Baseline Toxicity Model to Identify the Specific and Nonspecific Effects of Per- and Polyfluoroalkyl Substances in Cell-Based Bioassays

High-throughput screening is a strategy to identify potential adverse outcome pathways (AOP) for thousands of per- and polyfluoroalkyl substances (PFAS) if the specific effects can be distinguished from nonspecific effects. We hypothesize that baseline toxicity may serve as a reference to determine the specificity of the cell responses. Baseline toxicity is the minimum (cyto)toxicity caused by the accumulation of chemicals in cell membranes, which disturbs their structure and function. A mass balance model linking the critical membrane concentration for baseline toxicity to nominal (i.e., dosed) concentrations of PFAS in cell-based bioassays yielded separate baseline toxicity prediction models for anionic and neutral PFAS, which were based on liposome-water distribution ratios as the sole model descriptors. The specificity of cell responses to 30 PFAS on six target effects (activation of peroxisome proliferator-activated receptor (PPAR) gamma, aryl hydrocarbon receptor, oxidative stress response, and neurotoxicity in own experiments, and literature data for activation of several PPARs and the estrogen receptor) were assessed by comparing effective concentrations to predicted baseline toxic concentrations. HFPO–DA, HFPO–DA-AS, and PFMOAA showed high specificity on PPARs, which provides information on key events in AOPs relevant to PFAS. However, PFAS were of low specificity in the other experimentally evaluated assays and others from the literature. Even if PFAS are not highly specific for certain defined targets but disturb many toxicity pathways with low potency, such effects are toxicologically relevant, especially for hydrophobic PFAS and because PFAS are highly persistent and cause chronic effects. This implicates a heightened need for the risk assessment of PFAS mixtures because nonspecific effects behave concentration-additive in mixtures.

Table S6.Distribution ratios between medium and water (Dmedium/w) and distribution ratios between BSA and water (DBSA/w) of 11 PFAS.Table S7.Volume fractions of protein and lipid in medium used in the four cell-based HTS bioassays.Table S8.Volume fractions of protein and lipid in cells used in the four cell-based HTS bioassays.Table S9.Free and nominal concentrations of 11 PFAS related to baseline toxicity.
Table S10.Distribution ratios between cell and water (Dcell/w) of 11 PFAS with four cell lines.Table S11.Chemical information of anionic and neutral PFAS.
Table S12.Cell responses of 24 PFAS in four cell-based HTS bioassays.
Table S13.Maximum            The nominal concentration in the medium is the total molar amount of chemicals (ntot) divided by the total volume (Vtot), which is composed of medium (Vmedium) and cells (Vcell) (Eq.S1).The free concentration Cfree,medium is the concentration in the aqueous phase of the medium (Eq.S2).
The Cfree,medium relates to Cnom by Eq.S3.ffree,medium is the fraction of freely dissolved chemical in the medium.
ffree,medium is defined by a mass balance equation (Eq.S4), where chemicals are bound to components of medium (nbound,medium) and cell (nbound,cell) in the bioassay system.Protein and lipid in the medium and cells are the major sorptive phases.The distribution ratio between medium and water (Dmedium/w) is defined by concentration of chemical bound to the protein and lipid in the medium (Cbound,medium) divided by the freely dissolved concentration in the medium Cfree,medium as Eq.S5.
Analogously, the distribution ratio between cell and water (Dcell/w) can be calculated by Eq.S6.At steady state, the free concentration in the cytosol Cfree,cell can be assumed to be equal to the Cfree,medium.
and calculation details for free concentrations of PFAS in the medium is as Henneberger et al. 1 Step 4: Cytotoxicity.Cell plate was imaged with IncuCyte S3 after dosing with PFAS.The cytotoxicity was determined by comparing the confluency of exposed cells and un-exposed cells after 24 h PFAS exposure.Table S3.Experimental conditions of (a) BioSPME 96-Pin Device for the medium binding assay and (b) the C18-SPME fiber for cell and structural protein binding assay.Text S3.Experimental procedure of measuring cell binding assay of PFAS.
Step 1: Cells were detached with trypsin and collected as a pellet after centrifugation.Four cell pellets (HEK293H, MCF7, H4lle and SH-SY5Y) were resuspended with phosphate buffered saline (PBS) at a density of 10 7 cells/mL.Cell suspension was transferred to a 5 mL plastic vial (0030119401, Eppendorf) and homogenized by ultrasonic shattering (Sonoplus 2070, Germany) in an ice-water bath.The cell homogenate was diluted with PBS to a density of 2.5 × 10 6 cells/mL.PFAS stock solutions were diluted with PBS.
Step 2: 100μL cell homogenate and 100μL PFAS solution were added and vortexed in a 1.5mL HPLC vial with insert (7648146, 765116, Labsolute).Cell homogenates in each sample contained approximately 1.25× 10 6 cells.100μL PFAS solution and 100μL PBS were mixed and were used for the measurements of total molar amount (ntot), which was used to calculate the total concentration (Ctot) in the cell homogenate as listed in Table S2.
Step3: Samples of PFBA, PFHxA, PFHpA, PFOA, HFPO-DA, PFHxS and 6:2 FTSA were prepared in screw vials with insert and 520 nL C18-coated fibers were used.Samples of PFNA, PFUnA, PFOS and PFOSA were prepared in crimp vials with insert and 173 nL C18-coated fibers were used.The experimental conditions are listed in Table S3b.These samples were used to derive the distribution ratios between cell and water (Dcell/w) as Qin et al.Text S4.Experimental procedure of structural protein binding assay of PFAS.
Step 1: Structural protein is not dissolved in PBS but a homogenous suspension at density of 100 mg/mL can be obtained after high-speed vortex (3 × 3 min).The pH value of suspension was adjusted gradually to 7.4 with sodium hydroxide.11 PFAS solution were prepared in PBS individually.
Step 2: 500 μL PFAS solution and 500 μL structural protein were mixed in 1.5 mL vials (7654554, 7663230, Labsolute).500 μL PFAS solution and 500 μL PBS were mixed and were used for the measurements of total molar amount (ntot), which were used to calculate the total concentration (Ctot) in the protein suspension as listed in Table S2.
Step  S3b.These samples were used to derive the distribution ratios between structural protein and water (DSP/w).On the third day after 24 h of PFAS exposure, for the three reporter gene cell lines, the cytotoxicity was also analyzed by comparing confluency of the cells before and after 24 h of exposure.In case of the PPARγ-GeneBLAzer, the production of the β-lactamase reporter protein was measured with ToxBLAzer™ DualScreen Kit (Invitrogen™ K1138).The fluorescence at excitation of 409 nm and emission of 460 nm for blue light and 530 nm for green light were read with an Infinite® M1000 plate reader (Tecan, USA).
The reporter protein luciferase of AREc32 and AhR-CALUX was quantified by bioluminescence with substrates prepared with D-luciferin (ABD-12506, AAT Bioquest).For SH-SY5Y cell lines, neurite length of differentiated SH-SY5Y cells was quantified by phase-contrast imaging using an IncuCyte S3.Then, Nuclear Green LCS1 (ab138904, Abcam) and propidium iodide (81845, Sigma Aldrich) were used to stain the total cells and death cells for one hour.
The inhibitory concentrations triggering 10% cytotoxicity (IC10) and 10% of maximum effect (EC10) were calculated by 10% divided a slope of a linear concentration response curve (CRC). 3For antagonism of PPARγ, the suppression ratio SPR of 20% is often used and ECSPR20 were calculated by 20% divided a slope of a linear CRC. 2 For AREc32, the concentration causing an induction ratio of 1.5, ECIR1.5, was derived from a linear CRC through the intercept IR 1. 4      Therefore, the prediction of Dlip/w of PFOSA was done with N=8 and of 6:2 FTSA with N=6 by Eq.S10.    1) against measured distribution ratios between liposome and water (Dlip/w, Table 1) for anionic PFAS (Eq.14) and neutral organic chemicals from literature 8 (Eq.16).The empty symbols are 6:2 FTSA and PFOSA, which were not included in the regression (Eq.14) because their Dlip/w were predicted.(b) Linear relationship of distribution ratios between structural protein and water (DSP/w, Table 1) against Dlip/w for anionic PFAS (Eq.15) and neutral organic chemicals from literature 9 (Eq.17).
6:2 FTSA and PFOSA (empty symbols) were not included in the regression (Eq.15).(c) Contributions of cell binding to the baseline toxicity prediction with generic cell models.Cells were excluded and only medium was considered in the model for anionic PFAS (Eq.19) and model for neutral chemicals (Eq.20).
(d) Contributions of distribution to medium lipids to the baseline toxicity prediction models.

Figure S2 .
Figure S2.Experimental workflow of BioSPME 96-Pin Device used in PPARγ-GeneBLAzer reporter gene assay to measure distribution ratio of PFAS between medium and water (Dmedium/w) and free concentration (Cfree,medium) of PFAS, as well as the inhibitory concentration at 10% cytotoxicity (IC10).

Figure S3 .
Figure S3.Experimental workflow of C18-SPME used in cell binding assays to measure distribution ratio of PFAS between cell and water (Dcell/w).

Figure S4 .
Figure S4.Experimental workflow of C18-SPME used in structural protein binding assays to measure distribution ratio of PFAS between structural protein and water (DSP/w).

Figure S5 .
Figure S5.Experimental workflow of high throughput screening of PFAS in 384-well plates.

Figure S8 .
Figure S8.Information of lipid and protein binding and PFAS structures.

Figure S10 .
Figure S10.Relationships of protein and lipid binding and distribution of medium and cells in the baseline toxicity prediction models.

f 1 1+n
free,medium = n free,medium n tot = n free,medium n free,medium +n bound,medium +n free,cell !n bound,cell = bound,medium n free,medium + n free,cell n free,medium !n bound,cell n free,medium (S4)

Figure S2 .
Figure S2.Experimental workflow of BioSPME 96-Pin Device used in PPARγ-GeneBLAzer reporter gene assay to measure distribution ratio of PFAS between medium and water (Dmedium/w) and free concentration (Cfree,medium) of PFAS, as well as the inhibitory concentration at 10% cytotoxicity (IC10).

Figure S3 .
Figure S3.Experimental workflow of C18-SPME used in cell binding assays to measure distribution ratio of PFAS between cell and water (Dcell/w).

Figure S4 .
Figure S4.Experimental workflow of C18-SPME used in structural protein binding assays to measure distribution ratio of PFAS between structural protein and water (DSP/w).

Figure S5 .
Figure S5.Experimental workflow of high throughput screening of PFAS in 384-well plates.

Figure S6 .
Figure S6.Medium binding isotherms of 11 PFAS.These isotherms were analyzed with a Freundlich-type model 2 to derive regression equations between log Dmedium/w against log Cw as shown in Table S6.Concentration unit of PFAS in the water phase (Cw) is micromolar [μmol/Lw], where Lw is liter of water.IC10,free related to cytotoxicity were derived from PPARγ-GeneBLAzer reporter gene assays as shown in Figure S7.

Figure S7 .
Figure S7.Cytotoxicity of 11 PFAS in the PPARγ-GeneBLAzer reporter gene assay.Inhibitory concentration IC10,free were derived from the concentration-response curves at 10% cytotoxicity with measured free concentrations in the medium Cfree,medium.Values of cytotoxicity more than 40% were excluded from the linear fitting (hollow circle).

Figure S9 .
Figure S9.Relationship between nominal (Cnom) and measured free (Cfree,medium) concentrations of PFAS in the PPARγ-GeneBLAzer assay.Cfree,medium were predicted concentration-dependently by the mass balance model (MBM) from Cnom with distribution ratio between medium and water (Dmedium/w) and cell and water (Dcell/w) measured in this study (Eq.3), or with distribution ratio between BSA and water (DBSA/w), between structural protein and water (DSP/w) and liposome and water (Dlip/w) (Eq.6).Inhibitory concentration of 10% cytotoxicity IC10,nom or IC10,free were derived from concentration-response curves at 10% cytotoxicity with Cnom or measured Cfree,medium.

Figure S10 .
Figure S10.Relationships of protein and lipid binding and distribution of medium and cells in the baseline toxicity prediction models.(a) Linear relationship of measured distribution ratios between bovine serum albumin and water (DBSA/w, Table1) against measured distribution ratios between liposome and water (Dlip/w,

Figure
Figure S11 continued.Agonistic mode and antagonistic mode of PPARγ-GeneBLAzer reporter gene assays of 24 PFAS.

Figure
Figure S11 continued.Agonistic mode and antagonistic mode of PPARγ-GeneBLAzer reporter gene assays of 24 PFAS.

Figure
Figure S11 continued.Agonistic mode and antagonistic mode of PPARγ-GeneBLAzer reporter gene assays of 24 PFAS.

Figure
Figure S11 continued.Agonistic mode and antagonistic mode of PPARγ-GeneBLAzer reporter gene assays of 24 PFAS.

Figure
Figure S11 continued.Agonistic mode and antagonistic mode of PPARγ-GeneBLAzer reporter gene assays of 24 PFAS.

Table S1 .
Purchase information of 24 PFAS used in cell-based bioassays.

Table S2 .
Total concentrations Ctot of 11 PFAS measured by LCMS.Ctot were used in medium for the PPARγ-GeneBLAzer reporter gene assay, in cell homogenates for the cell binding assay and in chicken protein suspension for structural protein binding as shown in FigureS2, S3 and S4.

Table S4 .
Information on (a) cells and (b) medium used for the four cell-based HTS bioassays.

Table S5 .
Maximum concentrations of 24 PFAS in four cell-based HTS bioassays.

Table S6 .
2istribution ratios between medium and water (Dmedium/w) and distribution ratios between BSA and water (DBSA/w) of 11 PFAS.Regression equations between log Dmedium/w and DBSA/w against log Cw were derived using a Freundlich-type model.2Theconcentration unit of PFAS in the water phase (Cw) is micromolar [μmol/L].

Table S7 .
Volume fractions of protein and lipid in medium used in the four cell-based HTS bioassays.The generic medium was defined for a common experimental condition using 10% FBS.

Table S8 .
Volume fractions of protein and lipid in cells used in the four cell-based HTS bioassays.The generic cell was defined based on average values measured from four cell lines.

Table S9 .
Free and nominal concentrations of 11 PFAS related to baseline toxicity.IC10,free,baseline were calculated with Eq.8 in main manuscript and IC10,nom,baseline were predicted by the mass balance model (MBM, Eqs. 3 or 6).

Table S11 .
10emical information of anionic and neutral PFAS.24PFAS a were used in this study and 16 PFAS b were from Evans et al.10Distribution ratios of PFAS between liposome and water (Dlip/w) were from literature or predicted with Eqs.S9, S10 and S11 as shown in FigureS8.IC10,free,baseline were calculated with Eq.8 in main manuscript and IC10,nom,baseline were predicted by the baseline toxicity generic models (Eq.11,