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(Mon810) in Rats As a Model

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(Mon810) in Rats As a Model A PROTEOMICS-BASED APPROACH TO STUDYING THE IMPACT OF TRANSGENIC MAIZE (MON810) IN RATS AS A MODEL Asmaa Ali Al-Harbi A thesis submitted for the degree of Doctor of Philosophy (Ph.D.) at Newcastle University School of Biology, Faculty of Science, Agriculture and Engineering October 2015 Table of Contents List of Figures ............................................................................................................. vii List of Tables .................................................................................................................. x List of Appendices ....................................................................................................... xii Acknowledgements ....................................................................................................... xv Declaration .................................................................................................................. xvi Abstract ...................................................................................................................... xvii List of Abbreviations ................................................................................................ xviii Chapter 1. General Introduction ................................................................................... 1 1.1 Genetically modified crops .................................................................................... 1 1.1.1 Types of GM crops ......................................................................................... 2 1.1.2 Commercially growing GM crops .................................................................. 3 1.1.3 Advantages of GM-derived foods .................................................................. 3 Pest resistance ...................................................................................................... 3 Herbicide tolerance .............................................................................................. 4 Disease resistance ................................................................................................ 9 Cold tolerance ...................................................................................................... 9 Drought tolerance/salinity tolerance ................................................................... 9 Nutrition ............................................................................................................. 10 Pharmaceuticals ................................................................................................. 11 Phytoremediation ............................................................................................... 11 1.1.4 Concerns regarding GM foods for human use.............................................. 12 Allergenicity ....................................................................................................... 12 Unknown effects on human health ..................................................................... 12 1.1.5 Environmental concerns ............................................................................... 13 Pesticide resistant crops could be toxic to non-target organisms ..................... 13 Reduced effectiveness of pesticides .................................................................... 13 All of these environmental hazards are compounded by genetic pollution ....... 13 1.1.6 Genetically modified maize/corn .................................................................. 14 Definition of MON 810 ...................................................................................... 14 History of MON 810 ........................................................................................... 16 MON810 is a group of tens of varieties ............................................................. 17 The European stalk borer .................................................................................. 17 i Mode of action of Bt Cry toxins ......................................................................... 18 Yield advantages for the cultivation of the MON810 crop ................................ 19 1.2 Omic based technologies in food safety .............................................................. 22 1.2.1 Assessing the safety of whole foods ............................................................. 23 1.2.2 Can–omics of novel food/feed inform a safety assessment? ........................ 25 Transcriptomics ................................................................................................. 25 Proteomics ......................................................................................................... 26 Metabolomics ..................................................................................................... 30 1.2.3 Use of proteomic based technologies to study toxicology and animal response to stress ................................................................................................... 31 1.3 Aims of the project .............................................................................................. 33 Chapter 2. Materials and Methods .............................................................................. 35 2.1 Materials .............................................................................................................. 35 2.2 Methods ............................................................................................................... 36 2.2.1 Effects of different maize lines on weight gain and food consumption of male Wistar rats ..................................................................................................... 36 Test materials ..................................................................................................... 36 Diet formulation for rat feeding trials ............................................................... 37 Compositional analyses of rat diets ................................................................... 37 Analysis for nutritional composition and environmental contaminants ............ 38 Animals and housing .......................................................................................... 38 Experimental design: 7-day and 28-day Rat Feeding Trials ............................. 38 2.2.2 Isolation of small intestine epithelial cells ................................................... 39 2.2.3 Protein separation and identification ............................................................ 41 Protein extraction from isolated rat intestinal epithelial cells .......................... 41 Determination of protein concentration............................................................ 41 Cleaning up and preparing the samples for 2-D electrophoresis ...................... 44 Two-dimensional gel electrophoresis (2-DE) .................................................... 45 First dimensional electrophoresis (Isoelectric Focusing (IEF)) .................... 45 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ................................. 46 SDS-PAGE molecular weight standards, broad range .................................. 47 Visualisation of proteins .................................................................................... 50 Image acquisition and analysis .......................................................................... 50 Mass spectrometry based identification of proteins of interest ......................... 51 ii Tryptic in gel digestion for liquid chromatography-mass spectrometry (LC-MS/MS) ................................................................................................... 51 Nanoflow HPLC electrospray tandem mass spectrometry (nLC-ESI- MS/MS) ........................................................................................................... 51 Database search for protein identification ........................................................ 52 2.2.4 In vitro effects of transgenic maize (Bt maize MON810) expressing Cry1Ab protein (Bacillus thuringiensis toxin) on mammalian epithelial cells ..... 52 Test material ...................................................................................................... 52 Protein extraction of maize (MEB307Bt) Cry1Ab MON810 ............................. 53 Immunoassy by Western blotting of test material .............................................. 53 Cell culture methods .......................................................................................... 53 Isolation of epithelial cells from rat small intestine ....................................... 54 Establishment in culture of epithelial cells isolated from rat small intestine and the commercial human epithelial cell line HCT116 ................. 54 Cell count and viability count......................................................................... 55 Subculturing of cells ....................................................................................... 56 Cell staining ................................................................................................... 56 Cryopreservation ............................................................................................ 58 Cell viability assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay) ................................................................. 58 Calibration of growth curve through the Growth Cell Cycle ........................ 59 Determination of exposure time of Bt and non-Bt maize extracts for both IE cells
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