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A high-throughput toxicity screen of 42 per- and polyfluoroalkyl substances (PFAS) and functional assessment of migration and gene expression in human placental trophoblast cells

Bevin E. Blake, Brittany P. Rickard, and Suzanne E. Fenton
DOI: https://doi.org/10.22427/NTP-DATA-025-00001-0001-000-9

High-Throughput Toxicity Screen

Table 1. Dose-response curve EC50

Dose-response curve EC50 estimates for JEG-3 cell viability, proliferation, and mitochondrial membrane potential (MMP) after 24 hr exposure to PFAS.

Figure 1. Dose-response modeling results

Dose-response modeling results obtained from JEG-3 cells exposed to 42 different PFAS congeners for 24 hours corresponding to (A) viability, (B) proliferation, and (C) mitochondrial membrane potential (MMP).

Figure 2. Examples of Chemical Structures and Corresponding Phase Contrast - effects on Cellular Viability

Examples of chemical structures and corresponding phase contrast live cell images obtained from JEG-3 cells after 24 hours exposure to PFAS with most pronounced effects on cellular viability

Figure 3. Examples of Chemical Structure and Corresponding Phase Contrast - effects on Cellular Proliferation

Examples of chemical structures and corresponding phase contrast live cell images obtained from JEG-3 cells after 24 hours exposure to PFAS with most pronounced effects on cellular proliferation

Figure 4. Examples of Chemical Structure and Corresponding Phase Contrast - effects on Mitochondrial Membrane Potential

Examples of chemical structures and corresponding phase contrast live cell images obtained from JEG-3 cells after 24 hours exposure to PFAS with most pronounced effects on mitochondrial membrane potential.

Supplementary Table 2. Raw Values for Dose-Response Curve EC50

Raw values for dose-response curve EC50 estimates from JEG-3 cell viability, proliferation, and mitochondrial membrane potential (MMP) assays after exposure to individual PFAS

Supplementary Table 3. Rough binning of PFAS bioactivity in JEG-3 cells

Rough binning of PFAS bioactivity in JEG-3 cells using lowest concentration (µM) at which the mean response value exceeded the mean control value ± 2*SD

Supplementary Figure 2. Representative phase contrast live cell images and raw viability relative luminescence units

Representative phase contrast live cell images and raw viability relative luminescence units (RLU) in JEG-3 cells after 24 hours exposure to media only or vehicle control (2% methanol, 98% media)

Supplementary Figure 3. Representative images of JEG-3 cells after 24 hours exposure

Representative images of JEG-3 cells after 24 hours exposure to (A) 0 µM, (B) 100 µM, and (C) 150 µM menadione (positive control for cell death).

Supplementary Figure 4.Dose-response model fits for cell viability after 24 hours exposure to PFAS

Dose-response model fits for cell viability after 24 hours exposure to PFAS. CAS numbers are shown in the grey header of individual plots.

Supplementary Figure 5. Dose-response model fits for cell proliferation after 24 hours exposure to PFAS

Dose-response model fits for cell proliferation after 24 hours exposure to PFAS. CAS numbers are shown in the grey header of individual plots.

Supplementary Figure 6. Dose-response model fits for cell mitochondrial membrane potential

Dose-response model fits for cell mitochondrial membrane potential (MMP) after 24 hours exposure to PFAS.

Scratch Wound/Migration Assay

Figure 5. JEG-3 cell migration after 24 hours of exposure to select PFAS

. JEG-3 cell migration after 24 hours of exposure to select PFAS, assessed using a scratch wound assay (pictured in Figure S6).

Supplementary Figure 7. Effects of PFAS on JEG-3 cell migration

Effects of PFAS on JEG-3 cell migration during wound healing after 0, 18, 36, and 72 hours of exposure.

Supplementary Figure 8. JEG-3 cell migration

JEG-3 cell migration after 48 hours of exposure to select PFAS, assessed using a scratch wound assay

Supplementary Figure 9. JEG-3 cell migration after 72 hours of Exposure

JEG-3 cell migration after 72 hours of exposure to select PFAS, assessed using a scratch wound assay.

NanoString Gene Expression

NanoString heatmap

Figure 6. Heatmap illustrating gene expression changes in a set of 46 genes evaluated in JEG-3 cells after exposure to PFOA or GenX for 24 hours.

NanoString data

Spplementary Table 4. Gene expression data expressed as fold change over untreated control from JEG-3 cells after exposure to PFOA or GenX

Supplemental Information

Supplementary Figure 1. Experimental workflow

Experimental workflow to conduct efficient high-throughput toxicity screening of PFAS using JEG-3 cells

Supplementary Table 1. List of PFAS tested

List of PFAS tested in the JEG-3 placental cell line, and their unique identifiers, molecular formulas and weights.