The utility of trout hepatic cells in the prediction of xenobiotic bioaccumulation and environmental persistence by Chibuzor Uchea A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences College of Life and Environmental Sciences University of Birmingham February 2013 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract The need to protect aquatic organisms from the toxic potential of environmental contaminants with minimal use of animal testing has been highlighted by the introduction of a number of regulatory directives around the world. This thesis proposes the potential of 3D cell culture models to ‘bridge the gap’ between the use of existing subcellular and in vivo systems for the testing of compounds for potential persistence, bioaccumulation and toxicity, improving the predictive power of in vitro screening procedures. In this project, a novel trout hepatocyte spheroid model was optimised and using metabolic, fluorescence, transport and gene expression assays, the utility of this system in such studies as a superior alternative to currently used subcellular fractions and traditional hepatocyte monolayer culture was demonstrated. Hepatocytes cultured as spheroids remained viable for up to 40 days, exhibiting significantly greater functional xenobiotic metabolism and transport compared to other in vitro systems. Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were also measured in spheroids compared to hepatocytes cultured using traditional techniques. These features of spheroids present a profile more closely representative of that of the whole liver. Together with the potential advantages of screening highly persistent compounds that require long term incubation and possible use in the assessment of the toxicity of compounds exhibiting chronic effects, this system successfully enriches current in vitro testing strategies. Acknowledgements The past four years have proved an incredibly testing, yet rewarding period and there are many people that I must thank for making the latter the part that stands out the most. Firstly, my principal supervisor Kevin Chipman for his time, guidance, belief and support- especially in my application for the policy placement scheme and for allowing me to pursue my own areas of interest which were not within the original project plans. Huge thanks also go to Annette Evans for always helping me to locate Kevin and organising our meetings. I acknowledge the BBSRC and AstraZeneca for funding the project. I am grateful to my external supervisor Stewart Owen, the founder and architect of our award winning in vitro fish ecotoxicology team, for keeping in good contact, helping to push things forward and especially for his 'golden nuggets' and extend my gratitude to all at Brixham Environmental Laboratory for their useful input and suggestions. Unfortunately, Timothy Schulz-Utermoehl left the project prematurely, however I thank him for his contribution and for inviting me to come and work for a few weeks at Alderley Park. In addition, I would like to thank Sunil Sarda for all of his help while I was in Macclesfield. I also thank Mark Viant for his contribution to discussions during the project. During my time in Birmingham, I have been very fortunate to work with some of the brightest, kindest and funniest people I have met. I would like to thank all members of the 4th floor, past and present who have helped to make long days, nights and weekends in the lab so much more enjoyable. There is an excellent feel of camaraderie in our collective and my thanks go to Paul Coleman, Alex Gavin, Gregory Genta-Jouve, Bob Harris, Martin Jones, Zahra Khan, Farhat Khanim, Wilber Sabiti, Mohammed Shuwaikan (who will be on my coaching staff when I become a Premier League manager), Andy Southam, Louise Stone, Lorna Thorne, Ralf Webber, Tom White (with whom I am looking forward to entering the gel production business), Tim Williams and Jinkang Zhang. I especially thank Huw Jones, Rachael Kershaw and Nadine Taylor for relaxing, science free tea breaks and finally Leda Mirbahai for allowing me to constantly bug her with questions, teaching me many techniques and being a great person to sit next to in the office and lab. I like to think I've rewarded her well by talking to her about sport and letting her steal my pens! I also extend my thanks to my friends in the Schools of Chemistry and Engineering, especially Danny Brown, Vinh Doan, Fracncesco Dorigatti, Jose Manuel Ramirez Leon and Jimmy Roussel (the most English Frenchman I have ever met- I introduced him to cricket and he loves it!) for providing me with endless entertainment and advice. I thank my friends and team mates at Harborne Rugby Club for helping me to relieve stress on Saturday afternoons, loads of laughs, pitch memories and scars that I will never forget! Thanks also to all at the National Assembly for Wales. I had a wonderful time working there during my placement and am grateful to my colleagues in the Health and Social Policy team for showing me the ropes and helping me to get a few of my reports published. Special thanks must go to the wonderful Katarzyna Koczuła for teaching me how to use FACS, introducing me to NMR, keeping me well fed (especially with pierniczki), encouraging me to be more artistic, ensuring that I did not become a complete recluse while writing up and teaching me to always believe in the power of the 4 leaf clover. Finally I would like to thank my engine room. I am so blessed to have the most amazing family who have always supported and encouraged me. I thank my siblings Ogor, Nneka and Ndubuisi for their calls and visits, introducing me to great music which helped me through writing and for the character building wind-ups and banter growing up. To Baby O, this book is the reason why Uncle Chi had always gone, I hope that people find it as interesting as you find the book about the London Eye! I'd also like to thank my extended family and friends at home who always ask after me, for their thoughts and prayers. I reserve the biggest thank you for my parents, Andrew and Frances, for their cards and letters, unending love, always providing me with a safe haven (laden with food!), reassurance on the numerous occasions on which I thought that I could not make it and being truly inspirational. iv Contents Chapter 1: General Introduction ............................................................................................................... 1 1.1 Rationale ............................................................................................................................................. 2 1.2 Pharmaceuticals and other chemicals in the environment .......................................................... 2 1.2.1 Pharmaceuticals in the environment ....................................................................................... 3 1.2.2 Routes of environmental exposure of pharmaceuticals ........................................................ 7 1.2.3 Emerging non-pharmaceutical environmental contaminants ............................................. 9 1.3 Environmental risk assessment of compounds ........................................................................... 11 1.4 Metabolism ....................................................................................................................................... 14 1.5 Cytochrome P450s ........................................................................................................................... 19 1.5.1: Hepatic rainbow trout CYPs .................................................................................................. 20 1.5.1.1 CYP1A ..................................................................................................................................... 22 1.5.1.2 CYP2 ........................................................................................................................................ 23 1.5.1.3 CYP3 ........................................................................................................................................ 24 1.6 Glucuronide formation ................................................................................................................... 24 1.7 Sulfate conjugation .......................................................................................................................... 27 1.8 Excretion ........................................................................................................................................... 28 1.9: ABC efflux transporters ................................................................................................................. 32 1.9.1. Multidrug resistance protein (MDR1) .................................................................................. 36 1.9.2 Bile salt export pump (BSEP) .................................................................................................