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Heavy Metal Toxicity in Human Lung Fibroblasts and Inhibition of Human Topoisomerase-I As a Potential Mechanism

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Heavy Metal Toxicity in Human Lung Fibroblasts and Inhibition of Human Topoisomerase-I As a Potential Mechanism HEAVY METAL TOXICITY IN HUMAN LUNG FIBROBLASTS AND INHIBITION OF HUMAN TOPOISOMERASE-I AS A POTENTIAL MECHANISM GEORGIA KIOUMOURTZI BSc A thesis submitted to the University of Lancaster for the degree of Master of Science (by research) in Biomedical Science Supervised by Dr Sarah Allinson June 2015 Division of Biomedical and Life Sciences Faculty of Health and Medicine Lancaster University DECLARATION I declare that this thesis is my own work submitted for the degree of Masters (by Research) in Biomedical Science at Lancaster University. This work has not been previously submitted to another University or Institute of Learning for the award of a higher degree. Words: 34.000 ΑCKNOWLEDGEMENTS First and foremost Ι would like to thank my supervisor Dr Sarah Allinson for her constructive criticism and academic guidance throughout this year. With her immeasurable patience and support I would not have had such development as I did during this project. I would like to extend my thank to my second supervisor Dr. Niki Copeland for always having time for me; answering my queries and helping find solutions to instant problems during experiments and to all my colleagues in the lab with whom lab-work was so much easier. I am extremely grateful to my family and friends and especially to my sisters, as with them everything is more enjoyable; and my partner Dimitris Afouxenidis and my dear friend Ioannis Tsitsimpelis, for the vast emotional support and encouragement they have provided me over the last year. Without them I would not have finished this challenging year. Finally, I want to express my sincere gratitude to my mentor and beloved friend Dr Androniki Papoutsi for advising my academic choices and teaching me how to be a better scientist and person. Without her inspiration I would not have tried for the best me. This project is dedicated to her. CONTENTS ABSTRACT ............................................................................................................................. - 1 - ABBREVIATIONS .................................................................................................................. - 2 - 1. INTRODUCTION ................................................................................................................ - 5 - 1.1. Introduction to heavy metals ................................................................................... - 6 - 1.2. Metal genotoxicity/ carcinogenicity ........................................................................ - 8 - 1.3. Cadmium .................................................................................................................... - 8 - 1.3.1. Cadmium kinetics in human body ..................................................................... - 9 - 1.3.2. Health problems following cadmium exposure ............................................. - 10 - 1.3.3. Genotoxic effects of cadmium .......................................................................... - 13 - 1.3.4. Cadmium Toxicity and Carcinogenesis ........................................................... - 18 - 1.4. Lead .......................................................................................................................... - 21 - 1.4.1. Lead kinetics in human body ........................................................................... - 22 - 1.4.2. Health Effects following lead exposure .......................................................... - 23 - 1.4.3. Genetics and related effects ............................................................................. - 27 - 1.4.4. Toxicity and carcinogenesis of lead ................................................................ - 28 - 1.5. Copper ...................................................................................................................... - 29 - 1.5.1. Copper kinetics in human body ....................................................................... - 30 - 1.5.2. Copper transport and pools ............................................................................. - 30 - 1.5.3. Human health effects from copper deficiency or excess ............................... - 33 - 1.6. Zinc ........................................................................................................................... - 36 - 1.6.1. Zinc roles in cellular level. ............................................................................... - 37 - 1.6.2. Zinc transport and cellular pools .................................................................... - 39 - 1.6.3. Human health effects from zinc deficiency or excess .................................... - 40 - 1.7. Topoisomerases ....................................................................................................... - 43 - 1.7.1. Topoisomerase-I structure and mechanism .................................................. - 46 - 1.7.2. Topoisomerase-I cleavage complexes and its inhibitors .............................. - 50 - 1.8. Aim of study ............................................................................................................. - 53 - 2. MATERIALS AND METHODS ......................................................................................... - 54 - 2.1. Cell culture ............................................................................................................... - 57 - 2.1.1. Clonogenic survival assays .............................................................................. - 57 - 2.1.2. Immunofluorescence assay of γ-Η2ΑΧ formation ......................................... - 59 - 2.2. Western blot ............................................................................................................ - 60 - 2.2.1. Sample preparation .......................................................................................... - 60 - 2.2.2. Samples running and transferring .................................................................. - 60 - 2.2.3. Antibody Blocking ............................................................................................ - 60 - 2.3. Topoisomerase inhibition by heavy metals .......................................................... - 61 - 2.3.1 Topo-I substrate preparation ........................................................................... - 61 - 2.3.2. Substrate titration ............................................................................................ - 62 - 2.3.3. Topoisomerase-I enzyme titration ................................................................. - 63 - 2.3.4. Topo-I oligonucleotide cleavage assay ........................................................... - 63 - 2.3.5. Topoisomerase-I relaxation assay .................................................................. - 64 - 2.4. Topoisomerase I expression in bacteria ................................................................ - 65 - 2.4.1. Transformation of bacteria with plasmid is induct of protein expression .. - 65 - 2.5. Topoisomerase I expression in mammalian cells ................................................. - 67 - 2.5.1. Preparation of vector ....................................................................................... - 67 - 2.5.2. Transfection of mammalian cells .................................................................... - 68 - 2.5.3. Purification of His-Tagged protein from transfected mammalian cells ....... - 69 - 3. RESULTS .......................................................................................................................... - 70 - 3.1. Clonogenic assay of MRC5 cells treated with cadmium, lead, zinc and copper .. - 71 - 3.2. Immunofluorescence assay of γH2AX.................................................................... - 77 - 3.2.1. The role of histone H2AX in the DNA repair mechanisms ............................ - 77 - 3.3. Inhibition of Topo-I function .................................................................................. - 82 - 3.3.1. Topoisomerase-I oligonucleotide cleavage assay .......................................... - 83 - 3.3.2. Topoisomerase-Ι cleavage function inhibition by heavy metals .................. - 88 - 3.3.3. Plasmid relaxation assay.................................................................................. - 96 - 3.3.4. Relaxation assay metal titration ...................................................................... - 98 - 3.4. Bacterial expression of recombinant human Topo-I .......................................... - 101 - 3.5. Attempted expression of Topo-I in mammalian cells ......................................... - 106 - 4. DISCUSSION .................................................................................................................. - 108 - 4.1. Cadmium ................................................................................................................ - 110 - 4.2. Lead ........................................................................................................................ - 111 - 4.3. Copper .................................................................................................................... - 112 - 4.4. Zinc ......................................................................................................................... - 113 - 4.5. Topoisomerase-I expression ................................................................................ - 114 - 5. CONCLUSION ...............................................................................................................
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