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THE ROLE of GLUTATHIONE in ALLERGIC CONTACT DERMATITIS. Sandrine Spriggs

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THE ROLE of GLUTATHIONE in ALLERGIC CONTACT DERMATITIS. Sandrine Spriggs THE ROLE OF GLUTATHIONE IN ALLERGIC CONTACT DERMATITIS. This thesis is submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor of Philosophy by Sandrine Spriggs November 2017 Declaration I declare that the work presented in this thesis is all my own work and has not been submitted for any other degree. …………………………………………………………………………….. Sandrine Spriggs (MSc) Acknowledgements Firstly, I would like to thank Dr Dean Naisbitt for giving me the opportunity to carry out this piece of research at the University of Liverpool under his supervision. Dr Naisbitt and his team have made sure that I felt very welcome during each of my visits to Liverpool. I am very grateful for all the time they have spent to sort out the technicalities of me carrying out experiments in their lab in the short time allocated. Equally, I would like to thank Dr Neil Kitteringham for his time and advice during the writing of the publications associated with this thesis. Secondly, all my heartfelt thanks to Unilever Safety and Environmental Assurance Centre (SEAC) for sponsoring this piece of research and especially to Dr Maja Aleksic, my external supervisor. Her guidance has been much appreciated as she has helped me improving my scientific project design skills greatly. I would also like to mention Mr Richard Cubberley and Dr Steve Gutsell, who jointly covered Dr Aleksic’s supervising role while she was on maternity leave. I reserve a special thanks to all my Unilever colleagues with experimental toxicology skills, especially Mrs Annette Furniss and Mrs Penny Jones, who kindly shared they invaluable experience of skin cells and skin related 3D models and overviewed my cell culture training. I also wish to thank my family for all their various degrees of involvement in my student life. First of all to my husband Danny, for his constant support throughout my six years of study. During the past few years, our relationship has blossomed and we have now become a very happy family. My father, for being an inspiration to me and fully endorsing my wishes to become a scientist in a field he knew very little about. To my in-laws in the UK, thank you so much for being there for me when I am in need of comfort and reassurance. And last but not least, I need to thank my newly born son Xavier, for keeping incredibly quiet day and night thus allowing me to get the final touches to this manuscript. Contents Abbreviations ............................................................................................................................ i Publications ............................................................................................................................. iv Abstract ................................................................................................................................... vi Chapter 1: General Introduction .............................................................................................. 1 Chapter 2: Materials and Methods. ....................................................................................... 45 Chapter 3: Modulation of GSH oxido-reduction mechanism in the HaCaT cell line. ............. 66 Chapter 4: GSH conjugation as clearance mechanism of dinitrohalobenzenes in the HaCaT cell line. .................................................................................................................................. 99 Chapter 5: GSH conjugation of model sensitisers of various potencies: Case study for DNCB, DPCP and DEM. .................................................................................................................... 123 Chapter 6: Effect of multiple dosing of DNCB on the Reconstructed Human Epidermis model. .................................................................................................................................. 155 Chapter 7: General Discussion. ............................................................................................ 181 Bibliography. ........................................................................................................................ 190 Abbreviations 2-AAPA 2-acetylamino-3-[4-(2-acetylamino-2- carboxyethylsulfanylthiocarbonylamino) phenylthiocarbamoylsulfanyl] propionic acid ABC ATP binding cassette ACD Allergic contact dermatitis AOP Adverse outcome pathway ATP Adenosine triphosphate BA Benzaldehyde BCNU 1,3-Bis(2-chloroethyl)-1-nitrosourea (or carmustine) BSO L-buthionine sulfoximine COMT Catechol-O-methyl transferase CYP Cytochrome P450 mono-oxygenase DEM Diethyl maleate DMEM Dulbecco's modified Eagle's medium DNBB 1-bromo-2,4-dinitrobenzene DNCB 1-chloro-2,4-dinitrobenzene DNFB 1-fluoro-2,4-dinitrobenzene DNP-SG S-(2,4-dinitrophenyl)glutathione DPCP Diphenylcyclopropenone EC3 Effect concentration 3 (estimated concentration of a chemical required to produce a 3-fold stimulation of draining lymph node cell proliferation compared with concurrent controls, expressed as a %) EC50 concentration of reagent able to deplete 50% of the reduced GSH available in GSH depletion assay ESI Electrospray ionisation FCS Foetal calf serum GCL Glutamate-cysteine ligase GPx Glutathione peroxidase i GSH Reduced glutathione GR Glutathione reductase GSSG Glutathione disulphide GST Glutathione s-transferase HaCaT Human Adult low Calcium high Temperature HP Hydrogen peroxide IAA Iodoacetamide LC Liquid chromatography LLNA Local lymph node assay MIE Molecular initiating event MRM Multiple reaction monitoring mRNA Messenger ribonucleic acid MRP Multidrug resistance- associated protein MS Mass spectrometry MTT 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (or methyl tetrazolium) NADPH Nicotinamide adenine dinucleotide phosphate NAT N-acetyltransferase NHEK Normal human epidermal keratinocytes NMR Nuclear magnetic resonance Nrf2 Nuclear factor erythroid 2-related factor 2 OECD Organisation for Economic Co-operation and Development PA Phenylacetaldehyde PAPS 3'-Phosphoadenosine-5'-phosphosulfate PBS Phosphate buffer saline RHE Reconstructed Human Epidermis ROS Reactive oxygen species RT Retention time SAM S-adenosylmethionine SNAr Aromatic nucleophilic substitution reaction ii TMT Thiol methyltransferase UDPGA Uridine diphosphoglucoronic acid UGT UDP-glucoronosyl transferase UPLC Ultra performance liquid chromatography UV Ultra Violet iii Publications Published papers Sandrine Jacquoilleot, David Sheffield, Adedamola Olayanju, Rowena Sison-Young, Neil R. Kitteringham, Dean J. Naisbitt, Maja Aleksic. Glutathione metabolism in the HaCaT cell line as a model for the detoxification of the model sensitisers 2,4-dinitrohalobenzenes in human skin. Toxicology Letters. 237(1): p. 11-20. 2015 Sandrine Spriggs, David Sheffield, Adedamola Olayanju, Neil R Kitteringham, Dean J Naisbitt, Maja Aleksic. Effect of repeated daily dosing with 2,4-dinitrochlorobenzene on glutathione biosynthesis and Nrf2 activation in Reconstructed Human Epidermis. Toxicological Sciences. 154 (1): p. 5-15. 2016 Manuscripts in preparation Sandrine Spriggs, Maja Aleksic, Richard Cubberley, Paul Loadman, Dean J Naisbitt and Antonia Wierzbicki. Glutathione clearance of DEM, DNCB, and DPCP in human skin models, a case study. This draft publication will use the results from chapter 4 and 5 and include some results obtained during a collaboration with Bradford University. Richard Cubberley, Sandrine Spriggs, Paul Loadman, David Sheffield and Antonia Wierzbicki. A study of inter-individual variability in the Phase II metabolism of xenobiotics in human skin. This draft publication will use the results a collaboration with Bradford University, where significant input and advice was given for GSH metabolism in skin and an active participation to writing of the manuscript was made. Abstracts Sandrine Jacquoilleot, Maja Aleksic, Richard Cubberley, Steve Gutsell, Neil R. Kitteringham, Dean J. Naisbitt. The role of Glutathione as a clearance system in skin. Oral presentation, 2nd Skin Metabolism Meeting- Skin Forum in Nice, France (October, 2013). Sandrine Jacquoilleot, Maja Aleksic, Dean J. Naisbitt. Understanding the role of skin metabolism in Allergic Contact Dermatitis: Glutathione as a clearance system. Oral iv presentation, 25th Meeting of the European Research Group on Experimental Contact Dermatitis (ERGECD 2014) in Paris, France (June, 2014). v Abstract Allergic Contact Dermatitis is a skin condition that affects up to twenty percent of the North American and Western European population. The molecular initiating event (MIE) of this type IV delayed hypersensitivity is the formation of antigenic species by covalent modification of endogenous proteins. Cysteine residues, though not the most prevalent residues in skin proteins, are prone to react with small electrophilic molecules and thus play a crucial role in protein haptenation. The role of cysteine is also very prominent in removal of electrophiles. Glutathione (GSH) is the most prominent antioxidant in cells and contains a cysteine residue, providing a free reactive thiol for GSH conjugation of electrophiles and their subsequent removal. It is also the co-factor of an important set of enzymes involved in the metabolic clearance system, glutathione S-transferases (GST). However, it remains unclear whether the reactivity of small exogenous electrophiles with intracellular GSH could be a key factor in determining the epidermal bioavailability of sensitising chemicals. GST activity has been demonstrated both in skin and in most in vitro skin equivalents but so far studies
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