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Some Pages of This Thesis May Have Been Removed for Copyright Restrictions Some pages of this thesis may have been removed for copyright restrictions. If you have discovered material in Aston Research Explorer which is unlawful e.g. breaches copyright, (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please read our Takedown policy and contact the service immediately ([email protected]) Title page Evaluating the biological effects of electronic cigarettes using a novel in-house designed aerosol delivery system and an in-vitro co-culture model of the human airways Pranav Vasanthi Bathrinarayanan Doctor of Philosophy Aston University June 2018 ©Pranav Vasanthi Bathrinarayanan, 2018 Pranav Vasanthi Bathrinarayanan asserts his moral right to be identified as the author of this thesis This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without proper acknowledgement. 1 Aston University Evaluating the biological effects of electronic cigarettes using a novel in-house designed aerosol delivery system and an in-vitro co-culture model of the human airways Pranav Vasanthi Bathrinarayanan Doctor of Philosophy June 2018 Summary Although electronic cigarettes (ECs) have been widely promoted as safer alternatives to tobacco cigarettes, limited scientific data is currently available on their possible health effects. The current study aims to investigate the potential effects of ECs using a novel in- house designed cigarette/EC aerosol delivery system and physiologically relevant 2D and 3D in-vitro human airway models. Submerged cultures of BEAS 2B and CALU 3 bronchial epithelial cells were used to investigate the effects of nicotine and its oxidative metabolite cotinine. Results demonstrated that neither nicotine nor cotinine had any significant impact on the bronchial epithelial cell viability or IL-6/IL-8 pro-inflammatory mediators’ production. Further, treatment of submerged cultures of a number of airways related cell types to extracts of commercially available ECs of different nicotine strength, flavourings and brands showed that while the differing nicotine strengths had no impact on the cell viability, flavourings significantly influenced cell viability, with strawberry and cherry flavoured ECs demonstrating the highest cytotoxicity. Moreover, same flavours from different brands produced different effects on cells. Finally, a co-culture human airways model consisting of CALU 3 bronchial epithelial cells and MRC-5 pulmonary fibroblasts cultured at air-liquid- interface were treated to whole cigarette smoke (WCS) and EC vapour (ECV) at different exposure times (7 m, 1 h, 2 h, 3 h, 4.5 h and 6 h) as per the ISO:3308 standard smoking regime using an in-house designed bespoke, automated aerosol delivery system. Results demonstrated that while WCS caused a significant reduction in cell viability post 7 min exposure, ECV produced cytotoxic effects only at exposure times ≥ 3 h. A significant increase in oxidative stress and IL-6/IL-8 production was observed post 3 h ECV treatment, both of which are hallmark characteristics of airway inflammatory conditions like chronic obstructive pulmonary disorder. Overall, results from the current study suggest that ECs have the potential to cause substantial airways damage, and as yet, cannot be regarded as safe alternatives to tobacco cigarettes. Importantly, a standardised testing method is urgently required in order to elucidate on the long-term health effects of ECs. Key words: E-cigarettes, co-culture, human airways, CALU 3, MRC-5 2 Dedication Dedicated to all the eminent scientists of the past, both the heard and the unheard, on whose sturdy shoulders of original thought, hard-work, sacrifice, dedication and rational thinking, I stand upon today. At the face of the unknown and at the depth of mystery, you dared to smile. Thank you, great people. 3 Acknowledgements First and foremost, I wish to thank my wonderful supervisor Dr. Laura Leslie. What a lovely journey it has been! It is unimaginable to think of my PhD without your support, encouragement, advice (both life and PhD), vegan energy bars, Christmas goodies, free- tickets to Electric cinema and British indoors athletics and everything else you have knowingly and unknowingly done to help me complete my PhD journey and become a better person overall. I truly am thankful to you beyond words and hope to be in great touch in years to come! Next, I would like to thank the ever-ebullient, emollient, gregarious and energetic Dr. Lindsay Marshall. How could I have completed my PhD if not for your round-the-clock support?!? Though you could have left me stranded in the middle of my PhD, you offered as much support (or even more!) as you would have, had you been in the university. I hope to carry forward this mentor-mentee partnership in the years to follow. Thank you! Next, I would like to thank Dr. James Brown. Firstly, for readily agreeing to take me into your custody (supervise my PhD) without any question or hesitation when Lindsay left. Secondly and most importantly, for introducing me to the ‘Helping Birmingham Homeless’ sessions. What started as a sporadic voluntary service has now transformed into a life-long goal for me. What you have taught me is nothing short of life-changing. Thank you! I would like to specifically thank the School of EAS for providing me with a fully-funded studentship to pursue my PhD and for magnanimously supporting me financially, especially towards my conferences. Dr. Philip Davies especially has been a great mentor to me and I cannot but thank him enough for all his timely help and support. I would also like to extend my thanks to all the lovely, humble staffs of the EAS school office. Special mention to Sandra Mosley, Carol Wilson, Helen Yard and Gez Cooney who have guided me in many aspects right from the beginning of my PhD. Now, to my friends. My special thanks to soon to be Dr. Ross Pallett. It was not a coincidence that we started our PhD journey together, for birds of same feather flock together! Thank you for being such a great companion, scientific advisor and a wise friend during the past 4 years. And I am SORRY for everything that you went through during your PhD. You are the most courageous and resilient person I’ve ever met. Be the same. Special thanks to Arnold Mabiala for being my only late-night office mate! Thanks to Dr. Shruti Sharma for being the ever-carefree, nonchalant and the occasionally-wise friend during my times at Aston. You are awesome! Thanks to Nupur, Vinod Nadella and Khaled for being 4 great friends especially during the beginning of my PhD when I was quite lonely. Thanks to Omkar Joshi, Navin and Jono for being the brilliant mentors that you guys have always been! Special thanks to my newly-found brothers (office-mates) namely Blake, Moid, Mito, Kemal, Abdel Nasir, Ahmad Fikri. You guys made working in office so much enjoyable (though we work at different shifts!) Thanks to the people who will always be close to my heart- Hadrian Vivek, Karthik, Ranjith, Prachya and Namra for not losing touch with me. Special thanks to Gautham Kumar ji whom I admire and look upto so much in my life. I don’t want to thank and dwarf David Finny, Binu, Nishant, Vicky, Surendar, Peppin. You guys are my life. Now the difficult bit. I really do not know how to appreciate my dear Amma, Appa and Nivi. The incessant love, care, kindness and prayers which you guys have showered on me since my birth and especially during my PhD life is something I cannot express in words. I am eternally indebted to all three of you. I am sorry for all the sacrifices and the suffering which you guys underwent silently without drawing my attention- I noticed every bit of it. Nivi, I am also sorry for not growing up with you. I hope it is all worth it. And lastly…thank you Ramakrishna, Ramana and Shiva. Tat Twam Asi. 5 Table of Contents Title page .......................................................................................................................... 1 Summary ........................................................................................................................... 2 Dedication ......................................................................................................................... 3 Acknowledgements ........................................................................................................... 4 Table of Contents .............................................................................................................. 6 List of Figures.................................................................................................................. 15 List of Tables ................................................................................................................... 20 List of abbreviations ........................................................................................................ 22 List of units ...................................................................................................................... 25 1 Structure of the thesis .............................................................................................. 26 2 Literature review and background research .............................................................. 28 2.1 Traditional cigarette smoking............................................................................
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