Document ba1qLkBMdRM7X0kprz1r3ynJ1

Toxicological activity profiling and potency ranking of per- and polyfluoroalkyl substances (PFAS) using ToxProfiler Bas ter Braak, Liesanne Loonstra-Wolters, Kim Elbertse, Giel Hendriks, Amer Jamalpoor Toxys, Leiden BioScience Park, The Netherlands Fraction PI positive cells GFP level normalized GFP-readout Introduction Per- and polyfluoroalkyl substances (PFAS), also known as "the forever compounds", have the tendency to accumulate in organisms and their environment. Despite extensive in vitro and in vivo testing of PFAS, there is a remaining concern about the human safety of these chemicals. ToxProfiler is a unique human-based reporter assay that provides an extensive quantitative toxicological profile of novel chemicals and drugs. The assay contains seven fluorescent reporter genes to visualise the major cellular stress response pathways responsible for cellular/organ toxicity. In this study, we have applied ToxProfiler to gain insight into: 1. Biological activity of 13 often used PFAS. 2. Potency ranking and identification of the primary toxicological mode-of-action of PFAS. 3. Relationship between in vitro toxicity and structure-activity relationship (carbon chain length) of PFAS. ToxProfiler: Experimental design Oxidative stress Cell cycle stress SRXN1 p21 ER stress CHOP Autophagy LC3 Ion stress MT1X Protein stress HSPA1B Inflammation ICAM1 CDDO-me Cisplatin Tunicamycin Amiodarone CadCl2 CadCl2 TNF 1. Concentration range finding Goal: Determine appriopriate concentration range Endpoint: Propidium iodide (cytotoxicity) 2. ToxProfiler reporter assay Goal: Visualize ToxProfiler reporter inductions Endpoint: Live cell confocal imaging 3. Image analysis and data processing Goal: Quantify GFP responses on single cell level Output: Concentration response plots/heatmaps/ clusterings/potency ranking. Compounds 1 2 3 4 5 6 7 8 9 10 C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Seed cells (384-well) and test 14 conc. Concentration Compounds 1 2 3 4 5 6 7 8 + -1 2 3 1 4 2 5 3 6 4 7 5 1 6 2 7 3 1 4 2 5 3 6 4 7 5 6 7 p21-GFP LC3-GFP SRXN1-GFP MT1X-GFP CHOP-GFP MT1X-GFP ICAM1-GFP Seed reporter lines (7x) and test 7 conc. Addition of S9 (metabolism) is optional. Raw confocal image Nuclei Cytoplasm Automated image segmentation and quantification. 0.1 POD: 140 uM Determine cytotoxcity and select 7 conc. 1.0 0.5 Measure GFP reporter activity at 24 hrs. 2x SD 0.0 10 100 Concentration (uM) 1000 Concentration modeling, visualisation of data, clustering and ranking. 0.0 0 0.1 1 10 100 Concentration (M) 1000 Cellular stress induction by PFAS Oxidative stress SRXN1 Cell cycle stress p21 ER stress CHOP Autophagy LC3 PFOA 464 M GenX 2154 M PFBS 2154 M Representative ToxProfiler confocal images of three example PFAS. Pictures were generated using the automated Operetta CLS microscope setup at 24 hours after the exposure. PFOA, GenX and PFOS were chosen because these compounds are often used as model PFAS. Quantification of stress response activation by PFAS Autophagy (LC3-GFP) Ion stress (MT1X-GFP) ER stress (CHOP-GFP) Cell cycle stress (p21-GFP) Inflammation (ICAM1-GFP) Protein stress (HSPA1B-GFP) Cell death (Propidium Iodide) Oxidative stress (SRXN1-GFP) 3 PFOA 2 3 GenX 2 3 PFBS 2 1.0 1.0 1.0 0.5 0.5 0.5 0.0 10 -0.5 100 1000 Concentration (M) 0.0 10 -0.5 100 1000 Concentration (M) 0.0 10 -0.5 100 1000 Concentration (M) Example of ToxProfiler concentration response curves. Concentration response plotting allows to compare amplitide of the reporter activations (shift along y-axis) as well as the potency (shift along x-axis.) Point of departures and potency ranking of PFAS Compound name CAS # FTOH_8:2 PFDA PFOA_1 PFOA_2 PFBS 6:2 FtS PFHpA GenX FTOH_6:2 PFOS PFHxA PFPeA HFBA 678-39-7 335-76-2 335-67-1 3825-26-1 375-73-5 2706-90-3 375-85-9 27619-97-2 647-42-7 1763-23-1 307-24-4 13252-13-6 375-22-4 Carbon Length Hepatotox RPF (Bil, Concentration 2021) range ToxProfiler Oxidative stress (SRXN1) Cell cycle ER stress Autophagy stress (CHOP) (LC3) (p21) Ion stress (MT1X) Protein stress (HSPA1B) Inflammation (ICAM1) Cytotoxicity (PI) ToxProfiler rank C10 0,04 1 - 100 M 68 C10 4 < RPF < 10 5 - 464 M 3000 150 93 C8 1 10 - 1000 M 120 300 215 215 C8 1 10 - 1000 M 150 300 300 215 C4 0,001 100 - 10000 M 900 1500 150 C5 0.01 < RPF <0.05 10 - 1000 M 560 650 830 170 C7 0.01 < RPF < 1 46 - 4642 M 418 511 232 C8 ND 46 - 4641 M 557 603 278 696 C8 0,02 5 - 464 M 330 C8 2 10 - 1000 M 600 C6 0,01 50 - 5000 M 750 700 C6 ND 100 - 10000 M 1300 900 1200 C4 0,05 100 - 10000 M 6800 2154 215 150 720 300 710 580 790 1500 1 150 2 660 3 500 4 1500 5 740 6 464 7 1000 8 9 750 10 1077 11 1600 12 7400 13 Heatmap clustering of PFAS toxicity finger prints Dendrogram Compound PFHxA PFPeA HFBA PFOS 6:2 FtS FTOH_6:2 FTOH_8:2 PFDA PFOA_1 PFOA_2 PFHpA GenX PFBS SRXN1 Concentration level 765432 1 p21 CHOP Stress type Oxidative Stress Cell Cycle Stress LC3 ER Stress Autophagy Stress type MT1X HSPA1B ICAM1 Reporter PI Ion Stress Protein Stress Inflammation Cytotoxicity Effect level 1 0.8 0.6 0.4 0.2 0 Heatmap with hierarchical clustering of ToxProfiler reporter activation profiles of PFAS. The intensity of the red color of the heatmap is representative for the GFP reporter induction. The dendrogram on the left represents similarity scoring of the different chemicals. Note that PFAS are screened in a equitox level and not necessary on an equimolar fashion. Conclusions ToxProfiler is a novel reporter assay that can be applied in early mechanistic toxicity testing which can unravel the toxicological MoA and help with the risk assessment of chemicals (e.g. PFAS). In general, ToxProfiler data suggests that PFAS with longer carbon chains (FTOH 8:2 and PFDA) are more potent than PFAS with shorter carbon lenghts (HFBA). This is concordant with the Calculated Relative Potency Factors (RPF) of PFAS regarding their hepatotoxicity. Majority of the tested PFAS strongly activated ER stress. Autophagy, oxidative stress and cell cycle stress were also induced by most of the tested PFAS. Activation of these ToxProfiler readouts is correlated with Drug Induced Livery Injury (DILI). PFAS with shorter carbon lengh (C4; PFBS and HFBA) strongly induced autophagy. GenX technology was developed to replace PFOA. A GenX derived compound was indeed found to less potently induce stress signalling. Nevertheless, the tested GenX derived compound is a strong ER stress inducer at high concentrations (>250 M). You can download this poster by scanning the QR code here: