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Physical and chemical interactions between bile pigments and polyaromatic mutagens Hung Trieu Hong BSc. and Master in Organic Chemistry A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 School of Chemistry and Molecular Biosciences Abstract A major cause of cancer in humans is exposure to mutagenic compounds. This raises the question of how humans can be protected from these environmental mutagens. Bile pigments (BPs) such as biliverdin, unconjugated bilirubin and protoporphyrin and their derivatives have recently been found to act as antioxidants and inhibit the mutagenic effects of several known environmental mutagens including 2-aminofluorene, benzo[α]pyrene, and 2-amino-1-methyl-6-phenylimido[4,5- b]pyridine.

Despite these promising results, very little is known about the mechanisms by which this inhibition is achieved. Understanding these mechanisms would be useful for future drug development. Therefore, this PhD thesis aims to explore physical and chemical interactions between BPs and mutagens. Effects of BPs on the bioavailability and metabolism of mutagens were also examined in vitro using the colorectal adenocarcinoma (Caco-2 cell) monolayer model and the human liver S9 fraction.

The physical interactions between mutagens and BPs were examined using three different methods: NMR, UV and effects of bioavailability. The results of the comparison of the NMR spectra of mutagens in the absence and presence of BPs showed very little changes in the chemical shifts of the protons and the changes that did occur were the result of acid/base interactions between the BPs and mutagens. The UV spectrum of each mutagen was measured in the presence and absence of varying concentrations of BPs, and there were no changes to the UV spectra of any of the compounds. Strong physical interactions or aggregation of compounds can also affect their absorption across cell monolayers and so the apparent permeability of mutagens across Caco-2 cell monolayers in the presence and absence of BPs were measured.

The results indicated that BPs increased the permeability of the mutagens slightly and effected how much of the compounds remained in tight association with the monolayer but the effects were small. These experiments provided evidence to suggest that physical interactions and aggregations are unlikely to be a major contributing mechanism of the inhibitory effects of BPs on environmental mutagens. Chemical reactions between BPs and the DNA modifying metabolites of mutagens (epoxides) were studied using styrene epoxide as a model for the reactive metabolites. Styrene epoxide is commercially available, stable and less toxic than the reactive metabolites of the mutagens.

Competitive reactions were performed in which BPs and their derivatives were placed in solution with guanine and allowed to react with styrene epoxide. These reactions showed that BPs and their dimethyl esters are more reactive to the epoxide than guanine. Bile pigments primarily react through their carboxylic acid groups with the mono- and di-styrene epoxide esters being the major products isolated form the reactions. The pyrrole rings in bilirubin also showed some evidence of ii reaction with styrene epoxide though the products were too unstable to isolate.

Thus, it was clear that BPs can effectively scavenge reactive metabolites, but the free carboxylic acids were significantly more effective at this than the dimethyl ester derivatives. This is not reflected in the anti-mutagenic activities of the compounds. Also, the ubiquitous nature of carboxylic acid groups in the cellular environment makes it unlikely that this reaction with activated epoxides would be unique to BPs. For these reasons we concluded that it is unlikely that chemical scavenging of reactive metabolites is the sole or even major mechanism of the inhibition of BPs.

Another possible mechanism of action of the BPs is that they inhibit the formation of the DNA modifying metabolites of the mutagens. We investigated this by performing in vitro experiments in which mutagens were co-incubated in the human liver S9 fraction in the presence and absence of BPs. The results indicated BPs were inhibitors of the metabolism of benzo[a]pyrene and 2-amino-1- methyl-6-phenylimido[4,5-b]pyridine by liver enzymes. The order of inhibitory effectiveness was bilirubin > protoporphyrin > biliverdin.

Molecular modelling studies which examined the docking of the various BPs into the active sites of published crystal structures of the enzymes known to be responsible for the metabolism of the mutagens, suggested BPs could bind to the active sites of CYP1A1, 1A2, 1B1 and 3A4. In summary, we conducted a series of experiments to evaluate the likely mechanisms of the inhibitory effects of BPs on known environmental mutagens. There are several theories postulated to explain the anti-mutagenic effects of BPs including the physical -stacking driven aggregation of BPs with the polyaromatic mutagens, the chemical scavenging of BPs towards reactive metabolites, and the inhibition of BPs of the P450 mediated activation of the mutagens. We have systematically tested each of these and found that the latter appears to be the most likely mechanism to explain the effects reported.

In broader terms, this research will aid in understanding how BPs inhibit mutagenesis and thus may lead to the development of synthetic compounds that could decrease the risk to humans exposed to these environmental mutagens. iii 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 policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. 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. iv Publications during candidature Peer Reviewed Papers: Mölzer, C., Bilirubin and related tetrapyrroles inhibit food-borne mutagenesis: A mechanism for antigenotoxic action against a model epoxide. Manuscripts to be submitted to the journal Hong H., Abu-Bakar, A'edah., De Voss, James J. Exploring the inhibitory effects of endogenous bile pigments on 2-Amino-1-methyl-6- phenylimidazo[4,5-b]pyridine and Benzo[α]Pyrene metabolic activation by cytochrome P450 enzymes., De Voss, James J.

investigation into the physical interactions between 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine and bile pigments., De Voss, James J. Preliminary examinations into the mechanism of reactions that may contribute to the inhibition of mutagenic epoxide agents by natural bile pigments. Conference abstracts, poster and oral presentations: Hung, H., The evaluation of physical and chemical interactions between bile pigments and polyaromatic mutagens, 8th Annual Research Students Symposium School of Chemistry and Molecular Biosciences, The University of Queensland, Australia, November 2012. Abstract and poster presentation., Mechanistic evaluation of chemical interactions between dipyrroles, tetrapyrroles and polyaromatic mutagens.

9th Annual Research Students Symposium School of Chemistry and Molecular Biosciences, The University of Queensland, Australia, November 2013. Abstract and poster presentation., Systhesis dipyrroles and the evaluation chemical interaction mechanisms between dipyrroles, tetrapyrroles and v polyaromatic mutagens, BBOCS 2013 - Brisbane Biological & Organic Chemistry Symposium, December 2013. Abstract and poster presentation., Exploring the mechanism of tetrapyrroles’ inhibition of environment mutagens, Heterocyclic and synthetic conference, University of Florida, ARKAT USA, March 2014. Abstract and poster presentation.

Publications included in this thesis Mölzer, C., Bilirubin and related tetrapyrroles inhibit food-borne mutagenesis: A mechanism for antigenotoxic action against a model epoxide. Included as Appendix-C. Chapter IV includes some of the work described in this publication. Contributor Statement of contribution Hung H.

Designed experiments (50%) Wrote and edited paper (50%) Edited the paper (40%) Huber, H. Supervision and discussions (5%) Edited the paper (2%) Steyrer, A. Supervision and discussions (5%) Edited the paper (2%) Ziesel, G. Supervision and discussions (5%) Edited the paper (2%) Wallner, M.

Supervision and discussions (5%) Edited the paper (2%) Blanchfield, J. Supervision and discussions (50%) vi Edited the paper (12%) Bulmer, A. Supervision and discussions (10%) Edited the paper (10%) Wagner, K. Supervision and discussions (20%) Edited the paper (10%) vii Contributions by others to the thesis A/Prof.

Joanne Blanchfield: Conception and design of the project, supervision, discussion, assistance with interpretation of data, and thesis editing; Prof. James De Voss: Conception and design of the project, supervision, discussion, assistance with interpretation of data, and thesis editing; Dr. Andrew Bulmer: Conception and design of chapter VI and chapter II, supervision, discussion, assistance with interpretation of data, and thesis editing; Dr. Abu-Bakar, A'edah: Conception and design of chapter VI, supervision, discussion, assistance with interpretation of data, and thesis editing.

Statement of parts of the thesis submitted to qualify for the award of another degree None viii Acknowledgements Firstly, I would like to thank my principal supervisor, Associate Professor Joanne Blanchfield who allowed me the opportunity to study in this interesting and challenging project. I am extremely grateful to you for your support in all aspects of my academic development. I especially appreciate your valuable criticism and expert guidance during my research candidature and assistance in editing this thesis. I also wish to thank my co-advisor, Professor James De Voss who always provides useful advice and constructive criticism.

James provided me with advanced facilities and also helped me to correct data analysis in my reports. I sincerely wish to thank Dr Andrew Bulmer from the School of Medical Science, Griffith University and Dr Abu-Bakar A'edah from the National Centre for Environmental Toxicology for allowing me the opportunity to work in your laboratories. I am extremely grateful to you for your invaluable advice, editing my thesis, and the provision of technical support during my time working on the human liver S9 project. I am also grateful to Professor Mary Garson for her constructive criticism, knowledgeable feedback and helping me to develop critical thinking skills.

Thank you to past and present laboratory members and research groups, Joanne Blanchfield and James De Voss who provided much assistance during my working time in the laboratory. I would also like to thank Dr Tri Le and Mr Graham McFarlane for their NMR and MS expertise. Thank you to the general staff within SCMB and International Student Services who provided me with excellent service. Very special thanks to my dear friends who have remained by my side during my study time at The University of Queensland.

Finally, warmest thanks and appreciation to my family, my college, the School of Chemistry and Molecular Biosciences and The University of Queensland for providing finance and supporting my spirit during my studies. I specially thank my wife, Anh, my dear little sons who have brought me great inspiration and motivation to complete my difficult project. I wish to express my deep gratitude to my mum and dad for unlimited encouragement and support for my studies at all levels. Hung Trieu Hong July, 2015 ix Keywords Anti-mutagens, bile pigments, unconjugated bilirubin, styrene epoxide, caco-2 cell monolayer, chemical interactions, physical interactions, metabolism, inhibition.

Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 030503, Organic Chemical Synthesis, 40%; ANZSRC code: 030499, Medicinal and Biomolecular Chemistry not elsewhere classified, 50%; ANZSRC code: 060199, Biochemistry and Cell Biology not elsewhere classified, 10% Fields of Research (FoR) Classification FoR code: 0305, Organic Chemical, 40%; FoR code: 0304, Medicinal and Biomolecular Chemistry, 50%; FoR code: 0601, Biochemistry and Cell Biology, 20% x Table of Contents Abstract .ii Declaration by author .