title: Metabolomics analysis of the toxicity pathways of triphenyl phosphate in HepaRG cells and comparison to oxidative stress mechanisms caused by acetaminophen.
abstract: Since the publication of REACH guidelines, the need for in vitro tools for toxicity testing has increased. We present here the development of a hepatotoxicity testing tool using human HepaRG cell cultures and metabolomics. HepaRG cells were exposed to either 4mM acetaminophen (APAP) as reference toxicant for oxidative stress or 50 μM triphenyl phosphate (TPHP) as toxicant with unknown toxicity pathways (TPs). After 72 h exposure, cells were subjected to quenching and liquid-liquid extraction which resulted in a polar and an apolar fraction. Analysis of fractions was performed by ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-QTOF-MS). Significantly up or down regulated metabolites were selected by univariate statistics prior to identification. In order to obtain robust and specific TP biomarkers, the experiment was also repeated using a different culture medium composition to assess which metabolites show consistent changes. Potential biomarkers belonging to different TPs were found for APAP and TPHP. For APAP, the biomarkers were related to a decrease in unsaturated phospholipids, and for TPHP to an accumulation of phosphoglycerolipids and increase of palmitoyl lysophosphatidylcholine. This first proof-of-concept opens new perspectives for the analysis of other (reference) toxicants with different TPs and it can be used to expand the in vitro tool for hepatotoxicity screening of various compounds.
authors: Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium. Electronic address: nele.vandeneede@uantwerpen.be.
authors: Nele
authors: N
authors: Van den Eede
authors: Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium.
authors: Matthias
authors: M
authors: Cuykx
authors: Department of In Vitro Toxicology and Dermato-cosmetology, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Belgium.
authors: Robim M
authors: RM
authors: Rodrigues
authors: Department of Mathematics & Computer Science, University of Antwerp, Middelheimlaan 1, 2020 Antwerp, Belgium.
authors: Kris
authors: K
authors: Laukens
authors: Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium.
authors: Hugo
authors: H
authors: Neels
authors: Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium. Electronic address: adrian.covaci@uantwerpen.be.
authors: Adrian
authors: A
authors: Covaci
authors: Department of In Vitro Toxicology and Dermato-cosmetology, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Jette, Belgium.
authors: Tamara
authors: T
authors: Vanhaecke
mesh_terms: D000082:Acetaminophen / Q000633:toxicity*; D018712:Analgesics, Non-Narcotic / Q000633:toxicity; D015415:Biomarkers; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D055432:Metabolomics / Q000379:methods*; D010755:Organophosphates / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects*; D010968:Plasticizers / Q000633:toxicity
chemical_list: D018712:Analgesics, Non-Narcotic; D015415:Biomarkers; D010755:Organophosphates; D010968:Plasticizers; D000082:Acetaminophen; C005445:triphenyl phosphate
keywords: Acetaminophen; Biomarker; HepaRG; Hepatotoxicity; Metabolomics; Triphenyl phosphate