CYTOTOXICITY OF HEXAVALENT CHROMATE COMPOUNDS IN CH310T1/2 CELLS AND CYTOMODULATION BY SODIUM ARSENITE AND METHANOL EXTRACT OF Rauvolfia vomitora (Afzel) IN MICE. By KAZEEM AKINYINKA AKINWUMI BSc., MSc. Biochemistry, Ibadan. A thesis in the Department of BIOCHEMISTRY Submitted to the Faculty of Basic Medical Sciences in partial fulfillment of the requirements of the award of the Degree of DOCTOR OF PHILOSOPHY of the UNIVERSITY OF IBADAN FBRUARY, 2015 UNIVERSITY OF IBADAN LIBRARY CERTIFICATION I certify that this work was carried out by KAZEEM AKINYINKA AKINWUMI in the Cancer Research and Molecular Biology Laboratories, Department of Biochemistry, University of Ibadan. ------------------------------------ -------------------------------- Supervisor Date Oyeronke Odunola, PhD Director, Cancer Research and Molecular Biology Laboratories, Department of Biochemistry University of Ibadan. ii UNIVERSITY OF IBADAN LIBRARY DEDICATION The thesis is dedicated to Almighty God for His mercy and favours throughout my educational pursuit iii UNIVERSITY OF IBADAN LIBRARY ACKNOWLEDGEMENTS All praises and adoration are due to God almighty; that is able to do all things. I thank God for His favours, mercies and protection throughout the duration. This thesis would not have been possible without the help and encouragement of so many people. I express my gratitude to the Head of Department, Prof. O.O. Olorunsogo for his fatherly advice and providing enabling environment in the Department, which aided the completion of this work. I would like to thank my supervisor and mentor, Dr. Oyeronke Adunni Odunola who nurtured me academically from B.Sc. to Ph.D. I appreciate the cordial relationship I enjoyed throughout my stay in the laboratory. I am grateful for her love, kindness, encouragement and support over the period of my stay. I also thank her for the guidance and taking time out to correct this thesis. May God reward you abundantly. My appreciation goes to the former Director of the Cancer Research and Molecular Biology, Prof. A.O. Uwaifo for stimulating my interest in Cancer Research and more importantly, introducing me to Dr. Joe Landolph in whose laboratory a substantial part of this work was carried out. I am equally gratefully to Prof. E.O. Faronmbi for his care, advice and support. My sincere thanks go to Drs. Gbadegesin, Owunmi and Adaramoye for their invaluable comments and suggestions that greatly improved the quality of this work. Also, my special thanks go all my Lecturers in the Department for the knowledge imparted that provided a formidable foundation for this degree. All the non-teaching staff including Mr Isiaka and Eric is appreciated. My sincere gratitude goes to the Vice Chancellor of Bells University, Prof. I. A. Adeyemi and the management staff for allowing me to go on study leave with pay to University of Southern California, Los Angeles in order to carry out my research. The Executive Secretary, National University Commission, Prof Julius Okojie, who through the NUC provided the air ticket to and from Los Angeles, is appreciated. I also thank Mr. Mayaki, the Director of Protocol and Special Duties in NUC for his moral support and encouragement. I am very grateful to my host and mentor at the Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, USA, Dr. Joseph R. Landolph. iv UNIVERSITY OF IBADAN LIBRARY His daily guidance, support, mentorship, encouragement and extremely kindness are worthy of emulation. His family members, Alice, Joe III and Louis Landolph are also appreciated for their hospitality and friendship throughout my stay in Los Angeles. I appreciate the assistance given to me by members of the Laboratory like Prethi, Sara, Sid, Shira, Megan, Sophia, Shelly, Will, Oliver, Ibukun, Laureen, Ahmed, Khalid and Justin. Douglas Hauser, Anthony Rodriguez, Ernesto and Eric Baron are appreciated for teaching me electron microscopy. Many thanks goes to Dr. John Mata & his laboratory members at the Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific , University of Health Sciences, Lebanon, Oregon USA. The genes expression studies reported in this thesis were done in his laboratory. I also appreciate the hospitality and warm embrace received from the Harley as well as Maggie and Jessse Mata. I acknowledge without reservation the encouragement and support of Ayo and Labake Akerele, High Chief (Dr.) Olufemi Olaifa, Prof. F.A.A. Adeniyi, Prof. L. A. Bamidele, Ayo Adedoja, Lekan Ilesanmi, Eco, Bode, Ismail Adeyemo, Kola Bada, Ali Gafar, Taofeeq Yaqeen and my colleagues at Bells University of Technology. Dami, Debbie, Aminat, Jumoke, and Ola Olajumoke are all appreciated for their support and secretariat assistance. My family members, Washi, Hammed, Zainab, Hafeez and Mutiat are appreciated for their forbearance, support and prayers. Finally, I thank my parents, for encouraging me to tread this path. I appreciate your prayers, love and care that contributed immensely to my academic success. My prayer is that you both live long to enjoy the fruit of your labour. v UNIVERSITY OF IBADAN LIBRARY ABSTRACT Exposure to certain hexavalent chromate compounds (HCC) causes lung and colon cancers. Their mechanisms of cytotoxicity are unclear, but believed to be affected by ascorbate and particle size. However, their role is not clearly defined. Co-exposure with sodium arsenite (SA) is common, but its effect on HCC toxicity is unknown. Current therapy has side effects, necessitating the search for antidote from unexplored natural products such as Rauvolfia vomitora (RV). This study therefore investigates the effect of particle size and ascorbate on cytotoxity of selected HCC [lead chromate (PbCrO4), barium chromate (BaCrO4), strontium chromate (SrCrO4) and potassium dichromate (K2Cr2O7)] in C3H10T1/2 cells and cytomodulatory effects of SA and RV in mice. The effect of ascorbate, dehydroascorbate and particle size on HCC cytotoxicity in C3H10T1/2 cells was determined by measuring survival fraction and yield of foci by microscopy. Actin and cellular ultrastructure disruption and induction of cell death were assessed by electron and fluorescent microscopy. The molecular mechanisms of cytotoxicity and transformation were 2 evaluated in eighty-four cell death genes using real time (RT ) gene array, while cell cycle analysis was done by flow cytometry. Leaves of RV were air dried, powdered and extracted with methanol. Forty male mice (20-25g) were divided into 8 groups of 5 Swiss albino mice each and treated with water (control), RV (275 mg/Kg), SA (2.5 mg/kg), K2Cr2O7 (12 mg/Kg), SA + K2Cr2O7, RV + SA, RV + K2Cr2O7, RV + SA + K2Cr2O7. Rauvolfia vomitora was given orally for seven days, while K2Cr2O7 and SA were administered on day seven. Serum aspartate and alanine aminotransferases (AST and ALT), catalase, glutathione-S-transferase (GST), glutathione and malondialdehyde (MDA) levels were determined by spectrophotometry. Micronucleated polychromatic erythrocytes (mPCEs) were evaluated by microscopy. Data were analysed using ANOVA and Student‟s t- test at p= 0.05. Survival fraction of control cells was 1.0, treatment with PbCrO4 and ≤ 12.5 µM ascorbate or ≤ 2 µM dehydroascorbate decreased it to 0.4. The 15-20 µM ascorbate and 3-4 µM dehydroascorbate reversed it to 0.7. Exposure of cells to small (≤ 3 µm) and large particles (≤ 8 µm) of PbCrO4, BaCrO4 and SrCrO4 resulted in a dose-dependent decrease in survival. The total foci were higher for PbCrO4 (3.8) with large particles and BaCrO4 (6.6) with small particles. Phagocytosis of particles was time-dependent. The HCC treatment led to G2/M and S phase arrest, anucleation, actin disruption and mixed cell death. Thirty-four cell death genes including Bax and Casp3 were up-regulated by 4 folds and six including Bcl-2 and Traf2 were down- regulated in treated cells. Twenty-one anti-apoptotic and autophagy genes including Atg5 and Bcl-2 were up-regulated in PbCrO4 transformed cells. The K2Cr2O7 and/ or SA significantly increased mPCEs, AST, ALT, catalase and MDA levels while glutathione and GST were reduced. The RV restored the markers towards normal values. Cytotoxicty of chromate compounds is particle size and ascorbate dependent. The cytotoxicity might be due to actin disruption, micronuclei induction and cell cycle arrest. Methanol extract of Rauvolfia vomitora modulated the toxicity in mice. Keywords: Hexavalent chromate compounds, Sodium arsenite, Rauvolfia vomitora, Cytotoxicity Word counts: 494 vi UNIVERSITY OF IBADAN LIBRARY TABLE OF CONTENTS Title Page Abstract i Certification ii Acknowledgements iii Dedication v Table of contents vi List of figures xv List of tables xvii Abbreviation xxi CHAPTER ONE 1.1 Introduction 1 1.2 Justification 5 1.3 Aim of study 9 1.3.1 Specific Objectives 9 CHAPTER TWO LITERATURE REVIEW 2.1 Cancer 10 2.2 Carcinogenesis 11 2.3 Reactive oxygen species 12 2.4 Antioxidant 13 2.4.1 Biomarkers of oxidative stress 21 2.5 Cell death 23 2.6 Apoptosis 23 2.6.1. Mechanisms of apoptosis 25 vii UNIVERSITY OF IBADAN LIBRARY 2.6.2. Apoptosis and cancer 26 2.6.3 Assays for apoptosis 29 2.6.3.1 Cytomorphological alterations 30 2.6.3.2 DNA fragmentation 31 2.6.3.3 Detection of caspases 32 2.6.3.4 Apoptosis PCR microarray 32 2.6.3.5 Membrane alterations 33 2.6.3.6 Detection of apoptosis in whole mounts 34 2.6.3.7 Mitochondrial assays 34 2.7 Necrosis 35 2.8 Autophagy 36 2.8.1 Types of a autophagy 40 2.8.2 Regulation of autophagy 41 2.8.3 Autophagy and oxidative Stress 42 2.8.4 Detection of autophagy 43 2.8.4.1 Transmission electron microscopy 43 2.8.4.2 Monitoring autophagy by fluorescence microscopy 43 2.8.5 Autophagy and tumor cell survival 44 2.9 Hexavalent chromate 44 2.9.1 Physicochemical properties of chromium and its principal ions 45 2.9.2 Uses of chromium compounds 46 2.9.3 Contamination of the environment by hexavalent chromate 47 2.9.4 Routes of exposure 51 2.9.5 Toxicokinetics 52 viii UNIVERSITY OF IBADAN LIBRARY 2.9.6 Metabolism of chromate VI compounds 54 2.9.7 Health effects of hexavalent chromate exposure 55 2.9.7.1 Nonmalignant effects 56 2.9.7.2 Malignant effects 58 2.9.7.3 Genotoxicity 58 2.10 Arsenic 66 2.11 Cancer chemoprevention 67 2.12 Rauvolfia vomitora (Afzel) 72 2.12.1 Classification 72 2.12.2 Habitat and distribution 74 2.12.3 Description 74 2.12.4 Functional uses 74 2.12.5 Medicinal uses 74 2.12.6 Phytochemical constituents 75 CHAPTER THREE MATERIALS AND METHODS 3.0 Chemicals 77 3.1 C3H/10T½ Cl 8 (10T½) mouse embryo cell culture model 77 3.2 Determination of plating efficiencies of the cells 78 3.3 Plant collection and extraction 78 3.4 Experimental animals 79 3.5 Investigation of the cytotoxicity of lead chromate (PbCrO4) in the presence of ascorbate or dehydroascorbate 79 ix UNIVERSITY OF IBADAN LIBRARY 3.6 The effect of particle size on the cytotoxicity by PbCrO4, BaCrO4 and SrCrO4 using clonogenic assay 82 3.7 Phagpoytic uptake of PbCrO4, BaCrO4 and SrCrO4 by CH310T½ cells 83 3.8 Scanning electron microscopy of CH310T½ cells treated with PbCrO4, BaCrO4 and SrCrO4 84 3.9 Transmission electron microscopy of CH310T½ cells treated with PbCrO4, BaCrO4 and SrCrO4 86 3.10 Cell cycle distribution of CH310T½ cells exposed to PbCrO4, BaCrO4 and SrCrO4 88 3.11 Assessment of caspase activation in CH310T½cells exposed to PbCrO4, BaCrO4 AND SrCrO4 89 3.12 Quantitative assessment of extent of apoptosis and necrosis in CH310T½ cells exposed to PbCrO4, BaCrO4 and SrCrO4 93 3.13 Assessment of induction of autophagy in CH310T½ cells treated with PbCrO4, BaCrO4 and SrCrO4 94 3.14 Assessment of actin disruption in CH310T½ cells exposed to PbCrO4, BaCrO4 and SrCrO4 96 3.15 Assay for morphological transformation induced by PbCrO4, BaCrO4 and SrCrO4 98 3.16 Expression of apotosis, autophagy and necrosis related gene in PbCrO4, BaCrO4 and SrCrO4 treated cells. 101 3.16.1 Isolation of Total RNA and RT-PCR Profiling 103 3.17 The effect of sodium arsenite and methanolic extract of Rauvolfia vomitoria on the toxicity potassium dichromate in mice. 103 3.17.1 Administration of test substances 103 3.17.2 Effect of methanolic extract of Rauvolfia vomitoria on K2Cr2O7 alone and in combination with sodium arsenite induced micronuclei formation 105 3.17.3 Preparation of bone marrow smears 106 3.17.4 Slide preparation 106 3.17.5 Staining 106 x UNIVERSITY OF IBADAN LIBRARY 3.18 Evaluation of alanine aminotransferase (ALT) and aspartate amino transferace (AST) in mice preexposed to Rauvolfia vomitoria before administration of K2Cr2O7 and sodium arsenite 107 3.19 Preparation of liver homogenate 108 3.20 Evaluation of malondialdehyde level in the hepatocyte of mice pretreated with Rauvolfia vomitoria prior to administration of K2Cr2O7 alone and in combination with SA. 108 3.21 Protein determination 110 3.22 Catalase activity in the liver of mice treated with methanolic extract of Rauvolfia vomitoria before exposure to K2Cr2O7 alone and in combination with sodium arsenite. 113 3.23 Effect of pretreatment of mice with Rauvolfia vomitoria on reduced glutathione (GSH) levels before exposure to K2Cr2O7 alone and in combination with sodium arsenite 114 3.24 Glutathione- S- transferase activity in the liver of mice treated with methanol extract of Rauvolfia vomitoria before exposure to K2Cr2O7 alone and in combination with sodium arsenite 115 3.25 Statistical analysis 117 CHAPTER FOUR 118 EXPERIMENT AND RESULTS 118 4.1 Experiment 1: Effect of ascorbate or dehydroascorbate on lead chromate cytotoxicity 118 4.2 Experiment 2: The effect of particle size on the cytotoxicity by PbCrO4, BaCrO4 and SrCrO4 using clonogenic assay. 127 4.3 Experiment 3: Phagocytic uptake of PbCrO4, BaCrO4 and SrCrO4 by CH310T ½ cells 141 4.4 Experiment 4: Scanning electron microscopy of CH310T ½ treated with PbCrO4, BaCrO4 and SrCrO4. 149 xi UNIVERSITY OF IBADAN LIBRARY 4.5 Experiment 5: Transmission electron microscopy of CH3 10T ½ cells treated with PbCrO4, BaCrO4 and SrCrO4. 151 4.6 Experiment 6: Cell cycle distribution of CH310T ½ cells exposed to PbCrO4, BaCrO4 and SrCrO4 171 4.7 Experiment 7: Assessment of caspase activation and cytotoxicity in CH3 10T ½ cells exposed to PbCrO4, BaCrO4 and SrCrO4. 176 4.8 Experiment 8: Quantitative assessment of the extent of apoptosis and necrosis in CH310T ½ cells exposed to PbCrO4, BaCrO4 and SrCrO4 180 4.9 Experiment 9: Assessment of induction of autophagy in chromate treated cells. 185 4.10 Experiment 10: Assessment of actin disruption in CH310T ½ cells exposed to PbCrO4, BaCrO4 and SrCrO4 194 4.11 Experiment 11: Assay for morphological transformation induced by PbCrO4, BaCrO4 and SrCrO4 202 4.12 Experiment 12: Expression of apotosis, autophagy and necrosis gene in PbCrO4, BaCrO4 and SrCrO4 treated cells 207 . 4.13 Experiment 13: Effect of pretreatment of mice with Rauvolfia vomitoria on reduced glutathione (GSH) levels before exposure to K2Cr2O7 alone and in combination with sodium arsenite. 222 CHAPTER FIVE 5.1 Discussion 229 xii UNIVERSITY OF IBADAN LIBRARY CHAPTER SIX 6.1 Conclusion 247 6.2 Contributions to knowledge 247 Reference 248 Appendix 290 xiii UNIVERSITY OF IBADAN LIBRARY LIST OF FIGURES Fig1.1 Common sources of exposure to hexavalent chromate in the environment 3 Fig 1.2 Major steps in uptake, metabolism, and formation of DNA damage by Cr (VI) 7 Fig 2.1 Three stages model of carcinogenesis and the level of carcinogenic effect vs. level of free radicals at various stages of carcinogenic process 14 Fig 2.2 The hallmarks of cancer 15 Fig 2.3 Environmentally induced cell death and transformation 16 Fig 2.4 Some commonly encountered reactive oxygen and nitrogen reactive species 17 Fig 2.5 ROS formation and lipid peroxidation process 18 Fig 2.6 ROS/RNS role in the carcinogenic process 19 Fig 2.7 Chemical structures of some antioxidant 20 Fig 2.8 Schematic representation of apoptotic events 27 Fig 2.9 Some common morphologic features of apoptosis and nercosis 37 Fig 2.10 Structures of chromate and dichromate 46 Fig 2.12 Metabolism of hexavalent chromate 57 Fig 2.13 Reduction schemes for Cr (VI) 64 Fig 2.14 Interference of different stages of carcinogenesis by phytochemicals 69 Fig 2.15 Chemopreventive phytochemicals and their dietary source 71 Fig 2.16 Rauvolfia vomitora (Afzel) Plant 73 Fig 3.1 Caspase-3/7 cleavage of the luminogenic substrate containing the DEVD substrate 91 Fig 3.2 Principle of the viability /cytotoxicity assay 92 Fig 3.3 Schematic depiction of the autophagy pathway in a eukaryotic cell 95 Fig 3.4 Structure of rhodamine phalloidin 97 Fig 3.5 Morphological transformation assay in-vitro 99 Fig 3.6 Type II and Type III foci observed in 10 T½ cells treated with hexavalent chromate compounds 100 xiv UNIVERSITY OF IBADAN LIBRARY Fig 3.7 Protocol chart of RT-PCR array (Qiagen) 102 Fig 3.8 Protocol for the administration of test substances 104 Fig 4.1 Survival curve of 10T½ cells treated with lead chromate in the presence of ascorbate 121 Fig 4.2 Survival curve of 10T½ cells treated with lead chromate in the presence of dehydroascorbate 123 Fig 4.3 Survival curve of CH310T½cells treated with lead chromate in the presence of reduced concentrations of ascorbate 125 Fig 4.4 Effect of reduced concentration of dehydroascorbate on the survival of 10T½cells treated with PbCrO4 for 48hrs 126 Fig 4.5 Electron micrographs showing the effect of time of sonication on the size of lead chromate particles 130 Fig 4.6 Comparison of particle size distribution of lead chromate 131 Fig 4.7 Electron micrographs showing the effect of sonication on BaCrO4 particles 132 Fig 4.8 Comparison of particle size distribution of barium chromate sonicated for 0 and 20 minutes 133 Fig 4.9 Survival fraction curve for CH310T½ cells treated with small and large particles of PbCrO4 for 48hrs 134 Fig 4.10 Cytotoxicity of sonicated and unsonicated BaCrO4 in 10T½ cells 137 Fig 4.11 Cytotoxicity of sonicated and unsonicated BaCrO4 in 10T½ cells 139 Fig 4.12 Micrographs showing the effect of insoluble chromate treatment on 10T ½ cells 144 Fig 4.13 Assessment of anucleation in the log phase CH310T ½ cells exposed to PbCrO4 or BaCrO4 or SrCrO4 for 48h 145 Fig 4.14 Formation of phagocytic vacuoles by 10T½ cells in the log phase growth exposed to PbCrO4 or BaCrO4 or SrCrO4 for 48h 146 Fig 4.15 Thick section (1µm) from chromate treated 10T ½ cells 147 Fig 4.16 SEM micrograph of cells treated with PbCrO4 150 xv UNIVERSITY OF IBADAN LIBRARY Fig 4.17 TEM images of 10T ½ cells showing phagocytic uptake of PbCrO4 153 Fig 4.18 Time dependent increase in internalization of lead chromate 154 Fig 4.19 Energy Dispersive X-rays (EDX) analysis of PbCrO4 treated 10T½ cells 155 Fig 4.20 Phagocytosed SrCrO4 particles in the cytoplasm of 10T ½ cells 156 Fig 4.21 The hexavalent chromate particles in the cytoplasm and nucleus. 157 Fig 4.22 The effect of chromate treatment on the mitochondria of control and treated cells 158 Fig 4.23 The effect of chromate treatment on the endoplasmic recticulum (ER) 159 Fig 4.24 The distorted and high number of lysosome in chromate treated cell 160 Fig 4.25 Chromate treatment led to the formation of lipid droplet 161 Fig 4.26 Increase in frequency of focal degeneration in chromate treated cells 162 Fig 4.27 Different forms of myelin figures observed in chromate treated cells 163 Fig 4.28 Disruption of cytoskeletal structure in chromate treated cells 164 Fig 4.29 Typical features of necrosis observed in the chromate treated cells 168 Fig 4.30 Different features of apoptosis frequently observed in the chromate treated cells 169 Fig 4.31 Autophagic features observed in the chromate treated cells. 170 Fig 4.32 The effect of PbCrO4 treatment on viability, caspase 3/7 activity and cytotoxicity to cultured CH310T½ cells 177 Fig 4.33 The effect of BaCrO4 treatment on viability, caspase 3/7 activity and cytotoxicity to cultured CH310T½ cells 178 Fig 4.34 The effect of SrCrO4 treatment on viability, caspase 3/7 activity and cytotoxicity to CH310T½ cells 179 Fig 4.35 The percentage apoptotic and necrotic cells in 10 CH310T½ cells treated with PbCrO4 for 48h hours 182 Fig 4.36 The percentage apoptotic and necrotic cells in CH310T½ cells treated with BaCrO4 for 48h hours 183 xvi UNIVERSITY OF IBADAN LIBRARY Fig 4.37 The percentage apoptotic and necrotic cells in CH310T½ cells treated with SrCrO4 for 48h hours 184 Fig 4.38 Induction of autophagy in hexavalent chromate treated cells 187 Fig 4.39 Percentage cell expressing the LC3B-GFP puncta after BacMam LC3B-GFP transduction in CH310T½ cells exposed to PbCrO4 188 Fig 4.40 Percentage cell expressing LC3B-GFP puncta after BacMam LC3B-GFP transduction in CH310T ½ cells exposed to BaCrO4 189 Fig 4.41 Percentage cell expressing LC3B-GFP puncta after BacMam LC3B-GFP transduction in CH310T½ cells exposed to SrCrO4 190 Fig 4.42 The effect of hexavalent chromate treatment on actin filament in CH310T½ cells 195 Fig 4.43 Reduction of actin flourescence intensity and disruption actin fibre by PbCrO4 196 Fig 4.44 Dose dependent decrease in the relative TRITC-phalloidin fluorescence of PbCrO4 and BaCrO4 treated cells 198 Fig 4.45 Dose dependent decrease in the relative TRITC-phalloidin flourescence of SrCrO4 treated cells 199 Fig 4.46 Time dependent decrease in the relative TRITC-phalloidin flourescence of PbCrO4, BaCrO4 and SrCrO4 treated cells 200 Fig 4.47 Possible mechanism of anucleation and cell remodelling in CH310T1/2 cells treated with hexavalent chromate compounds 201 Fig 4.48 Fold change in apoptosis related genes after exposure of CH310T½ cells to 2.5µg/ml PbCrO4 208 Fig 4.49 Fold change in anti-apoptotic gene expressions after treatment of CH310T½ cells to 2.5 µg/ml PbCrO4 209 Fig 4.50 Differential changes in the expression of autophagic related genes after treatment of CH310T½cells to 2.5 µg/ml PbCrO4 210 Fig 4.51 Differential Expression of genes associated with necrosis after exposure of CH310T ½ cells to 2.5 µg/ml PbCrO4 211 xvii UNIVERSITY OF IBADAN LIBRARY Fig 4.52 Fold increase in pro apoptotic genes after treatment of CH310T ½ cells with 2.5 µg/ml BaCrO4 213 Fig 4.53 Fold change in anti -apoptotic gene expression after treatment of CH310T ½ cells with 2.5 µg/ml BaCrO4 214 Fig 4.54 Fold change in autophagic related gene expression profile after exposure of CH310T ½ cells to 2.5 µg/ml BaCrO4 215 Fig 4.55 Fold change in expression of genes that regulate necrosis after BaCrO4 exposure 216 Fig 4.56 Fold changes in genes that regulate apoptosis in PbCrO4 transformed cell line, PbCr3 218 Fig 4.57 Fold changes in anti-apoptotic genes in PbCrO4 transformed cell line, PbCr3 219 Fig 4.58 Expression profile of autophagy related genes in PbCrO4 transformed cell line, PbCr3 220 Fig 4.59 Fold changes in genes that control necrosis in PbCrO4 transformed cell line, PbCr3 221 xviii UNIVERSITY OF IBADAN LIBRARY LIST OF TABLES Table 2.1 Comparison of morphological features of apoptosis and nercosis. 38 Table 2.2 Chemical and physical properties of selected hexavalent chromium compounds 49 Table 2.3 Uses of chromate compounds 50 Table 2.4 Level of daily chromium intake by human from different routes of exposure. 51 Table 2.5 Major alkaloids and their bioactivities isolated from the root of Rauwolfia vomitoria. 76 Table 3.1 Treatment of CH310T ½ cells with PbCrO4 and / ascorbate or dehydroascorbate 81 Table 3.2 Protocol for protein standard curve 112 Table 4.1 Experimental setup of lower concentration of ascorbate and dehydroascorbate 122 Table 4.2 Toxicity parameters of large and small particles of PbCrO4 in CH310T ½ cells. 135 Table 4.3 Toxicity parameters of large and small particles of BaCrO4 in CH310T½ cells 138 Table 4.4 Toxicity parameters of unsonicated and sonicated SrCrO4 in CH310T ½ cells 140 Table 4.5 Percentage cells with internalized particles in cells exposed to PbCrO4 or BaCrO4 for 6- 48h. 148 . Table 4.6 Alteration in ultrastructures of CH310T½ cells treated with PbCrO4 . 165 Table 4.7 Alteration in ultrastructures of CH310T½ cells treated with BaCrO4. 166 Table 4.8 Alteration in ultrastructures of CH310T½ cells cells treated with SrCrO4 167 Table 4.9 Effect of PbCrO4 treatment on cell cycle kinetics of CH310T½ cells 173 Table 4.10 Effect of BaCrO4 treatment on cell cycle kinetics xix UNIVERSITY OF IBADAN LIBRARY of CH310T½ cells 174 Table 4.11 Effect of SrCrO4 treatment on cell cycle kinetics of CH310T½ cells 175 Table 4.12 Autophagic vacuoles observed in cells exposed to lead chromate. 191 Table 4.13 Autophagic vacuoles observed in cells treated with barium chromate 192 Table 4.14 Autophagic vacuoles observed in cells exposed to strontium chromate. 193 Table 4.15 The yield of foci in CH310T½ cells treated small (S) and large (U) PbCrO4. 204 Table 4.16 The yield of foci in CH310T½ cells treated small (S) and large (U) BaCrO4 205 Table 4.17 The yield of foci in CH310T1/2 cells treated with small (S) particles size of SrCrO4 206 Table 4.18 Frequency of micronucleated polychromatic erythrocytes (mPCEs) in polychromatic erythrocytes in of test and control animals. 226 Table 4.19 Serum alanine amino transferase (SALT) and serum aspartate amino transferase (SAST) in test and control animals. 227 Table 4.20 Oxidative stress parameters in test and control animals. 228 ABBREVIATIONS ALT - Alanine aminotransferase xx UNIVERSITY OF IBADAN LIBRARY AO - Acridine orange AST - Aspartate aminotransferase ATM - Ataxia telangiectasia-mutated gene ATP - Adenosine triphosphate BacMam - Baculovirus with a mammalian promoter BAL - British anti lewisite Bis-AAF-R110 Bis-alanylalanyl-phenylalanyl-rhodamine 110 BME - Basal eagle medium CAT - Catalase CHK - Checkpoint kinase CHO - Chinese hamster ovary CIN - Chromosomal Instability CYD - Cytochalasin D DD - Death domain DED - Death effector domain DFFA - DNA fragmentation factor A DISC - Death-inducing signaling complex DMPS - 2,3-dimercaptopropane 1-sulphonate DNA - Deoxyribonucleic Acid DPBS - Dulbecco‟s Phosphate Buffered Saline 1X DTNB - 5, 5-dithiobis 2-nitrobenzoic acid EDTA - Ethylenediaminetetraacetic Acid EDX - Energy-dispersive X-ray analysis EPA - Environmental Protection Agency ESR - Eectron spin resonance FBS - Fetal bovine Serum FCS - Fetal calf serum xxi UNIVERSITY OF IBADAN LIBRARY FP - Fluorescent protein FRIN - Forestry Research Institute of Nigeria GF-AFC - Glycylphenylalanyl-aminofluorocoumarin GFP - Green fluorescent protein GST - Glutathione S-Transferases GPx - Glutathione Peroxidase GSSG - Glutathione Disulphide GSH - Reduced Glutathione GRd - Glutathione-Reductase Enzyme HPV - Human papillomavirus IARC - International Agency for Research on Cancer LSCM - Laser scanning confocal microscopy LUTH - Lagos University Teaching Hospital MCA - 3-methyl cholanthrene MDA - Malondialdehyde MMC - Mouse mesangial cells MN - Micronucleus MOM - Mitochondrial outer membrane MOMP - mitochondrial outer membrane permeabilization MPCEs - micronucleated polychromatic erythrocytes MPT - Mitochondrial permeability transition MTOR - Mammalian target of rapamycin NADH - Reduced form of nicotinamide adenosine dinucleotide NBS - Nile blue sulfate NR - Neutral red NSCs - Neural stem cells PCE - Polychromatic erythrocytes xxii UNIVERSITY OF IBADAN LIBRARY PCR - Polymerase chain reaction PI - Propidium iodide PI3K - Phosphatidylinositol-3-kinase RNA - Ribonucleic acid ROS - Reactive oxidative Species RV - Rauvolfia vomitora SA - Sodium arsenite SHE - Syrian hamster embryo SOD - Superoxide Dismutase TBA - 2-thiobarbituric acid TBARS - Thiobarbituric acid reactive substances TEM - Transmission electron microscopy TNF - Tumor necrosis factor TOR - Target of rapamycin TRITC - Tetramethylrhodamine TUNEL - Terminal dUTP Nick End-Labeling USC - University of Southern California PCD - Programmed Cell Death NER - Nucleotide Excicion Repair GFP - Green Flourescent Protein xxiii UNIVERSITY OF IBADAN LIBRARY CHAPTER ONE 1.0 INTRODUCTION Cancer, a category of diseases that are characterized by the uncontrolled growth and spread of abnormal cells in the body is a major threat to human existence. In the year 2000 only, cancer accounted for 6.2 million deaths, 22.4 million persons living with cancer and 10.1 million new cases where discovered globally (Vincent et al., 2004). Africa is not left out of the monster ravaging lives. International Agency for Research on Cancer (IARC), reported that about 681,000 new cancer cases and 512,400 cancer deaths occurred in 2008 in Africa and these data are expected to be doubled by the year, 2030 (Ferlay et al.,2008). Cancer incidence has been on the increase in the continent probably due to the adoption of western ways of life and diets as well as industrialisation. Interestingly, exposure to pollutants, toxicants and contaminants from these industries increases the age specific incidence of cancer and is estimated to increase the global burden of cancer annually to 15 million by 2020 with a lot of new cases from Africa (Klehues et al., 2003). Hexavalent chromate is one of these pollutant and contaminants. Regulatory bodies across the globe have not only declared it a human carcinogen, but one of the 33 compounds that possess the greatest potential health threat in urban areas (OSHA, 2006; NTP, 2005; IARC, 1990). Chromium is a wide spread element in the environment. It is found naturally in rocks, soil, plants,animals, volcanic dust and gases (Saygi et al., 2008; Li et al., 2009; Pouzar et al., 2009; Wise et al., 2009). It exists mainly in two forms, Cr (III) and Cr (VI). Cr (III) occurs naturally, but Cr (VI) is rarely found in nature. It is produced mainly from commercial and industrial processes. The Chromium (III) is essential for carbohydrate and lipid metabolism, while the Cr (VI) can be toxic and carcinogenic. Human beings get exposed to hexavalent chromate via water, food and by direct contact to the skin (Son et al., 2010). In addition, millions of workers worldwide are estimated to be occupationally exposed to chromate (VI) compounds in an array of industries such as pigment production, chrome plating, stainless steel welding, cement, textile, printing, photography, paint, wood preservatives, lithography plastics, ceramics, glass and leather tanning (Cheng et al., 2014; Urbano et al, 2008). Also, effluents from various industries such as plating, tanning, painting, pigment production and metallurgy contain hexavalent chromate that may contaminate natural waters (Cheng et al., 2014; Tziritis et al., 2012; Cossich 1 UNIVERSITY OF IBADAN LIBRARY et al., 2002; Liu et al., 2001; Fishbein, 1981). Furthermore, defective organ prostheses manufactured from chrome alloys also accounts for human exposure to the toxic hexavalent chromium (Michel et al., 1991). Non-occupational exposure to Cr occurs from automobile emissions, fossil fuel combustion, waste incineration and cigarette smoke (Tian et al., 2012; Cong et al., 2010). It has been estimated that cigarettes produced in the United States contain 0.24–6.3 mg Cr/kg (ASTDR, 2000; IARC, 1990). In the environment, elevated levels of chromate (VI) have been reported ipppn areas near landfills, hazardous waste disposal sites, chromate industries and highway. Moreover, the Environmental Protection Agency EPA (1999) 5 noted that approximately, 1.7 X 10 tons of Cr is released into the atmosphere every year. Consequently therefore, there is widespread inadvertent inhalation of chromate by the general populace especially in the developing countries. A summary of the different sources of human exposure to chromate (VI) is shown in Figure 1.1. Chronic and acute exposure to hexavalent chromium results in many undesirable effects including, hepatic failure, anaemia, thrombocytopenia, spontaneous abortions, ulcers, dermal damage, gastrointestinal bleeding, renal failure, intravascular haemolysis, liver damage, respiratory disturbances, coma and even death (Lin et al., 2009; Das and Mishra, 2008; IARC, 1990). Moreover, the International Agency for Research on Cancer (IARC, 1990) has classified chromium (VI) as human carcinogen based on the weight of evidences from experimental studies. For instance, consistent associations have been found between employment in the chromium industries and significant risk for respiratory cancer (Landolph, 1994; Biedermann and Landolph, l990; IARC, 1994; Gibbs et al., 2000). More recent studies also disclosed excess risk of lung cancer death resulting from occupational exposure to Cr (VI) compounds (Kuo et al., 2006; Park et al., 2004; Gibb et al., 2000). Cr (VI) carcinogenesis in humans follows a linear, no-threshold dose-response curve (De Flora, 2000). Furthermore, statistical studies have found a 3 25% risk of dying from lung cancer with the permissible exposure of 52μg/m in 1971 (Gibbs et 3 al., 2000), so this standard was lowered to 5μg/m by U.S. Occupational Safety Health Administration in 2006 (OSHA, 2006). Unfortunately, this new standard still expects 10-45 deaths for every 1,000 exposed workers (Gibb et al., 2000). 2 UNIVERSITY OF IBADAN LIBRARY Figure 1.1: Common Sources of Exposure to Hexavalent Chromate in the Environment 3 UNIVERSITY OF IBADAN LIBRARY Similarly, the genotoxicity and mutagenicity of chromium compounds have all been demonstrated in man and animals (Stohs and Bagchi, 1995; Mount and Hockett, 2000). Many animal experiments and in vitro experiments have further supported these epidemiology reports by showing that Cr (VI) compounds induce DNA damage, cytotoxicity, and neoplastic growth (NIOSH, 2005). Once inside cell, hexavalent chromate can be reduced to the Cr (III) under physiological conditions, producing reactive intermediates, including Cr (V), Cr (IV), and Cr (III). These active intermediates may bind directly to DNA forming stable DNA-chromium complexes, DNA strand breaks, DNA-DNA cross links (Hodges et al., 2001). Similarly, chromium metabolism has been shown to be accompanied by the generation of reactive oxygen species (ROS) that presumably trigger oxidative damage to DNA (Zhang et al., 2001) and consequent cell deatb (D‟Agostini et al., 2002). It has also been reported that Cr (VI) induced oxidative damage in tissue either through production of free radicals/ lipid peroxidation or by alteration of cellular antioxidant capacity (Quinteros et al., 2008). For instance Cr (VI) treatment was reported to increase the level of lipid peroxidation in rat and mice (Arreola- Mendoza et al., 2006; Perez et al., 2004). Moreover, recent studies showed that Cr (VI) alter the activities of antioxidant enzymes, including glutathione peroxidase, catalase and superoxide dismutase (Fatima and Mohamood, 2007; Luca et al., 2007). Furthermore, generation of reactive oxygen species (ROS), stimulation of lipid peroxidation and alteration of antioxidant reserves have been suggested to be major contributors to Cr (VI)-exposure related diseases (Luca et al., 2007; Valko et al., 2006 ). Despite progress in understanding of Cr (VI) toxic potential, the specific mechanisms of Cr (VI)- induced toxicity and carcinogenesis is still complex and not clearly elucidated. For instance, epidiemiology studies have repeated showed that exposure to insoluble chromate (VI) compounds causes cancers of the respiratory system including lung cancers, whereas this has not been convincingly demonstrated in vitro. Disparities in results have also been obtained in different cells exposed to hexavalent chromate. Although, hexavalent chromate toxicity are thought to be affected by particle sizes and the type as well as the amount of intracellular reductant present at the time of exposure, different results have been reported by different researchers. Furthermore, the toxicity of hexavalent chromate may also be compounded by co- 4 UNIVERSITY OF IBADAN LIBRARY exposure with other environmental toxicant such as arsenic and cigarette smoke. Moreover, phytochemicals such as antioxidant in diet and medicinal plants may also affect toxicological outcome of hexavalent chromate exposure. This work therefore investigates the effect of particle size and ascorbate on cytotoxity of selected hexavalent chromate compounds [lead chromate (PbCrO4), barium chromate (BaCrO4), strontium chromate (SrCrO4)] in C3H10T½ cells. In addition, the cytomodulation of potassium dichromate (K2Cr2O7) and sodium arsenite toxicity by methanol extract of Rauvolfia vomitora, a medicinal plant that is traditionally used for treating tumours was also investigated in mice. 1.1 JUSTIFICATION Despite the overwhelming evidences from epidemiological and animal studies concerning the strong potency of insoluble hexavalent chromates as human carcinogen, effort to establish the carcinogenesis of hexavalent chromate compounds in vitro has only yielded limited success. For instance, Patierno et al. (1988) reported that PbCrO4 induced a weak morphological transformation in CH310T½ cells. The weak transformation was ascribed to a number of factors including particle size that limits its uptake (Patierno et al., 1988). The particle size distribution of inhaled aerosols has important consequences for deposition in the lung. Penetration of an inhaled particle through the airways generally increases with decreasing particle size for particles. Chromate particles ≤ 3 µM are deposited in the bronchial tree where chromium- induced cancers occur (IARC, 1990). Moreover, workers in chromate related industries are exposed to different sizes of the compounds, but the role of particle size in chromate (VI) toxicity has not been reported. Inadequate or lack of ascorbate in culture has also been suggested to result in low mutation and morphological transformation in chromate treated cells (Holmes et al., 2008). Ascorbate is the fastest and main reductant of hexavalent chromate, generating reactive intermediates and free radicals (Fig 1.2) that can interact with cellular macromolecules (Costa and Klein, 2008; Zhitkovich et al., 2005). In vivo, ascorbate levels are quite high (about 1 mM). However, the levels of ascorbate in tissue culture media are quite low and therefore inadequate to reduce 5 UNIVERSITY OF IBADAN LIBRARY chromate. The only source of ascorbate in tissue culture media is supplied by fetal bovine serum (FBS). The level of ascorbate supplied by 10 % FBS usually use in tissue culture is only about 50 μM , which is 20 times lower than that found in vivo (Zhitkovich, 2005). Therefore, experiments on mutagenesis and other toxic effects of hexavalent chromium in tissue culture may underestimate its mutagenic, genotoxic, and cell-transforming activities (Zhitkovich, 2005; Costa and Klein, 2008). Addition of ascorbate or dehydroascorbate to culture media of hexavalent chromate treated cells would therefore mimic in vivo condition and may produce toxicity data there are more relevant to human exposure. The mode of action for the carcinogenicity observed with chromate needs to be more clearly understood in order to reduce the risks associated with human interaction with chromate compounds. The lack of clear mechanistic information regarding chromate cytotoxicity is believed to be partly due to the number of different oxidation states of this metal that may play a role in its carcinogenicity. In addition multiple oxidation and binding pathways have been proposed to account for the wide assortment of DNA lesions observed in cellular systems. Chromate (VI) toxicities are complicated by co-exposure with other environmental toxicant like cigarette smoke, UV and other heavy metals (Salnikow and Zhitkovich, 2008). For instance, arsenic is another carcinogenic heavy metal that is concomitantly inhaled or ingested with hexavalent chromate. Co-exposure to chromate and arsenic is common in drinking water, cigarette smoke, beverages and some fungicides (Maduabuchi et al, 2007). However, there is dearth of information concerning the toxic effect of co-exposure to both metals. 6 UNIVERSITY OF IBADAN LIBRARY Figure 1.2: Major steps in uptake, metabolism, and formation of DNA damage by Cr (VI) (Zhitkovich et al., 2005) 7 UNIVERSITY OF IBADAN LIBRARY Despite the numerous sources of exposure to chromium and its attendant health effects, there is still no known safe and effective antidote for preventing or treating chromate toxicity. The use of metal chelators such as ethylenediaminetetraacetic acid (EDTA), British Anti Lewisite (BAL), sodium 2,3-dimercaptopropane 1-sulfonate (DMPS) and meso 2,3-dimercaptosuccinic acid have been proposed. However, metal chelators have been reported to possess side effects that limit their use. For instance, vomiting, headache, lachrymation, and salivation, profuse sweating, chest and abdominal pain, anxiety and decreased plasma concentration of essential metal like Zn, Mo and Cu are associated with chelation treatment (Shi et al., 2004). The search for a safe and effective antidote against chromate (VI) and arsenic toxicity had led scientist to exploit the use of dietary antioxidants and medicinal plants with antioxidant properties in attenuating the toxic effects caused by exposure to heavy metals (Guha et al., 2009; Arreola et al., 2006; Perez et al.,2004, Fatima and Mohamood, 2007). The usefulness of Rauvolfia vomitora (RV), a herb that is traditionally used to manage cancer related problems in the management of hexavalent chromate and arsenite toxicities is not known. Rauvolfia vomitoria is a medicinal plant that is widely distributed in the humid tropical secondary and low land forests of Africa (Sofowora, 1993). It belongs to the family Apocynaceae and grows to a height of about 15m. Among the Yoruba speaking people of Nigeria, it is popularly known as “Asofeyeje” meaning bearing fruits for the birds, while the Igbo people in Nigeria and Ashantes of Ghana call it Akanta and Pempe respectively. The plant has found wide applications in traditional medicine across the world. Traditionally, different part of the plant found wide applications in the treatment of many diseases such as mental illness, tumours, liver problems, hypertension and fever (Amole et al., 2009; Akpanabiatu et al , 2009; Bemis, 2006). In addition, its tissue lipid lowering-effect, blood pressure lowering, antipyretic, analgesic, haematinic, aphrodisiac, purgative, dysenteric, abortive, insecticidal, anti psychotic, anticonvulsant properties have all been documented (Amole et al, 2009; Obembe,1994; Principe , 1989). Extract from the plant have also been reported to inhibit the growth of bacterial, viral, fungal and parasitic pathogens (Amole et al, 1993). Preliminary investigations show that the plant is rich in antioxidant, flavonoids, and polyphenols and could protect against CCl4 induced hepatotoxicity (Akinwumi et al., 2011). 8 UNIVERSITY OF IBADAN LIBRARY However to date, no information exists in literature about the effect of consumption of Rauvolfia vomitora against hexavalent chromate and arsenic intoxification. Therefore, a study was undertaken in this thesis to investigate the effect of pretreatment of mice with methanol extract of RV on the clastogenic and hepatotoxic potential of potassium dichromate and sodium arsenite. 1.3 Aim of the Study The overall aim of this work is to investigate the cytotoxity of three hexavalent chromate compounds [lead chromate (PbCrO4), barium chromate (BaCrO4), and strontium chromate (SrCrO4),] in C3H10T½ cells and cytomodulatory effects of sodium arsenite and Rauvolfia vomitora (Afzel) on potassium dichromate (K2Cr2O7) indued toxicity in mice. 1.3.1. Specific Objectives Specifically this work was designed to investigate: (i) The effect of ascorbate or dehydroascorbate on the cytotoxicity of PbCrO4 in CH310T½ cells. (ii) The effect of particles size on the cytotoxicity and transformation of CH310T½ cells treated with PbCrO4, BaCrO4 and SrCrO4. (iii) The effect of PbCrO4, BaCrO4 and SrCrO4 treatment on cell death, actin disruption and cell cycle kinetics. (iv) The mRNA expression profile of cell death related genes in chromate treated and transformed cells. (v) The effect of Rauvolfia vomitora (RV) on potassium chromate (VI) toxicity alone and in combination with sodium arsenite by monitoring liver function, oxidative stress and clastogenic markers in mice. 9 UNIVERSITY OF IBADAN LIBRARY CHAPTER TWO LITERATURE REVIEW 2.1 Cancer Cancer remains a major health problem and is responsible for one in eight deaths worldwide. (Berghe, 2012). There were 12.7 million new cancer cases and about 21,000 cancer deaths per day worldwide in 2008 (Ferlay et al., 2010). Also, an estimated 68.5% increase in cancer incidence and 73.7% cancer deaths is expected by the year 2030 if urgent intervention strategies are not taken(ACS, 2010; Ferlay et al., 2010 ). A recent survey of the global incidence of cancer shows that the age-adjusted cancer incidence in the Western world is above 300 cases per 100,000 population, whereas that in Asian countries is less than 100 cases per 100,000 (Messinaand and Hilakivi-Clarke, 2009). Observational studies have suggested that lifestyle risk factors such as tobacco, obesity, alcohol, sedentary lifestyle, high-fat diet, radiation, infections and exposure to environmental toxicant are major contributors to the high incidence of cancer. In addition, an increase in cancer cases was observed among immigrants from Asian to Western countries (Anand et al., 2008; Shu et al., 2009).Thus, a good percentage of cancer deaths may be prevented by modifying the diet composition (i.e. content of fiber, polyphenols, fat/oil, protein, spices, cereals, etc.) and regular physical exercise (Tennant et al., 2010; Anand et al., 2008; Boffetta et al., 2010). Diet modifies the genome and determines stress adaptative responses, metabolism, immune homeostasis and the physiology of an organism (Huang et al., 2011; vel Szic et al., 2010) All cancers involve the malfunction of genes that control cell growth, division, repairs and death (Vogelstein and Kinzler, 2004; Hanahan and Weinberg, 2000). Only a minority of cancers are caused by germline mutations, while the vast majority (90%) is linked to somatic mutations and environmental factors (Anand et al., 2008). Damage to genes may be due to internal factors, such as hormones or the metabolism of nutrients within cells, or external factors, such as tobacco, chemicals, and sunlight. Most cancers evolve through multiple changes resulting from a combination of hereditary and environmental factors. Certain types of cancer can be prevented by eliminating exposure to tobacco and other carcinogen that initiate or accelerate cancer development. Cancer is treated with surgery, radiation, chemotherapy, hormones, and immunotherapy. However, it well known that many treatment regimens cause mild to 10 UNIVERSITY OF IBADAN LIBRARY devastating and even fatal side effects. For instance, bevacizumab the best known drug for colorectal cancer can have terrible side effects, including gastrointestinal perforations, serious bleeding, severe hypertension, clot formation, and delayed wound healing. Therefore, there is renewed effort to discover new anticancer agents with less toxic effects from natural products such as plants. 2.2 Carcinogenesis Carcinogenesis is a multistep process by which a normal cell is transformed into cancer (Vogelstein and Kinzler, 2004; Hanahan and Weinberg, 2000). Three main stages are involved: initiation, promotion and progression (Figure 2.1). During initiation, normal cells are exposed to stress (ROS) and/or a carcinogen (Figure 2.1). Initiation results in an increased capacity of the cells to survive or decrease capacity to die. This initial event results in a selective growth advantage that allows cells to accumulate more mutations or epigenetic changes that facilitate “selection” of cells through clonal selection and expansion. As carcinogenesis progresses, the neoplastic cells consistently acquire essential alterations in cell physiology which are known as the “hallmarks of cancer” (Hanahan and Weinberg, 2000). These include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis (Fig 2.2). Underlying these hallmarks are genomic instability and inflammation. Genomic instability generates the genetic diversity that expedites their acquisition, while inflammation fosters multiple hallmark functions. In addition, reprogramming of energy metabolism and evasion of immune destruction were recently added to the list (Hanahan and Weinberg, 2011). Furthermore, tumors exhibit another dimension of complexity. They contain a repertoire of recruited, presumably normal cells that contribute to the acquisition of hallmark traits by creating the „„tumor microenvironment‟‟ (Hanahan and Weinberg, 2011). Environmental carcinogen represents a key contributor to human cancers. Exposure to many carcinogen such as heavy metals and other carcinogenic chemicals have detrimental effects on health and are considered to contribute substantially to the increasing global incidence of cancer and other public health diseases (Figure 2.3). They are well known to mediate a wide variety of toxic effects such as cell cycle arrest and DNA damage or genotoxicity. However, oxidative stress induced by a surge in cellular Reactive Oxygen Species (ROS) production and 11 UNIVERSITY OF IBADAN LIBRARY dysregulation of cell death are two leading mechanisms in the etiology, progression and promotion of several human cancers (Rodgrio et al., 2009). 2.3 Reactive Oxygen Species Reactive oxygen species (ROS) are implicated as possible underlying pathogenic mechanisms in the initiation and progression of cancer. A free radical can be defined as any molecular species capable of independent existence that contains an unpaired electron in an atomic orbital (Lobo et al., 2010). Most radicals are unstable and highly reactive. They can either donate an electron to or accept an electron from other molecules, therefore behaving as oxidants or reductants (Cheeseman and Slater, 1993). The most important oxygen-containing free radicals in many disease states are hydroxyl radical, superoxide anion radical, hydrogen peroxide, oxygen singlet, hypochlorite, nitric oxide radical, and peroxynitrite radical (Figure 2.4). These highly reactive species are capable of damaging biologically relevant molecules such as DNA, proteins, and carbohydrates (Young and Woodside, 2001). In addition, ROS could initiates fatty acid peroxidation in the nucleus and membranes of cells (Figure 2.5). Injury to the macromolecules by ROS in generally termed oxidative stress. Oxidative stress results when production of ROS exceeds the capacity of cellular antioxidant defenses to remove these toxic species (Shen et al., 2011; Limon-Pacheco and Gonsebatt, 2009). Oxidative stress usually leads to cell damage and homeostatic disruption. Free radicals including reactive oxygen species and reactive nitrogen species are generated by the human body by various endogenous systems, exposure to different physiochemical conditions, or pathological states; and thus, they participate in the pathogenesis of many of diseases including cancer. The different mechanisms by which reactive oxygen and nitrogen species, oxide and their biological metabolites initiates carcinogenesis is summarised in Figure 2.6. The initiation, promotion, and progression of cancer have been linked to the imbalance between ROS and the antioxidant defense system. ROS has been shown to cause at least 100 different types of DNA lesions, including base modifications, singe-strand breaks and double- strand breaks and interstrand crosslinks (Xu et. al., 2014; Cadet et. al., 1997). In addition, numerous investigators have proposed participation of free radicals in carcinogenesis, mutation, and transformation. It is clear that their presence in biosystem could lead to mutation, 12 UNIVERSITY OF IBADAN LIBRARY transformation, and ultimately cancer (Lobo et al., 2010). A summary of the different mechanism employed by ROS / RNS in inducing cancer is presented in Figure 2.7. The oxidative stress and cancer promoting activities of radicals can be decreased by antioxidant. 2.4 Antioxidant An antioxidant is a molecule that is stable enough to donate an electron to a rampaging free radical in order to neutralize it and reducing its capacity to damage cells. These antioxidants delay or inhibit cellular damage mainly through their free radical scavenging property (Halliwell, 1995). Antioxidants are mainly responsible for converting or transforming free radicals into molecules that are less harmful in the cell. They therefore decrease oxidative stress induced carcinogenesis by a direct scavenging of ROS and/or by inhibiting cell proliferation secondary to the protein phosphorylation. Antioxidants may also act as hydrogen donor, electron donor, peroxide decomposer, singlet oxygen quencher, enzyme inhibitor, synergist and metal-chelating agents (Krinsky et al., 1992). Both enzymatic and nonenzymatic antioxidants exist in the intracellular and extracellular environment to detoxify ROS (Lobo et al., 2010). Non-enzymatic antioxidants include low-molecular-weight antioxidants (Figure 2.7), which can interact with free radicals and terminate the chain reaction before vital molecules are damaged. These include glutathione, ubiquinol, and uric acid produced during normal metabolism in the body. Others such as carotenoids, flavonoids, vitamin E (α-tocopherol), vitamin C (ascorbic acid), β-carotene, selenium and copper, are supplied in diet. Water-soluble molecules, such as vitamin C, are potent radical scavenging agents in the aqueous phase of the cytoplasm, whereas lipid soluble forms, such as vitamin E and carotenoids, act as antioxidants within lipid environments. Selenium, copper, zinc, and manganese are also important elements, since they act as cofactors for antioxidant enzymes. Selenium is considered particularly important in protecting the lipid environment against oxidative injury, as it serves as a cofactor for glutathione peroxidase (GSH-Px) (Halliwell and Gutteridge, 1999). 13 UNIVERSITY OF IBADAN LIBRARY Figure. 2.1: Three stages model of carcinogenesis and the level of carcinogenic effect vs. level of free radicals at various stages of carcinogenic process (inset A) (Valko et al., 2006). . 14 UNIVERSITY OF IBADAN LIBRARY Figure 2.2: The Hallmarks of Cancer (Hanahan and Weinberg, 2011) 15 UNIVERSITY OF IBADAN LIBRARY Figure 2.3: Environmentally induced cell death and transformation (Rodgrio et al., 2009) 16 UNIVERSITY OF IBADAN LIBRARY Figure 2.4: Some commonly encountered reactive oxygen and nitrogen reactive species (Ríos-Arrabal et al., 2013) 17 UNIVERSITY OF IBADAN LIBRARY Figure 2.5: ROS formation and the lipid peroxidation process (Jomovaa and Valko, 2011). 18 UNIVERSITY OF IBADAN LIBRARY ROS/RNS Tumour promoting Inflammation DNA and Biomolecules Angiogenesis Cellular Transformation On cogenes Supression Immune response (growth factors) tumour genes Differentiation Genetic Instablity Proliferation Tissue invasion/ metastasis Apoptosis Cell cycle Repair Cell Death Sensescene Autophagy Figure 2.6: ROS/RNS role in the carcinogenesis process (Ríos-Arrabal et al., 2013) 19 UNIVERSITY OF IBADAN LIBRARY Figure 2.7: Chemical Structures of some antioxidants. (a) Ascorbic Acid, (b) VitaminE, (c) β- Carotene, (d) Flavonoid, (e) Anthocyanin, (f) Polyphenol. 20 UNIVERSITY OF IBADAN LIBRARY Examples of enzymic antioxidant include peroxisomal catalase (CAT), superoxide dismutase (SOD), glutathione S-transferases (GST) and glutathione peroxidase (GPx). Many of the enzymatic and non enzymatic antioxidants are exploited as bio-markers of oxidative stress in the body. 2.41 BIOMARKERS OF OXIDATIVE STRESS (A) Glutathione Glutathione is also an important antioxidant compound responsible for maintaining intracellular redox homeostasis. This redox balance is altered under hypoxia conditions, as in the case of tumors, with the production of ROS and NO•. Glutathione exists in reduced (GSH) and oxidized (glutathione disulphide, GSSG) states. In its reduced state, it sequesters ROS, which is transformed and recycled by the action of the glutathione-reductase enzyme (GRd). The electron source used by this enzyme is coenzyme NADPH, which is mainly derived from the phosphate pentose pathway. The main protective roles of glutathione against oxidative stress as summarized by Masella et al. (2005) are presented below: (a) Glutathione is a cofactor of several detoxifying enzymes against oxidative stress, e.g. glutathione peroxidase (GPx), glutathionetransferase etc; (b) GSH participates in amino acid transport through the plasma membrane; (c) GSH scavenges hydroxyl radical and singlet oxygen directly, detoxifying hydrogen peroxide and lipid peroxides by the catalytic action of glutathione peroxidase; (d) Glutathione is able to regenerate the most important antioxidants, Vitamins C and E, back to their active forms; glutathione can reduce the tocopherol radical of Vitamin E directly, or indirectly, via reduction of semidehydroascorbate (B) SUPEROXIDE DISMUTASE (SOD) Superoxide dismutases (SODs) are metal-containing proteins that catalyze the removal of superoxide, generating water peroxide as a final product of the dismutation. Three isoforms have been found in all eukaryotic cells, but their location differs inside the cell. The copper-zinc SOD isoform is present in the cytoplasm, nucleus, and plasma, while the manganese SOD isoform is primarily located in mitochondria (Limon-Pacheco and Gonsebatt, 2009). Iron SOD is found in 21 UNIVERSITY OF IBADAN LIBRARY the stroma of chloroplast in addition to prokayotic organisms. SOD is nuclear encoded and SOD genes have been shown to be sensitive to environmental stresses presumably due to increased •− ROS formation. SOD destroys O2 with remarkably high reaction rates, by successive oxidation and reduction of the transition metal ion at the active site in a “Ping-Pong” type mechanism (Mates et. al.,1999). (C) CATALASE The intracellular level of H2O2 is regulated by a wide range of enzymes, the most important being catalase and peroxidases. Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long. It contains four porphyrin heme (iron) groups that allow the enzyme to react with the hydrogen peroxide. It is a common enzyme found in nearly all living organisms exposed to oxygen and it is usually located in the peroxisome (Alberts et al., 2002). It efficiently catalyzes the decomposition of hydrogen peroxide to water and oxygen (Valko et al., 2006; Chelikani, 2004). Catalase has one of the highest turnover rates for all enzymes: one molecule of catalase can convert approximately 6 million molecules of hydrogen peroxide to water each minute and is a very important enzyme in protecting the cell from oxidative damage by reactive oxygen species (Valko et al., 2006). Infact, decreased capacity of a variety of tumours for detoxifying hydrogen peroxide is linked to a decreased level of catalase (Valko et al., 2006) (D) Glutathione Transferases The glutathione transferases (GSTs; also known as glutathione S-transferases) are major phase II detoxification enzymes found mainly in the cytosol. In addition to their role in catalyzing the conjugation of electrophilic substrates to glutathione (GSH), these enzymes also carry out a range of other functions. They have peroxidase and isomerase activities, they can inhibit the Jun N-terminal kinase and thus protecting cells against H2O2-induced cell death (Sheehan et al., 2001). They can bind non-catalytically to a wide range of endogenous and exogenous ligands. Cytosolic GSTs of mammals have been particularly well characterized, and were originally classified into Alpha, Mu, Pi and Theta classes on the basis of a combination of criteria such as 22 UNIVERSITY OF IBADAN LIBRARY substrate/inhibitorspecificity, primary and tertiary structure similarities and immunological identity (Sheehan et al., 2001). 2.5 CELL DEATH Cell death is a sequence event that culminates in cessation of biological activity of the cell. It is one of the most crucial events in the evolution of disease in any tissue or organ. It results from diverse causes, including ischemia infections, toxins, and immune reactions. Cell death is also a normal and essential process in embryogenesis, the development of organs, and the maintenance of homeostasis. Cell death plays a crucial role in many physiological processes and diseases. It is a process that is both complementary and antagonistic to cell division for the maintenance of tissue homeostasis (Dunai et al., 2011). Three major types of cell death have been described based on cell morphology: apoptosis, autophagic cell death, and necrosis / necroptosis (Chunlan, 2013). 2.6 APOPTOSIS Apoptosis, also referred to as type I cell death. Apoptosis is characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms. One of the early events during apoptosis is cell dehydration. Loss of intracellular water results in condensation of the cytoplasm followed by a change in cell shape and size. Cells that are originally round may become elongated and smaller. Another change, perhaps the most characteristic feature of apoptosis, is condensation of nuclear chromatin. The condensation starts at the nuclear periphery, and the condensed chromatin often takes on a concave shape resembling a half-moon, or sickle. The condensed chromatin has a uniform, smooth appearance, with no evidence of any texture normally seen in the nucleus. DNA in condensed (pycnotic) chromatin exhibits hyperchromasia, staining strongly with fluorescent or light absorbing dyes. The nuclear envelope disintegrates; lamin proteins undergo proteolytic degradation, followed by nuclear fragmentation (karyorrhexis). Many of the nuclear fragments stain uniformly with DNA dyes and are scattered throughout the cytoplasm. The nuclear fragments, together with constituents of the cytoplasm (including intact organelles), are then packaged and enveloped into apoptotic bodies. The 23 UNIVERSITY OF IBADAN LIBRARY apoptotic bodies are then shed from the dying cell. Appearance of membrane blebs on the plasma membrane is another important characteristics feature of apoptosis. Biochemical changes such as the activation of caspases and/or endonucleases are important characteristics in the process of typical apoptosis induction (Arends et al., 1990; Patel et al., 1996). Caspase-3 is a key apoptotic executioner caspases, being activated by proteolytic cleavage due to caspase-8 and caspase-9 (Annunziato et al., 2003; Qu and Qing, 2004; Son et al., 2006). Although internucleosomal fragmentation is a hallmark of apoptosis, recent studies have revealed that cellular DNA is not always fragmented into nucleosomal ladders in apoptotic cells, thus apparently depending on cell type (Oberhammer et. al., 1993). Other features of apoptosis include mobilization of intracellular ionized calcium (McConkey et al., 1989), activation of trans glutaminase which cross-links cytoplasmic proteins (Piacentini et al.,1995), loss of microtubules (Endersen et al., 1995), loss of asymmetry of the phospholipids on the plasma membrane leading to exposure of phosphatidyl serine on the outer surface (Hale et al., 1996 ; Evenson, et al.,1993), and other plasma membrane changes which precondition remnants of the apoptotic cell to become a target for phagocytic cells. The duration of apoptosis may vary, but generally is short, even shorter than the duration of mitosis (Darzynkiewicz et al., 1997). Many of these changes appeared to be unique to apoptosis, and have become markers used to identify this mode of cell death biochemically, by microscopy or cytometry (Darzynkiewicz et al., 1997). Apoptosis plays a critical role in a number of physiological functions such as fetal development or tissue homeostasis regulation (Siegel, 2006). In addition to these physiological roles, considerable evidence suggests that changes in apoptosis play a major role in the development of various diseases. Indeed, in cancer, inhibition of apoptosis leads to anarchic division of cells and favors tumor development (Debunne et al., 2011). In contrast, over activation of apoptosis contributes to the aggravation of various cardiovascular diseases such as atherosclerosis, myocardial, infarction, and heart failure (Fadeel and Orrenius, 2005). Mitochondria play an important task in apoptosis induced both by caspase-dependent and - independent pathways. Bcl-2 and Bcl-xL are the major anti-apoptotic proteins integrated in mitochondrial membrane. These proteins prevent apoptosis through either hetero dimerization 24 UNIVERSITY OF IBADAN LIBRARY with pro-apoptotic proteins, or their direct pore forming effects on the outer membrane of mitochondria (Gross et al., 1999; Harris and Thompson, 2000; Nechushtan et al., 2001). Many apoptogen induce apoptosis by reducing the cellular levels or activity of the antiapoptotic Bcl-2 family such as Bcl-2 and Bcl-xL 2.6.1. Mechanisms of Apoptosis The mechanisms of apoptosis are highly complex and sophisticated, involving an energy- dependent cascade of molecular events (Fig 2.7). There are two main apoptotic pathways: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. Recent evidences show that the two pathways are linked and that molecules in one pathway can influence the other (Igney and Krammer, 2002). Additionally, T-cell mediated cytotoxicity and perforin-granzyme pathway can also activate the killing of cells (El More, 2007). In the intrinsic pathway, which is more ancient, stimuli such as cellular damage or lack of essential factors cause mitochondrial outer membrane permeabilization (MOMP), a step that is controlled by the proapoptotic and antiapoptotic proteins of the Bcl-2 family. The critical molecules are Bax and Bak, which, following activation, assemble in the lipid pore, forming MOMP (Chipuk et al., 2006; Youle and Strasser, 2009). Once the MOMP is functional, the released cytochrome c binds to the cytosolic protease-activating factor-1 (Apaf-1), forming the apoptosome, the caspase-9 activating complex, that recruits and activates the initiator caspase-9, whereas Smac/Diablo antagonizes the X-chromosome-linked inhibitor of apoptosis (XIAP) thereby ensuring propagation of apoptosis (Pop and Salvesen, 2009). In the extrinsic apoptotic pathway, activation of the initiator caspases-8 and -10 is mediated by the extracellular ligands that bind to death receptors on the cell surface. The death receptors are transmembrane proteins belonging to the tumor necrosis factor (TNF) receptor superfamily. Ligand binding triggers a conformational change in the cytoplasmic domain of the receptor exposing the death domain (DD) that recruits the adaptor proteins via their own DD. In addition to the DD, some adaptor proteins contain a death effector domain (DED). The complex between the death receptor and the adaptor protein's DED is called the death-inducing signaling complex (DISC), which recruits the initiator caspase-8 to DISC, thereby enabling its activation (Oberst et 25 UNIVERSITY OF IBADAN LIBRARY al., 2010). The execution phase downstream of caspase-8 activation is cell-type specific. In type I cells, the amount of DISC-processed caspase-8 allows for the direct activation of sufficient amounts of the executioner caspases-3 and -7 to finalize apoptosis. However, in type II cells the DISC-mediated caspase- 8 activation is insufficient for efficient apoptosis triggering and caspase-8, rather than directly activating the executioner caspases activates Bid, thereby creating an amplification loop through recruitment of the mitochondrial pathway to help finishing up dysfunctional or superfluous cells. The perforin/granzyme pathway can induce apoptosis via either granzyme B or granzyme A. The extrinsic, intrinsic, and granzyme B pathways converge on the same terminal, or execution pathway (Figure 2.8). This pathway is initiated by the cleavage of caspase-3 and results in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, cross-linking of proteins, formation of apoptotic bodies, expression of ligands for phagocytic cell receptors and finally uptake by phagocytic cells. The granzyme A pathway activates a parallel, caspase-independent cell death pathway. 2.6.2. Apoptosis and Cancer. The normal mechanisms of cell cycle regulation are dysfunctional in cancer cells. This is usually caused by over proliferation of cells and/or decreased removal of cells (King and Cidlowski, 1998). In fact, suppression of apoptosis during carcinogenesis is thought to play a central role in the development and progression of some cancers (Kerr et al., 1994). There are a variety of molecular mechanisms that tumor cells use to suppress apoptosis. Tumor cells can acquire resistance to apoptosis by the expression of anti-apoptotic proteins such as Bcl-2 or by the down- regulation or mutation of pro-apoptotic proteins such as Bax. The expression of both Bcl-2 and Bax is regulated by the p53 tumor suppressor gene (Miyashita, 1994). Certain forms of human B cell lymphoma have over expression of Bcl-2, and this is one of the first and strongest lines of evidence that failure of cell death contributes to cancer (Vaux et al., 1988). Another method of apoptosis suppression in cancer involves evasion of immune surveillance (Smyth et al., 2001). Certain immune cells (T cells and natural killer cells) normally destroy tumor cells via the perforin/granzyme B pathway or the death-receptor pathway. In order to evade immune 26 UNIVERSITY OF IBADAN LIBRARY destruction, some tumor cells will diminish the response of the death receptor pathway to FasL produced by T cells. This has been shown to occur in a variety of ways including down regulation of the Fas receptor on tumor cells. Other mechanisms include expression of nonfunctioning Fas receptor, secretion of high levels of a soluble form of the Fas receptor that will sequester the Fas ligand or expression of Fas ligand on the surface of tumor cells (Cheng et al., 1994; Elnemr et al., 2001). In fact, some tumor cells are capable of a Fas ligand-mediated “counterattack” that results in apoptotic depletion of activated tumor infiltrating lymphocytes (Koyama et al., 2001). The ataxia telangiectasia-mutated gene (ATM) has also been shown to be involved in tumorigenesis via the ATM/p53 signaling pathway . The ATM gene encodes a protein kinase that acts as a tumor suppressor. ATM activation, via ionizing radiation damage DNA, stimulates DNA repair and blocks cell cycle progression. One mechanism through which this occurs is ATM dependent phosphorylation of p53 (Kurz and Lees-Miller, 2004). p53 then signals growth arrest of the cell at a checkpoint to allow for DNA damage repair or can cause the cell to undergo apoptosis if the damage cannot be repaired. This system can also be inactivated by a number of mechanisms including somatic genetic/epigenetic alterations and expression of oncogenic viral proteins such as the HPV, leading to tumourigenesis. Other cell signaling pathways can also be involved in tumor development. For example, upregulation of the phosphatidylinositol 3- kinase/AKT pathway in tumor cells renders them independent of survival signals. In addition to regulation of apoptosis, this pathway regulates other cellular processes, such as proliferation, growth, and cytoskeletal rearrangement. 27 UNIVERSITY OF IBADAN LIBRARY Figure 2.8: Schematic representation of apoptotic events. (El more, 2007) 28 UNIVERSITY OF IBADAN LIBRARY Alterations of various cell signaling pathways can result in dysregulation of apoptosis and lead to cancer. The p53 tumor suppressor gene is a transcription factor that regulates the cell cycle and is the most widely mutated gene in human tumorigenesis (Wang and Harris, 1997). The critical role of p53 is evident by the fact that it is mutated in over 50% of all human cancers. The p53 gene can activate DNA repair proteins when DNA has sustained damage and hold the cell cycle at the G1/S regulation point on DNA damage recognition. It can also initiate apoptosis if the DNA damage proves to be irreparable. Tumourigenesis can occur if this system goes awry. If the p53 gene is damaged, then tumor suppression is severely reduced. The p53 gene can be damaged by radiation, chemicals and viruses such as the Human papillomavirus (HPV). People who inherit only one functional copy of this gene will most likely develop Li–Fraumeni syndrome, which is characterized by the development of tumors in early adulthood. 2.6.3. Assays for Apoptosis Complex signaling cascade and its multiple regulation points are exploited in the assessment of apoptosis. There are a large variety of assays available, but each assay has advantages and disadvantages which may make it acceptable to use for one application but inappropriate for another application (Watanabe et al., 2002; Otsuki et al., 2003). Therefore, when choosing methods of apoptosis detection in cells, tissues or organs, understanding the advantages and disadvantages of each assay is crucial. Based on methodology, apoptosis assays, can be classified into six major groups: (i) Cytomorphological alterations (ii) DNA fragmentation (iii) Detection of caspases, cleaved substrates, regulators and inhibitors (iv) Membrane alterations (v) Detection of apoptosis in whole mounts (vi) Mitochondrial assays. 29 UNIVERSITY OF IBADAN LIBRARY 2.6.3.1 Cytomorphological Alterations The evaluation of hematoxylin and eosin-stained tissue sections with light microscopy allows the visualization of apoptotic cells. Although a single apoptotic cell can be detected with this method, confirmation with other methods may be necessary. The morphological events of apoptosis are rapid and the fragments are quickly phagocytized. Considerable apoptosis may occur in some tissues before it is histologically apparent. Also, this method detects the later events of apoptosis, so cells in the early phase of apoptosis will not be detected. Semi-ultrathin sections from an epoxy-resin-embedded block can be stained with toluidine blue or methylene blue to reveal intensely stained apoptotic cells when evaluated by standard light microscopy. This methodology depends on the nuclear and cytoplasmic condensation that occurs during apoptosis. The tissue and cellular details are preserved with this technique and surveys of large tissue regions are distinct advantages. However, smaller apoptotic bodies will not be detected and healthy cells with large dense intracellular granules can be mistaken for apoptotic cells or debris. Additionally, there is loss of antigenicity during processing so that immunohistological or enzyme assays cannot be performed on the same tissue. However, this tissue may be used for transmission electron microscopy (TEM). Transmission electron microscopy is considered the gold standard for confirmation of apoptosis. This is because categorization of an apoptotic cell is irrefutable if the cell contains certain ultrastructural morphological characteristics (White and Cinti, 2004). These characteristics are: (1) electron-dense nucleus (marginalization in the early phase); (2) nuclear fragmentation; (3) intact cell membrane even late in the cell disintegration phase; (4) disorganized cytoplasmic organelles; (5) large clear vacuoles; and (6) blebs at the cell surface. As apoptosis progresses, these cells will lose the cell-to-cell adhesions and will separate from neighboring cells. During the later phase of apoptosis, the cell will fragment into apoptotic bodies with intact cell membranes and will contain cytoplasmic organelles with or without nuclear fragments. Phagocytosis of apoptotic bodies can also be appreciated with TEM. The main disadvantages of TEM are the cost, time expenditure, and the ability to only assay a small region at a time. Other disadvantages include the difficulty in detecting apoptotic cells due to their transient nature and the inability to detect apoptotic cells at the early stage. 30 UNIVERSITY OF IBADAN LIBRARY 2.6.3.2 DNA Fragmentation The DNA laddering technique is used to visualize the endonuclease cleavage products of apoptosis (Wyllie, 1980). It involves extraction of DNA from a lysed cell homogenate followed by agarose gel electrophoresis. These results in a characteristic “DNA ladder” with each band in the ladder separated in size by approximately 180 base pairs. This methodology is easy to 6 perform, has a sensitivity of 1 × 10 cells (i.e., level of detection is as few as 1,000,000 cells), and is useful for tissues and cell cultures with high numbers of apoptotic cells per tissue mass or volume, respectively. However, it is not recommended in cases with low numbers of apoptotic cells. There are other disadvantages to this assay. DNA fragmentation occurs in the later phase of apoptosis. Therefore, the absence of a DNA ladder does not eliminate the potential that cells are undergoing early apoptosis. Additionally, DNA fragmentation can occur during preparation making it difficult to produce a nucleosome ladder. Furthermore, necrotic cells can also generate DNA fragments. The TUNEL (Terminal dUTP Nick End-Labeling) method is used to assay the endonuclease cleavage products by enzymatically end-labeling the DNA strand breaks. Terminal transferase is used to add labeled UTP to the 3‟-end of the DNA fragments. The dUTP can then be labeled with a variety of probes to allow detection by light microscopy, fluorescence microscopy or flow cytometry. The assays are available as kits and can be acquired from a variety of companies. This assay is also very sensitive, allowing detection of a single cell via fluorescence microscopy or as few as 100 cells via flow cytometry. It is also a fast technique and can be completed within 3 hours. The disadvantages are cost and the unknown parameter of how many DNA strand breaks are necessary for detection by this method. This method is also subject to false positives from necrotic cells and cells in the process of DNA repair and gene transcription. For these reasons, it should be paired with another assay. 2.6.3.3 Detection of Caspases, Cleaved Substrates, Regulators and Inhibitors There are more than 13 known caspases (procaspases or active cysteine caspases) that can be detected using various types of caspase activity assays (Gurtu et al., 1997). There are also immunohistochemistry assays that can detect cleaved substrates such as PARP and known cell 31 UNIVERSITY OF IBADAN LIBRARY modifications such as phosphorylated histones (Love et al., 1999; Talasz et al., 2002). Fluorescently conjugated caspase inhibitors can also be used to label active caspases within cells (Grabarek et al., 2002). Caspase activation can be detected in a variety of ways including western blot, immunoprecipitation and immunohistochemistry. Both polyclonal and monoclonal antibodies are available to both pro-caspases and active caspases. One method of caspase detection requires cell lysis in order to release the enzymes into the solution, coating of microwells with anti-caspases; followed by detection with a fluorescent 5 labelled substrate. Detection of caspase activity by this method usually requires 1 × 10 cells. This technique allows selection for individual initiator or execution caspases. It also allows for rapid and consistent quantification of apoptotic cells. The major disadvantage is that the integrity of the sample is destroyed thereby eliminating the possibility of localizing the apoptotic event within the tissue or determining the type of cell that is undergoing apoptosis. Another disadvantage is that caspase activation does not necessarily indicate that apoptosis will occur. Moreover, there is tremendous overlap in the substrate preferences of the members of the caspase family, affecting the specificity of the assay. 2.6.3.4 Apoptosis PCR microarray Apoptosis PCR microarray is a relatively new methodology that uses real-time PCR to profile the expression of at least 112 genes involved in apoptosis (Hofmann et al., 2001; Vallat et al., 2003). These PCR microarrays are designed to determine the expression profile of genes that encode key ligands, receptors, intracellular modulators, and transcription factors involved in the regulation of programmed cell death. Genes involved in anti-apoptosis can also be assessed with this methodology. Comparison of gene expression in cells or tissues can be performed between test samples and controls. This type of assay allows for the evaluation of the expression of a focused panel of genes related to apoptosis and several companies offer apoptosis pathway- specific gene panels. Hierarchical cluster analysis of genes can reveal distinct temporal expression patterns of transcriptional activation and/or repression. However, interpretation of the results can be confounded by the large number of analyzed genes and by the methodological complexity. This methodology uses a 96-well plate and as little as 5 nanograms of total RNA. 32 UNIVERSITY OF IBADAN LIBRARY Every mRNA, or transcript, is labeled with a marker, such as a fluorescent dye. A real-time PCR instrument is used for expression profiling. The location and intensity of the resulting signals give an estimate of the quantity of each transcript in the sample. The microarray test should be combined with a different methodology to confirm 2.6.3.5 Membrane Alterations Externalization of phosphatidyl serine residues on the outer plasma membrane of apoptotic cells allows detection via Annexin V in tissues, embryos or cultured cells. Once the apoptotic cells are bound with FITC-labeled Annexin V, they can be visualized with fluorescent microscopy. The advantages are sensitivity (can detect a single apoptotic cell) and the ability to confirm the activity of initiator caspases. The disadvantage is that the membranes of necrotic cells are labeled as well. Therefore a critical control is to demonstrate the membrane integrity of the phosphatidylserine-positive cells. Since loss of membrane integrity is a pathological feature of necrotic cell death. Necrotic cells will stain with specific membrane-impermeant nucleic acid dyes such as propidium iodide and trypan blue. Likewise, the membrane integrity of apoptotic cells can be demonstrated by the exclusion of these dyes. The transfer of phosphatidylserine to the outside of the cell membrane will also permit the transport of certain dyes into the cell in a unidirectional manner. As the cell accumulates dye and shrinks in volume, the cell dye content becomes more concentrated and can be visualized with light microscopy. This dye-uptake bioassay works on cell cultures, does not label necrotic cells, and has a high level of sensitivity (can detect a single apoptotic cell). 2.6.3.6 Detection of Apoptosis in Whole Mounts Apoptosis can also be visualized in whole mounts of embryos or tissues using dyes such as acridine orange (AO), Nile blue sulfate (NBS), and neutral red (NR) (Zucker et al., 2000). Since these dyes are acidophilic, they are concentrated in areas of high lysosomal and phagocytotic activity. The results would need to be validated with other apoptosis assays because these dyes cannot distinguish between lysosomes degrading apoptotic debris from degradation of other debris such as microorganisms. Although all of these dyes are fast and inexpensive, they have certain disadvantages. AO is toxic and mutagenic and quenches rapidly under standard conditions whereas NBS and NR do not penetrate thick tissues and can be lost during preparation 33 UNIVERSITY OF IBADAN LIBRARY for sectioning. Lyso- Tracker Red is another dye that acts in a similar way; however this dye can be used with laser confocal microscopy to provide 3-dimensional imaging of apoptotic cells. This dye is stable during processing, penetrates thick tissues and is resistant to quenching. This dye can be used for cell culture as well as whole mounts of embryos, tissues, or organs. 2.6.3.7. Mitochondrial Assays Mitochondrial assays and cytochrome c release allow the detection of changes in the early phase of the intrinsic pathway. Laser Scanning Confocal Microscopy (LSCM) creates submicron thin optical slices through living cells that can be used to monitor several mitochondrial events in intact single cells over time (Darzynkiewicz et al., 1999). Mitochondrial permeability transition 2+ (MPT), depolarization of the inner mitochondrial membrane, Ca fluxes, mitochondrial redox status, and reactive oxygen species can all be monitored with this methodology. The main disadvantage is that the mitochondrial parameters that this methodology monitors can also occur during necrosis. The electrochemical gradient across the mitochondrial outer membrane (MOM) collapses during apoptosis, allowing detection with a fluorescent cationic dye. In healthy cells this lipophilic dye accumulates in the mitochondria, forming aggregates that emit a specific fluorescence. In apoptotic cells the MOM does not maintain the electrochemical gradient and the cationic dye diffuses into the cytoplasm where it emits a fluorescence that is different from the aggregated form. Other mitochondrial dyes can be used that measure the redox potential or metabolic activity of the mitochondria in cells. However, these dyes do not address the mechanism of cell death and should be used in conjunction with other apoptosis detection methods such as a caspase assay. Cytochrome c release from the mitochondria can also be assayed using fluorescence and electron microscopy in living or fixed cells (Scorrano et al., 2002). However, cytochrome c becomes unstable once it is released into the cytoplasm (Goldstein et al., 2000). Therefore a non-apoptotic control should be used to ensure that the staining conditions used are able to detect any available cytochrome c. Apoptotic or anti- apoptotic regulator proteins such as Bax, Bid, and Bcl-2 can also be detected using fluorescence and confocal microscopy (Tsien, 1998; Zhang et al., 2002). However, the fluorescent protein tag may alter the interaction of the native protein with other proteins. Therefore, other apoptosis assays should be used to confirm the results. 34 UNIVERSITY OF IBADAN LIBRARY 2.7 NECROSIS Necrosis, also referred to as type III cell death is characterized by an increase in cell volume, the swelling of organelles and the rupture of the plasma membrane and is largely considered an accidental type of cell death (Kroemer and Levine, 2008). During necrosis, the mitochondria swell with apparently intact nuclei, and no DNA fragmentation. Necrosis is an uncontrolled and passive process that usually affects large fields of cells whereas apoptosis is controlled and energy-dependent and can affect individual or clusters of cells. However, Necroptosis, a regulated necrotic cell death triggered by broad caspase inhibition in the presence of death receptor ligands and is characterized by necrotic cell death morphology and the activation of autophagy (Degterev et al., 2005). Necrotic cell injury is mediated by two main mechanisms; interference with the energy supply of the cell and direct damage to cell membranes. Some of the major morphological changes that occur with necrosis include cell swelling; formation of cytoplasmic vacuoles; distended endoplasmic reticulum; formation of cytoplasmic blebs; condensed, swollen or ruptured mitochondria; disaggregation and detachment of ribosomes; disrupted organelle membranes; swollen and ruptured lysosomes; and eventually disruption of the cell membrane (Kerr et al., 1972; Majno and Joris, 1995). This loss of cell membrane integrity results in the release of the cytoplasmic contents into the surrounding tissue, sending chemotatic signals with eventual recruitment of inflammatory cells. Apoptotic cells do not release their cellular constituents into the surrounding interstitial tissue and are quickly phagocytosed by macrophages or adjacent normal cells, there is essentially no inflammatory reaction (Savill and Fadok, 2000; Kurosaka et al., 2003). It is also important to note that pyknosis and karyorrhexis are not exclusive to apoptosis and can be a part of the spectrum of cytomorphological changes that occurs with necrosis (Cotran et al., 1999). Some of the major morphological features of apoptosis and necrosis are presented in table 2.1 and shown in Figure 2.9. 2.8 Autophagy Autophagic cell death also referred to as type II cell death. Autophagy (also called autophagocytosis) is evolutionary conserved intracellular system that enables cells to degrade and recycle cellular components (Mihalache, 2012). The word autophagy derives from Greek and means to eat oneself. It is also referred to as type II cell death, is a degradative lysosomal 35 UNIVERSITY OF IBADAN LIBRARY pathway that is characterized by the accumulation of cytoplasmic material in the vacuoles for bulk degradation by lysosomal enzymes. Distinctive feature of type II PCD (or autophagic cell death) is the presence of autophagosomes and autophagolysosomes (Barth et. al., 2011; Ogier- Denis and Codogno, 2003; Edinger and Thompson, 2004), which are the executors of self- degradation. Autophagosomes are spherical structures consisting of double bilayered membranes with diameters of about 500 nm (Noda, 2009). Other morphology features associated with autophagy are marked modifications of the mitochondrial structure such as condensation of the mitochondrial matrix, dilatation of the intercristal spaces and budding of the outer mitochondrial membranes (a morphologic sign of mitochondrial fission), which may be critical for the segregation of dysfunctional mitochondria and their subsequent autophagic removal. Other authophagy related ultra structural modifications includes: dilatation of the rough endoplasmic reticulum and golgi complex, presence of cytoplasmic lipid droplets and peripheral chromatin condensation. 36 UNIVERSITY OF IBADAN LIBRARY Figure 2.9: Some common morphologic features of apoptosis and necrosis (Darzynkiewicz et al, 1997) 37 UNIVERSITY OF IBADAN LIBRARY Table 2.1: Comparison of morphological features of apoptosis and necrosis Apoptosis Necrosis Single cells or small cluster of cells Often contiguous cells Cell shrinkage and convolution Cell swelling Pyknosis and Karyorrhexis Karyolysis, pyknosis and karyorrhexis Intact cell membrane Disrupted cell membrane Cytoplasm retained in apoptotic bodies Cytoplasm released No inflammation Inflammation usually present 38 UNIVERSITY OF IBADAN LIBRARY The phenomenon of autophagy occurs in a wide range of eukaryotic organisms from yeast to mammals during starvation, cell and tissue development, and cell death (Levine and Klionsky, 2004). Autophagy is critical to the process of embryo development because some animal models have shown that the lack of autophagy leads to arrest, delays or defects in development (Adastra et al., 2011). The physiological role of autophagy enables cellular homeostasis by removing damaged or surplus organelles in order to ensure survival (Yang et al., 2008). In addition, amino acids and fatty acids that are produced from the degradation of component of the cells in autolysosomes can be used for protein synthesis or be oxidized by the mitochondrial electron transport chain to produce ATP for cell survival under starvation conditions (Levine and Yuan, 2005). Paradoxically, autophagy can also lead to cell death by depleting the cell's organelles and critical proteins (Zhang et al., 2011). It also plays a role in cells of the immune system. In addition, autophagy represents an adaptive response to cellular and environmental stress, such as nutrient deprivation or growth factor withdrawal (Park et al., 2013; Moreau et al., 2010). Other stress factors such as pathogens, chemicals, radiation, hypoxia, and ROS might induce autophagy. Dysregulation of autophagy has been implicated in a wide range of disease processes, including aging-related disorders, infection and immunity, inflammation, cancer and cardiomyopathy (Levine and Kroemer, 2008; Chiarelli et al., 2012). Recent studies have linked autophagy with neurodegenerative disease such as Alzheimer‟s and Huntington‟s disease ( Chiarelli et al., 2012). The molecular mechanisms of autophagosome formation are conserved in evolution and involve several autophagy related genes. One of these genes is atg5, whose product, autophagy related gene 5 (ATG5) conjugates with ATG12, to generate an E3 ubiquitin ligase-like enzyme required for autophagy (Geng et al., 2008). Mice deficient in the atg5 gene die on the first day after birth (Kuma et al., 2004). In addition to the ATG5–ATG12 conjugate, the ATG8 (LC3) conjugation system is also essential for autophagosome formation (Geng et al., 2008; Tacham and Simon, 2010). Once formed, autophagosomes then merge with lysosomes to form autolysosomes whose contents are degraded by lysosomal hydrolases (Geng et al., 2008). 39 UNIVERSITY OF IBADAN LIBRARY 2.8.1 Types of Autophagy In mammalian cells, three types of autophagy are recognised depending on the way of delivery of material subjected to degradation to lysosomes, and include macroautophagy, microautophagy and chaperone-mediated autophagy (Klionsky and Emr, 2000; Cuervo, 2004). Macroautophagy plays a major role in intracellular degradation and is often used as a synonym for autophagy (Yoshimori, 2004). During macroautophagy, portions of cytoplasm, and even entire organelles, are sequestered in a double-membrane organelle called autophagosome. Such organelles are subjected to a number of subsequent changes including membrane transformation, acidification, and, finally, fusion with lysosomes or late endosomes, resulting in the formation of autophagolysosomes or amphisomes, respectively. Although macroautophagy is generally considered a non-selective process, there have been reports of selective autophagy of peroxisomes and mitochondria (Yokota, 1993). The origin and nature of the autophagic sequestering membrane (phagophore) is not well understood. However, it has been proposed that phagophore formation occur either by de novo synthesis, or by utilisation of pre-existing cytoplasmic membranes. The process when lysosomes sequester cytoplasmic components in invaginations of lysosomal membranes is termed microautophagy (Dice, 2000). Digestion of the internalised material occurs after disappearance of the vesicle membrane. Microautophagy accounts for the degradation of peroxisomes and some cytosolic proteins. Chaperone-mediated autophagy is a selective pathway responsible for degradation of certain cytosolic proteins after their direct transport through the lysosomal membrane by means of molecular chaperones (Dice, 2000; Cuervo, 2004). Proteins selected for this type of autophagy, contain a specific amino acid sequence (KFERQ: lysinephenylalanine- glutamate-arginine- glutamine), which is recognised by a heatshock type chaperone protein, and is transported to the lysosomal-associated membrane protein type 2a (LAMP-2a) for translocation into the lysosomal lumen (Cuervo, 2004). 2.8.2 Regulation of Autophagy The exact mechanism of regulating autophagy-induced is unclear. However, a number of agents and conditions have been reported to regulate autophagy. Nutrient deprivation is a potent inducer 40 UNIVERSITY OF IBADAN LIBRARY of autophagy, while amino acids, being the final product of autophagic degradation of proteins, act as negative feedback regulators (Blommaart et al., 1997). An important part of nutrient dependent regulation pathway is the serine/threonine kinase mammalian TOR (target of rapamycin). Inhibition of MTOR by rapamycin induces autophagy even in presence of amino acids (Blommaart et al., 1997). Although the exact mechanism of amino acid regulation of MTOR activity is not yet understood, it has been shown that activation of MTOR by nutrients induces phosphorylation of proteins involved in the initial step of autophagy, resulting in their disassembling and inhibition of autophagy (Levine and Klionsky, 2004; Meijer and Codogno, 2004). MTOR has also been found to be sensitive to depletion of ATP (Dennis et al., 2001). In addition to nutrients, autophagy is also regulated by some hormones especially insulin and glucagon. Activation of the insulin receptor induces activity of class I phosphatidylinositol-3- kinase (PI3K), with consequent activation of a cascade of intermediate enzymes, finally resulting in upregulation of MTOR and inhibition of autophagy (Levine and Klionsky, 2004). A MTOR independent insulin receptor associated pathway has also be been described (Saeki et al., 2003). Activation of the pathway also results in inhibition of autophagy. Glucagon, on the other hand, activates autophagy by inhibiting MTOR via a protein kinase A-related mechanism (Kimball et al., 2004). Autophagosome formation is controlled mainly by class III phosphatidylinositol 3-kinase (PI3K) and Atg6, and elongation of the autophagosomal membrane is mediated by two ubiquitin-like conjugation systems: the conjugation of Atg12 to Atg5, which is localized together with Atg16 to the phagophore and, downstream of it, the Atg8 conjugation to phosphor ethanolamine (PE), which decorates both the phagophore and the autophagosomal membrane (Fujita et al., 2008; Hanada et al., 2007). Atg8–PE undergoes deconjugation by the Atg4 protease, a step regulated by ROS that allows recycling of this protein (Scherz-Shouval et al., 2008). Atg4 also is responsible for the priming of Atg8 by cleaving its C terminus, which exposes a glycine residue (Kirisako et al., 2000). Although a number of additional proteins, such as heterotrimeric G proteins, Ras, protein kinases A and B were reported to regulate autophagy, their role and mechanisms of action are not yet well understood (Levine and Klionsky, 2004; Meijer and Codogno, 2004). 41 UNIVERSITY OF IBADAN LIBRARY 2.8.2 Autopagy and Oxidative Stress It is generally accepted that ROS induce autophagy (Xu, et al., 2006; Matsui et al., 2007), and that autophagy, in turn, serves to reduce oxidative damage (Marino, 2004; Scherz-Shouval et al., 2007; Pelicano et al., 2004). Many cellular stresses can cause induction of autophagy such as endoplasmic reticulum stress or mitochondrial dysfunction (Marin˜o, 2004; Matsui et al., 2007) Oxidative stress has been shown to induce autophagy under starvation and ischemia/reperfusion conditions ( Matsui et al., 2007; Scherz-Shouval et al., 2007) Under oxidative stress, reactive . . oxygen species (ROS) including free radicals such as superoxide (O2 ), hydroxyl radical (HO ) and hydrogen peroxide (H2O2) are generated at high levels inducing cellular damage and cell death (Pelicano et al., 2004). This cell death often involves induction of apoptosis through caspase activation. Blockage of caspase activation causes degradation of catalase and increased ROS generation leading to cell death. The degradation of catalase is mediated by autophagy indicating a role for autophagy in caspase-independent cell death (Yu et al., 2006). Furthermore, ROS contribute to caspase-independent cell death in macrophages (Shouval et al., 2007). Under starvation conditions, ROS production is increased and is required for induction of autophagy (Scherz-Shouval et al., 2007). The mechanism involved in oxidative stress induced autophagy- related cell death is unknown. 2.8.4 Detection of Autophagy 2.8.4.1. Transmission Electron Microscopy Autophagy was first described by transmission electron microscopy (TEM) about half century ago (Glick et al., 2010; Ashford, 1962). Till date, TEM remains one of the most widely used and sensitive techniques to detect the presence of autophagic vesicles (Eskelinen, 2008; Klionsky et al., 2008). TEM has recently been used to great effect in combination with tomographical approaches to identify regions of the endoplasmic reticulum as the likely origin of autophagosomes in mammalian cells (Hayashi-Nishino, 2009; Yla-Anttila et al., 2009). TEM characterizes autophagy qualitatively, as early autophagic compartments (autophagosomes) containing morphologically intact cytosol or organelles, or as late, degradative autophagic structures (autolysosomes) containing partially degraded cytoplasmic as well as organelle material. The major limitation of TEM in analyzing autophagy is that it is not objectively quantitative. Thus, while TEM remains an important qualitative approach to monitor steady state 42 UNIVERSITY OF IBADAN LIBRARY levels of autophagy and to gain structural insight to the unique inter-relationship between phagophore membranes and other organelles, additional techniques are needed in conjunction with TEM to quantify steady-state levels of autophagy and autophagic flux. 2.8.4.2. Monitoring Autophagy by Fluorescence microscopy In addition to electron microscopy, earlier studies of autophagy relied heavily on cell staining and fluorescent microscopy. In particular, the over-expression of GFP–LC3, in which GFP (green fluorescent protein) is expressed as a fusion protein at the amino terminus of LC3, was widely used to measure autophagy (Kadowaki and Karim, 2009). These studies were limited, however, by several issues: (a) counting GFP-positive punctate structures in order to quantify relative levels of autophagy is a laborious and arguably subjective task, despite the use of computer software; (b) overexpressed GFP–LC3 can be incorporated into protein aggregates independent of autophagy; (c) transfection procedures used to introduce exogenous GFP–LC3 has been shown to induce autophagy; (d) GFP–LC3 is sensitive to acid pH and ceases to fluoresce once autophagosomes fuse with the lysosome, resulting in the inability to look at end- stages of autophagy ( Kuma et al., 2007; Kimura et al., 2007). 2.8.5 Autophagy and Tumor Cell Survival The predominant role of autophagy in cancer cells is to confer stress tolerance, which serves to maintain tumor cell survival (Degenhardt et al., 2006). Knockdown of essential autophagy genes in tumor cells has been shown to confer or potentiate the induction of cell death (White and DiPaola, 2009). Cancer cells have high metabolic demands due to increased cellular proliferation, and in vivo models, exposure to metabolic stress was shown to impair survival in autophagy-deficient cells compared with autophagy- proficient cells (Mathew et al., 2009). Basal levels of autophagy were increased in human pancreatic cancer cell lines and tumor specimens, and they were shown to enable tumor cell growth by maintaining cellular energy production (Yang et al., 2011). Cancer cells that survive chemotherapy and/or radiation activate autophagy to enable a state of dormancy in residual cancer cells that may contribute to tumor recurrence and progression (Lu et al., 2008). Inhibition of autophagy in tumor cells has been shown to enhance the efficacy of 43 UNIVERSITY OF IBADAN LIBRARY anticancer drugs supporting its role in cytoprotection. Recent report indicate that human cancer cell lines bearing activating mutations in H-ras or K-ras have high basal levels of autophagy even in the presence of abundant nutrients (Guo et al.,2011). Suppression of essential autophagy proteins was shown to inhibit cell growth in those cells. Indicating that autophagy maintains tumor cell survival and suggesting that blocking autophagy in tumors that are addicted to autophagy, such as Ras-driven cancers, may be an effective treatment approach. 2.9 HEXAVALENT CHROMATE Chromium was discovered in 1797 as part of the mineral crocoite, used as pigment due to its intense coloration. It derived its name from the Greek word “χρώμα” (chroma- color) (Santos and Rodriguez, 2012). Chromium is the 21st most abundant element in Earth's crust with an average concentration of 100 ppm, ranging in soil between 1 mg/kg and 3000 mg/kg; in sea water from 5 μg/L to 800 μg/L and in rivers and lakes between 26 μg/L and 5.2 mg/L (Santos and Rodriguez, 2012). Normally, Cr is mined from chromate but native deposits are not unheard off. One of the most interesting characteristics of this metal is its hardness and high resistance to corrosion and discoloration. The importance of these proprieties resulted among others in the usage of this metal in the development of stainless steel, which together with chrome plating and leather tanning, are the most important applications of this element (Santos and Rodriguez , 2012).. These industries are also the main sources of Cr pollution in the environment. Chromium is highly soluble under oxidizing conditions and forms, exhibiting a wide range of possible oxidation states (from -2 to +6), The trivalent [Cr (III)] and hexavalent [Cr(VI)] are the most stable forms. Naturally occurring chromium is usually present as Cr(III), while hexavalent chromium in the environment is almost entirely derived from human activities. Hexavalent chromium is especially of great interest not only because of its toxic effects on industrial workers, but the high risk of exposure to hexavalent chromium that still exists for the general public. 2.9.1 Physicochemical properties of chromium and its principal ions Chromium (atomic number 24, relative atomic mass 51.996) occurs in each of the oxidation states from -2 to +6, but only the 0 (elemental metal form), +2, +3 and +6 states are common. Divalent chromium (+2) is unstable in most compounds as it is easily oxidized to the trivalent form by air. Only the trivalent Cr(III) and hexavalent Cr(VI) forms are more common and important for human health (Skovbjerg et al., 2006). 44 UNIVERSITY OF IBADAN LIBRARY The relationship between the hexavalent and trivalent states of chromium is described by the equation: 2- + - 3+ Cr2O7 + 14H + 6 e 2Cr + 7H2O + 1:33 eV 6+ 3+ 3+ Considering the high electric potential of conversion of Cr to Cr , Cr rarely occur in biological systems. However, reduction of Cr (VI) occurs spontaneously in the organism unless present in an insoluble form. For instance, in blood, Cr(VI) is rapidly reduced to Cr (III). Thus, once Cr (VI) has penetrated the membrane of the red blood cell it is reduced to Cr (III). Cr (III) becomes bound to cellular constituents making it unable to leave the erythrocyte. Chromium (VI) exists in solution as ionic species which are dependent on pH. At basic and neutral pH, Cr (VI) is largely in chromate form. The dichromates species becomes dominat at lower pH. Some physicochemical properties of some selected hexavalent chromate compounds are summarized in Table 2.2. Both chromate and dichromate have tetrahedral arrangements of coordinated oxygen groups (Figure 2.10). Hexavalent chromate is a potent oxidant at highly acidic pH and any organic molecule with oxidizable groups can promote its reduction to Cr (III). Increasing pH enhances the stability of Cr (VI), particularly at near neutral and alkaline pH. The strong pH effect on Cr(VI) reduction is demonstrated by the findings that human gastric juice was capable of reducing approximately 70% of Cr(VI) after a 30-min incubation at pH 1.4 but was 2+ completely ineffective at pH 7.0 (Donaldson et al., 1996). The presence of Fe is important for abiotic reduction of Cr (VI) under anaerobic conditions in soil and underground water. Uptake and reduction by microorganisms is another detoxification process for Cr (VI), although it is limited by the ability of resistant bacteria striving in contaminated water to rapidly extrude Cr(VI) (Branco et al., 2008). Figure 2.10: Structure of chromate and dichromate. 45 UNIVERSITY OF IBADAN LIBRARY 2.9.2 Uses of Chromium Compounds The resistance of chromium to ordinary corrosive agents at room temperature makes it useful in electroplating and protective coating. It is also used in non-ferrous and ferrous alloys. Ferrous alloys, mainly stainless steels, account for most of the consumption (Shadreck and Mugadza, 2013). These steels have a wide range of mechanical properties as well as being corrosion and oxidation resistant (Bielicka et al., 2005). In addition, chromium and its compounds are very useful in everyday life, as presented in Table 2.2. It is used on a large scale in many different industries, including metallurgical, electroplating, production of paints and pigments, tanning, wood preservation, chromium chemicals production, and pulp and paper production (Zayed and Terry, 2003). 2.9.3 Contamination of the Environment by Hexavalent Chromate Industrial activities associated with the direct release of Cr (VI) into the soil and water is the most important source of Cr (VI) contamination in the atmosphere. Pollution with various forms of Cr results from its numerous uses in the chemical industry, production of dyes, wood preservation, leather tanning, chrome plating, manufacturing of various alloys, and many other applications and products (IARC,1990; ASTDR,2000). Incineration and emissions from cars create ambient pollution with Cr (VI)- and Cr(III)-containing particles, which leads to low-level inhalation exposures by large segments of the general population and increases Cr levels in surface waters (Zhitkovich, 2011). Chromate can travel over significant distances if introduced into a water source. The permissible level of chromium was lowered to 0.1 ppm due to the emergence of new information about amounts of chromium being released into the environment (Clifford and Chau, 1998). Between the years 1987 and 1993, there were 2,876,055 pounds of chromium released into bodies of water in the United States and 196,880,624 pounds released on land. The contamination stems from the mining, smelting, and wood treatment industries. Again, in 2006, the standard was further lowered from 52 to 5 μg/m3 of air as an 8 hour time-weighted average for hexavalent chromium. Some of the chromate may be reduced to Cr (III), but the portion that remains in the hexavalent form will be carried downstream (Fandeur, 2007). 46 UNIVERSITY OF IBADAN LIBRARY Large scale environmental pollution with Cr (VI) results from anthropogenic contamination of drinking water discharges of toxic Cr(VI) by cooling towers (Pellerin, 2000). Improper disposal of millions of tons of incompletely processed chromite ore also contribute to the pollution of the environment by hexavalent chromate (Stern et al., 1998). In addition, hundreds of the large toxic waste sites in the U.S. known as Superfund sites contain Cr as a major contaminant (ASTDR, 2000). Furthermore, the presence of Cr(VI) in drinking water can also result from the oxidation of naturally occurring Cr(III) by Mn(III/IV) oxides in birnessite (Oze et al., 2007), a common mineral that coats weathered grains and fractures in Cr-rich ultramafic rocks and serpentinites that are enriched with chromite [FeCr(III)2O4]. In addition to birnessite, the presence of two other Mn (IV) oxide containing minerals, asbolane and lithiophorite, has also been associated with the formation of Cr(VI) from natural Cr(III) (Fandeur et al.,2009). Examination of four minerals made of Mn oxides (birnessite, cryptomelane, todorokite, and hausmannite) showed that birnessite had the highest ability to oxidize Cr (III) under laboratory conditions. Furthermore, environmental contamination with Cr (III) can also generate Cr (VI) through oxidation reactions with water chlorination products, Ca and Mn oxides as well as photoxidation (Apte et al., 2006; Dai et al., 20003; Pilay et al., 2003; Clifford and Chau, 1988). 47 UNIVERSITY OF IBADAN LIBRARY 48 UNIVERSITY OF IBADAN LIBRARY Table 2.2: Uses of Chromate Compounds 49 UNIVERSITY OF IBADAN LIBRARY 2.9.4 Routes of Exposure. Occupational and non occupational exposure to chromate occurs through air, drinking water and direct skin contact (Son et al, 2010). The degree of exposure via the three routes is summarised in table 2.3. Air The bronchial tree is the primary target organ for carcinogenic effects of chromium (VI). Inhalation of chromium-containing aerosols is therefore a major concern with respect to exposure to chromium compounds. The retention of chromium compounds from inhalation, 3 based on a 24-hour respiratory volume of 20 m in urban areas with an average chromium 3 concentration of 50 ng/ m , is about 3–400 ng. Individual uptake may vary depending on concomitant exposure to other relevant factors, e.g. tobacco smoking, and on the distribution of the particle sizes in the inhaled aerosol. Chromium has been determined as a component of cigarette tobacco produced in the United States, its concentration varying from 0.24 to 6.3 mg/kg (IARC,1990), but no clear information is available on the fraction that appears in mainstream tobacco smoke Table 2.3: Levels of daily chromium intake by humans from different routes of exposure 50 UNIVERSITY OF IBADAN LIBRARY Drinking-water The concentration of chromium in water varies according to the type of the surrounding industrial sources and the nature of the underlying soils For instance, an analysis of 3834 tap- water samples in representative cities of the United States showed a chromium concentration ranging from 0.4 to 8 μg/litre (EPA, 1984) Food The daily chromium intake from food is difficult to assess because studies have used methods that are not easily comparable. The chromium intake from typical North American diets was found to be 60–90 μg/day (Pellerin et al., 2000) and may be generally in the range 50–200 μg/day. The chromium content of British commercial alcoholic beverages was reported to be slightlyhigher than that of wines produced in the United States, namely 0.45 mg/litre for wine, 0.30 mg/litre for beer, and 0.135 mg/litre for spirits (EPA,1984). 2.9.5. Toxicokinetics Although studies on the biokinetics of chromium are limited, it is believed that the rate of uptake in the airways is greatly governed by the size distribution of the inhaled particles and by the water solubility of the compounds (ATSDR, 2012). Large particles (> 10 μm) of inhaled Cr (VI) compounds are deposited in the upper respiratory tract, while smaller particles can reach the lower respiratory tract. Some of the inhaled Cr (VI) is reduced to trivalent chromium (Cr [III]) in the epithelial or interstitial lining fluids within the bronchial tree. The extracellular reduction of Cr (VI) to Cr (III) limits the cellular uptake of chromium because Cr(III) compounds cannot enter cells as readily as Cr(VI) compounds. Cr (VI) compounds are tetrahedral oxyanions (Figure 2.9) that can cross cell membranes. Cr (III) compounds are predominantly octahedral structures to which the cell membrane is practically impermeable. Absorption of water-soluble chromium (VI) compounds occurs rapidly by inhalation exposure although the extent of uptake is difficult to quantify. An estimate of pulmonary absorption, after chromium (III) chloride deposition in the lungs indicates that approximately 5% is absorbed within a few hours (EPA, 1984). A number of studies on the fate of chromium (VI) and chromium (III) following intratracheal administration have provided some information on 51 UNIVERSITY OF IBADAN LIBRARY pulmonary retention and absorption of chromium compounds (Edel and Sabbioni, 1985; Weigand et al., 1984). Chromium(III) is retained to a greater extent in the lungs than is chromium(VI) (Edel and Sabbioni, 1985) Chromates with low water solubility are mainly cleared to the gastrointestinal tract, whereas more soluble chromates are absorbed into the blood (Weigand et al., 1984). Absorption of chromium in the gastrointestinal tract is not greater than5% after oral exposure (EPA). Studies on the uptake of chromium (VI) compounds in the gastrointestinal tract show that that the rate of uptake is to is heavily dependent on the solubility of the compounds (IARC, 1990). Data from in vitro studies indicate that gastrointestinal juices are capable of reducing chromium (VI) to chromium (III). However, data from in vivo studies are insufficient to demonstrate whether this reduction process has the capacity to eliminate any differences in absorption between ingested chromium(VI) and chromium(III) compounds (EPA, 1984). Pulmonary cells have been shown in vitro to have some capacity to reduce hexavalent chromium. However, this capacity is low compared to that of liver cells (WHO, 2009). Tests on female experimental animals showed that absorbed chromium (III) and chromium (VI) can be transported to a limited extent to the fetus in utero. However, available data do not allow quantitative estimates of fetal exposure. In humans, the chromium concentration in the tissues of newborn babies has been found to be higher than that found later in life. There is a significant decline in concentration in children until about 10 years of age. Subsequently, there is a slight increase in the lung tissue concentration, but a slight decrease in other tissues. Chromium is transported by the blood and distributed to other organs. The most significant retention occurs in the spleen, liver, kidney and bone marrow (WHO, 2000; Weber, 1983). Studies conducted by the National Toxicology Programme (NTP) in male rats and female mice orally exposed to chromium (VI) for 2 years also showed dose-related and time-dependent increases in total chromium concentrations in red blood cells, plasma, and in several organs. The total chromium content of the red cells was higher than that of plasma (IARC, 1990). In both animals and humans, elimination of absorbed chromate from the body is biphasic, with a rapid phase, representing clearance from the blood, and a slower phase, representing clearance from tissues. The principal route of elimination is urinary excretion and it accounts for a little 52 UNIVERSITY OF IBADAN LIBRARY over 50%. Fecal excretion accounts for only 5%. The remaining chromium is deposited into deep body compartments, such as bone and soft tissue. Elimination from these tissues proceeds very slowly; the estimated half-time for whole-body chromium elimination is 22 days for chromium(VI) and 92 days for chromium(III) following intravenous administration (EPA, 1984). 2.9.6. Metabolism of Chromate VI compounds. Structural similarity of chromate ion to phosphate and sulfate (Figure 2.1) allows its easy entry through the general sulfate channels (Zhitkovich et al., 2005). As with abiotic reactions, cellular reduction of Cr (VI) yields thermodynamically stable Cr(III) (Levina et al., 2007; Ortega et al., 2005). Efficient uptake of Cr(VI) followed by Cr(III) trapping via its binding to macromolecules leads to a massive accumulation of Cr relative to its extracellular concentrations, ranging from 10- to 20-fold after 3-h exposures to about 100-fold after 24-h exposures (Reynolds et al., 2007; Messer et al., 2006). With the apparent exception of bacteria producing hyperoxidized Mn (III/IV), biological systems lack the ability to reoxidize Cr(III) to Cr(VI) (Murray et al., 2007). Extracellular reduction of Cr (VI) is a detoxification process that produces poorly permeable nontoxic Cr (III) (Fig. 2.3). Studies of reduction activities in tissue homogenates and biological fluids showed that ascorbate (Asc) was the principal biological reducer of Cr (VI), accounting for 80-95% of its metabolism (Standeven and Wetterhahn, 1992; Suzuki et al., 1991) A combined activity of Asc and small thiols glutathione (GSH) and cysteine is responsible for >95% of Cr (VI) reduction in vivo. Tissue concentrations of GSH and Asc are not usually dramatically different, and the predominant role of Asc stems from its very high rate of Cr(VI) reduction. At physiological 1mM concentration, t1/2 for Cr (VI) reduction by Asc was 1 min vs 60.7 min for GSH and 13.3min for Cys (Quievryn, 2003). Despite its slower rate of reduction, GSH is more important for Cr (VI) metabolism than Cys due to its higher cellular concentrations. Depending on the nature of the reducing agent, its concentration, and stoichiometry, Cr (VI) reduction reactions generate variable amounts of transient products such as Cr (V), Cr (IV), and sulfur- and carbon-based radicals (Lay et al., 1998; Stearn, 1994). The biological antioxidants, GSH-, cysteine-, and Asc derived radicals formed in Cr (VI) reactions are unreactive toward DNA (Guttmann et al., 2008). However, in the presence of H2O2, the Cr intermediate formed can 53 UNIVERSITY OF IBADAN LIBRARY catalyze Fenton-type reactions, generating highly reactive OH• radicals (Luo et al., 1996) Formation ROS and direct oxidizing abilities of Cr (V) are the two main processes contributing to the induction of oxidative stress in Cr (VI)-treated cells (Slade et al., 2007; Sugden et al., 2001) All the biological reducers convert Cr(VI) to Cr(III) with varying mechanisms of reduction. Kinetic analyses suggest that Cys acts almost exclusively as a one-electron reducer that is consistent with the presence of strong Cr (V) signal in Cys-driven reactions (Quievryn, et al., 2001). Reduction by GSH can proceed via either one- or two-electron reactions (Lay et al., 1998). Asc is a highly efficient two-electron donor, yielding Cr (IV) as the first reduction intermediate and dehydroascorbic acid as the oxidized product. The presence of Cr (V) is only detectable under non physiological conditions of equimolar or higher ratio of Cr (VI) to Asc (Lay et al., 1998). Insufficient amounts of Asc for the completion of Cr (VI) reduction in these reactions were responsible for a transient appearance of Cr (V), likely resulting from comproportionation of Cr (IV) and Cr (VI). Severe Asc deficiency of human and nonhepatic rodent cells in standard cultures raises concerns that studies with cultured cells may not accurately recapitulate genotoxic properties of Cr (VI) in vivo (Messer et al, 2007; Salnikow and Zhitkovich, 2008; Salnikow et al., 2004). The Asc depleted state of cultured cells results from the absence of the essential vitamin in the most common types of synthetic growth media. The additions of 10% serum to the media theoretically supplies only 10% of normal vitamin C levels. The actual levels of Asc in the growth media are lower due to its loss during the preparation and 0 storage of serum. The half-life of Asc at 37 C in cell culture media is 6-7 h (Bergsten, 1990). Recently fed cells can contain up to 50-60 μM vitamins C, but in many cases, its levels are dramatically lower or undetectable. Even when cells start with 50- 60 μM Asc, they become completely depleted of Asc after 24-48 h in culture (Karaczyn, 2006). Physiological concentrations of vitamin C in white blood cells and epithelial tissues are usually in the 1-2 mM range (Bergsten, 1990). 54 UNIVERSITY OF IBADAN LIBRARY 2.9.7 Health Effects of Hexavalent Chromate Exposure Exposure to hexavalent chromate is associated with cancer and non cancer effects.In addition exposure to hexavalent also lead to geneotoxicty, induction of micronuclei, oxidative stress and actin disruption. 2.9.7.1 Nonmalignant Effects Inhalation of Cr(VI) has been shown to cause ulceration of the nasal mucosa, perforation of the nasal septum, asthma, bronchitis, pneumonitis, inflammation of the larynx and liver, while exposure due to dermal contact of Cr(VI) compounds can induce skin ulcers, allergies, dermatitis, dermal necrosis and dermal corrosion (Bielicka et al., 2005). Chronic and acute exposure to chromate (VI) has been reported to cause kidney and liver damage, pulmonary congestion and edema, epigastric pain, erosion and discoloration of teeth, and perforated ear drums. Other effects of exposure to chromates include eye injury, leukocytosis, leukopenia, eosinophilia, (NIOSH, 2003; Johansen et al., 1994). Anemia, thrombocytopenia, infertility, birth defects, spontaneous abortions, ulcers, gastrointestinal bleeding, renal failure, intravascular haemolysis, liver damage, respiratory disturbances, coma and even death have all been reported. to be associated with chromate (VI) exposure (Lin et al., 2009; Loubieres et al., 1999; Bonde and Ernst, 1992). 2.9.7.2 Malignant Effects Particulate hexavalent chromium compounds are well established human respiratory toxins and carcinogens that are used commercially in welding, chrome plating, chrome pigmenting, leather tanning, and in the ferrochrome industry (Beaver, 2009). Occupational exposure to Cr (VI) in these industries and are associated with fibrosis, fibrosarcomas, adenocarcinomas and squamous cell carcinomas of the lung (Beaver, 2009). Epidemiological studies in the USA indicated a 10 to 30 fold- increased risk of lung cancer among workers in the chromate industry compared to the general population (Das and Mishra, 2008). A positive correlation was also found between the duration of exposure and lung cancer death (Das and Mishra, 2008). Upon inhalation, chromium 55 UNIVERSITY OF IBADAN LIBRARY particles accumulate at the bifurcations of the bronchi and the concentration of Cr in these regions of the lung can reach up to 15.8 mg/g tissue (dry weight) (Ishikawa et al., 1994a; Ishikawa et al., 1994b). Autopsy of the lungs of chromate workers show higher lung-Cr burdens correlate with increased lung tumor incidence (Ishikawa, et al., 1994b; Ishikawa, et al., 1994a). Epidemiological studies in Europe, Japan and the United States have consistently shown that workers in the chromate production industry have an elevated risk of respiratory disease including: fibrosis, perforation of the nasal septum, development of nasal polyps, hyperplasia of the bronchial epithelium, lung fibrosarcomas, adenocarcinomas and squamous cell carcinomas (IARC, 1990; Ishikawa, et al., 1994b; Ishikawa, et al., 1994a; Dalager et al., 1980). Animal studies of chromate exposure by inhalation or intratracheal/intrabronchial instillations illustrate that the slightly soluble and highly insoluble particulate Cr (VI) such as zinc, lead, strontium and sintered calcium chromate consistently induced lung tumors (Landolph et al., 1994; Levy et al., 1986; Hueper et al., 1959; Steffee and Baettjer, 1965). 2.9.7.3 Genotoxicity Inaddition, Cr (VI) is a potent genotoxin and initiates a variety of cellular and molecular damage that includes Cr-DNA adducts, DNA single and double strand breaks,, alkali labile sites, chromosomal aberrations, DNA-protein cross-links and apoptosis (Salnikow and Zhitkovich, 2008; Zhitkovich, 2005; O'Brien et al., 2003). The present understanding of Cr (VI)-induced genotoxicity is based on Wetterhahn‟s uptake-reduction model (Standeven and Wetterhahn, 1989): (i) the active transport of Cr(VI) into cells through anion channels for soluble chromates through phagocytosis for insoluble compounds such as PbCrO4); (ii) the intracellular reduction of Cr(VI) with the formation of potentially DNA-damaging Cr (V/IV) intermediates (which can be stabilized by intracellular ligands) and organic radicals; (iii) the formation of kinetically inert Cr(III) complexes, such as highly genotoxic DNA– Cr(III)–protein and DNA–Cr(III)–DNA cross-links, as a result of such reduction 56 UNIVERSITY OF IBADAN LIBRARY Figure 2.11: Metabolism of Hexavalent Chromate (Zhitkovich, 2011) 57 UNIVERSITY OF IBADAN LIBRARY The most common type of DNA damage associated with chromate (VI) exposure is Cr-DNA binding (adducts). It has been detected in reduction reactions in different cultured cells and in vivo (Zhitkovich et al., 2005). Cr-DNA adducts are a heterogeneous groupthat includes binary [Cr(III)-DNA] and several ternary [ligand- Cr(III)-DNA] adducts where the ligand can be Asc, GSH, cysteine, or histidine. All four ternary adducts have been detected in Cr (VI)-treated cells and are readily formed during in vitro Cr (VI) metabolism (Zhitkovich et al., 1995). Cr (VI) also causes the formation of protein-Cr(III)- DNA cross-links, which are rare lesions and whose main toxicological significance could lie not necessarily in the contribution to genotoxic responses but rather in the inhibition of gene-specific expression (Schnekenburger et al., 2007). Binary Cr-DNA adducts are the most frequent DNA modifications in the in vitro reductions of Cr(VI). In Cr(VI) reactions with Asc and GSH, binary adducts accounted for 75-95% of the total DNA-bound Cr (Guttmann et al., 2008; Quievryn et al., 2002).When normalized for recovery, Asc-Cr-DNA cross-links have been calculated to comprise 6% of Cr-DNA adducts in human A549 cells with restored Asc levels( Quievryn et al., 2002). Cys-Cr- DNA and GSH-Cr-DNA accounted for 24 and 17% respectively of all DNA adducts in hamster CHO cells (Zhitkovich et al., 1995) although these values were not been adjusted for recovery. Protein-Cr-DNA cross- links constitute only about 0.1% of total adducts immediately after Cr(VI) exposures (Zecevic , et al., 2010) but their relative amounts are likely higher at later post exposure times due to delayed formation and slower repair relative to those of small Cr-DNA adducts (Zecevic et al , 2010; Macfie et al., 2010 ). Replication of adduct-carrying shuttle-vectors in human cells showed that the most abundant adduct, the binary Cr-DNA conjugate, was weakly mutagenic, whereas four ternary adducts containing DNA-cross-linked Asc, GSH, cysteine, and histidine were strongly mutagenic (Quievryn et al., 2003; Voitkun et al., 1998). In vitro reduction of Cr (VI) by purified Asc, GSH, or cysteine also led to the production of mutagenic and replication-blocking DNA lesions, as revealed by analyses of replicated progeny of shuttle-vector plasmids propagated in human fibroblasts (Guttmann et al., 2008; Quievryn et al, 2003; Zhitkovich, et al, 2001). Asc-driven metabolism of Cr (VI) in vitro resulted in the strongest mutagenic responses, while reactions with GSH showed low yields of mutagenic damage and GSH-Cr-DNA adducts (Guttmann et al., 2008; Quievryn et al., 2003). These findings were corroborated by a strong potentiation of Cr (VI) mutagenicity in cells with restored Asc levels (Reynolds et al., 2007). Blocking of Cr-DNA 58 UNIVERSITY OF IBADAN LIBRARY binding during the reduction or dissociation of Cr-DNA adducts eliminated all mutagenic and replication-blocking responses in shuttle-vector plasmids incubated in Cr (VI) reactions containing Asc, GSH, or Cys (Guttmann et al., 2008; Reynolds et al., 2007; Quievryn et al, 2003). Thus, indicating a key role of Cr-DNA adducts in the mutagenicity and genotoxicity of Cr(VI) when metabolized by these three reductants. In agreement with these results, in vitro reduction reactions employing iron free reagents failed to generate detectable amounts of singlestrand breaks and abasic sites in DNA (Guttmann et al., 2008; Quievryn et al., 2003; Zhitkovich et al., 2001). Inhibition of in vitro replication on DNA templates damaged in Cr (VI)- Asc reactions was also dependent on Cr-DNA binding (O‟Brien et al., 2002). Involvement of Cr-DNA adducts in bacterial mutagenesis by Cr (VI) is indicated by higher yields of revertants in the Ames test using a NER-deficient Salmonella uvrA strain (Watanabe et al., 1998). Cr(VI) mutagenicity in transgenic lacI mice was inhibited by GSH depletion, which points to the importance of non-oxidative mechanisms and GSH-Cr-DNA adducts in mutagenic responses in vivo (Cheng et al., 2000). Cr (VI)-induced fold changes in the number of HPRT mutants were reported to be lower in NER-deficient clones of CHO cells grown under the standard Asc-deficient conditions which contrasts the positive role of NER in the removal of adducts and survival of CHO and human cells (O‟Brien, et al., 2005). The biological consequences of Cr-induced DNA damage include DNA and RNA polymerase arrest and mutagenesis/chromosomal abnormalities including induction of micronuclei (Reynolds et al., 2007; De Flora et al., 2006; Zhitkovich, 2005; Quievryn et al., 2003; Stearns et al., 1994, Suzuki, 1990; Borges et al., 1991). 2.9.7.4 Micronuclei Formation The micronucleus (MN) assay is an effective biomarker for the early detection of the changes related to cancer (Bonassi et al., 2011). Micronuclei originate from chromosome fragments or whole chromosomes that are not included in the main daughter nuclei during nuclear division (Maffei et al., 2014). Thus, MN are found in interphase cells as small, extranuclear bodies resulting from chromosome breaks and whole lagging chromosomes, which are not incorporated into the main nucleus during cell division. The formation of micronuclei during cell division process can be caused by chromosomal rearrangements, altered genome expression or 59 UNIVERSITY OF IBADAN LIBRARY aneuploidy, all of which are associated with the chromosome instability phenotype, often observed in cancer patients (Fenech, 2002). The hypothesis of an association between MN frequency and cancer development is supported by a number of observations, the most substantial of which include the high MN frequency in untreated cancer patients and in subjects affected by cancer-prone congenital diseases (Bolognesi et al., 2005; El-Zein et al., 2006; Iarmarcovai, et al., 2008). A number of international cohort studies have demonstrated that the MN frequency in the Peripheral Blood Lymphocytes of healthy subjects is a predictor of cancer risk (Bonassi et al. 2007; Fenech et al., 2011). MN therefore, is exploited in assessing structural and numerical chromosomal alterations in genotoxicity testing. The frequency MN can be used to screen drug candidates and other test chemicals for both clastogenic and aneugenic potential (Seager et al., 2014). Induction of micronuclei in vivo by hexavalent chromate appears to be dependent on the route of exposure. Several studies that have directly compared the micronucleus formation of Cr (VI) by administration in drinking water (or oral gavage) and ip injection suggest that only ip administration results in genotoxicity. Shindo et al. (1989) exposed two strains of mice (CD-1 and MS/Ae) to several concentrations of Cr (VI) via oral gavage and by ip injection. In both strains of mice, there was a dose-dependent decrease in polychromatic erythrocytes (PCEs) and an increase in micronucleated PCEs following ip injection of >10 mg/kg potassium chromate administration but not following gavage (Shindo et al., 1989). Similarly, De Flora et al. (2006) showed that ip administration of 17.7 mg/kg Cr (VI) to 8- month-old male BDF1 mice resulted in a significant increase in micronucleated PCEs, whereas gavage of the same dose did not. In another study, Bagchi et al. (2002), reported DNA damage in liver and brain tissue within 24–96 h after oral gavage of 6 mg/kg Cr (VI). Recently, it has been demonstrated that chromosome fragments that are not incorporated into the nucleus at cell division (micronuclei formation) is the one of the major manifestations of heavy metal toxicity in tissue culture and animals (Yih et al., 2007). There are several possible hypotheses on the pathways of MN formation. Cytogenetic alterations in chromium (VI)- exposed cells in culture and in vivo, such as increased frequencies of chromosomal breaks and micronuclei, are suggested to be due to DNA double-strand breaks, produced by a cell- replication-dependent mechanism in the G2 phase of the cell cycle. The induction ROS found in chromate (VI)-exposed mammalian and plant cells may attack purine and pyrimidine bases and deoxyribose in DNA. This can cause DNA strand breakage, which can increase the probability of 60 UNIVERSITY OF IBADAN LIBRARY chromosome/chromatid fragmentation. Thus, leading to MN formation. Chromate (VI) can binding to sulfhydryl groups and inactivate some important enzymes involved in DNA repair and expression, alter DNA repair mechanism, and cause an increase in MN frequency. Furthermore, some evidences suggest that chromate (VI) may interfere with microtubule assembly and spindle formation causes chromosomal lagging, chromosomal instability and possibly leads to a higher frequency of micronuclated cells (Reynolds et al., 2007; Salnikow and Zhitkovich, 2008). 2.9.7.5 Hexavalent chromate and Oxidative Stress In addition to intracellular reduction of Cr (VI) to Cr (III) by low molecular weight thiols such as glutathione (GSH) and cysteine, as well as antioxidants like ascorbate (Figure 2.13A) (O‟Brien et al., 2003; Zhitkovich, 2005). It has been hypothesized that the process of Cr (VI) cytotoxicity and carcinogenesis can be triggered by the dysregulation of gene expression, cellular redox state, and DNA damage as a result of oxidative stress. Oxidative stress is the favoring of cellular oxidant production over that of antioxidants, which leads to the formation of reactive oxygen species (ROS), and damage to cellular RNA, DNA, proteins, and lipids (Klaunig and Kamendulis, 2004). It involves reduction of Cr (VI) to Cr (V) by molecular oxygen, which results in the generation of reactive oxygen species (ROS) (Liu and Shi, 2001). This in turn leads to the formation of hydrogen peroxide that can be removed by catalase, conjugation with GSH (perhaps competing with GSH-mediated Cr (VI) reduction), as well as by Fenton reactions with iron. Peroxide can also undergo Fenton reactions with Cr (V), thereby reforming Cr (VI) and hydroxide radicals (Liu and Shi, 2001). Thus, sustained exposure to Cr (VI) might lead to oxidative stress (Figure 2.13B). While the direct relationship between DNA-reactive oxygen species and chromium-induced DNA damage is heavily debated and unclear, there have been several studies supporting the role of ROS in Cr (VI)-induced genotoxicity, cytotoxicity, and oxidative stress (Patlolla et al., 2009; Azad et al., 2010). Pretreatment with free radical scavenger such as flavin adenine dinucleotide or vitamin E were also shown to markedly decrease Cr (VI)- induced single-strand breaks and cytotoxicity (Sugiyama et al., 1989). Furthermore, Shi et al. (1999) suggests that ROS contributes to the early effects of Cr (VI)-induced apoptosis via a p53- independent mechanism, whereas the apoptotic effects could be blocked in the presence of ROS scavengers, such as catalase, aspirin and N-acetyl-L-cysteine. Moreover, gene expression analysis of Cr(VI)-treated A549 cells showed increases in glutathione peroxidase, CuZnSOD, and MT-II (protects cells from toxicity and oxidative stress) gene expression, while NADH 61 UNIVERSITY OF IBADAN LIBRARY ubiquinone oxidoreductase B18 subunit (a subunit of the mitochondrial respiratory chain complex I), glutathione peroxidase, and the sodium/potassium-transporting ATPase alpha 1 subunit (an ROS regulator) were up regulated in Cr(VI)-treated BEAS-2B cells (Andrew et al., 2003). Similarly, Cr (VI)-induced expression of the HO1 gene was also shown in human dermal fibroblasts and A549 (Joseph et al., 2008), however a report from O‟Hara and colleagues contradicts these data (O‟Hara et al., 2006). Generally, the relationship between oxidative stress and Cr (VI)-induced cytotoxicity, genotoxicity, and potential carcinogenesis remains unclear. This has been ascribed to the non- uniform treatment protocols, conflicting data and controversial methods used for detection of Cr (VI)- induced oxidative stress. For instance, Martin et al. (1998) demonstrated that the high valence chromium species, bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) [Cr(V)- EHBA], was able to independently induce the fluorescence of two dyes commonly used to detect ROS (2′,7′- dichlorofluorescin and dihydrorhodamine) in A549 cells that was not affected by treatment with radical scavengers (Martin et al., 1998). Thus indicating that 2, 7′-dichlorofluorescin and dihydrorhodamine are more appropriate for the qualitative detection of Cr (V), rather than ROS production in the presence of Cr (VI). In addition, total dose over time and dose rate, as well as the relative ratios and availability of intracellular reductants and anti-oxidants may also determine the response to cells to oxidative stress or otherwise. 2.9.7.6. Actin Disruption Actin is an important functional and structural protein common to all eukaryotic cells. The actin filaments are the major component responsible for the maintenance of global cell shape in fibroblast (Ujihara et al., 2008). The actin cytoskeleton has diverse function in the cell. It is essential for cell growth, maintenance of cell polarity and morphology, locomotion, phagocytosis, trafficking of organelles, signal transduction and cell division in fibroblasts and other cells. Disruption of the actin cytoskeleton elicits profound changes in cell survival and function. It may lead to disruption in signal transduction that may lead to cell death or enhanced survival. It has been reported that apoptotic signals trigger actin cytoskeletal changes that result in release from the extracellular matrix, membrane blebbing, and condensation into apoptotic bodies in a 62 UNIVERSITY OF IBADAN LIBRARY sequential fashion (Mills et al., 1999). These events are linked with stress fiber loss and cortical actin reorganization, actinomyosin contraction of the actin ring, and dissolution of polymerized actin, respectively (Mills et al., 1999). Actin reorganization and degradation has been viewed as a later consequence occurring only after a cell has committed to an apoptotic death. However, there is increasing evidence that perturbations of the actin cytoskeleton itself can initiate events that commit a cell to apoptosis. Loss of actin-based, integrin mediated cell matrix adhesion results in apoptosis in epithelial cells and other cell types (Frisch et al., 2001). Direct disruption of the actin cytoskeleton with cytochalasin D (CYD) or jasplakinolide induces apoptosis of airway epithelial cells (White et al., 2001), HL-60 cells (Rao et al., 1990), endothelial cells (Li et al., 2003). EL4 T lymphoma cells (Korichneva, 1999) and NIH3T3 cells (Korichneva and Hammerlig,1999). However, Ailenberg and Silverman, 2003 reported that cytochalsin D disruption of mouse mesangial cells (MMC) caused it to undergo apoptosis, but it promoted cell survival in NIH 3T3 cells. Although, it was reported that soluble hexavalent chromium interfere with cytoskeletal actin in murine fibroblasts and hepatocytes (Li et al., 1992; Gunaratnam and Grant, 2004), the role of actin disruption in the cytotoxicity of hexavalent chromate is CH310T ½ mouse embryonic fibroblast is yet to be studied. 63 UNIVERSITY OF IBADAN LIBRARY Figure 2.11: Reduction schemes for Cr (VI). (A) Reduction of Cr (VI) by low molecular weight ligands (L) (Zhitkovich, 2005). (B) Reduction of Cr (VI) by molecular oxygen (Liu and Shi, 2001). SOD, superoxide dismutase; Cys, cysteine; GSSG, oxidized GSH; GPx, glutathione peroxidase; Vc, ascorbate 64 UNIVERSITY OF IBADAN LIBRARY 2.10 Arsenic It is widely distributed in nature in many forms and its compounds are used extensively as components of herbicides, insecticides, rodenticides, food preservatives, and drugs (Mustafa et al., 2010; Baxley et. al., 1981). Inhalation exposure to arsenic also occurs in other industrial settings such as lead, copper, and zinc smelting as well as fossil fuel combustion in power plants and semiconductor industry. The trivalent (arsenite) or pentavalent (arsenate) has a wide distribution. The most common inorganic trivalent arsenic compounds are arsenic trioxide, sodium arsenite, and arsenic trichloride. The pentavalent inorganic compounds are arsenic pentoxide, arsenic acid, and arsenates, such as lead arsenate and calcium arsenate. Arsenite is less excreted from the body relative to arsenate. Thus, arsenites are considered more toxic and carcinogenic than arsenate (Huang 2004; Kreppel et al., 1993). Arsenite is extremely thiol- reactive. It can affect enzyme activities by binding to critical vicinal cysteinyl residues, such as those in the lipoamide of pyruvate dehydrogenase, tyrosine phosphatases, and enzymes involved in protein ubiquination. In general, arsenite is thought to be a sulfhydryl reagent having a high affinity mainly for vicinyl dithiols and thiols located near hydroxyls. Ingestion of arsenite in drinking water presents the greatest hazard and this has been associated with many cancer and non cancer effects in affected populations (Hua et al., 2004). Such effects include tumors at multiple sites including the skin, liver, lungs, urinary bladder and prostate (NRC, 1999; Lewis et al., 1999). The increase in cancer risk observed in epidemiological studies is attributed mainly to the presence of inorganic trivalent arsenic (arsenite). Similarly, arsenite exposure has been linked to endemic arsenic dermatosis along with hyperkeratosis, maningioma, diabetes, hypertension, embryotoxicities, spontaneous abortion, adverse pregnancy outcomes, gangrene, and blackfoot disease (Yang et al. 2003; Tseng et al., 2002.; Saha et al., 1995). An early event in arsenic carcinogenesis is molecular alterations both in humans and animals which manifests in a dose dependent chromosomal breaks and alterations (Barns et al. 2002; Vega et al., 1995). Recently, it was demonstrated that induction of micronuclei is one of the principal manifestations of arsenic toxicity in plants and animals (Odunola et al., 2007; Yi et al., 2007). Several reports have also implicated oxidative stress in arsenic-induced cytotoxicity and genotoxicity (Valko et al., 2006). Many other studies confirmed the generation of free radicals during arsenic metabolism in cells (Yamanaka et al., 2007). Interestingly, experimental 65 UNIVERSITY OF IBADAN LIBRARY evidences have shown that arsenic-induced generation of free radicals can cause cell damage and death through activation of oxidative sensitive signaling pathways (Kamat et al., 2005). Arsenic- mediated generation of reactive oxygen species is a complex process which involves the • − 1 generation of a variety of ROS including superoxide (O2 ), singlet oxygen ( O2), the peroxyl • • radical (ROO ), nitric oxide (NO ), hydrogen peroxide (H2O2), dimethylarsinic peroxyl radicals • • ([(CH3)2AsOO ]) and also the dimethylarsinic radical [(CH3)2As ]. The exact mechanism responsible for the generation of all these reactive species is not yet clear. In addition, it has been found that perturbation of signal transduction such as the JNK pathway occurred during arsenite induced malignant transformation. This confers resistant to apoptosis and thus, suggesting that apoptotic control mechanisms are disrupted as cells becomes transformed through arsenic exposure. This apoptotic disruption may allow damaged cells to inappropriately escape apoptosis and potentially proliferate, thereby providing initiating events in carcinogenic development. In addition, it has been found that perturbation of signal transduction such as the JNK pathway occurred during arsenite induced malignant transformation. This confers resistant to apoptosis and thus, suggesting that apoptotic control mechanisms are disrupted as cells becomes transformed through arsenic exposure. This apoptotic disruption may allow damaged cells to inappropriately escape apoptosis and potentially proliferate, thereby providing initiating events in carcinogenic development. 2.11. CANCER CHEMOPREVENTION The prevention of cancer is one of the most important public health and medical practices of the 21st century. Cancer chemoprevention encompasses the concepts of inhibition, reversal, and retardation of the cancer process. It aims to halt or reverse the development and progression of pre-cancerous cells through use of non-cytotoxic nutrients and/or pharmacological agents during the period between tumor initiation and malignancy. Cancer chemoprevention is regarded as a promising avenue for cancer control (De Flora et al., 2001). This strategy is based on the reduction of cancer incidence by increasing the public consumption of antimutagens and anticarcinogens common used. Chemopreventive agents can be divided into blocking and suppressing agents (Wattenberg, 1997). Blocking agents prevent carcinogens from reaching the target sites, undergoing metabolic activation or interacting with crucial cellular macromolecules such as DNA, RNA and proteins (Figure 2.13). For example, calcium can block bile acid uptake 66 UNIVERSITY OF IBADAN LIBRARY into colonic epithelial cells. On the other hand, suppressing agents, inhibit the malignant transformation of initiated cells at either the promotion or the progression stage (Figure 2.13). The cellular and molecular events modulated by these chemopreventive phytochemicals include carcinogen activation/detoxification by xenobiotic metabolizing enzymes; DNA repair; cell- cycle progression; cell proliferation, differentiation and apoptosis; expression and functional activation of oncogenes or tumour-suppressor genes; angiogenesis and metastasis; and hormonal and growth-factor activity (Surh, 2003). They lower the risk of human cancer development via their radical scavenging, antioxidant, anti-inflammatory and antiproliferative activities (Kwak and Kensler, 2010; Parys et al, 2010; Surh, 2003). Some other agents such as retinoids induce terminal differentiation in aberrant epithelial cells and thus removing them from the replicating pool. Furthermore, many of the bioactive agents‟ acts by altering signal transduction pathways (Surh, 2003). Cancer has no effective cure and conventional radiotherapy and chemotherapy with synthetic drugs used for treating cancer are limited by severe side effects such as immunosuppression, organ failure and infectious diseases which cause the death of patient after recovery from cancer (Barh, 2008). This has necessitated the search for chemopreventive agents from natural products including dietary and medicinal plant. These plants contain phytochemicals that are biologically active nonnutritive chemical compounds, which are responsible for the health promoting properties of varieties of medicinal plants. Several of these phytochemicals have been reported to inhibit the multistep process of carcinogenesis (Singh et al., 2006) (Figure 2.14) 67 UNIVERSITY OF IBADAN LIBRARY Figure 2.12: Interference of different stages of carcinogenesis by phytochemicals (Surh, 2003) 68 UNIVERSITY OF IBADAN LIBRARY Garlic alone contains 30 cancer preventing compounds including selenium. Broccoli contains indole-3-carbinol as well as phenethylisothio-cyanate, a sulfur-containing compound that is being studied in the prevention of cancer. Soy products contain phytoestrogens such as genistein that contribute to the putative breast- and prostate-cancer-preventive activity of soya bean (Surh, 2003). Topical application of capsaicin, a pungent component of hot chilli pepper (Capsicum annuum L.) inhibited PMA-induced mouse-skin tumour formation and activation of Nf-kb (Han et al., 2001). Both black and green tea contains an abundance of polyphenols such as catechins that have antioxidant and anti-cancer activity. Epigallocatechin gallate (EGCG), an antioxidant and chemopreventive polyphenol found in green tea has been shown to suppress malignant transformation in a PMA-stimulated mouse epidermal JB6 cell line by blocking activation of Ap1 or Nf-κb44 (Surh, 2003). Cur-cumin the most studied phytochemical and it is obtained from the spice, turmeric (Surh, 2003). It is both an anti-inflammatory agent and an antioxidant. In laboratory animals curcumin has been shown to inhibit colon, breast, and stomach cancer (Rukkumani et al., 2004). Although many herbs and species are been tested round the world for their cancer chemopreventive potentials, many are stilled unexplored or the scientific evidence of their traditional useage have not been properly investigated. One of such herbs is Rauvolfia vomitoria 69 UNIVERSITY OF IBADAN LIBRARY Figure 2.13: Chemopreventive phytochemicals and their dietary sources (Surh, 2003) 70 UNIVERSITY OF IBADAN LIBRARY 2.12. Rauvolfia vomitora (Afzel) 2.12.1 Classification Family: - Apocynaceae. Synonym(s):- Hylacium owariense Afzel, Rauvolfia Senegambia Common names: - (English): swizzle stick, poison devil's-pepper (Yoruba): Asofeyeje (Hausa): Wadda ` (Igbo): Ntu oku Organism type: tree, shrub Kingdom: Plantae-Plants Subkingdom: Tracheobionta-Vascular plants Superdivision: Spermatophyta-Seed plants Division: Magnoliophyta-Flowering plants Class: Dicotyledonae Subclass: Asteridae Order: Gentiales Family: Apocynaceae Genus: Rauvolfia Species: vomitoria 71 UNIVERSITY OF IBADAN LIBRARY Figure 2.14: Rauvolfia vomitora (Afzel) Plant (Taken at Omi Adio, Ibadan) 72 UNIVERSITY OF IBADAN LIBRARY 2.12.2 Habitat and Distribution. Rauvolfia vomitoria (RV) is a medicinal plant which native to in the humid tropical secondary forests of Africa (Sofowora,1993), but it has been introduce to Asia , Puerto Rico and Hawai'i . It naturally occurs in gallery forests, but is mostly found in forest re-growth where fallow periods are prolonged. RV is associated with palms, Trema guineensis and Combretum spp. RV is considered endangered species. 2.12.3 Description The generic name Rauvolfia commemorates a 16th century German physician, Leonhart Rauvolf, who travelled widely to collect medicinal plants. The specific epithet vomitoria refers to the purgative and emetic properties of the bark. Rauvolfia vomitoria. Rauvolfia vomitoria is a shrub or small tree up to 8 m tall. The leaves grow in whorls of three and are elliptic and pointed at the end, 5-12 cm long and 3-6 cm wide. Flowers are tiny, sweet-scented, pale greenish-white and somewhat hairy inside. The orange fruits are shaped like small balls, each containing a single seed. Flowering occurs between August and October, while fruiting occurs between October and December. 2.12.4 Functional uses The plant is rich nutrient such as crude protein (9.3-17%), crude fibre and ash (Ojo et al., 2012; Mecha et al, 1980). The sweet-scented flowers are frequented by bees and therefore, exploited in apiculture. It is used as firewood for in Sierra Leone. The bark has good yields of bast fiber. The latex extruded from the young stems is used in rubber production. A yellow pigment obtained from the bark is useful in making dye. The seeds are used in making decorative necklaces. In Gabon, the bark and root powder, are mixed with water or palm oil to kill fleas and vermin. 2.12.5 Medicinal Uses The plant has found wide applications in traditional medicine across the world. Traditional medicine practioner in Nigeria and other parts of Africa use different part of the plant in treating fever, general weakness, intestinal diseases, liver problems, mental illness, haemorrhoids, hypertension, snakebite and cholera (Amole, 2009; Bemis et al., 2006; Obembe et al., 1994; Akpanabiatu et al., 2009; Waterman, 1986). Decoctions of the leaves of RV have a powerful 73 UNIVERSITY OF IBADAN LIBRARY emetic and anti-swelling effect (Burkill, 1994). In addition, its tissue lipid lowering-effect, blood pressure lowering, antipyretic, analgesic, haematinic, aphrodisiac, purgative, dysenteric, abortive, insecticidal, anti psychotic, anticonvulsant properties have all been documented (Amole et al., 2009; Obembe et al., 1994; Principe, 1989). Extract from the plant have also been reported to inhibit the growth of bacterial, viral, fungal and parasitic pathogens (Amole et al., 2006). Furthermore, the plant has been reported to have anti-prostate cancer activity in both in vitro and in vivo model system. This mechanism of action has suggested to be via modulation of DNA damage and cell cycle control signaling pathways (Beljanski and Beljanski, 1999). Recently, Yu et al. (2013) reported that Rauvolfia vomitora inhibited the growth of 3 ovarian cancer cell lines and inhibited their colony formation in soft agar. In addition, it was shown that extract of RV suppressed tumour growth in mice (Yu et al., 2013). 2.12.6 Phytochemical Constituents The leaf of the plant is rich in alkaloids, flavonoids, tannin, saponins and tepernoid. (Ojo et al., 2012). Extensive studies carried out on its chemical properties showed that the plant contains more than 50 active indole alkaloids (Table 2.5), each possessing remarkable pharmacological activities (Yu et al., 2013; Iwu and Court, 1982; Pousset and Poisson, 1965). The main alkaloid present in Rauvolfia is the anti hypertensive and psychiatric agent, reserpine. The indole akaloids with yohimbane skeleton namely yohimbine, reserpine, rescinnamine, raucaffricine, ajmaline and ajmalicine have been isolated from the extract of RV (Yu et al., 2013). A bioactive carboline alkaloid, alstonine, present in the root and leaf were previously shown to have anti cancer activity (Pettit et al., 1994; Bemis et al., 2006). 74 UNIVERSITY OF IBADAN LIBRARY Table 2.5: Major alkaloids and their bioactivities isolated from the root of Rauvolfia vomitoria (Yu et al., 2013). 75 UNIVERSITY OF IBADAN LIBRARY CHAPTER THREE MATERIALS AND METHODS 3.1 EXPERIMENTAL ANIMALS Forty male Wistar strain albino mice approximately 8-10 weeks old with average weight of 20g obtained from the Animal House of the University of Lagos Teaching Hospital (LUTH), Idi- Araba, Lagos State, Nigeria were used for the experiment. They were housed five per cage with wood shaven bedding in polypropylene cages under standard environmental conditions of 50± 0 10% relative humidity, 29 ± 2 C temperature and 12 hours light and 12 hours dark cycle at the Experimental Animal House, Department of Chemical Sciences, Bells University of Technology, Ota, Ogun State, Nigeria. They were fed with mice pellet containing at least 20% protein, 3.5% fat, 9.0% fibre, 1.2% calcium, 0.7% phosphorus, vitamin, mineral per mix, antioxidant, antibiotics, carbohydrates etc from God First Feed Mill, Bodija, Ibadan, Nigeria and water ad libitum. A period of two weeks was allowed for the animals to acclimatize before the commencement of the experiment. Test and control substance were administered methanol extract of Rauvolfia vomotora (RV), potassium dichromate (K2Cr2O7) and sodium arsenite (SA) as summarized in Figure 3.8. The RV was given orally for seven days, while potassium dichromate K2Cr2O7 and SA were administered on day seven of the experiment. 3.2 PLANT COLLECTION AND EXTRACTION Fresh leaves of Rauvolfia vomotora (RV) were collected from the Botanical Garden, University of Ibadan, Ibadan. The leaves identified by Mr. O.S. Shasanya and Osiyemi O.A. at the herbarium in the Forestry Research Institute of Nigeria (FRIN), Ibadan and voucher specimen deposited at the same herbarium (Voucher No: FHI108901). The fresh leaves were air dried for 8 0 weeks at room temperature, after which they were completely dried in a solar drier at 40 C. It was then milled in a hammer- miller with mesh size 0.27µm. 100g of the powder obtained was soaked in 1000ml for 48 hours in 70% methanol and cold extraction carried out. The suspension 0 obtained was filtered and concentrated with a rotary evaporator under reduced pressure at 40 C. The percentage yield was 9.7 %. A dosage 275 mg/kg body weight corresponding to 1/25th of the LD 50 of RV (Amole et al., 1993) was dissolved in distilled water and injected intraperitoneally into test animals. 76 UNIVERSITY OF IBADAN LIBRARY Figure 3.1: Protocol for the administration of test substances. 77 UNIVERSITY OF IBADAN LIBRARY 3.3 DETERMINATION OF PLATING EFFICIENCIES OF CH310T½ CELLS Principle The plating efficiency of cells also known as clonogenic cell survival assay determines the ability of a cell to proliferate indefinitely, thereby retaining its reproductive ability to form a large colony or a clone (Munshi et al, 2005). Loss of reproductive integrity and the inability to proliferate indefinitely are the most common mechanism of cell death. Therefore, a cell that retains its ability to synthesize proteins and DNA and go through one or two mitoses, but is unable to divide and produce a large number of progeny is considered dead. This is referred to as loss of reproductive integrity or reproductive death and it is the end point measured with cells in culture. In contrast, a clonogenic cell is reproductively active, retains its ability to divide and proliferate into a large colony of cells. The ability of a single cell to grow into a large colony that can be visualized with the naked eye or microscope is a proof that it has retained its capacity to reproduce. The loss of this ability as a function of dose of radiation or chemotherapy agent is described by the dose-survival curve Reagents 10% Fetal Bovine Serum 50 ml fetal bovine serum was made up 500 ml with basal medium eagle (BME) 1% Crystal violet Stain 1 g of crystal violet is weighed and dissolved in 20 ml of methanol and made up to 100 ml with distilled water Procedure Once 10T½ cells from passage 7-8 had grown to 80% confluence, the standard steps of the plating efficiency protocol were conducted as follows: Medium was aspirated and 1ml of DPBS (Dulbecco‟s Phosphate Buffered Saline 1X) was added to the cells to remove serum-containing trypsin inhibitors. DPBS was aspirated and 1ml of trypsin was added back to each flask. Once a majority of the cells were detached from the vented flask, 1ml of medium containing 10% FBS was added to neutralize the trypsin. The cell suspension was then transferred to a centrifuge tube and centrifuged for 12 minutes at 3,000 rpm on an IEC-HN-S table top centrifuge. Next, the supernatant was aspirated, and the cell pellet was resuspended in 10 ml of BME containing 10% FBS. 1ml of the resuspended cell suspension was diluted 1:20 with PBS, aliquoted, and counted 78 UNIVERSITY OF IBADAN LIBRARY using a Coulter Counter Model Zf (Coulter Electronics, Hialeah, Florida). The cells were then diluted to a concentration of 200,000 cells/5 ml, serially diluted 1:10 three more times, and seeded onto the 60-mm tissue culture dishes at 200 cells/dish in 5 ml of BME containing 5% fetal bovineserum (FBS). Five dishes per treatment group were seeded for plating efficiency determination. This method has been published in previous studies (Reznikoff et al, l973a, b; Landolph and Heidelberger, l979; Miura et al, l987; Patierno et al, l988). Two days after seeding, the media were changed and cells were cultured for additional 8 days. Cells were then fixed with methanol for 30 minutes, the methanol was removed by aspiration, and finally, the cells were stained with 1% crystal violet in water for one hour as previously described by Reznikoff et al., l973a. Colonies containing 20 or more cells were counted and averaged in the five dishes. Relative survival was determined as a percentage of survival of cells. 3.4 PHAGOCYTIC UPTAKE DETERMINATION Principle The entry of a toxin into the cell is a vital step that determines whether or not the toxin will exert a carcinogenic effect on the cell through mutagenic or epigenetic mechanisms (Muñoz and Costa, 2012). Therefore, the varying toxicities associated with toxic compounds may be dependent on how they are internalized and the degree of internalization of their particles Reagents 70% Ethanol 70 ml of ethanol was mixed with 30 ml of distilled water. 1% Crystal violet Stain 1 g of crystal violet was dissolved in 20 ml of methanol and made up to 100 ml with distilled water Procedure The 10T½ cells were seeded at 2,000 cells in 60 mm dishes and treated after 24 hours. Two days later, cells were fixed with 70% ethanol, stained with 1 % crystal violet, and examined with a light microscope at 200x magnification. The amount of vacuolated and anucleated cells were scored in 100 cells in treated and control groups. Alternatively, 500,000 logarithmic growing cells were seeded in 75ml flask and allowed to adhere overnight. Cells were treated and harvested by trypsinisation , fixed with ½ strength karnovsky‟s fixative for 2 h at 4 °C and post fixed in 2% osmium tetra oxide for 1hr, and rinsed 79 UNIVERSITY OF IBADAN LIBRARY again three times in cacodylate buffer. The pellets were block stained in 1% uranyl acetate overnight. Cell pellets were washed in three changes of distilled water, gelatinized with 15% bovine serum albumin for 3 hours and dehydrated in graded ethanol. Subsequently, pellets were treated with a graded series of ethanol- propylene oxide as well as EPON-propylene oxide. Pellets were covered with EPON overnight and suspended in fresh pure EPON three times for at least three hours and embedded in beam capsules using pure EPON. Polymerization was thermally induced by incubating the samples overnight at 60°C. Semi thin plastic sections (1-2 µm) were cut with glass knives, stained uranyl acetate and evaluated for the percentage of cells containing chromate particle using a Scanscope Microscope. 3.5 SCANNING ELECTRON MICROSCOPY Principle The scanning electron microscope (SEM) uses a focused beam of high-energy electrons to generate a variety of signals at the surface of solid specimens. The signals derived from electron- sample interactions reveal information about the sample including external morphology (texture), chemical composition, and crystalline structure and orientation of materials making up the sample. The basic principle is that a beam of electrons is generated by a suitable source (e.g. tungsten filament or a field emission gun). The electron beam is accelerated through a high voltage (e.g. 20 kV) and pass through a system of apertures and electromagnetic lenses to produce a thin beam of electrons, then the beam scans the surface of the specimen and electrons emitted from the specimen are then collected by a suitably-positioned detector. Reagents 0.2% Sodium Cacodylate Buffer 42.8g of sodium cacodylate was dissolved in 1 liter of double distilled water. The mixture was stirred until the sodium cacodylate was completely dissolved. The pH was adjusted to 7.2. ½ strength Karnovsky’s fixative in cacodylate buffer 10% stock 20 ml of 2% paraformaldehyde was mixed with 5 ml of 2.5 % glutaraldehyde, 50 ml of 0.1M cacodylate buffer, 12.5ml of 200mg/ml Calcium Chloride and 12.5 ml of distilled water. The pH was adjusted to 7.2. 2% (w/v) paraformaldehyde A total volume of 10 ml of 16% formaldehyde is added to 70ml of 1M PBS. 80 UNIVERSITY OF IBADAN LIBRARY Ethanol 50, 75, 85, 95% absolute ethanol in distilled water and 100%. 2% osmium tetroxide 1 g OsO4 was dissolved in 50 ml of distilled water Hexamethyldisilizane- Ethanol 50%, 75%, 85%, 95%, hexamethyldisilizane in absolute ethanol and 100% Procedure Scanning electron microscopy was performed at the Electron Microscopy Facility of Cell and Tissue Imaging Core at USC/Norris Comprehensive Cancer Center, University of Southern California Los Angeles. Cells were seeded in a 25 ml flask and allowed to reach 70% confluence before treatment. Immediately following treatment, dishes containing the cells were rinsed twice in PBS and ½ strength karnovsky‟s fixative for 2h , after which they were rinsed in three changes of 0.1 M sodium cacodylate-buffer, (10 min each), post-fixed with 2% OsO4 for1 hour and rinse in 0.1 M sodium cacodylate buffer. Cells were later washed in three changes of water and dehydrated with ascending graded ethanol (50, 70, 85, 95 and 100%) as well as infiltrated with 50%, 75%, 85%,95%, hexamethyldisilizane- ethanol. Dishes were air-dried, cut with hot scapel and mounted on aluminum SEM stubs mounted on a stub with silver adhesive (Electron Microscopy Sciences, Hatfield, PA, U.S.A.). The cut sections of the dish were later gold coated in an Electron Microscopy Science Sputter Coater and examined in a JOEL JSM 6390LV Scanning Electron Microscope at 10KV. Reagents 70% ethanol 70 ml of absolute ethanol was mixed with 30 ml of distilled water 1% Crystal violet Stain 1 g of crystal violet was dissolved in 20 ml of methanol and made up to 100 ml with water. 3.6 TRANSMISSION ELECTRON MICROSCOPY Principle Transmission Electron Microscopy (TEM) is an imaging technique that uses electron beam to image a sample. High energy electrons, incident on ultra-thin samples allow for image 81 UNIVERSITY OF IBADAN LIBRARY resolutions that are on the order of 1-2Å. The high energy electrons (up to 300 kV accelerating voltage) are accelerated nearly to the speed of light. The electron beam behaves like a wave front with wavelength about a million times shorter than light waves. When an electron beam passes through a thin-section specimen of a material, electrons are scattered and sophisticated system of electromagnetic lenses focuses the scattered electrons into an image or a diffraction pattern, or a nano-analytical spectrum, depending on the mode of operation. Reagents 0.2% Sodium Cacodylate Buffer 42.8g of sodium cacodylate was dissolved in 1 liter of double distilled water. The mixture was stirred until the sodium cacodylate was completely dissolved. The pH was adjusted to 7.2. ½ strength Karnovsky’s fixative in cacodylate buffer 10% stock 20 ml of 2% paraformaldehyde was mixed with 5 ml of 2.5% glutaraldehyde, 50 ml of 0.1M cacodylate buffer, 12.5ml of 200mg/ml Calcium Chloride and 12.5 ml of distilled water. The pH was Adjust to 7.2. 15% bovine serum albumin 15 g of bovine serum albumin was dissolved in 100ml distilled water 2% (w/v) paraformaldehyde A total volume of 10 ml of 16% formaldehyde was added to 70ml of 1M PBS. 2% osmium tetroxide 1 g OsO4 is dissolved in 50 ml of distilled water Ethanol 50, 75, 85, 95% absolute ethanol in distilled water and 100%. Ethanol -propylene oxide 50, 75, 85, 95% propylene oxide in ethanol and 100%. EPON 812 resin -propylene oxide 50, 75, 85, 95% epon 812 resin in propylene oxide and 100%. Procedure Transmission electron microscopy was performed at the Electron Microscopy Facility of Cell and Tissue Imaging Core at USC/Norris Comprehensive Cancer Center, University of Southern 82 UNIVERSITY OF IBADAN LIBRARY California Los Angeles. Cells were seeded in a 75 ml flask and allowed to reach 70% confluence before treatment. Cells trypsinised and centrifuged at 5000 x g for 5minutes. Cell pellets were washed in three changes of distilled water, gelatinized with 15% bovine serum albumin for 3 hours and dehydrated in 50%, 70%, 85%, 95% and 100% ethanol. Subsequently, pellets were treated with a graded series of ethanol- propylene oxide as well as EPON-propylene oxide. Pellets were covered with EPON overnight and suspended in fresh pure EPON three times for at least three hours and embedded in beam capsules using pure EPON. Polymerization was thermally induced by incubating the samples overnight at 60 °C. Ultrathin sections (70–80 nm) were cut with a microtome diamond knife and mounted on copper grids, stained with uranyl acetate. Sections were observed using a transmission electron microscope (JEOL 2100) operated at 80 kV. Intracellular chromate particles were confirmed by energy-dispersive X-ray analysis (EDX) by Edax Inc. (Mahwah, USA). X- rays beam were focused on the portion of the cytoplasm and nucleus containing chromate particles. The EDX spectra of the elements in the particles were thereafter obtained. 3.7 DETERMINATION OF CELL CYCLE KINETICS Principle Measurement of cellular DNA content and the analysis of the cell cycle can be performed by flow cytometry. In addition to determining the relative cellular DNA content, flow cytometry also enables the identification of the cell distribution during the various phases of the cell cycle. Four distinct phases could be recognized in a proliferating cell population: the G1-, S- (DNA synthesis phase), G2- and M-phase (mitosis). Usually, a fluorescent dye such as popidium iodide that binds to the DNA is added to a suspension of permeabilized single cells or nuclei. Popidium iodide binds stoichiometrically to the DNA in a suspension of single cells or nuclei. The stained cells incorporate an amount of dye proportional to the amount of DNA. The stained material is then measured in the flow cytometer and the emitted fluorescent signal yields an electronic pulse with a height (amplitude) proportional to the total fluorescence emission from the cell. The fluorescence data are considered a measurement of the cellular DNA content. Samples are analyzed at rates below 1000 cells per second in order to yield a good signal for discrimination between singlets or doublets 83 UNIVERSITY OF IBADAN LIBRARY Reagents 70% ethanol 70 ml of absolute ethanol was mixed with 30ml of distilled water 0.05mg/ml of popidium iodide 0.5mg of popidium iodide was dissolved 10 ml of PBS 0.1 mg/mL of DNase-free RNaseA 1mg of RNase was dissolved in 10ml PBS Procedure Hundred thousands cells were seeded into 75 ml flask and allowed to grow to 70% confluence. The exponentially growing cells were treated and after treatment, cells were harvested and washed with PBS after a gentle centrifugation at 200 × g for 5 minutes. Cells were thoroughly resuspended in 0.5 mL of PBS and fixed in 70% ethanol for at least 2 hours at 4°C. Ethanol- suspended cells were then centrifuged at 200× g for 5 minutes and washed twice in PBS to remove residual ethanol. For cell cycle analysis, the pellets were suspended in 1 mL of PBS containing 0.05mg/mL of propidium iodide, 0.1 mg/mL of DNase-free RNase A and incubated in dark at room temperature for 1 hour . Cell cycle profiles were obtained using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and data were analyzed by ModFit LT software (Verity Software House, Inc., Topsham, ME). 3.8 ASSESSMENT OF CASPASE ACTIVATION Principle Caspase activation is an essential event in the apoptotic pathway. Caspase-3 is one of the important “executioner” caspase enzymes that trigger the cleavage of numerous proteins leading to the orderly breakdown of the cell. The assessment of caspase-3 activation is often used as a marker for apoptosis. Glo-apo-tox assay kit from Promega (Madison, WI USA) was used in this study. The Glo-apo-tox assay kit uses a luminogenic caspase-3/7 substrate that contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity, luciferase activity and cell lysis. Addition of the Caspase-Glo 3/7 Reagent results in cell lysis, followed by caspase cleavage of the substrate and generation of luminescent signal produced by luciferase (Figure 3.2). Luminescence is proportional to the amount of caspase activity present. In addition, the assay kits also simultaneously measures viability and cytotoxicity within the same assay well. This involves two proteases. The live-cell protease activity is restricted to intact viable cells and 84 UNIVERSITY OF IBADAN LIBRARY is measured using a fluorogenic, cell-permeant, peptide substrate (glycylphenylalanyl- aminofluorocoumarin; GF-AFC). The substrate enters intact cells where it is cleaved by the live- cell protease activity to generate a fluorescent signal proportional to the number of living cells. In contrast, the live-cell protease becomes dormant when there is damage to the cell membrane and leakage of cell content into the surrounding culture medium. A second, fluorogenic cell-impermeant peptide substrate (bis-alanylalanyl-phenylalanyl- rhodamine 110; bis-AAF-R110) is used to measure dead-cell protease activity that is released from cells that have lost membrane integrity (Figure 1). Bis-AAF-R110 is not cell-permeant, therefore no signal would be generated in intact, viable cells. The live- and dead-cell proteases produce different products, AFC and R110, which have different excitation and emission spectra, allowing them to be detected simultaneously. Reagent Glo-apo-tox assay assay kit Glo-apo-tox assay triplet assay kit from Promega (Madison, WI USA) Procedure The assays were carried out according to manufacturer‟s instruction. 15,000 logarithmic growing cells were seeded per well in 96-well plate , opaque-walled tissue culture plates with clear bottoms. Cells were treated and after the treatment, 20μl of Viability/Cytotoxicity Reagent containing both GF-AFC and bis-AAF-R110 substrate were added to all the wells, mixed briefly by orbital shaking at 300–500rpm for ~30 seconds and incubated for 60 minutes at 37°C. Fluorescence were measured at 400Ex/500Em and 485 Ex/520Em for viability and cytotoxicity respectively. 100μl of Caspase- Glo® 3/7 Reagent was added to all wells, mixed and incubated for 60 minutes at 25°C and luminescence measurement were read. Luminescence is proportional to the activity of caspase. 85 UNIVERSITY OF IBADAN LIBRARY Fig ure 3.2: Caspase-3/7 cleavage of the luminogenic substrate containing the DEVD substrate. Aminoluciferin is released after caspase cleavage, resulting in the luciferase reaction and the production of light (Promega, 2012) 86 UNIVERSITY OF IBADAN LIBRARY Figure 3.3: Principle of the Viability /Cytotoxicity Assay. The GF-AFC Substrate can enter live cells where it is cleaved by the live-cell protease to release AFC. The bis-AAF-R110 Substrate cannot enter live cells but instead can be cleaved by the dead-cell protease to release R110. (Promega, 2012) 87 UNIVERSITY OF IBADAN LIBRARY 3.9 ASSESSMENT OF APOPTOSIS AND NECROSIS Principle Cells were double stained the with acridine orange (AO) and propidium iodide (PI) and scored for apoptosis and necrosis as described by Ciapetti et al., 2002 with slight modification. AO and PI are intercalating nucleic acid-specific fluorochromes which emit green and orange fluorescence, respectively, when they are bound to DNA. Of the two, only AO can cross the plasma membrane of viable and early apoptotic cells. PI will only interact with the DNA of cells with damaged cell membrane. Therefore, viable cells are identified as green, while apoptotic cells were identified by their green nuclei with evident membrane blebbing or dense orange nuclei indicating DNA condensation. Necrotic cell have intact orange nuclei and sometimes with vacuoles. The assay provides a useful simultaneous quantitative evaluation for necrosis and apoptosis. Reagents Acridine Orange Stain 0.20 mg of acridine orange was dissolved in 20ml of PBS Popidium Iodide Stain 0.20 mg of popidium iodide was dissolved in 20ml of PBS Procedure Cell death in CH310T ½ cells was quantified using propidium iodide (PI) and acridine orange (AO) double-staining according to standard procedures and examined under fluorescence microscope (EVOS). Briefly 15,000 cells were cultured in slide chambers. Cells were allowed to attach overnight and treated. After treatment, cells were rinsed with phosphate buffered saline −1 (PBS) and resuspended in BME. 15µl of fluorescent dyes containing AO (10 μgml ) and PI −1 (10μgml ) were later added to each chamber and slides were observed with an inverted EVOS UV-fluorescence microscope equipped with a digital camera (Advanced Microscopy Group, Bothell, WA) within 30min before the fluorescent color starts to fade. The percentages apoptotic and necrotic cells were determined in >200 cells. 88 UNIVERSITY OF IBADAN LIBRARY 3.10 ASSESSMENT OF INDUCTION OF AUTOPHAGY Principle An investigation on the third form of cell death, autophagy was also carried out. This was monitored by imaging the distribution of LC3B protein in the cytoplasm. The LC3B protein plays a critical role in autophagy. Normally, this protein resides in the cytosol, but following cleavage and lipidation with phosphatidyl ethanolamine, LC3B associates with the phagophore. This localization can be used as a general marker for autophagic membranes (Figure.3.3). Premo Autophagy Sensors (LC3B-FP) BacMam 2.0 from Molecular Probes (Eugene, OR) was used in this work. The Premo™ Autophagy Sensor combines the selectivity of an LC3B- fluorescent protein (FP) chimera with the transduction efficiency of the BacMam 2.0 technology. BacMam reagents (insect Baculovirus with a Mammalian promoter) are safe to handle (Biosafety Level 1) because they are non-replicating in mammalian cells. They are also non-cytotoxic and ready- to-use. Unlike other expression vectors, BacMam reagents enable titratable and reproducible expression and offer high co-transduction efficiency. Multiple BacMam reagents can therefore be readily used in the same cell. Recent improvements made to the BacMam system, BacMam 2.0, enable efficient transduction in a wider variety of cells, including neurons and neural stem cells (NSCs). The two-step protocol for imaging autophagy involves simply adding the BacMam LC3B-FP to the cells and incubating them overnight for protein expression Reagent Premo Autophagy Sensors (LC3B-FP) BacMam 2.0 Assay Kit Premo Autophagy Sensors (LC3B-FP) BacMam 2.0 Assay Kit from Molecular Probes (Eugene, OR) was used in assessing the extent of autophagy. 89 UNIVERSITY OF IBADAN LIBRARY Figure 3.4 Schematic depiction of the autophagy pathway in a eukaryotic cell (Molecular Probes, 2010) d 90 UNIVERSITY OF IBADAN LIBRARY Procedure Ten thousand logarithm growing cells were placed in a 4 slide chamber and allowed to adhere overnight. Cells were later transduced with the LC3B protein for 24 hours in order to allow the expression of the LC3B protein. Thereafter, cells were treated and slides were observed with an inverted EVOS UV-fluorescence microscope equipped with a digital camera (Advanced Microscopy Group, Bothell, WA). The number of cells expressing LC3B-GFP puncta in 10 fields (>100 cells ) were scored and expressed as a percentage of the total cells in the test and control groups. 3.11 ASSESSMENT OF ACTIN DISRUPTION Principle The actin filaments are the major component responsible for the maintenance of global cell shape in fibroblast (Ujihara et al, 2008). Actin plays diverse role in cell growth, maintenance of cell polarity and morphology, locomotion and signal transduction. The transmission electron microscopy (TEM) observations that chromate exposure led to disruption of cytoskeleton informed the decision to stain cells with actin specific dye, TRITC phalloidin. Rhodamine phalloidin is the most widely used F-actin stain. It is a bicyclic peptide that is isolated from mushroom, Amanita phalloides toxin conjugated to the orange-fluorescent dye, tetra methylrhodamine (TRITC). It is commonly used in cellular imaging to specifically label F- actin in fixed cells, permeabilized cells, and cell-free experiments. The red fluorescent probe binds to F-actin with nanomolar affinity and very photostable. It binds in a stoichiometric ratio of about one phallotoxin per actin subunit in both muscle and nonmuscle cells. Reagents 3.7% formaldehyde 3.7 ml of formaldehyde was made up 100ml with PBS Phalliodin Stain 0.5 mg of tetramethylrhodamine (TRITC) phalloidin was dissolved in 100 ml 91 UNIVERSITY OF IBADAN LIBRARY Figure 3.5: Rhodamine Phalloidin Procedure Hundred thousand log phase cells were seeded into Lab-Tek II chamber slides and allowed to adhere overnight. Cells treated were and following treatment, cells were washed with phosphate buffered saline and fixed for 5 minutes in 3.7% formaldehyde solution in PBS. Subsequently, cells were washed three times in PBS and stained with a 50 µg/ml fluorescent phalloidin conjugate solution for 90 minutes at room temperature. Cells were later washed several times with PBS to remove unbound phalloidin conjugate. Cells were later observed with an inverted EVOS UV-fluorescence microscope and images captured with a digital camera (Advanced Microscopy Group, Bothell, WA). Images were loaded into Cell Profiler, a cell image analysis software, and then subjected to a “pipeline” of steps that were meant to calculate the mean intensity of the image. The pipeline begins by loading images with the appropriate names (Load Images) and converting them to grayscale (ColorToGray). The software then uses the Sobel method to detect “edges” within the image, providing boundaries for intensity calculation (EnhanceEdges). Next, a threshold is applied to the grayscale, bounded image, converting all pixels to either black or white (ApplyThreshold). Finally, the software measures the mean intensity of the image, and stores it in a spreadsheet. 3.12 MORPHOLOGICAL TRANSFORMATION ASSAY Assay for the incidence of focus formation was carried out following the method of Nesnow et al., 1980. 92 UNIVERSITY OF IBADAN LIBRARY Principle Exposure of cells to chemical carcinogen and other cancer causing agent can altered the growth control mechanism of normal cells into neoplastic transformed ones (Figure 3.5). In vitro cell transformation is the induction of phenotypic alterations in cultured cells that are characteristic of tumourigenic cells (Kakunaga and Kamasaki et al., 1985). Transformed cells with the characteristics of malignant cells have the ability to induce tumours in susceptible animals. These new characteristics include: I. Proliferation at higher rate and production of foci (discrete focal area of growth) on a dense monolayer of normal cells II. Anchorage independent i.e. transformed cells form colonies or grow in soft agar where as normal cells do not III. Transformed cells formed tumors after injection into susceptible animals Reagents Normal saline 0.9 g of NaCl was dissolved in 100 ml of distilled water. Giemsa stain Five grams of Giemsa powder was dissolved in 1ml of ethanol before diluting in 50 mls of distilled water. Procedure The 10T½ cells were grown to 70% confluence, trypsinized and seeded into 60-mm dishes in 5 ml medium at 2,000cells per dish. Twenty dishes were plated per concentration. Five days post seeding, cells were treated and the medium was then changed after treatment. The medium was subsequently changed once a week for 6 weeks. At the end of the sixth week of the transformation assay, the dishes were rinsed with 0.9% NaCl saline, fixed with methanol, stained with Giemsa and scored for foci under a dissecting microscope, according to standards methods ( Reznikoff et al, l973b; Landolph and Heidelberger, l979; Miura et al., l987; Patierno et al., l988; Landolph, l994). 93 UNIVERSITY OF IBADAN LIBRARY Figure 3.6: Morphological Transformation Assay In-vitro (Hall and Hei, 1986) 94 UNIVERSITY OF IBADAN LIBRARY A B Figure 3.7: Type II (a) and Type III foci (b) observed in 10 T½ cells treated with hexavalent chromate compounds 95 UNIVERSITY OF IBADAN LIBRARY 3.13 DETERMINATION OF THE EXPRESSION OF APOTOSIS, AUTOPHAGY AND NECROSIS RELATED GENES Principle 2 RT Profiler PCR Array is employed in the monitoring the expression profile of cell death related genes. Real-time RT-PCR is a reliable and very sensitive method for gene expression analysis. The method is very dynamic and it is used in the quantification of both rare and 2 abundant genes in biological samples. RT Profiler PCR arrays take advantage of the combination of real-time PCR performance and the ability of microarrays to detect the 2 expression of many genes simultaneously. RT Profiler PCR Arrays are designed to analyze a panel of genes related to a disease state or biological pathway. The arrays are provided in 96 or 384-well plates with microfluidic chips. 96-well plates contain primer assays for 84 pathway or disease-focused genes and 12 other housekeeping and control genes. Reagents RNeasy Mini Kit (Qiagen,Valencia, CA) PrimeScript™ RT Master Mix from Takara (Frederick, MD) SYBR® Premix Ex Taq II (Tli RNaseH Plus) Procedure One hundred thousand log phase growing cells were seeded in a 75 ml flask and cultured to 70% confluence before cells were harvested by trypsinisation. The harvested cells was then subjected to centrifugation at 5000 x g for 5minutes. Total RNA was prepared from the cell pellet using triazol and purification was carried out with RNeasy Mini Kit (Qiagen,Valencia, CA) according to the manufacturer's instructions. Complimentary DNA (cDNA) was then produced with 0.5μg of RNA template for each sample using the PrimeScript™ RT Master Mix from Takara (Frederick, MD) following the manufacturer‟s instruction. To complete the reaction, samples 0 0 0 were incubated for 15 minutes at 37 C, 5 secs at 85 C and then 4 C until samples were stored 0 at -80 C. The cDNA obtained was used with SYBR® Premix Ex Taq II (Tli RNaseH Plus) 2 from Takara for RT PCR profiling in 96-well Cell Death RT Profiler PCR arrays (PAMM 212Z from Qiagen ). The PCR mixture contains 2.75ml SYBR® Premix Ex Taq II (2X), 0.44ml cDNA, 0.11 ml ROX Reference Dye (50X) and 17 ml sterile distilled water. 50µl of reaction mixture was then dispensed into each well and relative quantification of RNA was 96 UNIVERSITY OF IBADAN LIBRARY determined by real time PCR using an Applied Biosystems 7300 Real Time PCR System and analyzed with 7300 System SDS software v 1.3.1. Each sample was subjected to 40 cycles with an initial incubation of 50° C for 2 min. followed by 90° C for 10 min and then 40 cycles of 95° C followed by 1 minute of 60° C. A manual Ct threshold of 0.0500 was used for all samples and the sample baseline was obtained from cycles 3 through 15. Data were analysed based on 2 ΔΔCt (manual) method using the RT Profiler PCR Arrays Data Analysis soft ware version 3.5 on Qiagen‟s website: http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php. For each gene, fold-changes were calculated as difference in gene expression between control and treated the cells or the transformed cell line. 97 UNIVERSITY OF IBADAN LIBRARY Figure 3.8: Protocol Chart of RT-PCR Array (Qiagen, 2012) 98 UNIVERSITY OF IBADAN LIBRARY 3.14 MICRONUCLEUS ASAY The frequency of micronuclei formation was determined by the method of Schmid et al., 1975. Principle A micronucleus (MN) is a small extra nucleus separated from the main one, generated during cellular division by late chromosomes or by chromosome fragments, due to its association with chromosomal aberrations. The mouse MN assay is the most widely used and best validated in vivo test for genotoxicity (Mavournin et al., 1990; Morita et al., 1997). The in vivo MN assay has become increasingly accepted as the model of choice for evaluating the genotoxic potential of chemicals and /or radiation. Micronucleus test is a more sensitive and statistically reliable technique when compared with chromosome aberrations test since the former comprises a significantly greater number of analyzed cells. The advantage of the MN test is in its ability to detect both clastogenic and aneugenic effects (Fenech, 2000). Recently, MN assay has been widely used in monitoring genetic damage in different tissue and cell types (Celik et al., 2005; Bonassi et al., 2007). The work of Schmid, 1973 led to an increasing and now almost universal selection of the polychromatic erythrocyte (PCE) as the cell choice. PCEs are very abundant, easily recognizable and the product of recent cell divisions. The protocol depends on the understanding of the behavior of the cells being studied. Several aspects of the biology of PCE have been insufficiently appreciated in the past. Enucleation of the matured erythrocyte takes place about 6 hours after mitosis, also young erythrocytes (PCEs) do not lose their ribosome for approximately 24hours after enucleation (Rifkind, 1976), and they stain a basophilic blue grey colour with Giemsa-stain. The PCEs are cells that have recently undergone DNA synthesis and mitosis. They are therefore easily distinguished by their color from any other cells on the slide. The PCEs are also somewhat larger and tend to have more diffused boundaries than the red blood cells (RBC).The erythrocytes are the only mammalian cells lacking a nucleus, a feature that makes a micronucleus obvious when present. MN assay is regarded as an important biomarker to predict the relative risk of cancer in upper aero-digestive tract (Bloching et al., 2000). 99 UNIVERSITY OF IBADAN LIBRARY Reagents 70% Methanol 70 ml of methanol was added into 30 ml distilled water. It was used to fix the slide. 10% Giemsa solution 5 g of Giemsa powder was dissolved in 1ml of ethanol before diluting in 50 ml of distilled water. 1:1 May Grunewald stain 20 ml of May-Grunewald stain was mixed with 20 ml distilled water Procedure The laboratory mice were sacrificed and femur was surgically extracted from each mouse. The epiphyses were removed; each of the femurs was flushed with 1ml of Fetal Bovine Serum (FBS) into an ependorff tube with syringe, and mixed thoroughly through tapping of the ependorff tube for proper dispersion of the cells. The samples were spun in a table centrifuge at 2000 rpm for 5mins, after which the supernatant was aspirated with syringe and needle while the pellet was re-suspended in 1 ml of FBS mixed properly and centrifuged again for 5 minutes in a table centrifuge at 2000 rpm. This process was repeated twice. Finally, a viscous suspension was obtained. The viscous suspension were dropped on a clean slide and smeared. Four slides were prepared for each mouse. The slides were allowed to dry completely on a slide tray after which each slide was fixed with 70% methanol and allowed to dry completely at room temperature. Each slide was placed in a coupling jar containing May-Greunwald stain for 3-4 minutes, and transferred immediately into another coupling jar containing May-Greunwald and distilled water in ratio 1: 1 for another 3-4 minutes. The slides were removed and rinsed in distilled water and allowed to dry completely. The slides were then stained in 10 % Giemsa for 4-5 minutes, rinsed and allowed to dry completely. 3.15 PREPARATION OF SERUM The blood samples collected were transferred into pre-labeled heparinized bottles to prevent coagulation and centrifuged at 2,000g for 20 minutes. The clear supernatants obtained were decanted and used immediately for the determination of plasma enzymes activity or stored at 0 –20 C until required. 10 0 UNIVERSITY OF IBADAN LIBRARY 3.16. DETERMINATION OF ALANINE AMINOTRANSFERASE (ALT) AND ASPARTATE AMINO TRANSFERASE (AST) ACTIVITIES Principle Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) are among the most sensitive test employed in the detection of acute liver damage .The increase in the activities of ALT and AST in plasma is indicative of liver damage and dysfunction. Thus, the enzymes are routinely used as markers of hepatic injury. The elevation of the ALT and AST may be due alteration in the permeability of liver membrane, which may lead to the leakage of these enzymes from the liver cytosol into the blood stream and disturbance in the biosynthesis of these enzymes. The transaminases were determined according to the method of Reitman and Frankel, 1957. ALT was measured by monitoring the concentration of pyruvate hydrazone formed with 2, 4- dinitrophenylhydrazine, while AST was measured by monitoring the concentration of oxaloacetate hydrazone formed with 2,4- dinitrophenylhydrazine Reagents Alanine aminotransferase (ALT) and aspartate aminotransferace (AST) assay Kits Randox alanine aminotransferase (ALT) and aspartate aminotransferace (AST) assay kits from Raandox (Randox Laboratories Ltd, UK) were used in this work 0.4 M NaOH 16 g of sodium hydroxide (BDH, England) pellets was dissolved in little distilled water and the solution made up to 1000 ml final volume. Procedure The levels of ALT and AST activity in the plasma was determined using the ALT kits (Randox Laboratories Ltd, UK) and following the method described by Reitman and Frankel (1957). 0.1ml of the sample was mixed with 5ml of Solution 1(Buffer; Phosphate buffer, L- Alanine or 0 L- Aspartate and α-Oxoglutarate) and incubated for exactly 30 minutes at 37 C. Thereafter, 0.5ml of Solution 2 (2, 4-dinitrophenylhydrazine) was added, mixed and allowed to stand for 0 exactly 20 mins at 20-25 C. 5.0 ml of NaOH was added and the absorbance of the sample was read against the reagent blank at 546 nm after 5 minutes. 10 1 UNIVERSITY OF IBADAN LIBRARY 3.17 PREPARATION OF LIVER HOMOGENATE Reagent Homogenising buffer (Tris HCl buffer) 3.9 g of Tris HCl in 5.75 g of KCl in distilled water and made up to 500 ml. The pH was adjusted to 7.4. Procedure The liver of each mouse was weighed and macerated in 10 times the volume of the actual weight of the liver using homogenate buffer (Tris buffer). The homogenate were spun for 10 minute at 10,000 rpm using a cold centrifuge. The supernant was collected and use for the determination of malondialdehyde and reduced glutathione (GSH) as well as catalase activity 3.18 DETERMINATION OF MALONDIALDEHYDE Principle Malondialdehyde (MDA) is used as a biomarker of oxidative stress because it is an end-product of lipid peroxidation and its levels indicate the degree of lipid peroxidation (Grotto et al., 2007). In this work, lipid peroxidation was determined using the method described by Esterbauer and Cheeseman, 1990. This method was based on the reaction between 2-thiobarbituric acid (TBA) and malondialdehyde, an end product of lipid peroxide during peroxidation. On heating at acidic pH, the product is a pink complex which absorbs maximally at 532nm and extractable into organic solvents such as butanol. Malondialdehyde (MDA) is often used to calibrate this test and thus the results are expressed as the amount of free MDA produced. Reagents 0.67% 2-Thiobarbituric acid 0.675g of 2-thiobarbituric acid was dissolved in 100 ml distilled water. 20% Trichloroacetic acid Twenty grams of trichloroacetic acid was dissolved in 100 ml of distilled water. 10 2 UNIVERSITY OF IBADAN LIBRARY Procedure The extent of lipid peroxidation in terms of thiobarbituric acid reactive substances (TBARS) formation was measured by mixing 0.5 ml of the tissue supernatant with 1 ml TCA (20%) and 2 ml TBA (0.67%) then heated for 1 hour at 100C. After cooling, the precipitate was removed by centrifugation. The absorbance of the sample was measured at 535 nm. Lipid peroxidation, in unit/mg protein was computed with a molar extinction coefficient of 1.56 5 x 10 M/cm MDA (unit/mg protein) = Absorbance Molar extinction x mg protein 3.19 PROTEIN DETERMINATION Principle The concentration of protein in the liver homogenate was determined by Biuret method as described by Weichselbaun (1946). Protein forms a coloured complex with cupric ions in an alkaline solution. The biuret reaction is a general one for the estimation of proteins, and it provides a relatively accurate measurement of serum protein. Compounds with two or more polypeptide bonds form dark blue-purple color in the presence of biuret reagent, which is characteristic of all proteins in the complex. The polypeptide specific biuret reaction occurs almost equally per given weight of all pure soluble proteins, since proteins are amino acids linked together by consecutive peptide bond. Proteins are about the only compound found in nature that has the polypeptide character required for biuiret assay. Thus, the biuret assay is remarkedly specific for protein. The dark blue colouration is apparently caused by the co- ordination complex of the copper atom and four nitrogen atoms, two from each of two peptide chains. 10% NaOH 50g of sodium hydroxide (BDH, England) pellets was dissolved in little distilled water and the solution made up to 500 ml final volume. 10 3 UNIVERSITY OF IBADAN LIBRARY Buiret reagent 1.5 g of copper sulphate was mixed with 6.0g of potassium sodium tartrate and dissolved in 500 ml of distilled water in a 1 liter standard volumetric flask. With constant shaking, 1 g of potassium iodide and 300 ml of 10% sodium hydroxide was added and then made up to 1 liter with distilled water. Stock bovine serum albumin (BSA) solution 2.5 g of BSA was dissolved in 100 ml of distilled water to give a stock of 2.5 mg/ml. Preparation Standard Curve Serial dilutions of stock BSA solutions were made by using varying concentrations. Biuret reagent was added to each diluted protein standard solution (stock BSA) and the mixture was allowed to stand at room temperature for 30mins before reading. The absorbances of the solutions were the read at 550 nm and a graph of absorbance against BSA Concentration (mg/ml) was then plotted. Determination of Protein Concentration in Sample Each homogenate sample were diluted twenty times, 1.5ml of the biuret reagent was added to 1ml of each diluted sample and allowed to stand for 30min at room temperature after which the absorbance was read at 550 nm 10 4 UNIVERSITY OF IBADAN LIBRARY Table 3.1: Standard curve for protein determination Test tube in Blank 0.125 0.25 0.50 1.00 1.50 2.00 2.50 duplicate Standard BSA - 0.05 0.10 0.20 0.40 0.60 0.80 1.00 - (2.5mg/ml) mg Saline (ml) 1.00 0.95 0.90 0.80 0.60 0.40 0.20 - - Sample (ml) - - - - - - - - 1.00 Biuret reagent (ml) 1.50 All (T U B E S ) Total volume (ml) 2.50 All (T U B E S ) Each test tube was mixed and allowed to stand at room temperature for 30 minutes. The absorbance was thereafter measured at 550 nm. 10 5 UNIVERSITY OF IBADAN LIBRARY 3.20 ESTIMATION OF CATALASE ACTIVITY Principle Catalase is an important first line antioxidant defense enzyme and its main function is the conversion of hydrogen peroxide to water. Catalase activity was determined according to the method of Aebi, 1983 by monitoring the decomposition of H2O2 as indicated by the decrease in the absorbance at 240 nm. The difference in the absorbance per unit time is a measure of the catalase activity Catalase 2H2O2 2H2 O + O2 Reagents 30 mM Hydrogen peroxide 6µl of H2O2 was completed to 2 ml with phosphate buffer pH 7.0 Phosphate Buffer pH 7.0 The phosphate was prepared by dissolving 3.58 g of Na2HPO4.12H2O and 1.19 g of NaH2PO2. 2H2O in distilled water and made to 900 ml. The pH was adjusted to 7.0, and distilled water was then added to make up to 1 liter. Procedure 0.1 ml of the supernatant fraction of the homogenate was mixed with 1.9 ml of the phosphate buffer, pH 7.0 in a quartz spectrophotometer cuvette. The reaction was started by adding 1 ml of hydrogen peroxide (H2O2) to the cuvette. The absorbance of the sample was read against distilled water immediately at zero time (A0) and was read again after 30 second (A30) at 240 nm. Catalase activity was expressed as µmol/ wet weight of the liver. Catalase activity was calculated by using the equation below: Catalase activity = K X 10 X log A0 T g fresh tissue A1 K = constant = 2.3 10 6 UNIVERSITY OF IBADAN LIBRARY T = time A0 = absorbance of the sample at zero time A1= absorbance of the sample at 30 secs 3.21 ESTIMATION OF REDUCED GLUTATHIONE (GSH) Principle Glutathione is also an important antioxidant compound responsible for maintaining intracellular redox homeostasis. Reduced glutathione (GSH) scavenges hydroxyl radical and singlet oxygen directly, detoxifying hydrogen peroxide and lipid peroxides by the catalytic action of glutathione peroxidase. Therefore, the level of the enzyme is altered during oxidative stress. The method of Beutler et al., 1963 was used in estimating the level of reduced glutathione in liver supernatants. This method is based upon the development of relative stable (yellow) colour when 5, 5-dithiobis 2-nitrobenzoic acid, (DTNB) is added to liver supernatant. The chromophoric product resulting from the reaction of Ellman‟s reagent with reduced glutathione, 2-nitro-5-thiobenzoic acid possess a molar absorption at 412nm. Reduced GSH is proportional to the absorbance at 412nm. Reagents Reduced Glutathione (GSH) 0.04g of GSH was dissolved in 100ml of phosphate buffer. Ellman’s Reagent 0.04g of 5, 5-dithiobis 2-nitrobenzoic acid (DTNB) was dissolved in 100ml of 0.1M phosphate buffer pH 7.4 Precipitating Agent 4% sulfosalicyclic acid (C6H6O6S.2H20) was prepared by dissolving 4g of sulfosalicyclic acid in 100ml of distilled water. 10 7 UNIVERSITY OF IBADAN LIBRARY Phosphate Buffer pH 7.0 3.58g of Na2HPO4.12H2O and 1.19g of NaH2PO2.2H2O were dissolved in distilled water and made to 900ml at the pH 7.0. The pH was adjusted to 7.0, and distilled water was then added to make up to 1 liter. Procedure 0.75 ml of the homogenate was deproteinized by the addition of 0.75 ml of 4% sulfosalicyclic acid. The mixture was spun at 14000g for 15 minute at 4C. 0.5 ml of the supernatant was added to 4.5 ml of Ellman‟s reagent. A blank was prepared by adding 0.4 ml of phosphate buffer with 0.1 ml of sulfosalicyclic acid. From this stock, 0.3ml of the diluted precipitating agent was taken and 4.5ml of Ellman‟s reagent was added. Reduced glutathione, concentration is proportional to the absorbance at 412nm. 3.22 ESTIMATION OF GLUTATHIONE-S-TRANSFERASE ACTIVITY Principle Glutathione–S–transferase activity was assayed spectro- photometrically using 1-chloro-2-4- dinitrobenzene (CDNB) and glutathione as described by Habig et al. (1974). This method is based on the fact that all known glutathione –S- transferase demonstrate a relatively high activity with 1-Chloro -2,4-dintrobenzene (CDNB) as the second substrate. Consequently, the conventional assay for glutathione –S- transferase activity utilizes 1-Chloro-2,4-dintrobenzene as substrate. Conjugation of the substrate with reduced glutathione (GSH) shifts the absorption maximum shifts to a longer wavelength. The absorption increase at the new wavelength (340nm) provides a direct measurement of the enzymatic reaction. Reagents GSH 40 mM 0.123 g of GSH was dissolved in 10 ml distilled water. CDNB 8 mM 0.162 g of CDNB was dissolved in 10 ml ethanol 2.5%. Phosphate buffer pH 6.5 1.375 g of KH2PO4 was dissolved in 100 ml of distilled water 1.799 g of Na2HPO4 was dissolved in 100 ml of distilled water 10 8 UNIVERSITY OF IBADAN LIBRARY The buffer was then made by mixing 68.2 ml KH2PO4 solution with 31.8 ml Na2 PO4 solution 0.33 N HCl 3 ml of HCl was mixed with 100 ml of distilled H2O. Procedure: 1 ml of homogenate was centrifuged for 3 min at 3000 rpm then 0.5 ml of supernatant was taken and diluted 1: 400 with distilled water. The following additions were done: Reagent Sample (µl) Blank (µl) Sample 500 500 GSH 250 250 CDNB 250 250 Buffer 1000 1000 HCl - 3000 0 Then, all tubes were incubated for 1 hour at 30 C, followed by addition of 3 ml HCl to the sample. The absorbance was measured at wave length 340 nm for each samples its blank. Calculation GST activity = O.D.Sample - BlankO.D. X 1 9.6 mg Protein = M mol / min / mg Protein. –1 Where 9.6 mM cm is the extension coefficient of 1- Chloro-2,4-dinitrobenzene. 3.23 STATISTICAL ANALYSIS Data were presented as mean and standard error of the mean. They were analysed by one-way th analysis of variance (ANOVA) using the 17 version of SPSS while differences between test and control groups were examined using Student‟s t- test. p < 0.05 was considered to be the level of significance. 10 9 UNIVERSITY OF IBADAN LIBRARY CHAPTER FOUR EXPERIMENTS AND RESULTS 4.1 EXPERIMENT 1: EFFECT OF ASCORBATE OR DEHYDROASCORBATE ON LEAD CHROMATE CYTOTOXICITY INTRODUCTION Ascorbate is the dominant biological reducing agent required for reductive activation of the toxicity and carcinogenic activity of Cr (VI) in vivo. In addition, Cr (VI) is usually present in the micromolar range when humans are exposed to it occupationally, whereas ascorbate is present at concentrations in the millimolar range under physiological conditions in humans. Inadequate or lack of ascorbate in culture has been suggested to result in low mutation and morphological transformation in chromate treated cells (Holmes et al, 2008). Therefore, in order to reproduce in vitro the conditions in mammalian cells in vivo, it is important to supplement cell culture medium with ascorbate. PROCEDURE Cells were cultured and seeded as previously described under “Materials and Methods” (Section 3.3., Page 78-79). Seeded cells were treated 24 hours later with 100µl of various concentrations of ascorbate or dehydroascorbate alone or with 25 µl of 1.62µg/ml lead chromate for 48 hours. The concentrations of ascorbate tested were 0.00625 mM, 0.0125 mM, 0.025 mM, 0.05 mM, 0.1mM, 0.25mM and 0.375 mM. For dehydroascorbate, the same concentrations were used as for ascorbate in addition to 0.5 mM. Control dishes were left untreated or were alternatively treated with 100µl phosphate buffer saline (PBS), 25µl acetone, 100µl PBS and 25µl acetone, or 1.62 µg/ml lead chromate. The medium was changed after treatment, but ascorbate or dehydroascorbate was again added at the same concentrations and volumes, with which the dishes were initially treated. Ten days post-seeding, dishes were fixed and stained as described earlier (Section 3.3., Page 78-79). Colonies containing 20 or more cells were counted and averaged in the five dishes. Relative survival was determined as a percentage of survival of cells. 11 0 UNIVERSITY OF IBADAN LIBRARY RESULTS The effect of ascorbate on the survival of CH310T½ treated with PbCrO4 is shown in Figure 4.1. The blue line shows the effect of ascorbate alone on the survival of 10T½ cells based on the average results of 3 experiments. The highest concentration of ascorbate that is non-cytotoxic was found to be 0.025 mM. Curiously, concentrations less than 0.025 mM, such as 0.0125 mM and 0.00625 mM appeared to cause some slight but measurable cytotoxicity. Above 0.025 mM, concentrations of 0.05 mM, 0.10 mM, 0.25 mM, and 0.375 mM reduced the survival of 10T½ cells in a dose-dependent manner, from a survival fraction of 1.0 to survival fractions of 0.82, 0.70, 0.30, and 0.10, respectively. The green rectangles show the survival fractions of CH310T½ cells after they were treated with 1.62 µg/ml of PbCrO4, which reduced the survival fraction of 10T½ cells from 1.0 down to 0.70 (70%). The red line shows the effect of ascorbate on the survival of CH310T½ cells treated with PbCrO4. The hypothesis in this experiment was that ascorbate inside cells would activate chromium (VI) compounds to become toxic to cells. The data can only be analysed up to concentrations of 0.025 mM ascorbate, since ascorbate itself becomes cytotoxic at higher concentrations. It was observed that at the lowest three concentrations of ascorbate (0.025 mM, 0.0125 mM, and 0.00625 mM), the survival fraction of cells treated with ascorbate and lead chromate decreased from 0.70 in cells treated with lead chromate only, down to 0.50, 0.45, and 0.55, respectively, in cells treated with these concentrations of ascorbate. Therefore, these three concentrations of ascorbate did decrease the survival of CH310T½ cells below that observed in cells treated with lead chromate only. This was consistent with our hypothesis; however, the decrease in cell survival was not dose- dependent with the ascorbate concentrations. Next, the effect of dehydro-ascorbate on the survival of CH310T½ cells treated with PbCrO4 was studied. As shown in Figure 4.2, treatment of CH310T½ cells with 1.62 µg/ml lead chromate reduced the survival of CH310T½ cells from 1.0 to 0.72. Dehydro-ascorbate was not cytotoxic up to a concentration of 0.05 mM. When dehydro-ascorbate was added to cells treated with lead chromate, it first decreased the cytotoxicity of lead chromate from 0.72 down to 0.40 at a concentration of 0.00625 mM of dehydro-ascorbate, i. e., this caused a pro-oxidant effect. Interestingly, higher concentrations of dehydro-ascorbate of 0.0125 and 0.0250 mM of dehydro- ascorbate actually increased the survival fraction of CH310T½ cells to 0.50 and to 0.65, 11 1 UNIVERSITY OF IBADAN LIBRARY respectively. This suggests that the initial reductive toxic effect becomes reversed at higher dehydro-ascorbate concentration. To demonstrate a dose-response dependent toxicity, there was need to lower the concentrations of dehydro-ascorbate, below 0.00625 mM dehydro-ascorbate, In the same vein, there was also need to repeat this experiment with concentrations of ascorbate between 0.0025 and 0.025 mM, to determine whether these concentrations of ascorbate can cause an anti-oxidant effect on the cytotoxicity of lead chromate, i. e., whether these concentrations reduce the cytotoxicity of lead chromate. To achieve this, the experimental set up was altered and the concentration of both ascorbate and dehydroascorbate were lowered as shown in appendix 1 (Table A1) The effect of lower concentrations of ascorbate on the survival of CH310T½ cells treated with PbCrO4 is shown in Figure 4.3. Figure 4.3 shows that when 1.62 µg/ml PbCrO4 was added to CH310T½ cells, it was cytotoxic as before, and reduced the survival fraction of CH310T½ cells to 0.70. Next, when ascorbate concentrations between 0.0025 to 0.025 mM were added to cells treated with non-cytotoxic concentrations of ascorbate, the survival fraction of the cells decreases to 0.40 as the concentration of ascorbate was increased from 0.0025 mM to 0.0125 mM, indicating a pro-oxidant effect. As the ascorbate concentration was increased from0.015 mM to 0.02 mM, the survival fraction actually increased to 0.70, indicating an anti-oxidant effect. The effect of lower concentrations of dehydroascorbate on the survival of CH310T½ cells treated with PbCrO4 is shown in Figure 4.4. In Figure 4.4, it can be seen that 1.62 µg/ml lead chromate reduced again the survival of CH310T½ cells from 1.0 to 0.70. Adding dehydro-ascorbate at concentrations from 0.00025 mM to 0.002 mM further decreased the survival of lead chromate- treated cells down to 0.45, indicating a pro-oxidant effect. Further increase in the concentration of dehydro-ascorbate to 0.003 and 0.004 mM increased the survival fraction back up to 0.50 and 0.70, conveying an anti-oxidant effect (Figure 4.4). Hence, again, it appeared that low concentrations of dehydro-ascorbate (0.00025 mM to 0.002 mM) increased the cytotoxicity of lead chromate, but increasing the concentrations of dehydro-ascorbate back up to 0.003 and 0.004 reversed the enhancement of the cytotoxicity (Figure 4.4). Conclusion 11 2 UNIVERSITY OF IBADAN LIBRARY Beyond 25 µM, ascorbate is cytotoxic to CH310T½. In addition, ascorbate or its oxidized form dehydroascorbate plays a paradoxical role in the toxicity of lead chromate. At low concentration ≤ 12.5 µM ascorbate or ≤ 2 µM dehydroascorbate), they act as proxidant by increasing the toxicity of lead chromate. In contrast at higher concentration (15-20 µM ascorbate and 3-4 µM dehydroascorbate), they act as anti-oxidant by deactivating lead chromate and decreasing its toxicity. 11 3 UNIVERSITY OF IBADAN LIBRARY Figure 4.1: Survival curve of 10T½ cells treated with PbCrO4 in the presence of ascorbate. 10T½ were treated simultaneously with suspension of PbCrO4 and ascorbate for48h. Blue line represents the survival curve of 10T½ cells treated with the higher concentrations of ascorbate alone while the red line represents the survival curve of the cells treated simultaneously with PbCrO4 and the same concentrations of ascorbate.The green rectangle on the y-axis represents the survival of the cells with with 1.62 µg/ml PbCrO4 alone. 11 4 UNIVERSITY OF IBADAN LIBRARY Figure 4.2: Survival curve of 10T½ cells treated with PbCrO4 in the presence of ascorbate. CH310T½ cells were treated simultaneously with suspension of PbCrO4 and dehydroascorbate for 48h. Blue line represents the survival curve of 10T½ cells treated with the higher concentrations of dehydroascorbate alone while the red line represents the survival curve of the cells treated simultaneously with PbCrO4 and the same concentrations of dehyroascorbate.The green rectangle on the y-axis represents the survival of the cells with 1.62 µg/ml PbCrO4 alone. 11 5 UNIVERSITY OF IBADAN LIBRARY Figure 4.3: Effect of ascorbate on the survival of 10T½ cells treated with PbCrO4 for 48hrs. Blue line represents the survival curve of 10T½ cells treated with the lower concentrations of ascorbate alone, while the red line represents the survival curve of the cells treated simultaneously with PbCrO4 and the same concentrations of ascorbate.The green triangle on the y-axis represents the survival of the cells with treated with 1.62 µg/ml PbCrO4 alone. 11 6 UNIVERSITY OF IBADAN LIBRARY Figure 4.4: Effect of dehydroascorbate on the survival of 10T½ cells treated with PbCrO4 for 48hrs. Blue line represents the survival curve of 10T½ cells treated with the lower concentrations of dehydroascorbate alone, while the red line represents the survival curve of the cells treated simultaneously with PbCrO4 and the same concentrations of dehyroascorbate. The green triangle on the y-axis represents the survival of the cells treated with 1.62 µg/ml of PbCrO4 alone. 11 7 UNIVERSITY OF IBADAN LIBRARY 4.2 EXPERIMENT 2: THE EFFECT OF PARTICLE SIZE ON THE CYTOTOXICITY BY PbCrO4, BaCrO4 and SrCrO4 USING CLONOGENIC ASSAY INTRODUCTION Workers in various chromate related industries are exposed to different particles sizes of particulate chromate. For instance, a spray painter is occupationally exposed to particulate chromate with reduced particle size when compared to that of a household painter. This in turn may affect the toxicity inherent from exposure to hexavalent chromate by two different workers. In addition, earlier work by Patierno et al. (1989) ascribed the weak transformation of CH310T1/2 mouse embryonic fibroblast by lead chromate to large particle size that limits its phagocytic uptake. However, comparative work with different particle size of chromate was not done. Moreover, the effect of particle size on the toxicity of particulate chromate VI compounds has not yet been investigated. In this experiment, the effect of particle size on the cytotoxicity of three insoluble chromate compounds to cultured CH310T½ mouse embryo cells was investigated. CH310T½ cells were exposed to both small (average particle size is ≤ 3 µm) and large particles (average particle size is ≤ 8 µm) of lead chromate (PbCrO4), Barium chromate (BaCrO4), and strontium chromate (SrCrO4) at concentrations ranging from 0.125 to 10 μg /ml for 48 hours. PROCEDURE 20 mg of each insoluble chromate compound was sonicated in 10 ml acetone, using a Braun Sonic Ultra Sonicator set at 150 watts for either 5, 10, or 20 minutes. The sonicated compounds and a control, containing an equal amount of unsonicated insoluble chromate compound in 10 ml acetone, were subsequently processed for electron microscopy. Twenty-five microliter of each insoluble chromate compound was briefly suspended in acetone and deposited on formvar- coated copper grid. The acetone was allowed to evaporate, and the grid was mounted on a stub. This was later gold coated in an Electron Microscopy Science Sputter Coater and examined in a JOEL JSM 6390LV Scanning Electron Microscope at 10KV. The particle diameter as observed with a JOEL JSM 6390LV Scanning Electron Microscope as measured with the Image J software for image processing and analysis. Briefly, Images from the electron microscope were loaded into ImageJ, an open-sourced image-processing software. The line tool was used to 11 8 UNIVERSITY OF IBADAN LIBRARY generate measurements of particle size along each particle‟s longest axis, giving values in raw pixels from the image. The line tool was also used to measure the image‟s scale bar, creating a unit conversion factor to change the units from pixels to microns. The CH310T½ cells were later exposed to the sonicated (small) and unsonicated (large) chromate compounds. The cells were thereafter seeded at 200 cells per 60 cell culture dish, five dishes per concentration of toxin studied, according to the plating efficiency protocol described earlier under “Materials and Methods” (Section 3.3., Page 78-79) . Twenty-four hours later, cells were treated with either unsonicated or sonicated insoluble chromate compounds. The concentrations of insoluble chromate compounds tested were between 0.5 µg/ml and 10 µg/ml. After 48 hours of exposure to toxin, the medium was changed, and cells were cultured for an additional 8 days th without toxin. On the 10 day, the medium was removed and the cells were rinsed once with isotonic phosphate-buffered saline. Cells were then fixed, stained and colonies counted as previously described (Section 3.3., Page 79-80) RESULTS Lead chromate particles were sonicated for 0, 5, 10 or 20 minutes. It was noted that the unsonicated particles of lead chromate were mostly in large particles aggregates < 8 µm in diameter. In previous work from the laboratory of Landolph, the particle size of unsonicated lead chromate particles as measured by light scattering was noted to be around 80 µm (Landolph, J. R., unpublished data). However, in this study using electron microscopy, it was observed that the size of particles of the compounds decreased with increasing time of sonication (Figure 4.5). After 5 minutes of sonication, approximately 50% of the particles had diameters < 8 um (Figure 4.5B). After 10 minutes of sonication, the average particles were 4.81 µm (Figures 4.5C and 4.6). After 20 minutes of sonication, the average particle sizes of the particles of lead chromate were lowered to 2.59 µm (Figures 4.5D and 4.6). In addition, more of the PbCrO4 particles went into suspension as the time of sonication was increased from 5 minutes to 10 minutes to 20 minutes. Based on this observation, lead chromate and the other insoluble chromate compounds used for the determination of the effect of particle size on cytotoxicity to CH310T½ cells were sonicated for 20 minutes. The mean particle size of BaCrO4 sonicated for 20 mins was 1.79 µm as compared to 2.19 µm for the unsonicated form (Figures 4.7. and 4.8). The mean particle size 11 9 UNIVERSITY OF IBADAN LIBRARY of sonicated PbCrO4 (2.59 µm) and BaCrO4 (1.79 µm) as well as unsonicated BaCrO4 (2.19 µm) are within the size range predicted for deposition in the bronchial tree where chromium-induced cancers usually occur (IARC, 1990). The cytotoxicity of large particles (unsonicated) PbCrO4 was first examined over a wide range of concentrations 0.5µg/ml to 10µg/ml. Treatment of CH310T½ cells with unsonicated PbCrO4 at concentrations of 0.5, 1.0, 1.62, 2.5 5.0, 7.5 and 10µg/ml resulted in a dose-dependent cytotoxicity and survival fractions of 0.87, 0.82, 0.71, 0.66, 0.08%, 0.04% and 0%, respectively (Figure 4.9). To compare the effect of particle size reduction on the cytotoxicity of lead chromate, CH310T½ cells were next treated with small particles (sonicated) of PbCrO4 at the same concentrations as the unsonicated particles. The dose-response relationship for cytotoxicity induced by sonicated lead chromate is presented in Figure 4.9. Treatment of 10T½ cells with sonicated particles of lead chromate reduced their survival at all doses in a dose-dependent manner. Sonicated lead chromate exposure led to survival fractions of 0.50, 0.40, 0.40, 0, 0, and 0 in cells exposed to 0.5, 1.0, 1.62,2.5, 5, 7.5, and 10 µg/ml respectively of lead chromate. The effect of particle size of lead chromate on the survival of 10T ½ cells, in comparison to that of the unsonicated lead chromate is shown in Figure 4.9. The results suggest that the smaller the lead chromate particles are in suspension, the higher their cytotoxicity to CH310T½ cells. The toxicity parameters obtained for both sonicated and unsonicated PbCrO4 is summarized below in Table 4.1 Next, the clonogenicity and the survival of 10T½ cells exposed to different concentrations of the large particles of BaCrO4 was assessed.The results are expressed as survival fraction of the no addition control, as shown in Figure 4.10. The 10T½ cells showed dose-dependent reduction survival after treatment with increasing concentrations of BaCrO4. Specifically, it resulted in 0.88, 0.47., 0.40, 0.38, 0.12%, 0%, 0%, and 0% clonogenic survival after treatment with 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 µg/ml BaCrO4 respectively. Next, cells were treated with small (sonicated) BaCrO4 to test the hypothesis that reduced particles size of BaCrO4 will reduce its clonogenic survival and enhance its cytotoxicity. The dose-response relationship for cytotoxicity induced by sonicated barium chromate is presented in Figure 4.10. Treatment of 10T½ cells with sonicated particles of barium chromate 12 0 UNIVERSITY OF IBADAN LIBRARY reduced their survival at all doses in a dose-dependent manner. Sonicated barium chromate exposure led to survival fractions of 0.43, 0.26, 0.23, 0.14, 0.08and 0.04 in cells exposed to 0.5, 1.0, 2.5, 5, 7.5, and 10 µg/ml of barium chromate, respectively. Over all, the effect of smaller particles of BaCrO4 on its cytotoxicity was noticeable at lower concentrations. The toxicity parameters obtained for both sonicated and unsonicated BaCrO4 is summarized in Table 4.2. The survival fraction of 10T ½ cells treated with different concentrations of unsonicated SrCrO4 is presented in Figure 4.11 Treatment of 10T½ cells with unsonicated SrCrO4 at concentrations of 0.125, 0.25, 0.5, 1.0, 2.5 and 5.0µg/ml resulted in a dose-dependent cytotoxicity and survival fractions of 0.74, 0.62, 0.37, 0.21, 0.03, and 0, respectively (Figure 4.11). Similarly, treatment of CH310T½ cells with sonicated SrCrO4 at concentrations of 0.125, 0.25, 0.5, 1.0, 2.5 and 5.0µg/ml resulted in a dose-dependent cytotoxicity and survival fractions of 0.70, 0.56, 0.42, 0.14, 0.07, 0, and 0, respectively (Figure 4.11). When the survival curve of the sonicated SrCrO4 was compared with the corresponding unsonicated form, the difference in cytotoxic effect was only apparent at higher doses (Figure 4.11). The toxicity parameters obtained for both sonicated and unsonicated SrCrO4 is summarized in Table 4.3. CONCLUSION Both the large and small forms of all the three hexavalent chromate induced dose dependent cytotoxicity in 10T½ cells .Generally, the order of toxicity of the hexavalent chromate compounds to 10T½ cells is: PbCrO4