Finding the target: Exploring the toxicity of perfluorohexane sulfonate (PFHxS) in zebrafish embryos using untargeted lipidomics and metabolomics

Mengmeng Xu

Research output: PhD ThesisPhD-Thesis - Research and graduation internal

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Abstract

Per- and polyfluoroalkyl substances (PFASs) are artificial chemicals with strong C-F bonds, with high thermal, chemical, and biological stability. Nowadays, the toxic potency of PFASs, especially PFOS and PFOA, has been increasingly assessed in in vivo and in vitro and epidemiological studies, but less is known about many other PFASs (chapter 1). In chapter 2, three comprehensive two-dimensional liquid chromatography combined with high resolution mass spectrometry (LC×LC-MS) methods were developed and used to unravel the lipidome of zebrafish embryos. The LC×LC-MS system could better separate lipids in zebrafish embryos than the one one-dimensional approaches (C18-MS, HILIC-MS, and PFP-MS) due to the two orthogonal LC columns tested in different orders (e.g., C18×HILIC, HILIC×C18, and HILIC×PFP). The performance of these methods was evaluated with the number of annotated lipids, showing that LC×LC-MS-based platforms, especially C18×HILIC, could annotate 1784 lipids, thus outperforming the one-dimensional C18-MS methods with 418 annotated lipids. In chapter 3, to enlarge the polar metabolome coverage in ZFE, the analytical platforms based on quadrupole (Q)-“time of flight” (TOF) mass spectrometry (QTOF) were optimized for polar and medium-polar metabolites. Different separation mechanisms, mobile phase compositions, and resuspension solvents were evaluated using retention behaviors and MS response of metabolite standards, as well as the feature number and number of annotated metabolites of ZFE samples. In positive ionization mode, zwitterionic and basic metabolites were mainly detected. The optimal LC-QTOF platform was achieved with a HILIC column at a neutral pH, with the highest number of annotated metabolites (n=151). In negative ionization, 218 metabolites, mainly zwitterionic and acidic species, were annotated using a PFP column with 0.02% acetic acid in the mobile phase. Besides, the resuspension solvents of the sample extracts should be similar to the initial mobile phase composition. In chapter 4, zebrafish embryos were exposed to PFHxS (0, 0.3, 1, 3, 10 μM), and samples were collected at 4, 24, 48, 72, and 120 hours post fertilization (hpf). The control groups showed different changes in the lipid patterns during embryonic development, which were associated with the lipid class and fatty acid composition. For example, levels of phosphatidylcholines (PCs) containing docosahexaenoic acid (DHA) chains increased in the embryo from 4 to 120 hpf. A similar increase was found for diacylglycerols (DGs) incorporated with polyunsaturated fatty acids (PUFAs). Furthermore, exposure to PFHxS, even at the lowest level of 0.3 μM, showed a strong dysregulation of the lipid metabolism in ZFE. PFHxS caused a decrease in levels of some polyunsaturated fatty acids (PUFAs), ether-phospholipids, and N-acylethanolamines (NAEs) and increased levels of oxidized lipids, which was possibly due to oxidative stress and inflammation. Besides, increased levels of acylcarnitines were observed, indicating dysregulation of fatty acid oxidation (FAO). Notably, PFHxS also remodeled the phospholipid composition, showing more omega-3 PUFAs (n3) and less omega-6 (n6) PUFAs incorporated into PCs and PEs compared to the control groups. In chapter 5, the effects of PFHxS (0, 0.3, 1, 3, and 10 μM) on ZFE during early development (4, 24, 48, 72, and 120 hpf) were studied using an untargeted metabolomics approach. Exposure to PFHxS dysregulated multiple metabolic pathways, such as FAO, sugar metabolism, and other metabolic pathways. Moreover, PFHxS led to oxidative stress, which correlated to the findings from the untargeted lipidomics study. Furthermore, comprehensive information was obtained on the changes in levels of essential metabolites (e.g., amino acids, nucleic acids, and sugars) during the early development stages (4, 24, 48, 72, and 120 hpf) of ZFE, which provided a better understanding of the changes of metabolite levels during the development of vertebrates. In chapter 6, the main results of this thesis were summarized.
Original languageEnglish
QualificationPhD
Awarding Institution
  • Vrije Universiteit Amsterdam
Supervisors/Advisors
  • Leonards, PEG, Supervisor
  • Legradi, JB, Co-supervisor
Award date6 Dec 2022
Publication statusPublished - 6 Dec 2022

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