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An Investigation Into Fatty Acid Ethyl Esters in Mouse Brains After Ethanol Treatment

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An Investigation Into Fatty Acid Ethyl Esters in Mouse Brains After Ethanol Treatment An Investigation into Fatty Acid Ethyl Esters in Mouse Brains After Ethanol Treatment: Detection, Quantification, and Potential Toxicity Thesis Presented by Terence Hall to The Bouvé Graduate School of Health Sciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Pharmaceutical Sciences with Specialization in Pharmacology NORTHEASTERN UNIVERSITY BOSTON, MASSACHUSETTS July 23rd, 2017 Page 1 of 121 Page 2 of 121 Page 3 of 121 Abstract Background: Mankind’s oldest drug, ethanol (alcohol) is largely cleared by oxidation in the liver. The proximal metabolite is acetaldehyde, but this reactive and volatile chemical is swiftly further oxidized to acetate which is incorporated into normal metabolic pathways. A small fraction of the acetaldehyde is well-known to be converted into alkaloid-like chemicals whose actions may contribute to the overall effects of alcohol intoxication. Importantly, however, small amounts of non-oxidative metabolites are also formed in numerous tissues including the liver, pancreas, and brain. For example, ethyl glucuronide and ethyl sulfate formed by classical phase II drug metabolizing pathways have been used as markers of ethanol consumption. One of the most prominent non-oxidative metabolic pathways involves condensation of ethanol with endogenous fatty acids, generating fatty acid ethyl esters (FAEEs). These compounds have previously been shown to be pharmacologically active and potentially toxic using both in vitro and in vivo animal models of ethanol use – primarily in the liver and pancreas. The potential role that these non-oxidative metabolites play in the brain, a tissue isolated from systemic circulation with limited oxidative metabolism of ethanol, has not yet been thoroughly explored. The brain is a known target for ethanol and fatty acid toxicity. One of the most abundant fatty acids in the brain is arachidonic acid, and the ethyl ester of arachidonic acid is ethyl arachidonate (AAEE). In studies of post mortem brain tissue from intoxicated accident victims the most prominent FAEE found is AAEE. However, AAEE has not been documented in brains from mice after ethanol administration, so that studies with this common model species were not available to explore the likely relevance of AAEE neurotoxicity in humans. Page 4 of 121 Methods: Male Swiss Webster mice were dosed orally with ethanol, with and without oxidative metabolism inhibitors, and tissues including including the cerebellum and the rest of the brain were harvested, flash frozen, and stored at -80°. Tissue extracts were analyzed by LC-MS/MS, and concentrations of fatty acid ethyl esters were quantified. Time courses of analytes were used to determine pharmacokinetic profiles of AAEE and other FAEEs. Ethanol, arachidonic acid and AAEE were tested using proliferation and LDH release and assays in neuronal and glial cell lines. Mechanistic studies were conducted with a caspase inhibitor. Results: A novel finding of this study was the detection of AAEE was in mouse brain tissues at levels up to 70% of the total of all five FAEEs measured (ethyl esters of arachidonic, stearic, palmitic, oleic, and linoleic acid). Inhibitors of either alcohol dehydrogenase or aldehyde dehydrogenase resulted in increased concentrations of FAEE, indicating that FAEE levels are negatively correlated with oxidative metabolism of ethanol. In cell proliferation assays, glial cells were generally more sensitive to the test compounds than neuronal cell lines, and AAEE was more toxic than either AA or ethanol alone. The toxicity of AA and AAEE was mitigated by pretreatment with Z-VAD-FMK, a caspase inhibitor, which suggests an apoptotic mechanism of cell death. Conclusions: These experiments demonstrate the presence and potential toxicity of FAEE’s in the mouse brain after ethanol administration. Conversion of even small fractions of ethanol doses to some metabolites may be pharmacologically and toxicologically relevant because of the very large amounts of ethanol that are often self-administered. Our previously undocumented findings are important because they validate studies in the mouse of a potential mechanism of ethanol neurotoxicity in human alcohol dependence and alcoholism. Furthermore, the increases in non-oxidative metabolites in the presence of dehydrogenase inhibitors suggests that in Page 5 of 121 individuals with compromised hepatic function there is an increased potential for FAEE presence in the brain and resulting neurotoxicity. Page 6 of 121 Table of Contents List of Abbreviations .................................................................................................................................... 9 List of Figures ............................................................................................................................................. 10 List of Tables .............................................................................................................................................. 12 Overview ..................................................................................................................................................... 13 The History of Ethanol use ..................................................................................................................... 14 The Cost of Abuse .................................................................................................................................. 15 Ethanol Metabolism ................................................................................................................................ 17 Oxidative Metabolism ............................................................................................................................. 17 Oxidative Metabolism in the Brain ..................................................................................................... 19 Ethanol Metabolism - Non-oxidative .................................................................................................. 22 Ethanol Metabolites and Biomarkers ...................................................................................................... 25 Analytical Methods ............................................................................................................................. 26 Ethanol toxicity ....................................................................................................................................... 27 Ethanol Toxicity – in the brain ........................................................................................................... 28 Arachidonic Acid .................................................................................................................................... 28 Fatty acid toxicity ................................................................................................................................... 30 FAEE in the human tissues ..................................................................................................................... 31 Fatty Acid Ethyl Esters in in vivo models of ethanol consumption/toxicity ...................................... 32 Specific Aims .............................................................................................................................................. 34 Materials and Methods ................................................................................................................................ 38 General Laboratory Equipment and Reagents: ....................................................................................... 38 Cell Lines ................................................................................................................................................ 40 Cell Proliferation Assays ........................................................................................................................ 40 Lactate dehydrogenase (LDH) assay ...................................................................................................... 40 In vivo studies ......................................................................................................................................... 41 FAEE Extraction from Tissue ................................................................................................................. 41 Software .................................................................................................................................................. 43 Statistical analysis ................................................................................................................................... 43 PK parameters ......................................................................................................................................... 43 Results ......................................................................................................................................................... 44 Specific Aim 1: To determine if AAEE and/or other fatty acid ethyl esters are present in the brain tissue of mice dosed acutely with ethanol. ........................................................................................................ 44 Specific Aim 2: To examine the impact of repeat dosing of ethanol on AAEE levels, and on relative abundance of AAEE in relation to other detectable ethyl esters of fatty acids present in mouse brain. 48 Page 7 of 121 Specific Aim 3: FAEE Method Validation ............................................................................................
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