Evaluation of 147 perfluoroalkyl substances for immunotoxic and other (patho)physiological activities through phenotypic screening of human primary cells

“Also of note was the lack of any PFAS with a liver toxicity signature assigned for both sample replicates at any concentration although liver toxicity in animals has been associated with a number of PFAS including PFOA and PFOS (Lau et al., 2007).”

“Extensive studies of several of the highly produced members of the class have demonstrated potential for adverse health consequences to humans as well as highly pervasive and persistent exposures (Wang et al., 2017; Cousins et al., 2019).”

“SRB was performed by staining cells with 0.1% SRB after fixation with 10% TCA and reading wells at 560 nm. Viability and proliferation of PBMC (T cells) was quantified by Alamar Blue reduction (Ahmed et al., 1994) for the SAg and BT systems.”

“Proteasome inhibitors are used therapeutically as anticancer agents and drive cancer cells into apoptosis at appropriate concentrations (Kisselev and Goldberg, 2001).”

“Profiles for PPARa agonists oleylethanolamide and gemfibrozil and PPARg agonists rosiglitazone and pioglitazone at concentrations exceeding their effective in vitro reported ones are shown in Figure 8 (Fu et al., 2003; De Filippis et al., 2015; Young et al., 1998; Sakamoto et al., 2000).”

“…Food Safety Authority extensively reviewed both animal studies and human epidemiological associations of PFAS (primarily PFOA and PFOS) and reported decreased T cell-dependent antibody responses and reduced antibody response to vaccinations as critical indicators of PFAS effects (EFSA, 2020).”

“Presumably, this activity results from the known mechanism of action for cyclosporin A: complexing with cyclophilin and subsequent inhibition of the phosphatase activity of calcineurin preventing nuclear translocation and activation of NFAT transcription factors (Matsuda and Koyasu, 2000).”

“The PPARs primarily regulate metabolism in many cell types, which impacts the differentiation, expansion, and fate commitment of immune cells (Christofides et al., 2021).”

“These concentrations exceed by two orders of magnitude the serum levels measured in the 2015–16 NHANES study, which reported geometric means of 3.8 nM PFOA and 9.4 nM PFOS for the general US population (CDC, 2019).”

“One chemical, ammonium perfluoro-2-methyl-3-oxahexanoate (GenX), was solubilized in H 2 O as it was known to be unstable in DMSO (Gaballah et al., 2020; Liberatore et al., 2020).”

“…contained all tested concentrations of dexamethasone, a glucocorticoid causing immunosuppression through activation of the glucocorticoid receptor resulting in transrepression (repression of gene activation by other transcription factors) of many inflammatory cytokine genes (Stahn et al., 2007).”

“…higher as, for example, those seen in the population exposed to heavily contaminated drinking water from industrial processes in West Virginia where geometric mean PFOA concentration was 193 nM with extremes up to 42 mM and mean PFOS of 44 nM ranging up to 1.5 mM (Steenland et al., 2009).”

“Similarity analysis at this concentration indicated a high correlation of the profile with that of omipalisib, a phosphoinositide 3-kinase inhibitor that blocks activity of the mammalian target of rapamycin complex 1 (mTORC1) (Knight et al., 2010).”

“Note that this concentration is greater than typical pharmacological exposures in patients treated with cyclosporin A as immunosuppressant therapy (with a maximum plasma concentration, Cmax, of < 1-2 mM) (Halloran et al., 1999).”

“Profiles for PPARa agonists oleylethanolamide and gemfibrozil and PPARg agonists rosiglitazone and pioglitazone at concentrations exceeding their effective in vitro reported ones are shown in Figure 8 (Fu et al., 2003; De Filippis et al., 2015; Young et al., 1998; Sakamoto et al., 2000).”

“The testing concentrations selected for this study were determined based on the ability to generate a universal solubilithe latter process known to be stimulated by inhibition of proteasome activity (Ji and Kwon, 2017).”

“Indeed, PFAS have been shown to have significant effects on several properties including serum protein binding and bioaccumulation that may impact actual cellular exposure levels (Yang, D. et al., 2020; Zhang, W. et al., 2020).”

“For instance, PPARa and PPARg are known molecular targets for PFAS, and among numerous bioactivities associated with these receptors in the literature are effects on the immune system, primarily categorized as anti-inflammatory and suggested as a mechanism for PFAS immunosuppression (Christofides et al., 2021; Peden-A…”

“Other studies have suggested that alterations in IgG secretion in mice exposed to PFOA may occur independently of systemic toxicity and stress-related corticosterone increases (DeWitt et al., 2009a), noting that IgG secretion in the BT system in BioMAP was unaffected by PFOA.”

“PPARs are also known to function in large complexes that include various co-activators and co-repressors that impart cell-type and tissue-specific effects, with considerable species differences including in immune responses (Yu and Reddy, 2007; Corsini et al., 2014).”

“This panel has been extensively used in pharmaceutical and consumer products research for characterization of product candidates (O’Mahony et al., 2018; Betts et al., 2018; Hammitzsch et al., 2015; Shah et al., 2017; Simms et al., 2021; Singer et al., 2019).”

“The other major correlated profile was that of the fumagillin analog TNP-40, which inhibits angiogenesis through irreversible inactivation of methionine aminopeptidase-2 (MetAP2), blocking endothelial cell proliferation in vitro and angiogenesis in vivo (Griffith et al., 1998).”

“0) (Filer et al., 2017) using methods to identify lowest observable effect concentration for BSK data rather than curve-fitting these data as is done for most other ToxCast data.”

“ALTEX 40(2), 2023 267 as well as contribute to risk analysis in facilitating grouping of PFAS from complex mixtures that may have additive or other interactive activities (Ojo et al., 2020).”

“…of PFAS, the OECD now defines PFAS as substances with a minimum of one perfluorinated carbon, which results in a list of PFAS that may approach 40,000 substances, with more stringent definitions of PFAS structures resulting in shorter lists in the thousands (Williams et al., 2022; OECD, 2021).”

“…as (patho)physiologically relevant screening assays for evaluating adverse effects was previously demonstrated through testing pharmaceuticals and clinical candidates as well as environmental chemicals in the EPA’s ToxCast program (Kleinstreuer et al., 2014; Berg et al., 2006; Singer et al., 2019).”

“PPARs are also known to function in large complexes that include various co-activators and co-repressors that impart cell-type and tissue-specific effects, with considerable species differences including in immune responses (Yu and Reddy, 2007; Corsini et al., 2014).”

“…in Table S1 1 , along with their average mass (g/mol), the analytical quality control grades on the solubilized chemical samples (stock samples), the final concentrations used in screening in the BioMAP assays, and the structural categories based on ChemoType ToxPrints (Yang, C. et al., 2015)”

“Extensive studies of several of the highly produced members of the class have demonstrated potential for adverse health consequences to humans as well as highly pervasive and persistent exposures (Wang et al., 2017; Cousins et al., 2019).”

“Two PFAS, 3-(perfluorooctyl)propanol and 3,3-bis(trifluoromethyl)-2-propenoic acid, matched the profile of bortezomib, a proteasome inhibitor that binds to the catalytic site of the 26S proteasome (Bonvini et al., 2007).”

“Here we used nominal cell culture medium concentrations; however, there are numerous parameters affecting actual exposure that need consideration (Armitage et al., 2014; Wambaugh et al., 2019).”

“Further, the BioMAP assays used human primary cells; even if these systems were responsive to PPAR agonists, the PPARa-linked liver toxicity for PFAS is argued as not relevant to human exposure (Corton et al., 2018).”

“PFAS are arranged from lowest molecular weight to highest and represented by their respective DSSTox substance identifiers, or DTXSIDs (Grulke et al., 2019).”

“Proteasome inhibitors are used therapeutically as anticancer agents and drive cancer cells into apoptosis at appropriate concentrations (Kisselev and Goldberg, 2001).”

“The European Food Safety Authority extensively reviewed both animal studies and human epidemiological associations of PFAS (primarily PFOA and PFOS) and reported decreased T cell-dependent antibody responses and reduced antibody response to vaccinations as critical indicators of PFAS effects (EFSA, 2020).”

“…useful in developing a read-across approach for data gap-filling; whether the PFAS were technically feasible to test based on physicochemical considerations (solubility/volatility); and whether the PFAS helped to represent structural diversity among this group of chemicals (Patlewicz et al., 2019).”

“Cell type-selective cytotoxicity patterns were previously observed in testing other chemical classes (Houck et al., 2009).”

“…in the 12-assay BioMAP assay suite and absence of the liver toxicity signature for these PPAR agonists is of interest given that PPAR-like liver effects have been shown in vivo for a number of PFAS including PFOA and PFOS (Perkins et al., 2004; Bjork et al., 2008; Bjork and Wallace, 2009).”

“Indeed, PFAS have been shown to have significant effects on several properties including serum protein binding and bioaccumulation that may impact actual cellular exposure levels (Yang, D. et al., 2020; Zhang, W. et al., 2020).”

“Here, too, crosstalk with the proteasome pathway has been shown through TXNL1, a thioreductase that plays a role in the transfer of misfolded nascent protein chain from the ribosome to the 26S proteasome, which was unprocessed upon MetAP2 inhibition (Andersen et al., 2009).”

“Reduced IgG is a reported effect in vivo for methotrexate and azathioprine when used for treatment of rheumatoid arthritis (Rackham et al., 2002; Kapetanovic et al., 2017; Levy et al., 1972).”

“For example, systemic toxicity and stress in the whole animal has been postulated as a mechanism of multiple immune suppression effects for PFOS and PFOA, and these are modes-of-action that may not be captured in the BioMAP panel (Loveless et al., 2008).”

“The European Food Safety Authority extensively reviewed both animal studies and human epidemiological associations of PFAS (primarily PFOA and PFOS) and reported decreased T cell-dependent antibody responses and reduced antibody response to vaccinations as critical indicators of PFAS effects (EFSA, 2020).”

“One chemical, ammonium perfluoro-2-methyl-3-oxahexanoate (GenX), was solubilized in H 2 O as it was known to be unstable in DMSO (Gaballah et al., 2020; Liberatore et al., 2020).”

“Here we used nominal cell culture medium concentrations; however, there are numerous parameters affecting actual exposure that need consideration (Armitage et al., 2014; Wambaugh et al., 2019).”

“…of PFAS, the OECD now defines PFAS as substances with a minimum of one perfluorinated carbon, which results in a list of PFAS that may approach 40,000 substances, with more stringent definitions of PFAS structures resulting in shorter lists in the thousands (Williams et al., 2022; OECD, 2021).”

“Evolving knowledge of instability of certain PFAS in DMSO (Liberatore et al., 2020; Zhang, C. et al., 2021) led to a stock quality and stability evaluation across the PFAS stocks employed in this work.”

“Preparation and co-culture of human primary cell types and methods for the systems were as previously described (Melton et al., 2013; Shah et al., 2017; Kunkel et al., 2004a).”

“Preparation and co-culture of human primary cell types and methods for the systems were as previously described (Melton et al., 2013; Shah et al., 2017; Kunkel et al., 2004a).”

“DMSO stock solutions were used as this is a universal solvent having good general small-molecule solvating capability and compatibility with high-throughput biological assays (Popa-Burke et al., 2004).”

“…as (patho)physiologically relevant screening assays for evaluating adverse effects was previously demonstrated through testing pharmaceuticals and clinical candidates as well as environmental chemicals in the EPA’s ToxCast program (Kleinstreuer et al., 2014; Berg et al., 2006; Singer et al., 2019).”

“IL-10 IL-10 is a cytokine that promotes B cell IgG production (Facciotti et al., 2020; Itoh et al., 1994).”

“This panel has been extensively used in pharmaceutical and consumer products research for characterization of product candidates (O’Mahony et al., 2018; Betts et al., 2018; Hammitzsch et al., 2015; Shah et al., 2017; Simms et al., 2021; Singer et al., 2019).”

“…acrylate and 1H,1H-perfluorooctyl acrylate showed similarity to auranofin, an antibiotic that can inhibit thioredoxin reductase, an enzyme that maintains cellular redox potential and whose expression can be repressed by the proteasome inhibitors bortezomib and carfilzomib (Fink et al., 2016).”

“The exception was for azathioprine and methotrexate, which clustered similarly, both with mechanisms of action as antimetabolites inhibiting cell cycle S phase (Bertino, 1973).”

“…as (patho)physiologically relevant screening assays for evaluating adverse effects was previously demonstrated through testing pharmaceuticals and clinical candidates as well as environmental chemicals in the EPA’s ToxCast program (Kleinstreuer et al., 2014; Berg et al., 2006; Singer et al., 2019).”

“This similarity search analysis relies on inference to data generated using BioMAP for a diverse set of chemicals (Berg, 2019; Berg et al., 2006, 2013).”

“The levels of cell surface (or secreted, indicated by the prefix “s”) biomarker endpoints were measured by ELISA as described (Shah et al., 2017; Melton et al., 2013).”

“5C), consistent with known activity of the glucocorticoids acting through the glucocorticoid receptor in aortic smooth muscle cells (Kumon et al., 2001).”

“IL-10 IL-10 is a cytokine that promotes B cell IgG production (Facciotti et al., 2020; Itoh et al., 1994).”

“Such activity may be consistent with reported anticancer effects of cyclosporin A at micromolar concentrations, with activity attributed to inhibition of mTORC1 signaling in prostate cancer cells (Lee et al., 2012).”

“Proteasome inhibitors have been previously reported to be immunosuppressive (Khalesi et al., 2021).”

“Side effects of proteasome inhibitors reported in clinical trials include anemia, gastrointestinal disorders, and peripheral neuropathy (Hungria et al., 2019).”

“The levels of cell surface (or secreted, indicated by the prefix “s”) biomarker endpoints were measured by ELISA as described (Shah et al., 2017; Melton et al., 2013).”

“Target mechanisms associated with the thrombosis signature include mTOR, AhR, V-ATPase, lysosomal function, CYP17A, PKC NOD2, estrogen receptor, H1R, HIF-1alpha, thyroid hormone receptor, OSM R (Berg et al., 2015).”

“This panel has been extensively used in pharmaceutical and consumer products research for characterization of product candidates (O'Mahony et al., 2018; Betts et al., 2018; Hammitzsch et al., 2015; Shah et al., 2017; Simms et al., 2021; Singer et al., 2019).”

“The NTP monograph did not include supporting in vitro/mechanistic data for IgG suppression but did report no suppression of IgM in either murine or human B cell lines, even at very high (750 mM) concentrations of PFOA (Levitt and Liss, 1986).”

“The profiles for PFAS chemicals were also analyzed for the presence of defined toxicity signatures from previous work with the BioMAP assay suite (Berg, 2019).”

“This panel has been extensively used in pharmaceutical and consumer products research for characterization of product candidates (O’Mahony et al., 2018; Betts et al., 2018; Hammitzsch et al., 2015; Shah et al., 2017; Simms et al., 2021; Singer et al., 2019).”

“Profiles for PPARa agonists oleylethanolamide and gemfibrozil and PPARg agonists rosiglitazone and pioglitazone at concentrations exceeding their effective in vitro reported ones are shown in Figure 8 (Fu et al., 2003; De Filippis et al., 2015; Young et al., 1998; Sakamoto et al., 2000).”

“Reduced IgG is a reported effect in vivo for methotrexate and azathioprine when used for treatment of rheumatoid arthritis (Rackham et al., 2002; Kapetanovic et al., 2017; Levy et al., 1972).”

“This panel has been extensively used in pharmaceutical and consumer products research for characterization of product candidates (O’Mahony et al., 2018; Betts et al., 2018; Hammitzsch et al., 2015; Shah et al., 2017; Simms et al., 2021; Singer et al., 2019).”

“The activity profile of PFOA shares some common features with that of dexamethasone, but also shows unique characteristics [41].”

“This finding corroborated our previous analysis in a single cardiomyocyte donor [22], as well as studies in other cell types [18, 19, 85].”

“…NAMs focus on multiple endpoints that cover a broad range of bioactivity including assays examining specific transcription factor activities [4]; panels that identify potential immunotoxicants [5]; developmental neurotoxicants [6]; thyroid targets [7]; and other bioactivity evaluations in progress.”

“In addition, a study of 147 PFAS identified a trend between molecular weight and bioactivity related to in vitro immunosuppression (Houck et al., 2023), and a study of 160 PFAS discovered that compounds with long carbon chains (8 or more carbons), high carbon to fluorine ratio, or containing a…”

“the immunotoxicity data on 146 PFass in human primary cells were collected from the literature (houck et al. 2023). this study defines immunosuppressive features as follows: t cell proliferation reduction feature with a significant response size of 20% (sag: Proliferation, log10Ratio < −0.1), or…”