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Dfp) Exposure in Fischer 344 Rat Brain

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Dfp) Exposure in Fischer 344 Rat Brain ‘OMIC’ EVALUATION OF THE REGION SPECIFIC CHANGES INDUCED BY NON-CHOLINERGIC DIISOPROPYLFLUOROPHOSPHATE (DFP) EXPOSURE IN FISCHER 344 RAT BRAIN A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Deirdre A. Mahle B.S., University of Dayton, 1987 _____________________________________ 2012 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL July 5, 2012 I HEREBY RECOMMEND THAT THE DISSERTATION PREPARED UNDER MY SUPERVISION BY Deirdre A. Mahle ENTITLED ‘Omic’ Evaluation of the Region Specific Changes Induced by Non-Cholinergic Diisopropylfluorophosphate (DFP) Exposure in Fischer 344 Rat Brain BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy. ______________________________ Nicholas V. Reo, Ph.D. Dissertation Director ______________________________ Gerald Alter, Ph.D. Director, Biomedical Sciences Ph.D. Program ______________________________ Andrew Hsu, Ph.D. Dean, Graduate School Committee on Final Examination ______________________________ Nicholas V. Reo, Ph.D. ______________________________ Adrian Corbett, Ph.D. ______________________________ James McDougal, Ph.D. ______________________________ James Olson, Ph.D. ______________________________ Michael Raymer, Ph.D. ABSTRACT Mahle, Deirdre A., PhD; Biomedical Sciences PhD program, Department of Biochemistry and Molecular Biology, Wright State University, 2012. ‘Omic’ Evaluation of the Region Specific Changes Induced by Non-Cholinergic Diisopropylfluorophosphate (DFP) Exposure in Fischer 344 Rat Brain Organophosphorous compounds (OPs) are a class of serine esterase inhibitors that have widespread application as pesticides, veterinary pharmaceuticals and chemical warfare agents. Environmental contamination is ubiquitous. The threat of exposure is a concern for both military and civilian populations. Acute inhibition of acetylcholinesterase by OPs triggers a cholinergic crisis that results in muscle flaccidity, paralysis, convulsions and death. At low doses OPs can alter neuronal differentiation, cell signaling, behavior and cognition through unknown mechanisms. An imbalance of reactive oxygen species may be implicated in the adverse effects of OPs. An integrated approach using both metabolomic and transcriptomic techniques was used to reveal some of the non-cholinergic effects of diisopropylfluorophosphate (DFP), a model OP, in rat brain. Adult male Fischer 344 rats were administered 1 mg/kg DFP or saline via subcutaneous injection at 10 mL/kg. Cortex, brainstem, cerebellum and hippocampus were collected at multiple time points ranging from 0.5 - 48 hr. Total RNA was isolated from each region for differential gene expression analysis using the Affymetrix 1.0 ST gene array at 1 hr post dose. Lipid and aqueous extracts were prepared from each brain region at 2 hr post dose, and profiles of small molecule metabolites, lipids and phospholipids were measured using multinuclear NMR spectroscopy. Because the dose was below the threshold for cholinergic iii toxicity, it was hypothesized that DFP exposure would up-regulate inflammatory pathways and down-regulate processes that result in cellular degradation, such as apoptosis, and that these changes would correlate with perturbations in the small molecule and lipid profiles as well as gene expression. All brain regions reached minimum acetylcholinesterase activity (40-55%) at 1- 2 hr post dose with the exception of cortex, which had minimum activity at 12 hr post dose. No brain region showed significant increases in lipid peroxidation. After 1 hr, pathways associated with prostaglandin D2 synthesis were up-regulated in cortex. Brainstem showed increased expression of genes associated with an inflammatory response and ascorbate transport. Cortex showed the most changes in the lipid profile with significant decreases in phosphatidylcholine, phosphatidylethanolamine, cholesterol, n3 and n6 fatty acids. The mitochondrial phospholipid cardiolipin was significantly decreased after 2 hr in brainstem. By evaluating the impact of low level OP exposure on the neuronal phenotype of specific brain regions, we hope to gain a greater understanding of the non-cholinergic mechanisms of action and sensitive target areas in order to improve the development of therapeutic targets for individuals exposed to OPs. iv TABLE OF CONTENTS I. INTRODUCTION ............................................................................................................. 1 1.1 Background .................................................................................................................. 1 1.2 Transcriptomics and metabolomics ............................................................................. 3 1.3 Cholinergic toxicity of OP compounds ........................................................................ 6 1.4 Non cholinergic toxicity of OP compounds ............................................................... 13 1.5 Altered phenotypes after OP exposure....................................................................... 15 1.6 OPs and oxidative stress ............................................................................................ 16 1.7 OPs and inflammation................................................................................................ 19 1.8 Diisopropylfluorophosphate ...................................................................................... 19 1.9 Selection of brain regions .......................................................................................... 21 1.10 Thesis focus and significance .................................................................................. 22 II. MATERIALS AND METHODS ..................................................................................... 23 2.1 Animals and treatments.............................................................................................. 23 2.2 Biochemical assays .................................................................................................... 26 2.3 RNA preparation ........................................................................................................ 28 2.3.1 RNA isolation ................................................................................................. 28 2.3.2 1st strand cDNA synthesis ............................................................................... 30 2.3.3 2nd strand cDNA synthesis .............................................................................. 32 2.3.4 Synthesis of cRNA using in vitro transcription (IVT) .................................... 32 2.3.5 Purification of cRNA ...................................................................................... 32 2.3.6 2nd cycle cDNA synthesis ................................................................................ 33 v 2.3.7 Fragmentation, labeling and hybridization of single stranded cDNA ............. 34 2.3.8 Data processing procedures............................................................................. 35 2.4 GeneChip analysis ..................................................................................................... 36 2.5 NMR sample preparation ........................................................................................... 38 2.6 NMR analysis ............................................................................................................ 40 2.7 NMR data analysis and quantification of specific metabolites .................................. 40 III. RESULTS ......................................................................................................................... 43 3.1 Basal regional differences .......................................................................................... 43 3.1.1 Basal AChE activity ........................................................................................ 43 3.1.2 Basal lipid profiles .......................................................................................... 44 3.1.3 Aqueous metabolite profiles ........................................................................... 47 3.2 Cortex......................................................................................................................... 48 3.2.1 Biochemical assays ......................................................................................... 48 3.2.2 Differential gene expression in cortex ............................................................ 50 3.2.3 Metabolic changes in cortex............................................................................ 53 3.3 Brainstem ................................................................................................................... 56 3.3.1 Biochemical assays ......................................................................................... 56 3.3.2 Differential gene expression in brainstem ....................................................... 57 3.3.3 Metabolic changes in brainstem ...................................................................... 59 3.4 Cerebellum ................................................................................................................. 62 3.4.1 Biochemical assays ......................................................................................... 62 3.4.2 Differential gene expression in cerebellum ..................................................... 64 3.4.3 Metabolic changes in cerebellum ...................................................................
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