSITY OF IBADAN LIBRARY 138 Carafoli, E. (1988a) Intracellular calcium regulation, with special attention to the role of the plasma membrane calcium pump. J, Cardiovasc. Pharmacol., 12, 577-584. Carafoli, E. (1988b) The intracellular homeostasis of calcium: An overview. Ann. N.Y. Acad. Sei., 551, 147-158. Carafoli, E. (1991) Calcium pump of the plasma membrane. Phvsioloqical Reviews.. 71, 129-153. Carafoli, E. and Penniston, J. T. (1985) The Calcium Signal. Scientific American. 253. 50-58. Castagna, M., Takai, Y. Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) Direct activation of calcium-activated phospholipid dependent protein kinase by tumour-pro- moting phorbolester. J. Biol. Chem., 257, 7847-7851. Cerrutti, P.A. (1985) Prooxidant States and tumour promotion. Science. 227. 375-381. Cerutti, P.A. (1987) Research on Carcinogenesis and anticarcinogenesis. Int. Cancer News. 5, 10-12. Cerutti, P.A., Larsson, R. and Krupitza, G. (1989) Mechanisms of oxidant carcinogenesis. UCLA Symposium “Genetic mechanisms in carcinogenesis and tumour progression” Keystone, Colorado, Jan. 21-27. pp. 1-14. Chafouleas, J.G., Bolton, W.E., Hidaka, H., Boyd, A. E. III and Means, A. R. (1982) Calmodulin and cell cycle.involvement in regulation of cell cycle progression. Cell, 28, 41-50. Cheung, W.Y. (1980) Calmodulin plays a pivotal role in cellular regulation. Science. 207. 19-26. Cobbold, P.H. and Rink, T.J. (1987) Fluorescence and bioluminescence measurement of cytoplasmic free clacium. Biochem. J.. 248. 313-328. Connors, T.A. (1988) The involvement of free radicals and lipid peroxidation in carcinogenesis. InrEicosanoids, lipid peroxidation and canceer. Nigam, E. (Ed.). Springer-Verlag Berlin Heidelberg pp. 143-151. Cooper, G.P., Suszkiw, J.B. and Manalis, R. S. (1984) Presynaptic effects of heavy metals. Imcellular and Molecular Neurotoxicology (T. Narahashi, Ed.) pp 1-21. Raven Press, New York. Corbett, J.R. (1974). DDT and related compounds. In: The Biochemical mode of action of pesticides. Academic Press, London/New York. pp. 169-180. Corrocher, R., Casaril, M., Bellisola, G., Gabrielli, G.B., Nicoli, N., Guidi, G.C. and De Sandre, G. (1986). Severe impairment of antioxidant System in human hepatoma. Cancer. 58. 1658-1662. Coulter, J.B.S., Hendrickse, R.G., Lamplugh, S.M., Macfarlane, S.B.J., Moody, J.B., Omer, M.I.A., Suliman, G.l. and Williams, T.E. (1986). Aflatoxins and Kwashiorkor: Clinical studies in Sudanese children. Trans. R. Soc. Trop. Med. Hyq.. 80. 945-951. UNIVERSITY OF IBADAN LIBRARY 139 Cova, L., Wild,C.P., Mehrotra, R., Turusov, V., Shirai, T., Lambert, V., Jacquet, C., Tomatis, L., Trepos, C. and Montesano, R. (1990). Contribution of aflatoxin B and hepatitis B virus infection in the induction of liver tumours in ducks. Cancer Res] 50, 2156-2163. Coward, W.A. (1971) The erythrocyte membrane in Kwashiorkor. Br. J. Nutr.. 25, 145-151. Darneil, J. Lodish, H. and Baltimore, D. (1986) Cancer ln:Molecular cell Biology. Scientific American Books pp. 1035-1080. Das, U.N., Padma, M., Sangeetha-Sagar, P., Ramesh, G and Koratkar, R. (1990) Stimulation of free radical generation in human leukocytes by various agents including tumour necrosis factor, is a calmodulin-dependent process. Biochem. Biophvs. Res. Comm.. 167,1030-1036. De Eds, F. (1968) Flavonoid metabolism. ln:Comprehensive Biochemistry, Florkin, M. and Stotz, E.H. (Eds.), vol.20, Amsterdam:Elsevier Publishing Co. pp.127-177. De-Vries, H.R., Lamplugh, S.M. and Hendrickse, R.G. (1987) Aflatoxins and Kwashiorkor in Kenya : a hospital based study in a rural area of Kenya. Ann. Trop. Paediatr.. 7, 249- 257. De-Vires, H.R., Maxwell, S.M. and Hendrickse, R. G. (1989) Foetal and neonatal exposure to aflatoxins. Acta Paediatr. Scand.. 78, 373-378. Dodge, J.T., Mitchell, Co., Hanahan, D.J. (1963) The preparation and Chemical charac- teristics of haemoglobin-free ghost of human erythrocytes. Arch. Biochim. Biophvs.. 100.119-130. Dubovsky, S.L., and Franks, K.D. (1983). Intracellular calcium ions in affective disorders: A review and an hypothesis. Biol. Psvchiatrv. 18, 781-797. Dunaif, G.E. and Campbel, T.C. (1987) Dietary protein level and aflatoxin B-induced preneoplastic hepatic lesions in the rat. J. Nutr.. 117. 1298-1302. 1 Durham, A.C.H. and Walton, J. M. (1982) Calcium ions and the control of proliferation in normal and cancer cells. Biosci. Rep.. 2, 15-30. Erickson, P.F., Seamon^JCB., Moore, B.W., Lasher, R.S. and Minier, L.N. (1980) Axonal transport of the Ca -dependent protein modulator of 3’, 5’-cyc!ic-AMP phosphodiester ase in the rabbit visual System. J. Neurochem.. 35, 242-248. 2+ Famulski, K. and Carafoli, E. (1982) Ca transporting activity of membrane fractions isolated from the post-mitochondrial supernatant of rat liver. Cell Calcium. 3, 263-281. Famulski, K. and Carafoli, E. (1984) Calmodulin dependent protein phosphorylation and calcium uptake in rat liver microsomes. Eur. J. Biochem.. 141. 15-20. Färber, E. (1980) The sequential analysis of liver cancer induction. Biochim. Biophvs. Acta. 605.149-166. Feuell, A.J. (1969) Types of mycotoxins in foods and feeds In:Aflatoxin: Scientific back ground, control and implications. Goldblatt, L.A. (Ed.) Academic Press, N.Y. pp.187- 236. UNIVERSITY OF IBADAN LIBRARY 140 Fiske, C.H. and Subbarow, Y. (1925) The colorimetric determination of phosphorus. J. Biol. Chem., 66, 375-400 2+ Fiskum, G. (1985) Intracellular levels and distribution of Ca in digitonin-permeabilized cells. Cell Calcium, 6, 25-37. Folkman, J. and Moscona, A. (1978) Role of cell shape in growth control. Nature, 273, 345- 349. Fouids, L. (1969) Inferences from epidermal carcinogenesis in rabbits and mice. ln:Neoplastic development. Academic Press N.Y. pp.44-46. Freeman, B.A. and Crapo, J.D. (1982). Biology of disease, free radicals and tissue injury. Lab. Invest., 47, 412-426. Fröhlich, D. R., Burris, T. E. and Brindley, W. A. (1989) Characterization of glutathione-s- transferases in a solitary bee, Megachile rotundata (FAB ) (Hymenoptera:Megachilidae) and inhibition by chalcones, flavone, quercetin and tridiphane-diol. Comp. Biochem. PhysioL. 94B, 661-665. Fullmer, C. S., Edelstein, S. and Wasserman, R. H. (1985). Lead-binding properties of intestinal calcium-binding proteins. J. Biol, Chem.. 260, 6816-6819. Gill, D.L., Ghosh, T.K. and Mullaney, J.M. (1989) Calcium signalling mechanisms in endo- plasmic reticulum activated by inositol-1,4,5-trisphosphate and GTP. Cell Calcium. 10, 363-374. Gamer, R.C. and Mclean, A.E.M. (1969) Increased susceptibility to carbon tetrachloride poisoning in the rat after pretreatment with oral phenobarbitone. Biochem. Pharmacol. J 8 , 645-650. Gillen, M.F., Banville, D., Rutledge, R.G., Narang, S. Seligy, V.L., Whitfield, J.F. and MacManus, J.P. (1987) A complete complementary DNA for the oncodevelopmental calcium- binding protein, oncomodulin. J. Biol. Chem.. 262, 5308-5312. Golden, M.H.N. and Ramdath, D. (1987) Free radicals in the pathogenesis of kwashiorkor. Proc, Nutr. Soc., 46, 53- 68 . Golden, M.H.N. (1988). The effects of malnutrition in the metabolism of children. Trans. Roy. Soc. Trop. Med, and Hvg.. 82, 3 -6 . Gorecka, A., Aksoy, M. O. and Hartshorne, D.J. (1976) The effect of phosphorylation of gizzard myosin on actin activaion. Biochem. Biophys. Res. Comm.. 71. 325-331. Graff, J.M., Young, T.N., Johnson, J.D. and Blackshear, P.J. (1989). Phosphorylation - regulated calmodulin binding to a prominent cellular Substrate for protein kinase C. Biol. chem.. 264. 21818-21823. Graziani, Y. (1977) Bioflavonoid regulation of ATPase and hexokinase activity in Ehrlich ascites cell mitochondria. Biochim. Biophys. Acta. 460. 364-373. U IVERSITY OF IBADAN LIBRARY 141 Greeb, J. and Shull, G.E. (1989) l^olecular cloning of a third isoform of the calmodulin- sensitive plasma membrane Ca +-transporting ATPase that is expressed predominantly in brain and skeletal muscle. J. Biol. chem.. 264. 18569-18576. 2+ Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) A new generation of Ca indicators with greatly improved fluorescence properties. J. Biol. Chem.. 260. 3440-3450. Harborne, J. B. and Williams, C. A. (1975). Flavone and flavonol glycosides. ln:The fla- vonoids. Harborne J. B. and Mabry, T.J. and Mabry, H. (Eds), Academic Press, New York. pp. 376-380. Hebbel, R.P., Shalev, O., Foker, W. and Rank, B.H. (1986) Inhibition of erythrocyte Ca2+- ATPase by activated oxygen through thiol-and lipid-dependent mechanisms. Biochim. Biophys Acta. 862, 8-16. Heilmann, C., Spamer, C. and Gerok, W. (1989) Mechanism of the calcium pump in the endoplasmic reticulum of liver: phosphoproteins as reaction intermediates. Cell Cal­ cium. 10, 275-287. Hendrickse, R.G., Coulter, J.B.S., Lamplough, S.M., Macfarlane, S.B.J., Williams, T.E., Omer, M.I.A. and Suliman, G.l. (1982) Aflatoxins and Kwashiorkor: a study in Sudanese children. Br. Med. J. 285. 843-846. Henzl, M. T. and Birnbaum, E. R. (1988) Oncomodulin and parvalbumin: Acomparison of their interactions with europium ion. J. Biol. Chem.. 263. 10674-10680. Herrmann, K. (1976) Flavonols and flavones in food plants: a review J. Food Techno!.. 11, 433-448. Hofmann, J., Fiebig, H. H., Winterhalter, B. R., Berger, D. P. and Grunicke, H. (1990). Enhancement of the antiproliferative activity of Cis-diaminedichloro platinum (II) by quercetin. Int. J. Cancer. 45, 536-539. Hofmann, J., Doppler, W., Jakob, A., Maly, K., Posch, L., Ueberall, F. and Grunicke, H. (1988). Enhancement of the antiproplatinum (II) and nitrogen mustad by inhibitors of protein Kinase C. Int. J. Cancer. 42, 382-388. Hunter, T. (1984) The proteins of oncogenes. Scientific American. 251. 60-69. Inoue, M., Kishimoto, A., Takai, Y. and Nishizuka, Y. (1977) Studies on a cyclic nucleotide- dependent protein kinase and its proenzyme in mammalian tissues. J. Biol Chem, 252, 7610-7616. Ito, N., Tatematsu, M., Nakanishi, K., Hasegawa, R., Takano, T., Imaida, K., Ogiso, T. (1980). The effects of various Chemicals on the development of hyperplastic liver nodules in hepatectomized rat treated with N-nitrosodiethylamine or N-2-fluorenylacetamide. Gann. 71, 832-842. Iwasa, Y., Iw as^l., Higashi, K., Matsui, K. and Miyamoto, E. (1982). Demonstration of a high affinity Ca -ATPase in rat liver plasma membranes. Biochem. Biophys. Res. Commun.. 105. 488-494. Jenks, W.P. (1989) How does a calcium pump pump calcium? J. Biol. Chem, 264, 18855- 18858. UNIVERSITY OF IBADAN LIBRARY 142 Kaplan, J. H. and Groses, J. N. (1972) Liver and blood cell catalase activity of tumour- bearing mice. Cancer Res.. 32, 1190-1194. Kessler, F., Bennardini, F., Bach, O., Serratosa, J., James, P., Caride, A. J., Gazzotti, P., P ens ion , J. T. and Carafoli, E. (1990). Partial purification and characterization of the Ca -pumping ATPase of the liver plasma membrane. J. Biol. Chem., 265. 16012- 16019. Khan, I and Grover, A.K. (1991) Expressior^of cyclic-nucleotide-sensitive and insensitivee isoforms of the plasma membrane Ca +-pump in smooth muscle and other tissues. Biochem. J .. 277. 345-349. Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S., and Nishizuka, Y. (1982) Calcium- activated, phospholipid-dependent protein kinase from rat brain. J. Biol. Chem.. 257, 13341-13348. Kitagawa, R. and Sugano, H. (1978) Enhancing effect of phenobarbital on development of enzyme-altered islands and hepatocellular carcinomas initiated by 3’-methyl-4-(dimethy- lamino) azobenzene or diethylnitrosamine. Gann. 69. 679-687. Kitagawa, R.; Hino, O., Nomura, K. and Sugano, H. (1984) Dose-response studies on promoting and anticarcinogenic effects of phenobarbital and DDT in the rat hepato- carcinogenesis. Carcinogenesis. 5. 1653-1656. Kitchin, K.T. and Brown, J.L. (1987) Biochemical effects of two Promoters of hepto- carcinogenesis in rats. Fd. Chem. Toxicol., 25, 603-607. Klaunig, J.E., Ruch, R.J. and Weghorst, C.M. (1990) Comparative effects of phenobarbital, DDT, and lindane on mouse hepatocyte gap junctional intercellular communication. Toxicol. Appl. PharmacoL 102, 553-563. Klein, J. (1982) Neoplasia and the immune System. ln:lmmunology-the Science of self- nonself discrimination. John Wiley and Sons, N.Y. pp.623-648. Klockner, U., and Isenber, G. (1987). Calmodulin antagonists depress calcium and potas- sium currents in ventricular and vascular myocytes. Amer. J. Physiol.. 253. H1601- H1611. Kozumbo, W., Trush, M. and Kensler, T. (1985) Are free radicals involved in tumour promotion? Chem-Biol. Interactions. 54. 199-207. Kuhnau, J. (1970) The flavonoids, a dass of semi-essential food components: Their role in human nutrition. Wld. Rev. Nutr. Diet. 24, 117-191. Lang, D. R. and Racker, E. (1974) Effects of quercetin and F inhibitor on mitochondrial ATPase and energy-linked reactions in submitochondrial pdrticles. Biochim. Biophys. Acta.. 333.180-186. LaPorte, D. C., Gidwitz, S. Weber, M. J. and Storm, D. R. (1979) Relationship between changes in the calcium dependent r egulatory protein and adenylate cyclase during Viraltransformation. Biochem. Biophys. Res. Comm.. 86. 1169-1177. UNIVERSITY OF IBADAN LIBRARY 143 Levine, L., Goldstein, S.M., Snoek, G.T., and Riga, A. (1984) Arachidonic acid metabolism by cells in culture : effects of tumour Promoters. In: Eicosanoids and cancer. Thaler- Dao, H., De Paulet, A.C., Paoletti, R (Eds.) Raven Press, New York. PP 115-125. Levine, L. (1988) Tumour Promoters and prostaglandin production. ln:Eicosanoids, lipid peroxidation and cancer. Nigam, E. (Ed.). Springer-Verlag Berlin Heidelberg pp.11-2. Loch-Caruso, r. and Trosko, J. E. (1985) Inhibited intracellular communication as a mechanistic link between teratogenesis and carcinogenesis. CRC Crit. Rev. ToxicoL 16,157-183. Loewenstein, W.R. (1979) Junctional intercellular communication and the control of growth. Biochim. Biophys. Acta, 560, 1-65. Long, R.M., and Moore, L. (1986) Elevated cytosolic calcium in rat hepatocytes exposed to carbon tetrachloride. J. Pharmacol. Exp. Ther., 238, 186-191. Lotersztajn, S., Hanoune, J. and Pecker, F. (1981) A high affinity calcium-stimulated Magnesium dependent ATPase in rat liver plasma membranes. J. Biol. Chem.. 256, 11209-11215. Lotersztajn, S. and Pecker, F. (1982). A membrane bound protein Inhibitor of the high affinity Ca-ATPase in rat liver plasma membranes. J. Biol. Chem.. 257. 6638-6641. Lowrey, K., Glende, E.A. Jr. and Recknagel, R.O. (1981a) Destruction of liver microsomal calcium pump activity by carbon tetrachloride and bromotrichloromethane. Biochem. Pharmacol.. 30.135-140. Lowrey, K., Glende, E.A. Jr., and Recknagel, R.O. (1981b) Rapid depression of rat liver microsomal calcium pump activity after administration of carbon tetrachloride or Biomotrichloromethane and lack of effect after ethanol. Toxic. Appl. Pharmacol.. 59 389-394. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, N.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem.. 193. 265-275. Mabry, T.J. and ulubelen, A. (1980) Chemistiy and Utilization of phenyl propanoids including flavonoids, coumarins and lignans. Aqric. Food Chem.. 28, 188: 196. MacLennan, Jg.H., Ejjrandl, C.J., Korczak, B. and Green, N.M. (1985) Amino-acid sequence of a Ca ++Mg +-dependent ATPase from rabbit muscle sarcoplasmic reticulum, de- duced from its complementary DNA sequence Nature. 316. 696-700. MacManus, J.P., Braceland, B.M. Rixon, R.H., Whitfield, J.F. and Morris, H.P. (1981) An increase in calmodulin during growth of normal and cancerous liver in-vivo. FEBS Lett.. 133. 99-102. MacManus, J.P., Watson, D.C. and Yaguchi, M. (1983) The complete amino acid sequence of oncomodulia parvalbumin-like calcium-binding protein from Morris hepatoma 5123 tc. Eur. J. Biochem.. 136. 9-17. UNIVERSITY OF IBADAN LIBRARY 144 Madhavan, T. V. and Gopalan, C. (1968) The effect of dietary protein on carcinogenesis of aflatoxin Arch. Pathol.. 85. 133-137. Mandel, H.G., Manson, M.M., Judah, D.J., Simpson, J.L., Green, J.A., Forrester, L.M., Wolf, C.R. and Neal, G.E. (1987). Metabolie basis for the protective effect of the antioxidant ethoxyquin on aflatoxin B hepatocarcinogenesis in the rat. Cancer Res.. 47, 5218- 5223. 1 Mannering, C. J. (1971) Properties of cytochrome P as affected by environmental factors: qualitative changes due to administration of p o l^ c lic hydrocarbons. Metabolism (Clin. Exp.).. 20, 228-245. Manson, M.M. (1983). Biphasic early changes in rat liver^glutamyl transpeptidase in re­ sponse to aflatoxin B . Carcinogenesis, 4, 467-472. Mathur, M. and Nayak, N.C. (1989) Effect of low protein diet on low dose chronic aflatoxin B^-induced hepatic injury in Rhesus monkey S. J. Toxicol. Toxin Reviews. 8, 265-273. McLean, A.E.M. and McLean, E.K. (1966) The effect of diet and 1,1,1 -trichloro-2, 2-bis-(p- chlorophenyl) ethane (DDT) on microsomal hydroxylating enzymes and on sensitivity of rats to carbon tetrachloride poisoning. Biochem. J.. 100, 564-571. Mes-Masson, A-M., Masson, S., Banville, D. and Chalifour, L. (1989) Expression of oncomodulin does not lead to the transformation or immortalization of mammalian cells in-vitro J.Cell Sei.. 94, 517-525. Metcalfe, J.C., Hesketh, R.T., and Smith, G.A. (1985). Free cytosolic Ca2+ measurement fluorine-labelled indicators using FNMR. Cell calcium. 6, 183-195. Michaelis, M. L., an^+Michaelis, E. K. (1983). Alcohol and local anesthetic effects on Na+- dependent Ca + fluxes in brain synaptic membrane vesicles. Biochem Pharmacol. 32, 963-969. Michell, R.FI. (1979) Inositol phospholipids in membrane function. Trends Biochem. Sei.. 128-131. Mirmomeni, FI.M. Suzangar, M., Wise, A., Messripour, M., Emami, H. (1979) Biochemical studies during aflatoxin B -induced liver damage in rats fed different levels of dietary protein. Int. J. Cancer. 24,1 471-476. 2+ Moore, P.B. and Kraus-Friedmann, N. (1983) Hepatic microsomal Ca -dependent ATPase: Calmodulin dependent and partial purification Biochem. J.. 214, 69-75. Moore, L., Davenport, G.R., and Landon, E.J. (1976) Calcium uptake of a rat liver microsomal subcellular fraction in response to in vivo administration of carbon tetra Chloride. J. Biol. Chem.. 251. 1197-1201. ’v Morgan, J. P. and Morgan, K. G. (1984) Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein. J. Physiol.. 351. 155-167. 2+ Murphy, A.N. and Fiskum, G. (1987) Abnormal Ca transport characteristics of hepatoma mitochondria and endoplasmic reticulum. In : Regulation of cellular calcium homeosta- sis. Pfeiffer, D. Ed. Plenum Press, pp 1-12. UNIVERSITY OF IBADAN LIBRARY 145 Mutus, B., Paimer, E.J. and MacManus, J.P. (1988) Disulfide-Iinked dimer of oncomodulin: comparison to caimodulin. Biochemistry. 27, 5615-5622. Nakajo, S., Hayashi, K., Nakaya, K. and Nakamura, Y. (1983) A simple procedure for the purification of caimodulin bound to membranes, caimodulin bound to the particulate fraction of AH-66 hepatoma Ascites cells. J. Biochem.. 93, 149-157. Nakamura, Y., Colburn, N.H. and Gindhart, T.D. (1985) Role of reactive oxygen in tumour promotion: implication of Superoxide anion in promotion of neoplastic transformation in J.B6 cells by TPA. Carcinogenesis. 6, 229-235. Nassi, P., Nediani, C., Liguri, G., Taddei, N., Ruggiero, M. art£ Ramponi, G. (1990) Effect of acylphosphatase on human erythrocyte membrane Ca +-ATPase. Biochem. Biophys. Res. Comm.. 168. 651-658. Niggli, V., Adunyah, E.S., Penniston, J.T. and Carafoli, E. (1981) Purified (Ca2+ + Mg2+)- ATPase of the erythrocyte membrane:Reconstitution and effect of caimodulin and phos- pholipid. J.Biol.Chem.. 256. 395-401. Nishizuka, Y. (1984) The role of protein Kinase C in cell surface Signal transduction and tumour promotion. Nature. 308, 693-698. Nishizumi, M. (1979) Effect of phenobarbital, dichlorodiphenyl trichloroethane and polychlo- rinated biphenyls on diethylnitrosamine induced hepatocarcinogenesis. Gann. 70, 835- 837. O’Brien, T.G. (1976) The induction of omithine decarboxylase as an early possibly obligatory event in mouse skin carcinogenesis. Cancer Res.. 36, 2644-2649. O’Connel, J.F., Klein-Szanto, A.J.P., DiGiovanni, D.M., Fries, J.W. and Slaga, T.J. (1986) Enhanced malignant progression of mouse skin tumours by the free radical generator benzoyl peroxide. Cancer Res.. 46, 2863-2865. 2+ 2+ Olorunsogo, O. O. (1989) Erythrocyte membrane (Ca +Mg ) - ATPase in human protein - energy malnutrition. Biosci. Rep.. 9, 359-368. Omura, T. and Sato, R. (1964) The carbon-monoxide binding pigment of liver microsomes. I. Evidence for its haemoprotein nature. J.Biol. Chem.. 239. 2370-2378 Orrenius, S. (1965) On the mechanism of drug hydroxylation in rat liver microsomes. J. Cell Biol.. 26, 713-723. Orrenius, S. and Ericsson, J.L.E. (1966) Enzyme-membrane relationship in phenobarbital induction of synthesis of drug-metabolizing enzyme System and proliferation of endo- plasmic membranes. J, Cell Biol.. 28. 181-198. Palmer, E.J., MacManus, J.P., and Mutus, B. (1990) Inhibition of glutathione reductase by oncomodulin. Arch. Biochem. Biophys.. 277. 149-154. Pamukcu, A.M., Talciner, S., Hatcher, J.F. and Bryan, G.T. (1980) Quercetin, a rat intestinal and bladdercarcinogen present in Bracken Ferm (Pteridium aquilinum). Cancer Res.. 40, 3468-3472. UNIVERSITY OF IBADAN LIBRARY 146 Parkin, D. M., Laara, E. and Muir, C. S. (1988), Estimates of the worldwide frequency of sixteen major cancers in 1980. Int. J. Cancer.. 41, 184-197. Parola, M., Albano, E., Autelli, R., Barrera, <£, Biocca, M.E., Paradisi, L. and Dianzani, M.U. (1990) Inhibition of the high affinity Ca +-ATPase activity in rat liver plasma membranes following carbon tetrachloride intoxication. Chem-Biol. Interactions. 73, 103-119. Pedersen, P.L. and Carafoli, E. (1987) lon-motive ATPases. I. Ubiquity, properties, and significance to cell function. Trends Biochem. Sei.. 12, 146-150. Peers, F.G. and Linsell, C.A. (1973) Dietary aflatoxins and liver cancer-a population based study in Kenya. Br. J. Cancer 27, 437-484. Penniston, J.T. (1983) Plasma membrane Ca?+-ATPase as active Ca2+ pumps. ln:Calcium and cell function (Vol.IV) Cheung, W.Y. Ed. New York. Academic Press, pp.100-149. Peraino, C., Fry, R.J.M. and Staffeldt, E. (1971) Reduction and enhancement by phenobarbital of hepatocarcinogenesis induced in the rat by 2-acetylaminofluorene. Cancer Res.. 31, 1506-1512. Peraino, C., Fry, R. J. M., Staffeldt, E. and Kisieleski, W. E. (1973) Effects of varying the exposure to phenobarbital on its enhancement of 2-acetylaminofluorene-induced hepatic tumourigeneis in the rat. Cancer Res., 33, 2701-2705. Peraino, C., Fry, R.J.M., Staffeldt, E., Christopher, J.P. (1975) Comparative enhancing effects of phenobarbital, amobarbital, diphenylhydantoin, and dichlorodiphenyltrichloro- ethane on 2-acetylaminofluorene induced hepatic tumourigenesis in the rat. Cancer Res., 35, 2884-2890. Patterson, S.D.P. (1973) Metabolism as a factor in determining the toxic action of the afla­ toxins in different animal species. Fd. Cosmet. Toxicol., 11. 287-294. Perchellet, J.P., Perchellet, E.M., Orten, D.K. and Scheider, B.A. (1986) Decreased ratio of reduced/oxidised glutathione in mouse epidermal cells treated with tumour Promoters. Carcinoqenesis. 7, 503-506. Percehllet, J. P., Abney, N. L., Thomas, R.M., Guislain, Y. L., and Percehellet, E. M. (1987) Effects of combined treatments with selenium, glutathione and Vitamin E on glutathione peroxidase activity, omithine decarboxylase induction and complete and multistage carcinogenesis in mouse skin. Cancer Res.. 47. 477-485. Perera, F.P. (1990) Carcinogens and human health: Part 1. Science. 250. 1644-1646. Peskin, A. V., Koen, Y. M. and Zbazsky, I. B. (1978) Superoxide dismutase and glutathione peroxidase activities in tumours. FEBS Lett.. 1, 41-45. Pitot, H.C., and Sirica, A.E. (1980) The stages of initiation and promotion in hepatocarcino­ genesis, Biochim. Biophvs. Acta. 605. 191-215. Poli, G., Albano, E. Dianzani, M.U., Melloni, E., Pontremoli, S., Marinari, U.M., Pronzato, M A and Cottalasso, D. (1988) Carbon tetrachloride-induced inhibition of protein Kinase C in isolated rat hepatocytes. Biochem. Biophvs. Res. Comm., 153, 591-597. UNIVERSITY OF IBADAN LIBRARY 147 Pounds, J.G. (1984) Effect of lead intoxication on calcium homeostasis and calcium- mediated cell function:A review. Neurotoxicology. 5, 295-332. Pounds, J.G., and Rosen,^.F. (1988) Contemporary issues in toxicology: Cellular Ca2+ homeostasis and Ca -mediated cell processes as critical targets for toxicant action: conceptual and methodological pitfalls. Toxicol. Appl. Pharmacol.. 93, 331-341. Preston, R.S., Hayes, J.R. and Campbell, T.C. (1976) The effect of protein deficiency on the in-vivo binding of aflatoxin to rat liver macromolecules. Life Sei., 19, 1191-1198. Raunio, H. and Pelkonen, 0 . (1983) Effect of polycyclic aromatic compounds and phorbol esters on ornithine decarboxylase and aryl hydrocarbon hydroxylase activities in mouse liver Cancer Res.. 43, 782-788. Rasmussen, H. (1970) Cell communication, calcium ion, and cyclic adenosine monophos- phate. Science, 170, 404-412. Rasmussen, H. (1989) The cycling of calcium as an intracellular messenger. Scientific American. 261,44-51. Rasmussen, H. (1990) The complexities of intracellular Ca signalling. Biol. Chem. Hoppe- Sevler. 371, 191-206. Rasmussen, H. (1986). The calcium messenger System (Part.1). N.Enql. J. Med.. 314, 1094-1101. Ratanasavanh, D., Tazi, A., Galteau, M.M. and Siest, G. (1979) Localization of gamma- glutamyltransferase in subceliular fractions of rat and rabbit liverieffect of phenobarbital. Biochem. Pharmacol., 28, 1363-1365. Read, C. (1991) Behind the face of malnutrition. New Scientist, 17, 38-42. Recknagel, R.O. (1983) A new direction in the study of CCI hepatotoxicity. Life Sei.. 33. 401-408. 4 Recknagel, R.O., Lowrey, K., Waller, R.L. and Glende, Jr., E.A. (1982) Chemical mecha- nisms and biological effects. ]m Biological reactive intermediates II. Snyder et aj. (Eds.). Academic Press, New York, pp. 619-631 Rixon, R.H. and Whitfield, J.F. (1976) The control of liver regeneration by para-thyroid hormone and calcium. J.Cell Physiol... 87, 147-156. Rizvi, T.A., Mathur, M. and Nayak, N.C. (1987). Effect of protein calorie malnutrition and cell replication on aflatoxin B -induced hepato-carcinogenesis in rats. J. Natl. Cancer Inst.. 79, 817-830. 1 Rojanapo, W., Tepsuwan, A., Kupradinum, P. and Chutimataewin, S. (1988) Modulation of hepatocarcinogenicity of aflatoxin B by the chlorinated insecticide DDT. ln:Eicosanoids, lipid peroxidation and cacner. Nigäm, , (Ed.). Springer-Verlag Berlin Heidelberg pp.327-338. Rous, P. and Kidd, J.G. (1941) Conditional neoplasms and subthreshold neoplastic States: A study of the tar tumours of rabbits. J.Exp. Med.. 73, 365-389. UNIVERSITY OF IBADAN LIBRARY 148 Sato, Y., Fujii, S., Fujii, Y. and Kaneko, T. (1990) Antiproliferative effects of glutathione-s- transferase Inhibitors on the K562 cell line. Biochem.Pharmacol.. 39, 1263-1266. Schulsinger, D.A., Root, M.M. and Campbell, T.C. (1989) Effect of dietary protein quality on development of aflatoxin B -induced hepatic preneoplastic lesions. J.Natl. Cancer Inst.. 81, 1241-1245. 1 Scribner, J.D. and Mottet, N.K. (1981) DDT acceleration of mammary gland tumours induced in the male Sprague-Dawley rat by 2-acetamido phenanthrene. Carcinoqenesis. 2, 1235-1239. Seidler, N.W., Jona, X M-> and Martonosi. A- (1989) Cyclopiazonic acid is a specific inhibitor of the Ca +-ATPase of sarcoplasmic reticulum. J. Biol. Chem.. 264. 17816- 17823. Shabad, L.M., Kolesnichenko, T.S. and Nikonova, T.V. (1972) The effect of transplacental administration of DDT in organ cultures of foetal mouse lung tissue. Int. J. Cancer. 9, 365-373. Shah, G.M. and Bhattacharya, R.K. (1986) Modulation by plant flavonoids and related phe- nolics of microsome catalyzed adduct formation between benzo(a) pyrene and DNA. Chem.-Biol. Interations. 59. 1-15. Shank, R.C., Bhamarapravati, N., Gordon, J.E., Wogan, G.N. (1972) Dietary aflatoxins and human liver cancer. IV. Incidence of primary liver cancer in two municipal populations of Thailand. Food cosmet. Toxicol., 10, 171-179. Shimada, T.t Kreiser, D.M. and Williams, G.M. (1981) Effect of the tumour promoter pheno- barbital on memb rane mediated properties of adult rat liver derived epithelial cells in culture. In-Vitro. 17, 224-226. Shull, G.^. and Greeb, J. (1988) Molecular cloning of two isoforms of the plasma membrane Ca -transgorting ATPase from rat brain: Structural and functional domains exhibit simi- larity to N a\ K+- and other cation transport ATPases. J. Biol. Chem.. 263, 8646-8657. Silverstone, H. and Tannenbaum, A. (1951) Proportion of dietary protein and the formation of spontaneous hepatomas in the mouse. Cancer Res. 11, 442-446. Simons, T.J.B. (1986) Cellular interactions between lead and calcium. Brit. Med. Bull.. 42, 431-434. Suolinna, E-M., Lang, D.R. and Racker, E. (1974) Quercetin an artificial regulator of the high aerobic glycolysis of tumour cells. J. Natl. Cancer Instit., 53. 1515-1519. Smallwood, J.l, Gugi, B. and Rasmussen, H. (1988) Regulation of erythrocyte Ca2+-pump activity by protein kinase C. J.Biol. Chem., 263, 2195-2202. Smith, G.D., Ding, J .L and Peters, T.J. (1979) A senstitive fluorimetric assay for^-glutamyl transferase. Anal. Biochem., 100, 136-139 Sommer, E.W., Blum, J.K., Berger, M.C. and Berchtold, M.W. (1989) A chemically trans form ed rat fibroblast ce ll line expresses high levels o f oncom odulin. FEBS Leg.. 2o7. UNIVERSITY OF IBADAN LI RARY 149 Stärket, P.E., Hoek, J.B., and Färber, J.L. (1986) Calcium-dependent and calcium-indepen­ dent mechanisms of irreversible cell injury in cultured hepatocytes. J. Biol. Chem. 261, 3006-3012. Streb, H. Irvine, R.F., Berridge, M.J. and Schulz, I. (1983) Release of Ca2+ from a non­ mitochondrial intra-cellular störe in pancreatic acinar cells by inositol-1,4,5, trisphosphate. Nature. 306, 67-69. Strehler, E. E.; Strehler-Page, M-A; Vogel, G. and Carafoli, E. (1989) mRNAs for plasma membrane calcium pump isoforms differing in their regulatory domain are generated by alternative splicing that involves two internal donor sites in a single exon. Proc. Natl. Acad. Sei. USA. 86, 6908-6912. Strehler, E. E.; James, P., Fischer, R.; Ham, R.; Vorherr, T.; Filoteo, A.G.; Penniston, J. T.; and Carafoli, E. (1990). Peptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membrane J. Biol. Chem.. 265. 2835- 2842. Sugimura, T. (1979) Naturally occuring genotoxic carcinogens. In: Naturally occuring carcinogens-mutagens and modulators of carcinogenesis. Miller, E.C., Miller, J.A., Hirono, I., sugimura, T. and Takayama, S. (Eds). Baltimore, University Press, pp.241- 261. Suresh, K.J., Thomas, A.P., Williams, R.J., Irvine, R.F. and Williamson, J.R. (1984) Myo- Inositol, 1,4,5-trisphosphate. J.Biol. Chem.. 259. 3077-3081. Takai, Y., Kishimoto, A., Kikkawa, U., Mori, T., and Nishizuka, Y. (1979) Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated phospho lipid-dependent protein kinase System. Biochem. Biophvs. Res. Comm., 91,1218-1224. Tannenbaum, A. and Silverstone, H. (1953) Nutrition in relation to cancer. Adv. Cancer Res.. 1. 451-501. Tarjan, R. and Kemeny, T. (1969) Multigeneration studies on DDT in mice. Food Casmet. ToxicoL 7,215-222. Tate, S.S. and Meister, A. (1978) Serine-borate complex as a transition-state inhibitor of )f glutamyl transpeptidase. Proc. Natl. Acad. Sei. U.S.A.. 75. 4806-4809 2+ 2+ Thastrup, O. (1990) RoleofCa -ATPase in regulation of cellular Ca signalling as studied with the selective microsomal Ca +-ATPase inhibitor thapsigargin. Agents and Actiohs. 29,8-15. Thastrup, O., Cullen, P.J., Drobak, B.K., Hanley, M.R. ar$ Dawson, A.P. (1990) Thapsigargin, a tumour promoter, disch^rges intracellular Ca Stores by specific inhibition of the endoplasmic reticulum Ca -ATPase. Proc. Natl. Acad. Sei USA.. 87, 2466-2470. Thomas, J.H. and Gillham, B. (1985) Carcinogenesis. In: W ll’s Biochemical Basis of Medicine. pp. 1-378. Troll, W. and Wiesner, R. (1985) The role of oxygen radicals as a possible mechanism of tumour promotion. Ann. Rev. Pharmacol. Toxicol.. 25, 509-528. UNIVERSITY OF IBADAN LIBRARY 150 Trump, B.F. and Berezesky, I.K. (1985) The role of calcium in cell injury and repair: A hypothesis. Surv. Svnthe. Pathol. Res., 4. 248-256. Turusov, v.s., Day, N.E., Tomatis, L., Gati, E. and Charles, R.T. (1973) Tumours in CF.1 mice exposed for six consecutive generations to DDT. J.Natl. Cancer Inst.. 51, 983- 997. Uenishi, K., Criss, W.E., and Kakiuchi, S. (1980) Calcium-activatable phosphodiesterase and calcium-dependent modulator protein in transplantable hepatoma tissues J.Biochem.. 87, 601-607. Van Eldik, L.J. and Burgess, W.H. (1983) Analytical subcellular distribution of calmodulin and calmodulin-binding proteins in normal and virus-transformed fibroblasts. J.Biol. Chem.. 258. 4539-4547. 2+ Veigl, M.L., Sedwick, W.D. and Vanaman, T.C. (1982) Calmodulin and Ca in normal and transformed cells. Fed. Proc.. 41. 2283-2288. Veigl, M.L., Vanaman, T.C., and Sedwick, W.D. (1984) Calcium and Calmodulin in cell growth and transformation. Biochim. Biophys. Acta. 738. 21-48. Verma, A.K., Filoteo, A. Stanford, D.R., Wieben, E.D., Penniston, J.T., Strehler, E.E., Fischer, R., Heim, R., Vogel, G., Matthews, S., Strehler-Page, M.-A., James, P., Vorherr, T., Krebs, J. and Carafoli, E. (1988) Complete primary structure of a human plasma membrane calcium pump. J.Biol. Chem.. 263. 14152-14159. Voegtlin, C. and Thompson, J.W. (1936) Lysine and malignant growth. I. The amino acid lysine as a factor controllinq the qrowth rate of a typical neoplasm. Public Health Rep. 51, 1429-1436. Wang, C.Y., Chiu, C.W., Pamukcu, A.M. and Bryan, G.T. (1976) Identification of carcino- genic trannin isolated from bracken fern (Pteridium aquilinum). J.Natl. Cancer Inst.. 56, 33-36. Ward, J. M., Sontag, J. M. Weisburger, E. K. and Brown, C. A. (1975) Effect of lifetime exposure of aflatoxin B̂ in rat. J. Natl. Cancer Inst.. 55. 107-113! Watanabe, K. and Williams, G.M. (1978) The enhacement of rat hepatocellular altered foci by the liver tumour promoter phenobartial: Evidence that foci are precursors of neo plasms and that the Promoter acts on carcinoqen induced lesions. J.Natl. Cancer Inst 61,1311-1314 Watterson, D.M., Van Eldik, L.J., Smith, R. E. and Vanaman, T.C. (1976) Calcium-dependent regulatory protein of cyclic nucleotide metabolism in normal and transformed chicken embryo fibroblasts. Proc. Natl. Acad. Sei. U.S.A.. 73. 2711-2715. Weber, K. and Osbom, M. (1969) The reliability of molecular weight determination by dodecyi sulphate polyacrylamide gel electrophoresis. J.Biol. Chem.. 244. 4406-4412. Weinberg, R.A. (1983) A molecular basis of cancer. Scientific American. 249. 102-116. UNIVERSITY OF IBA AN LIBRARY 151 Weisburger, J.H. and Horn, C. (1982) Nutrition and cancer: Mechanisms of genetoxic and epigenetic carcinogens in nutritional carcinogenesis. Bull. N.Y. Acad. Med.. 58 296- 312. Welsh, M. J., Dedman, J. R., Brinkley, B. R. and Means, A. R. (1978). Calcium-dependent regulator protein : localization in mitotic apparatus of eukaryotic cells. Proc. Natl. Acad. Sei. U.S.A.. 75, 1867-1871. Welsh, M.J., Dedman, J.R. and Brinkley, B.R. (1979) Tubulin and calmodulin. J.Cell Biol. 81, 624-634. White, J., White, F. R. and Mider, G. B. (1947) Effect of diets deficient in certain amino acids on the induction of leukemia in dba mice. J. Natl. Cancer Inst., 7, 199-202. Whitfield, J.F., Boynton, A.L., MacManus, J.P., Sikorska, M. and Tsang, B.K. (1979) The regulation of cell proliferation by calcium and cyclic AMP. Mol. Cellular Biochem.. 27, 155-179. WHO/UNEP joint meeting of govemment-designated experts on environmental health-related issues, GEMS global results (1988) Chemical contaminants in food. Sentinel. 5, 6-7. Williams, G.M., Telang, S. and Tong, C. (1981) Inhibition of intercellular communication between liver cells by the liver tumour promoter 1,1,1, trichloro-2, 2-bis(p-chlorophenyl) ethane Cancer Lett, 11.339-344. Williams, G. M. (1983) Epigenetic effects of liver tumour Promoters and implications for health effects. Environ. Health perspectives, 50, 177-183. Williams, G.M. (1980) Classification of genotoxic and epigenetic hepatocarcinogens using liver culture assays. Ann. N.Y. Acad. Sei.. 349. 273-282. Williams, G.M., Ohmori, T., Katayama, S. and Rice, J.M. (1980) Alteration by phenobarbital of membrane-associated enzymes including gamma-glutamyltranspeptidase in mouse liver neoplasms. Carcinogenesis. 1, 813-818. Williams, G.M. and Numoto, S. (1984) Promotion of mouse liver neoplasms by the organo- chlorine pesticides chlordane and heptachor in comparison to dichlorodiphenyltrichloro- ethane. Carcinoqensis. 5, 1689-1696 Willingham, M.C., Wehland, J., Kleen, C.B., Richert, N.D., Rutherford, A.V. and Pastan, I.H. (1983) Ultrastructural immunocytochemical localization of calmodulin in cultured ce s. J. Histochem. cvtochem.. 31, 445-461. Wogan, G.N. (1966) Chemical nature and biological effects of the aflatoxins. Bacte^: Rev.. 30, 460-470. Wogan, G. N. (1969) Metabolism and biochemical effects of aflatoxins. In: Aflatoxins: Scientific background, control and implications. Goldblatt, L.A. (Ed.) Academ'c -'ess N.Y. pp.151-186. Wollenweber, E. and Dietz, V. H. (1981) Occurence and distribution of free flavoroc aglycones in plants. Phvtochemistry. 26, 869-932. UNIVERSITY OF IBADAN LIBRARY 152 Yanagi, s., Kazuyuki, S. and Yamamoto, N. (1981). Induction by phenobarbital of ornithine decarboxylase activity in rat liver after initiation with diethylnitrosamine. Cancer Lett.. 12, 87-92. Yoshida, M., Sakai, T., Hosokawa, N., Marui, N., Matsumoto, K., Fujioka, A., Nishino, H. and Aoike, A. (1990) The effect of quercetin on ceil cycle progression and growth of human gastric cancer cells. FEBS Lett.. 260, 10-13. UNIVERSITY OF IBADAN LIBRARY Appendix 1 Diet 41B 10mm [441] Calculated Analysis Nutritional Information Vitamins & Trace Elements Added by Supplementation C rude Oil % 2.82 Iron m g /k g 50 .0 0 C rude P rote in % 16.18 C opper m g /k g 5.00 C rude Fibre % 6.35 Total Ash % 9.06 M anganese rn g /k g 50 .00 Zinc m g /k g 15.00 lodine m g /k g 0.50 N eutra l D e te rg e n t Fibre % 22.00 Hem icellu lose % 14.9 C oba lt m g /k g - A cid D e te rg e n t Fibre % 7.1 Selenium m e g /k g - S ta rch % 36.11 Sugars % 3.04 G ross Energy M j/k g 15.5490 D igestib le Energy M j/k g 12.2455 M e tabo lisab le E nergy M j/k g 10.1399 E ssentia l F a tty A cids % 1.3815 Vitam in A lU /k g 15000.00 V itam in D lü /k g 2 0 0 0 .0 0 Vitam in E m g /k g 70 .00 D.C.P % 13.2952 Lysino % 0.6594 Vitam in K m g /k g 10 00 M olh lon lno % 0.2752 V itam in B1 m g /k g 5.00 M ethionine & C ystine % 0 .4 873 Threonine % 0.5127 Vitam in B2 m g /k g 6.00 Tryp tophan % 0.1793 % Vitam in B6 m g /k g 7.50A rgonine 0 .9 9 9 0 Vitam in B12 m e g /k g 7.50 Folie Acid m g /k g 2 00 Calcium % 1.3665 N icotin ic A cid m g /k g 12.00 Phosphorus % 0.9301 % P a n lo th e n ic Acid m g /k g 15 00 A va ilab le P hosphorus 0.5461 Sodium % 0.4686 Choline m g /k g 1000.00 P otassium % 0.7240 Biotin m e g /k g 2 0 0 0 0 Salt (NaCI) % 1.1386 M agnesium % 0.2734 Vitam in C m g /k g Notes 1. The s p e c ifica tio n s qu o ted above are tho se at the time o f prin ting and are on ly in ten ded as a guide. C hanges in grow ing cond itions will a lte r thus e ffe c tin g the va lues given above. W hen th is o ccu rs it will be re c o rd e d on the bag label o r an am mended e x te n d e d an a lys is sheet will be issued. 2. Values in the le ft hand set o f figures are to ta l ca lcu la te d values. 3. F igures fo r tra c e e lem ents and Vitamins are the am ounts added by supplem entation . 4. 1 MJ = 2 3 3 .0 0 5 K cal 5. 1 U o f V itam in A =■ 0.344 mcg puro V itam in A A co ta to . 6. -1 U o f V itam in D = 0 .0 25 mcg pure V itam in D 2 /D 3 7. 1 J o f V itam in E = 1mg DL A lpha T ocophero l A c e ta te . 8. F ir th e r analytical 'Information can be prov ided fo r b a lc h e s as an extended analysis. © . Pilsbury's Ltd. September 1987 UNIVERSITY OF IBADAN LIBRARY Appendix II Composition of SDS’s low Protein diet Nutritional Information Vitamins and Trace elements (%) Added bv supplementation Volume 100.00 Iran 0.071 g/kg Mositure 11.212 Copper 0.015 ii Crude Fat 2.733 Marganese 0.062 ii Crude Protein 5.253 Zinc 0.037 ii Crude Fibre 1.760 Cobalt 0.517 mg/kg Ash 5.202 lodine 0.645 n Total Dietry Fibre 4.574 Solenium 0.119 ii Starch 66.593 Retinol 1.598 i i Sugar 3.615 Cholecalciferol 0.025 i i Digestibe Crude Fat 2.574 X-Tocopherol 0.051 g/kg Digestible Crude Protein 4.555 Vitamin B1 7.029 mg/kg Calcium 0.705 Vitamin B2 6.253 n Phosphorus 0.542 Vitamin B6 5.931 i i Phytate Phophorus 0.052 Vitamin B12 5.499 meg/kg Sodium 0.238 Folie Acid 0.928 mg/kg Chlorine 0.341 Nicotinic Acid 23.584 i i Magnesium 0.168 Pantothenic Acid 13.658 i i Potasium 0.698 Choline 0.400 g/kg Lysine 0.208 Inositol 0.693 II Methionine & Cystine 0.130 Biotin 0.093 mg/kg UNIVERSITY OF IBADAN LIBRARY