NMR ANALYSES SHOW TCDD ELICITS DIFFERENCES IN HEPATIC METABOLISM IN FEMALE C57BL/6 MICE AND SPRAGUE-DAWLEY RATS A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By MEGHAN KATHERINE MAKLEY B.S. University of Dayton, 2004 2008 Wright State University WRIGHT STATE UNIVERSITY SCHOOL OF GRADUATE STUDIES Date of defense: 12-11-08 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Meghan Katherine Makley ENTITLED NMR Analyses Show TCDD Elicits Differences in Hepatic Metabolism in Female C57BL/6 Mice and Sprague- Dawley Rats BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. ______________________ Nicholas V. Reo, Ph.D. Thesis Director ______________________ Steven J. Berberich, Ph.D. Department Chair Committee on Final Examination ____________________ Nicholas V. Reo, Ph.D. ________________________ Darrell E. Fleischman, Ph.D. ________________________ Lawrence J. Prochaska, Ph.D. _____________________ Joseph F. Thomas, Jr., Ph.D. Dean, School of Graduate Studies ABSTRACT Makley, Meghan Katherine. M.S., Department of Biochemistry and Molecular Biology, Wright State University, 2009. NMR Analyses Show TCDD Elicits Differences in Hepatic Metabolism in Female C57BL/6 Mice and Sprague-Dawley Rats. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) elicits tissue-, sex-, and species- specific effects. This study compares the hepatic response to an oral dose of TCDD in immature ovariectomized (i.o.) C57BL/6 mice (30 µg/kg) and i.o. Sprague-Dawley rats (10 µg/kg), at 72, 120, and 168 h post-dose. Hepatic lipid extracts were analyzed by 13C and 31P NMR, and aqueous extracts by 1H and 31P NMR. Consistent with increased lipid content in mice (p≤0.05), TCDD induced increases in hepatic triacylglycerides (TAG), cholesterol, and fatty acids. Principal component analysis of 13C spectra show treatment groups separate in mice, but not rats. Mice showed decreases in the lactate/pyruvate ratio and dihydroxyacetone phosphate, consistent with decreased cytosolic NADH/NAD+ ratio and upregulated TAG synthesis. TCDD-treated rats exhibited decreased levels of sphingomyelin, and three-fold increase in phosphocholine, suggesting TCDD activates sphingomyelinase. Both species showed similar decreases in cardiolipin, indicating oxidative stress. These observations are compared with hepatic histopathology and gene expression findings. iii TABLE OF CONTENTS Page I. INTRODUCTION . 1 II. MATERIALS AND METHODS . 6 A. MATERIALS . 6 Chemicals . 6 Animals . .6 NMR Spectroscopy Analysis . 6 B. METHODS . 6 Animal Protocol . 6 Dual-Phase Extraction Procedure . 7 NMR Spectroscopy of Liver Extracts . .8 1. NMR Spectroscopy of Lipid Extracts . 9 Preparation of Cs2EDTA Salt . 9 2. NMR Spectroscopy of Aqueous Extracts. 10 Spectral Data Analysis. 10 Data Processing for PCA . 11 Quantification . .12 1H NMR Spectroscopy of Aqueous Extracts . 14 iv Statistical Analysis . 14 III. RESULTS . .16 A. LIPID EXTRACTS . .16 1. Effects of TCDD on Mean Total Hepatic Lipid Content . 16 iv Summary of Mean Total Hepatic Lipid Content . 17 2. PCA for Hepatic Lipids in Mice and Rats Analyzed by 13C NMR . 19 3. Hepatic Lipids Analyzed by 13C NMR Spectroscopy . 19 13C NMR Spectrum of Hepatic Lipids . 21 a. Effects of TCDD on Mean Hepatic TAG and Cholesterol 22 1.) Hepatic TAG . 22 2.) Hepatic Cholesterol . 22 b. Effects of TCDD on Mean Hepatic n3 and n6 FAs . 24 1.) Hepatic n3 FAs . 24 2.) Hepatic n6 FAs . 24 Summary of Lipid Quantities Analyzed by 13C NMR . 25 4. Hepatic Phospholipids Analyzed by 31P NMR Spectroscopy . 28 31P NMR Spectrum of Hepatic Phospholipids . 28 a. Effects of TCDD on Mean Hepatic Cardiolipin and PtdS .29 1.) Hepatic Cardiolipin . 29 2.) Hepatic PtdS . 30 b. Effects of TCDD on Mean Hepatic PtdC and PtdE . 31 1.) Hepatic PtdC . 31 2.) Hepatic PtdE . 32 c. Effects of TCDD on the Mean Hepatic PtdC/PtdE Ratio . 33 d. Effects of TCDD on the Mean Hepatic PtdS/PtdE Ratio. 34 e. Effects of TCDD on Mean Hepatic SphM . 35 v Summary of Phospholipid Quantities Analyzed by 31P NMR. 36 B. AQUEOUS EXTRACTS. 39 1. Effects of TCDD on Mean Total Hepatic Aqueous Content . 39 Summary of Mean Total Hepatic Content of Aqueous Extracts . 40 2. Hepatic Metabolites Analyzed by 31P NMR Spectroscopy of Aqueous Extracts . 42 31P NMR Spectrum of Hepatic Aqueous Metabolites . 42 a. Effects of TCDD on Mean Hepatic DHAP and G3P . 43 1.) Hepatic DHAP . .43 2.) Hepatic G3P . .43 b. Effects of TCDD on the Mean Hepatic G3P/DHAP Ratio44 c. Effects of TCDD on Mean Hepatic G6P . .45 d. Effects of TCDD on Mean Hepatic Pcho and Peth . .46 1.) Hepatic Pcho . 46 2.) Hepatic Peth . 47 e. Effects of TCDD on Mean Hepatic GPC and GPE . 48 1.) Hepatic GPC . 48 2.) Hepatic GPE . 48 Summary of Metabolite Quantities Analyzed by 31PNMR of Aqueous Extracts . 49 f. Effects of TCDD on the Mean Hepatic SphM/Pcho Ratio52 3. Hepatic Metabolites Analyzed by 1H NMR Spectroscopy of Aqueous Extracts . 54 a. Effects of TCDD on Mean Hepatic Lactate and Pyruvate 54 vi 1.) Hepatic Lactate . 54 2.) Hepatic Pyruvate . 55 b. Effects of TCDD on the Mean Hepatic Lactate/Pyruvate .56 Summary of Metabolite Quantities Analyzed by 1H NMR of Aqueous Extracts . 57 IV. DISCUSSION . 59 A. Increased Levels of Hepatic TAG Suggest TAG Synthesis is Upregulated in Mice . 59 TAG Synthesis in Mice – Comparison to Genomic Effects . .62 TCDD Induces FA Uptake and Metabolism . 63 B. Increased Levels of Hepatic Cholesterol Suggests Cholesterol Uptake in Mice . 64 Cholesterol Uptake in Mice – Comparison to Genomic Effects . 65 C. Activation of Sphingomyelinase in Rats . .65 Sphingomyelin Pathway – Comparison to Genomic Effects . 70 D. TCDD Induces Oxidative Stress in Both Mice and Rats . 71 Oxidative Stress – Comparison to Genomic Effects . 72 V. CONCLUSIONS . 74 VI. REFERENCES . 75 APPENDICES. 81 APPENDIX A – Abbreviations. 81 APPENDIX B – Experimental Protocol. 83 APPENDIX C – Quantification . 84 APPENDIX D – ANOVA . 86 vii LIST OF FIGURES Figure Page 1 Mean hepatic lipid content . .17 2 PCA of rats and mice combined . 19 3 13C NMR spectrum of lipids . 20 4 Example of a 13C NMR spectrum of lipids . 21 5 Hepatic TAG and cholesterol . 23 6 Hepatic n3 and n6 FAs . 25 7 31P NMR spectrum of phospholipids . 28 8 Hepatic cardiolipin and PtdS . 31 9 Hepatic PtdC and PtdE . 32 10 PtdC/PtdE ratio . 34 11 PtdS/PtdE ratio . 34 12 Hepatic SphM . 35 13 Mean hepatic aqueous content . ..