Validation of an In Vitro Mutagenicity Assay Based on Pulmonary Epithelial Cells from the Transgenic MutaMouse: Intra-Laboratory Variability and Metabolic Competence By: Joleen Hanna, B.Sc. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science In Biology Specializing in Chemical and Environmental Toxicology Supervisor: Dr. Paul White (University of Ottawa) Thesis Advisory Committee: Dr. Frances Pick (University of Ottawa) Dr. Iain Lambert (Carleton University) University of Ottawa Ontario, Canada March 2018 © Joleen Hanna, Ottawa, Canada, 2018 Abstract: Genetic toxicity tests used for regulatory screening must be rigorously validated to ensure accuracy, reliability and relevance. Hence, prior to establishment of an internationally- accepted test guideline, a new assay must undergo multi-stage validation. An in vitro transgene mutagenicity assay based on an immortalized cell line derived from MutaMouse lung (i.e., FE1 cells) is currently undergoing formal validation. FE1 cells retain a lacZ transgene in a λgt10 shuttle vector that can be retrieved for scoring of chemically-induced mutations. This work contributes to validation of the in vitro transgene (lacZ) mutagenicity assay in MutaMouse FE1 cells. More specifically, the work includes an intra-laboratory variability study, and a follow-up study to assess the endogenous metabolic capacity of FE1 cells. The former is essential to determine assay reliability, the latter to define the range of chemicals that can be reliably screened without an exogenous metabolic activation mixture (i.e., rat liver S9). The intra- laboratory variability assessment revealed minimal variability; thus, assay reproducibility can be deemed acceptable. Assessment of metabolic capacity involved exposure of FE1 cells to 5 known mutagens, and subsequent assessment of changes in the expression of genes involved in xenobiotic metabolism; induced transgene mutant frequency (±S9) was assessed in parallel. The results revealed that the FE1 cell line is capable of mobilising several Phase I and Phase II gene products known to be involved in the bioactivation of mutagens. Collectively, the results presented support the contention that the FE1 cell mutagenicity assay can be deemed reliable and reproducible. Consequently, the assay is an excellent candidate for continued validation, and eventual establishment of an OECD (Organization for Economic Cooperation and Development) Test Guideline. II Acknowledgements: Firstly, I must give an immense thank to my supervisor Dr. Paul A. White for being so enthusiastic and encouraging of my accomplishments, and supportive and approachable when things are difficult. I have learned and grown an incredible amount in your lab and can’t thank you enough for giving me the opportunity to take on this thesis. Thank you to my committee members Dr. Iain Lambert and Dr. Frances Pick for your helpful feedback and contributions, I am grateful to have you as advisors. My sincerest thank you to Rebecca Maertens for taking me under your wing, being so helpful and kind, letting me cry at you, all the edits and the countless little things you do to be supportive; I feel very fortunate to have you to depend on. Thank you to Julie Cox for all the tips, training, practice seminars, editing and most of all the pep talks and encouragement; you have been my biggest cheerleader and I am so grateful for your generosity. Thank you Matt Meier and Richard Webster for being such wonderful office mates, I’ve learned so much by just being in the same room with you. Thank you to my brilliant and impressive peers Jennifer Keir, Eunnara Cho, Marc Beal, Sarah Labib, Nikolai Chepelev, Francina Webster, John Wills, Clotilde Maurice and Alexandra Long for all their training, helpful hints and suggestions, being a wonderful audience for presentations, and all the laughs, rants and snacks. You have all been so supportive, willing to help and you made coming to the lab an easier option than moping at home; I can’t thank you enough for your friendship and encouragement. Thanks to Webster’s for hosting awesome parties. And a big thank you to all the people at MSD who provide an inspirational place to work and learn. III This research could not have been completed without the financial support from NSERC’s CREATE-REACT program, Health Canada’s Research Affiliate Program and the University of Ottawa. Thank you Samuel Clemont-Dupont, John Gingerich, Alexandra Long and Rebecca Maertens for your contributions as Operators 2-5 in the intra-laboratory variability study. I need to thank my friends and family for all their optimism and encouragement over the last 2 years. I am so grateful to my brothers, step-dad and family members for all their support in hard times, celebration in good and for making sure I don’t take things too seriously. An enormous thank you to my Mom for showing me what hard work and a strong woman look like; you continuously motivate me by watching you achieve the impossible. Thank you to my Dad for thinking I’m a rock star and instilling the Hanna confidence in me (or attitude depending on who you ask); I wish you were here but I know you would be the most proud. To Olaf, my fur-baby, thank you for the endless cuddles and keeping me smiling through the long days. To my forever, Jordan, the last two years have been some of our hardest, but swooning over you takes most of the stresses away. Thank you for your patience, love and support and for making sure that my diet consists of more than breakfast cereals. Thanks everyone! IV Table of Contents: Abstract.......................................................................................................................................... II Acknowledgements ...................................................................................................................... III Table of Contents .......................................................................................................................... V List of Tables ….......................................................................................................................... VIII List of Figures……........................................................................................................................... X List of Abbreviations .................................................................................................................. XIII Statement of Contributions....................................................................................................... XVII Chapter 1: Introduction ………………………………………………………………………………………………………..... 1 1.1 Brief History of Genotoxicity Assessment …………………………………………………………………………… 1 1.2 The Organisation for Economic Cooperation and Development (OECD) Test Guideline Program …………………………………………………………………………………………………………………………….. 7 1.3 Detection of Gene Mutations …………………………………………………………………………………………….. 9 1.3.1 In Vivo Transgenic Rodent (TGR) Systems .………………..………..…………………………………… 11 1.3.2 The MutaMouse FE1 Cell Line …..…………………………………………..………………………………… 13 1.3.2.1 Morphological Features and Growth Characteristics ………………………………….….. 13 1.3.2.2 Genetic and Biochemical Features ….……………………………………………………………… 14 1.3.2.3 Performance of the MutaMouse FE1 Cells Mutagenicity Assay ….………………….. 15 1.4 Organisation for Economic Cooperation and Development (OECD) Test Guideline Validation ………………………………………………………………………………………………………………………… 17 1.5 Requirement for the Development of In Vitro Alternatives …………………………………………….. 20 1.6 International Organizations Promoting the Development of In Vitro Assays ……………………. 22 1.7 Thesis Objectives ……………………………………………………………………………………………………………… 24 1.8 Tables and Figures …………………………………………………………………………………………………………… 25 1.9 References ………………………………………………………………………………………………………………………. 29 Chapter 2: Chapter 2: Intra-laboratory Variability in lacZ Mutant Frequency Values Generated Using the MutaMouse FE1 Cell in Vitro Transgene Mutation Assay ..……………………….………….. 33 2.1 Introduction …………………………………………………………………………………………………………………….. 35 2.2 Materials and Methods ……………………………………………………………………………………………………. 41 2.2.1 Chemicals …………….……..………………………………………………………………………………………….. 41 2.2.2 Cell Culture …………..………..……………………………………………………………………………………….. 42 2.2.3 FE1 Cell Transgene Mutation Assay …..……..……………………………………………………………… 42 2.2.4 Study Design ………………………………………..……..…………………………………………………………… 44 2.2.5 Data Analysis …..…………………………………..………………………………………………………………….. 45 2.3 Results …………………………………………………………………………………………………………………………….. 48 2.3.1 Analysis of Controls …………………………..…..……………………………………………………………….… 48 V 2.3.1.1 Negative Control Groups …….………………………………………………………………………….. 48 2.3.1.2 Positive Control Groups ……….…………………………………………………………………………. 49 2.3.2 Analysis of Test Chemicals ………..….………………………………………………………………………… 50 2.3.3 Comparison of Responses Across Operator and/or Test Date ….……………………………… 52 2.4 Discussion ………………………………………………………………………………………………………………………… 55 2.5 Tables and Figures …………………………………………………………………………………………………………… 71 2.6 Supplementary Data ………………………………………………………………………………………………………... 82 2.7 References ………….…………………………………………………………………………………………………………… 85 Chapter 3: Xenobiotic-induced Gene Expression Changes in MutaMouse FE1 Pulmonary Epithelial Cells …………………………………………………………………………………………………….…………………...…….. 92 3.1 Introduction …………………………………………………………………………………………………………………..… 94 3.2 Materials and Methods ………………………………………………………………………………………………..… 103 3.2.1 Chemicals ……………………………………….……………………………………………………………………. 103 3.2.2 Cell