Untargeted metabolomics to study ethanol-induced hepatotoxicity in HepaRG cells
Despite the high prevalence of alcoholic liver disease, its identification and characterization remain poor, especially in early stages such as alcoholic fatty liver disease and alcoholic steatohepatitis. Consequently, this implies diagnostic difficulties, limited therapeutic options and unclear mechanisms of action. Untargeted metabolomics concerns the holistic investigation of endogenous metabolites, low molecular weight (< 1500 Da) biomolecules, that provide information on the biochemical status of biological samples. As the metabolome is located at the most downstream level of cellular organization, metabolomics was used in this thesis in an in vitro set-up to identify biochemical biomarkers able to elucidate the mechanism of ethanol-induced hepatotoxicity. To enable untargeted metabolomics research, analytical platforms were developed using liquid chromatography-quadrupole-time-of-flight high-resolution mass spectrometry (LC-QTOF-HRMS) and coupling to drift tube ion mobility spectrometry was explored. A data analysis workflow was generated, and quality control measures were implemented through the metabolomics workflow. Excessive ethanol consumption disrupts lipid metabolism and initiates progressive intracellular lipid accumulation, resulting in alcoholic fatty liver disease. HepaRG cells (i.e., a human hepatic cell line) were exposed to ethanol at varying concentrations and durations to mimic this latter phenotype. Distinctive patterns between exposed and control cells were consistently observed, with intracellular upregulation of di- and triglycerides, downregulation of phosphatidylcholines and -ethanolamines, sphingomyelins, and S-adenosylmethionine, among others. Several intracellular metabolic patterns could be related to changes in the extracellular environment, such as increased intracellular hydrolysis of sphingomyelins, leading to increased phosphorylcholine secretion. Carnitine alterations highlighted the interplay between β-oxidation in mitochondria and peroxisomes. The combination of the metabolic fingerprints and footprints enabled a comprehensive investigation of the pathophysiology behind ethanol-induced hepatotoxicity. To improve in vitro simulation of alcoholic steatohepatitis, HepaRG cells were further exposed to a combination of ethanol and tumor necrosis factor alpha (TNF-α), an inflammation inducer. Co-exposure to TNF-α highlighted its importance in the upregulation of hepatic triglycerides and the downregulation of hepatic phosphatidylcholines and -ethanolamines. In addition, fatty acyl esters of hydroxy fatty acid-containing triglycerides were detected for the first time in human hepatocytes and their alterations showed a potentially important role during the progression of alcoholic steatohepatitis. Ethylated phosphorylcholine was observed as a possible new biomarker of ethanol exposure. Overall, subjecting the HepaRG liver cell line to metabolomics analyses proved to be a valuable tool to obtain mechanistic insights in ethanol-induced hepatotoxicity. Usage of this tool facilitates future in vivo research by pinpointing interesting biomarkers and metabolic pathways.
Antwerp : University of Antwerp & Vrije Universiteit Brussel , 2023
351 p.
Supervisor: Covaci, Adrian [Supervisor]
Supervisor: van Nuijs, Alexander L.N. [Supervisor]
Supervisor: Vanhaecke, Tamara [Supervisor]
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Creation 19.09.2023
Last edited 11.10.2023
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