Effects of Novel (Poly-) Ether PFAS an the Transcriptome of HepaRG cells

German Federal Institute for Risk Assessment [email protected] | bfr.bund.de/en Follow us Effects of Novel (Poly-) Ether PFAS an the Transcriptome of HepaRG cells Wiebke Alker1, Heike Sprenger1, Anna Rocchi2, Chiara Leo2, Rosa Giglio2, Albert Braeuning1, Thorsten Buhrke1 Funding: This project has received funding from the H2020 programm under Grant Agreement No. 101037509 Unpublished data, ©2025, Wiebke Alker, German Federal Institute for Risk Assessment, All rights reserved. 1German Federal Institute for Risk Assessment, 2Polo d’innovazione di Genomica, Genetica e Biologia Contact: [email protected] Abbreviations: PFAS: Perand polyfluoroalky substances, PFCA: perfluoroalkyl carboxylic acids, PFECA: perfluoroalkyl (poly-)ether carboxylic acids, PFESA: perfluoroalkyl (poly-)ether sulfonic acids, PFSA: perfluoroalkyl sulfonic acids Introduction Due to their high persistence in the environment and adverse health efffects in humans, the use of many legacy perand polyfluoroalkyl substances (PFAS) has been restricted. Thus, novel PFAS are increasingly used for numerous industrial applications, despite the fact that currently limited or no data is available on their toxicity. This study aims to examine the molecular mechanisms of action of novel PFAS by whole transcriptome analysis and by using differentiated HepaRG cells as a model for human hepatocytes. The focus is on the subgroup of monoand polyether PFAS, with both linear and branched structures, including PFAS congeners with either a carboxylic acid or a sulfonic acid functional group. Experimental setup Conclusions •Similar mechanism(s) of action in HepaRG cells for all tested PFAS, despite their structural differences •All tested PFAS activated lipid metabolism (regulated by PPARαand PPARγ) and xenobiotic metabolism (regulated by PXR) •Some PFAS inhibited cholesterol biosynthesis –no obvious association with PFAS structure •For all tested PFAS, IPA predicted an association between PFAS exposure and various liver diseases, e.g., cholestasis Results Figure 1: (A) Number of DEG; IPA comparison analysis of (B) canonical pathways, (C) upstream regulators and (D) downstream effects (tox functions) in PFAS-treated HepaRG cells at high concentration. Depending on the PFAS and its cytotoxicity, low concentration is 5, 10 or 25 µM, medium concentration is 25 or 100 µM and high concentration is 50,100 or 250 µM. A positive z-score (orange color) indicates an activation, a negative zscore (blue color) an inhibition of the respective canonical pathway or upstream regulator. In parallel, statistical significance is given via the gray-scaled adjusted p-value.No z-scores are available for the downstream effects. Due to limited space on the poster, only a selection of the most prominent affected canonical pathways, upstream regulators and downstream effects is shown. A PFAS selection •In total 33 PFAS •15 legacy PFAS: PFCA and PFSA •18 novel PFAS: PFECA and PFESA (linear/ branched, mono-/ polyether) In vitro experiments •Differentiated HepaRG cells •24 h incubation •3 non-cytotoxic concentrations •RNA isolation Transcriptome Analysis •Whole transcriptome analysis using RNA sequencing •Analysis of differentially expressed genes (DEG) cut-off criteria: |log2fold change| > 0.5, adjusted p-value < 0.05 •Data evaluation by Ingenuity Pathway Analysis (IPA) B C D Structures of some PFAS used in this study PFOA HFPO-DA (GenX) 8:2 Cl-PFESA Branched ADONA PFoxaOA PFO2DA PFO3DA PFHpS PF2EOESA Nafion-BP2