Characterisation of Bilirubin Metabolic Pathway in Hepatic Mitochondria Siti Nur Fadzilah Muhsain M.Sc. (Medical Research) 2005 Universiti Sains Malaysia Postgrad. Dip. (Toxicology) 2003 University of Surrey B.Sc.(Biomed. Sc.) 2000 Universiti Putra Malaysia A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 School of Medicine ABSTRACT Bilirubin (BR), a toxic waste product of degraded haem, is a potent antioxidant at physiological concentrations. To achieve the maximum benefit of BR, its intracellular level needs to be carefully regulated. A system comprising of two enzymes, haem oxygenase-1 (HMOX1) and cytochrome P450 2A5 (CYP2A5) exists in the endoplasmic reticulum (ER), responsible for regulating BR homeostasis. This system is induced in response to oxidative stress. In this thesis, oxidative stress caused accumulation of these enzymes in mitochondria — major producers and targets of reactive oxygen species (ROS) — is demonstrated. To understand the significance of this intracellular targeting, properties of microsomal and mitochondrial BR metabolising enzymes were compared and the capacity of mitochondrial CYP2A5 to oxidise BR in response to oxidative stress is reported. Microsomes and mitochondrial fractions were isolated from liver homogenates of DBA/2J mice, administered with sub-toxic dose of pyrazole, an oxidant stressor. The purity of extracted organelles was determined by analysing the expressions and activities of their respective marker enzymes. HMOX1 and CYP2A5 were significantly increased in microsomes and even more so in mitochondria in response to pyrazole-induced oxidative stress. By contrast, the treatment did not increase either microsomes or mitochondrial Uridine-diphosphate-glucuronosyltransferase 1A1 (UGT1A1), the sole enzyme that catalyses BR elimination through glucuronidation. In response to pyrazole-induced oxidative stress, BR oxidation was enhanced not only in microsomes but also in mitochondria. The reaction in both control and pyrazole treated microsomes were blocked by up to 70% with anti-CYP2A5 antibody. By contrast, no CYP2A5 antibody inhibition on BR oxidation was observed in control mitochondria but was 55% inhibited in treated mitochondria. Antibody against adrenodoxin reductase (AdxR) (a component of mitochondrial monooxygenase system) had no effect on the microsomal BR oxidation, but the reaction was inhibited by up to 50% in mitochondria i after pyrazole treatment. As in microsomes, the antibody had no effect in control mitochondria. Inhibition study with ascorbic acid — a scavenger of ROS — was undertaken to determine the contribution of ROS to BR oxidation. In control microsomes, a slight inhibition of 5% in BR degradation was noted which was augmented to 22% after pyrazole treatment. The inhibition was 100% in control mitochondria but limited to 50% after pyrazole treatment. Mass spectrometry analysis reveals that in microsomes, biliverdin (BV), the main metabolite of CYP2A5-dependant BR oxidation was formed. Meanwhile, mitochondria produced predominantly dipyrroles, the products of BR scavenging ROS. Pyrazole treatment caused mitochondria to yield relatively similar amount of BV and dipyrroles. Enzyme kinetic analysis showed that mitochondrial and microsomal CYP2A5 have an equally strong affinity towards BR. Additionally, no initiation of apoptosis, as indicated by the absence of cytosolic cytochrome C was observed in treated liver. Collectively, these observations suggest that: i) both HMOX1 and CYP2A5 are recruited into mitochondria to regulate BR metabolism during oxidative stress; (ii) CYP2A5- dependent BR oxidation in mitochondria is driven by the mitochondria-specific electron transfer chain; (iii) constitutive mitochondrial BR oxidation is driven un-enzymatically yet equally driven by ROS and CYP2A5 in response to oxidative stress; (iv) BR affinity to mitochondrial CYP2A5 is in the same range to that of microsomal CYP2A5; (v) mitochondrial BR is not eliminated by glucuronidation but recycled by CYP2A5 oxidation to BV; and (vi) mitochondrial targeting of enzymes crucial for BR homeostasis is not associated with initiation of mitochondria specific apoptosis. It is thus submitted that targeting of key BR regulatory enzymes to mitochondria is an adaptive response to oxidative stress, aimed at mitochondrial protection against oxidative damage. ii DECLARATION BY AUTHOR This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iii PUBLICATIONS DURING CANDIDATURE Peer-Reviewed Journal Article in Submission Muhsain, S.N.F., Lang, M.A., and Abu-Bakar, A. (2014). Mitochondrial targeting of bilirubin regulatory enzymes: An adaptive response to oxidative stress. Toxicology and Applied Pharmacology (under review), Appendix 8. Peer-Reviewed Conference Proceedings Muhsain, S.N.F., Lang, M.A., and Abu-Bakar, A. (2013). Bilirubin regulation in hepatic mitochondria. World Academy of Science, Engineering and Technology 74, 545. Conference Presentations Muhsain, S.N.F., Lang, M.A., Abu-Bakar, A. Bilirubin regulation in hepatic mitochondria. International Conference on Pharmacology and Pharmaceutical Sciences, 14th- 15th Feb 2013. Kuala Lumpur, Malaysia. Muhsain, S.N.F., Lang, M.A., Abu-Bakar, A. Cytochrome P450 2A5 a mitochondrial bilirubin oxidase? Annual Scientific Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (ASCEPT), 1st- 4th Dec 2013. Melbourne, Australia. iv PUBLICATIONS INCLUDED IN THIS THESIS Chapter 5 Muhsain, S.N.F., Lang, M.A., and Abu-Bakar, A. (2013) Mitochondrial targeting of bilirubin regulatory enzymes: An adaptive response to oxidative stressToxicology and Applied Pharmacology (under review as of 22/09/2014) Contributor Statement of contribution Designed experiments (20%) Muhsain, S.N.F., (Candidate) Conducted experiments (100%) Data analysis (60%) Wrote the paper (50%) Statistical analysis (100%) Project conceptualisation (20%) Lang, M.A. Designed experiments (40%) Wrote and edited paper (25%) Project conceptualisation (80%) Abu-Bakar, A. Designed experiments (40%) Data analysis and interpretation (40%) Wrote and edited paper (25%) v CONTRIBUTIONS BY OTHERS TO THE THESIS Significant contributions to this project were made by the following: Dr A’edah Abu Bakar and Professor Matti Lang – in the conceptualisation and design of the project based on their research works, as well as critically revising the thesis so as to contribute to the interpretation. Dr Wasantha Wickramasinghe – in the assistance of non-routine technical work. Mr Geoff Eaglesham – in the analysis of HPLC/MS-MS work. STATEMENT OF PARTS OF THE THESIS SUBMITTED TO QUALIFY FOR THE AWARD OF ANOTHER DEGREE None vi ACKNOWLEDGEMENTS First and foremost, thanks to God Almighty who has given me the strength and will to complete this thesis. I would also wish to express my gratitude to the many individuals who have supported me throughout this study. I am greatly indebted to my principal supervisor, Dr A’edah Abu Bakar, for her guidance, support and attention during the course of my PhD candidature. I truly appreciate the invaluable time, effort and patience that she has spent in supervising me. I would also like to thank my associate supervisor, Professor Matti Lang, who provided great ideas, assistance and encouragement. A special thanks to Universiti Teknologi Mara (UiTM), Malaysia for their financial assistance, without which I would not have been able to pursue my study. My gratitude also goes to National Research Centre for Environmental Toxicology (Entox) as well as Faculty of Health Sciences, University of Queensland, Australia for granting a scholarship during the final semester of my study. I am also indebted to Dr Wasantha Wickramasinghe (Entox, Coopers Plains Australia) for his advice and assistance in the laboratory. Thanks is also due to Mr Geoff Eaglesham (Entox, Coopers Plains Australia), without whose help in HPLC/MS-MS and Q-tof analysis, this thesis would not be complete. My sincere appreciation is extended to my many colleagues and friends, especially Nur Syafawati ,Amalina and Jarina for their endless support and