Abstract
A hydrophilic interaction liquid chromatography (HILIC)–ultra high-pressure liquid chromatography (UHPLC) coupled with tandem mass spectrometry (MS/MS) method was developed and applied to profile metabolite changes in human Huh-7 cells exposed to the potent aryl hydrocarbon receptor (AHR) ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Comparisons of sensitivity (limit of detection as low as 0.01 µM) and reproducibility (84% of compounds had an interday relative standard deviation (RSD) less than 10.0%; 83% of compounds had an intraday RSD less than 15.0%) were assessed for all the metabolites. The exposure of Huh-7 cells to the hepatotoxic carcinogen TCDD at low doses (1 nM and 10 nM for 4 h and 24 h, respectively) was reflected by the disturbance of amino acid metabolism, energy metabolism (glycolysis, TCA cycle), and nucleic acid metabolism. TCDD caused a significant decrease in amino acids such as serine, alanine, and proline while promoting an increase in arginine levels with 24 h treatment. Energy metabolism intermediates such as phosphoenolpyruvate and acetyl–CoA and nucleosides such as UMP, XMP, and CMP were also markedly decreased. These results support the application of HILIC–UHPLC–MS/MS for robust and reliable analysis of the cellular response to environmentally relevant toxicants at lower doses.
Highlights
Metabolomics aims to understand the metabolic dynamics in living systems and provide a readout of physiologic status [1,2]
107 polar metabolites including components of central carbon, amino acid, and nucleotide metabolism were quantitated by the developed hydrophilic interaction liquid chromatography (HILIC)–MS/MS using stable isotopes as internal standards
In addition to the conventional method validation, the HILIC–MS/MS method was validated with Huh-7 cells for the glycolytic pathway and applied on TCDD toxicology with
Summary
Metabolomics aims to understand the metabolic dynamics in living systems and provide a readout of physiologic status [1,2]. Metabolomics has impacted the fields of analytical and clinical chemistry, nutrition, drug discovery, and toxicology [3,4,5,6]. Metabolomics has been an important tool for providing a better view of toxicity mechanisms. Information obtained from metabolomic studies reflecting metabolic changes may be more relevant to classical toxicological endpoints rather than transcriptomics, as metabolic changes. Biofluids or tissues from in vivo experiments have been analyzed via metabolomics [15,16], but the development of robust and high-throughput profiling methods of intracellular metabolites using cell culture systems [17,18]
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