HEPATOCYTE MOLECULAR CYTOTOXIC MECHANISM STUDY OF FRUCTOSE AND ITS METABOLITES INVOLVED IN NONALCOHOLIC STEATOHEPATITIS AND HYPEROXALURIA By Yan (Cynthia) Feng A thesis submitted in the conformity with the requirements for the degree of Master of Science Graduate Department of Pharmaceutical Sciences University of Toronto © Copyright by Yan (Cynthia) Feng 2010 ABSTRACT HEPATOCYTE MOLECULAR CYTOTOXIC MECHANISM STUDY OF FRUCTOSE AND ITS METABOLITES INVOLVED IN NONALCOHOLIC STEATOHEPATITIS AND HYPEROXALURIA Yan (Cynthia) Feng Master of Science, 2010 Department of Pharmaceutical Sciences University of Toronto High chronic fructose consumption is linked to a nonalcoholic steatohepatitis (NASH) type of hepatotoxicity. Oxalate is the major endpoint of fructose metabolism, which accumulates in the kidney causing renal stone disease. Both diseases are life-threatening if not treated. Our objective was to study the molecular cytotoxicity mechanisms of fructose and some of its metabolites in the liver. Fructose metabolites were incubated with primary rat hepatocytes, but cytotoxicity only occurred if the hepatocytes were exposed to non-toxic amounts of hydrogen peroxide such as those released by activated immune cells. Glyoxal was most likely the endogenous toxin responsible for fructose induced toxicity formed via autoxidation of the fructose metabolite glycolaldehyde catalyzed by superoxide radicals, or oxidation by Fenton’s hydroxyl radicals. As for hyperoxaluria, glyoxylate was more cytotoxic than oxalate presumably because of the formation of condensation product oxalomalate causing mitochondrial toxicity and oxidative stress. Oxalate toxicity likely involved pro-oxidant iron complex formation. ii ACKNOWLEDGEMENTS I would like to dedicate this thesis to my family. To my parents, thank you for the sacrifices you have made for me, thank you for always being there, loving me and supporting me throughout my life. To my uncle, thank you for providing me with many professional advices. And to Brandon, thank you for always believing in me, even in the times when I stopped believing in myself. I am sincerely grateful to my supervisor, Dr. Peter J. O’Brien. Thank you for your constant guidance and support throughout my Master’s study at University of Toronto. Your enthusiasm and positive attitude for research have inspired me to overcome every challenge that I encountered during my study. Your insights and wisdom have always been helpful and led me to find the right path in my research. I would like to give thanks to the past and current students in the lab. Thank you Rhea, Qiang and Monica, for your friendship and company, and thank you for sharing your valuable knowledge and experiences with me. The current students, Luke, Kai, Stephanie and Sarah, thank you for all your helpful discussion and encouragement. I also want to thank the past members of the lab, for training me and helping me when I first started. All of you had made my experience extremely warm and memorable. I would like to express my appreciation to my advisory committee members, Dr. Bruce and Dr. Pennefather, for their guidance and support. Finally I would like to acknowledge Dr. Uetrecht and his lab for letting me use their plate reader. iii TABLE OF CONTENTS Abstract ii Acknowledgements iii Table of contents iv List of publications and abstracts v List of tables vi List of figures vii List of abbreviations viii-x Chapter 1. General Introduction 1 Chapter 2. Materials and Methods 21 Chapter 3. Hepatocyte Inflammation Model for Cytotoxicity Research: 26 Fructose or Glycolaldehyde as a Source of Endogenous Toxins. Chapter 4. Hepatocyte Study of the Molecular Cytotoxic Mechanisms of 40 Endogenous Toxins Formed by Hyperoxaluria Chapter 5. Conclusions and Future Perspectives 67 References. 76 Appendix I. Permissions 87 iv LIST OF PUBLICATION AND ABSTRACTS Publications: Chan, K., Lehmler, H.J., Sivagnanam, M., Feng, C.Y., Robertson, L., O’Brien, P.J. (2009) Cytotoxic effects of polychlorinated biphenyl hydroquinone metabolites in rat hepatocytes. J Appl Toxicol. In press. O’Brien, P.J., Feng, C.Y., Lee, O., Dong, Q., Mehta, R., Bruce, J., and Bruce, W.R. (2009). Fructose-derived endogenous toxins. Editors, O’Brien, P.J. and Bruce, W.R. Endogenous Toxins: Targets for Disease Treatment and Prevention. (pp173-204). WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Feng, C.Y., Wong, S., Dong, Q., Bruce, J., Mehta, R., Bruce, W.R., and O’Brien, P.J. (2009). Hepatocyte inflammation model for cytotoxicity research: fructose or glycolaldehyde as a source of endogenous toxins. Arch. Physiol Biochem. 115(2), 105-111 Abstracts: Feng, C.Y. and O’Brien, P.J. (2009) Rat hepatocyte molecular cytotoxic mechanism study of toxins formed by primary hyperoxaluria. Abstract presented at the National Health Research Day, University of Toronto, Toronto, Ontario. Nov 17, 2009. O’Brien, P.J., Feng, C.Y., and Tafazoli, S. (2009). Accelerated cytotoxic mechanism screening (ACMS) for idiosyncratic drug hepatotoxin. Abstract presented at 12th Canadian Society for Pharmaceutical Sciences Annual Symposium, Toronto, Ontario. June 3-6, 2009. Feng, C.Y. and O’Brien, P.J. (2009). Rat hepatocyte cytotoxicity model for primary hyperoxalurias. Abstract presented at the 6th Meeting of the Canadian Oxidative Stress Consortium, Winnipeg, Manitoba. May 7-10, 2009. (Runner-up for poster award) v LIST OF TABLES Table 1.1 Availability of high fructose corn syrup (HFCS) in the caloric sweetener supply in the United States. Table 3.1 Fructose cytotoxicity is markedly enhanced by non-toxic H2O2/Fe(II) in isolated rat hepatocytes. Table 3.2 The cytotoxicity of glycolaldehyde, a fructose metabolite, is markedly enhanced by non-toxic H2O2/Fe(II) in isolated rat hepatocytes. Table 3.3 Glycolaldehyde protein carbonylation is increased by Fenton’s reagent and inhibited by a glyoxal scavenger in cell free system. Table 4.1 Concentrations of each fructose metabolites studied that induce 50% cell death at 2 hours in isolated rat hepatocytes. Table 4.2 Glyoxylate induced reactive oxygen species (ROS) formation in isolated rat hepatocytes. Table 4.3 Endogenous glyoxylate reacts with mitochondrial oxaloacetate to form oxalomalate, a citric acid cycle inhibitor. Table 4.4 Concentrations of glycolate that induce 50% cell death at 2 hours in isolated rat hepatocytes under various stress conditions. Table 4.5 Hydroxypyruvate induced ROS formation and lipid peroxidation in primary rat hepatocytes Table 4.6 Oxalate induced ROS formation and lipid peroxidation in rat hepatocytes vi LIST OF FIGURES Figure 1.1 Simplified fructose to oxalate pathway and glycolysis pathway. Figure 1.2 The relationship between high fructose corn syrup, free fructose, total fructose intake and the prevalence of overweight and obese people in the United States. Figure 1.3 Superoxide is detoxified by the enzyme superoxide dismutase, and can react with nitric oxide forming a more reactive oxidant peroxynitrite. Figure 1.4 Detoxification of hydrogen peroxide could be catalyzed by catalase or reduced glutathione (GSH) peroxidise. Figure 1.5 The Haber-Weiss reaction. Hydrogen peroxide is oxidized by transition metals by Fenton’s reaction forming highly reactive hydroxyl radicals. Figure 1.6 Oxidation of guanine by hydroxyl radical forming 8-hydroxyguanine. Figure 1.7 Schematic of advanced glycation end-product formation. Figure 1.8 Schematic of lipid peroxidation formation. Figure 1.9 Structures of some metabolites on the fructose to oxalate pathway. Figure 3.1 The proposed molecular hepatotoxic mechanisms associated with fructose induced NASH as per the two-hit hypothesis. Figure 4.1 Glyoxylate induced cytotoxicity in primary rat hepatocytes measured by the trypan blue exclusion test at 120min and 180min. Figure 4.2 Hydroxypyruvate induced cytotoxicity in primary rat hepatocytes. Figure 4.3 Oxalate induced cytotoxicity in primary rat hepatocytes. Figure 4.4 Cysteine forms an adduct with glyoxylate. Figure 4.5 Glyoxylate condenses with oxaloacetate forming oxalomalate, an inhibitor of aconitase and NADP+ dependent isocitrate dehydrogenase. Figure 4.6 A simplified fructose to oxalate metabolic pathway. vii LIST OF ABBREVIATIONS ACMS Accelerated cytotoxic mechanism screening ADH Alcohol dehydrogenase AGT Alanine glyoxylate transaminase AGE Advanced glycation end-product ALDH Aldehyde dehydrogenase ALE Advanced lipoxidation end-product ANOVA Analysis of variance ATP Adenosine-5-triphosphate BHA Butylated hydroxyl anisole BMI Body-mass index BSA Bovine serum albumin CBA 2-carboxybenzaldehyde Cl- Chloride CML Carboxymethyllysine CO2 Carbon dioxide Cu Copper Cu(II) Cupric copper DETAPAC Diethylenetriaminepentaacetic acid DCFH-DA Dichlorofluorescein diacetate DCF Dichlorofluorescein DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DNPH 2,4-dinitrophenylhydrazine EDTA Ethylenediaminetetraacetic acid F.I. Fluorescent intensity Fe Iron viii Fe(II) Ferrous iron Fe(III) Ferric iron FOX Ferrous oxidation in xylenol orange assay G/GO Glucose / glucose oxidase GI system Gastrointestinal system GLUT4 Glucose transporter 4 GLUT5 Glucose transporter 5 GR Glyoxylate reductase GSH Glutathione (reduced) GSSG Glutathione (oxidized) H2O2 Hydrogen peroxide HCl Hydrochloric acid HFCS High fructose corn syrup HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPR Hydroxypyruvate reductase iNOS Inducible nitric oxide synthase JNK c-Jun N-terminal kinase pathway Concentration of the toxin lethal to 50% of the LC50 hepatocytes LDH Lactate dehydrogenase MDA Malondialdehyde Na+ Sodium NAD+ Nicotinamide