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

Main Article Content

Keith A. Houck, Katie Paul Friedman , Madison Feshuk, Grace Patlewicz, Marci Smeltz, M. Scott Clifton, Barbara A. Wetmore, Sharlene Velichko, Antal Berenyi, Ellen L. Berg
[show affiliations]


A structurally diverse set of 147 per- and polyfluoroalkyl substances (PFAS) was screened in a panel of 12 human primary cell systems by measuring 148 biomarkers relevant to (patho)physiological pathways to inform hypotheses about potential mechanistic effects of data-poor PFAS in human model systems. This analysis focused on immunosuppressive activity, which was previously reported as an in vivo effect of perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), by comparing PFAS responses to four pharmacological immunosuppressants. The PFOS response profile had little correlation with reference immunosuppressants, suggesting in vivo activity does not occur by similar mechanisms. The PFOA response profile did share features with the profile of dexamethasone, although some distinct features were lacking. Other PFAS, including 2,2,3,3-tetrafluoropropyl acrylate, demonstrated more similarity to the reference immunosuppressants but with additional activities not found in the reference immunosuppressive drugs. Correlation of PFAS profiles with a database of environmental chemical responses and pharmacological probes identified potential mechanisms of bioactivity for some PFAS, including responses similar to ubiquitin ligase inhibitors, deubiquitylating enzyme (DUB) inhibitors, and thioredoxin reductase inhibitors. Approximately 21% of the 147 PFAS with confirmed sample quality were bioactive at nominal testing concentrations in the 1-60 micromolar range in these human primary cell systems. These data provide new hypotheses for mechanisms of action for a subset of PFAS and may further aid in development of a PFAS categorization strategy useful in safety assessment.

Article Details

How to Cite
Houck, K. A. (2023) “Evaluation of 147 perfluoroalkyl substances for immunotoxic and other (patho)physiological activities through phenotypic screening of human primary cells”, ALTEX - Alternatives to animal experimentation, 40(2), pp. 248–270. doi: 10.14573/altex.2203041.

Ahmed, S. A., Gogal, R. M., Jr. and Walsh, J. E. (1994). A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: An alternative to [3H]thymidine incorporation assay. J Immunol Meth 170, 211-224. doi:10.1016/0022-1759(94)90396-4

Andersen, K. M., Madsen, L., Prag, S. et al. (2009). Thioredoxin Txnl1/TRP32 is a redox-active cofactor of the 26S proteasome. J Biol Chem 284, 15246-15254. doi:10.1074/jbc.m900016200

Armitage, J. M., Wania, F. and Arnot, J. A. (2014). Application of mass balance models and the chemical activity concept to facilitate the use of in vitro toxicity data for risk assessment. Environ Sci Technol 48, 9770-9779. doi:10.1021/es501955g

Berg, E. L., Kunkel, E. J., Hytopoulos, E. et al. (2006). Characterization of compound mechanisms and secondary activities by BioMAP analysis. J Pharmacol Toxicol Meth 53, 67-74. doi:10.1016/j.vascn.2005.06.003

Berg, E. L., Yang, J. and Polokoff, M. A. (2013). Building predictive models for mechanism-of-action classification from phenotypic assay data sets. J Biomol Screen 18, 1260-1269. doi:10.1177/1087057113505324

Berg, E. L., Polokoff, M. A., O’Mahony, A. et al. (2015). Elucidating mechanisms of toxicity using phenotypic data from primary human cell systems – A chemical biology approach for thrombosis-related side effects. Int J Mol Sci 16, 1008-1029. doi:10.3390/ijms16011008

Berg, E. L. (2019). Human cell-based in vitro phenotypic profiling for drug safety-related attrition. Front Big Data 2, 47. doi:10.3389/fdata.2019.00047

Bertino, J. R. (1973). Chemical action and pharmacology of methotrexate, azathioprine and cyclophosphamide in man. Arthritis Rheum 16, 79-83. doi:10.1002/art.1780160113

Betts, B. C., Bastian, D., Iamsawat, S. et al. (2018). Targeting JAK2 reduces GVHD and xenograft rejection through regulation of T cell differentiation. Proc Natl Acad Sci U S A 115, 1582-1587. doi:10.1073/pnas.1712452115

Bjork, J. A., Lau, C., Chang, S. C. et al. (2008). Perfluorooctane sulfonate-induced changes in fetal rat liver gene expression. Toxicology 251, 8-20. doi:10.1016/j.tox.2008.06.007

Bjork, J. A. and Wallace, K. B. (2009). Structure-activity relationships and human relevance for perfluoroalkyl acid-induced transcriptional activation of peroxisome proliferation in liver cell cultures. Toxicol Sci 111, 89-99. doi:10.1093/toxsci/kfp093

Bonvini, P., Zorzi, E., Basso, G. et al. (2007). Bortezomib-mediated 26S proteasome inhibition causes cell-cycle arrest and induces apoptosis in CD-30+ anaplastic large cell lymphoma. Leukemia 21, 838-842. doi:10.1038/sj.leu.2404528

CDC (2019). Fourth National Report on Human Exposure to Environmental Chemicals. Updated Tables, Volume One. doi:10.15620/cdc75822

Christofides, A., Konstantinidou, E., Jani, C. et al. (2021). The role of peroxisome proliferator-activated receptors (PPAR) in immune responses. Metabolism 114, 154338. doi:10.1016/j.metabol.2020.154338

Corsini, E., Luebke, R. W., Germolec, D. R. et al. (2014). Perfluorinated compounds: Emerging POPs with potential immunotoxicity. Toxicol Lett 230, 263-270. doi:10.1016/j.toxlet.2014.01.038

Corton, J. C., Peters, J. M. and Klaunig, J. E. (2018). The PPARα-dependent rodent liver tumor response is not relevant to humans: Addressing misconceptions. Arch Toxicol 92, 83-119. doi:10.1007/s00204-017-2094-7

Cousins, I. T., Ng, C. A., Wang, Z. et al. (2019). Why is high persistence alone a major cause of concern? Environ Sci Process Impacts 21, 781-792. doi:10.1039/c8em00515j

De Filippis, B., Agamennone, M., Ammazzalorso, A. et al. (2015). PPARα agonists based on stilbene and its bioisosteres: Biological evaluation and docking studies. MedChemComm 6, 1513-151. doi:10.1039/c5md00151j

DeWitt, J. C., Copeland, C. B. and Luebke, R. W. (2009a). Suppression of humoral immunity by perfluorooctanoic acid is independent of elevated serum corticosterone concentration in mice. Toxicol Sci 109, 106-112. doi:10.1093/toxsci/kfp040

DeWitt, J. C., Shnyra, A., Badr, M. Z. et al. (2009b). Immunotoxicity of perfluorooctanoic acid and perfluorooctane sulfonate and the role of peroxisome proliferator-activated receptor alpha. Crit Rev Toxicol 39, 76-94. doi:10.1080/10408440802209804

ECHA (2014). REACH, Proposal for a Restriction – Perfluorooctanoic acid (PFOA), PFOA Salts and PFOA-Related Substances. https://echa.europa.eu/documents/10162/e9cddee6-3164-473d-b590-8fcf9caa50e7

EFSA (2020). Risk to human health related to the presence of perfluoroalkyl substances in food. EFSA J 18, e06223. doi:10.2903/j.efsa.2020.6223

EPA (2000). Perfluorooctyl Sulfonates; Proposed Significant New Use Rule. https://www.gpo.gov/fdsys/pkg/FR-2000-10-18/pdf/00-26751.pdf

EPA (2019). EPA’s Per- and Polyfluoroalkyl Substances (PFAS) Action Plan. https://www.epa.gov/sites/default/files/2019-02/documents/pfas_action_plan_021319_508compliant_1.pdf

EPA (2020). Long-chain Perfluoroalkyl Carboxylate and Perfluoroalkyl Sulfonate Chemical Substances; Significant New Use Rule. https://www.govinfo.gov/content/pkg/FR-2020-07-27/pdf/2020-13738.pdf

EPA (2021). National PFAS Testing Strategy: Identification of Candidate Per- and Polyfluoroalkyl Substances (PFAS) for Testing. https://www.epa.gov/system/files/documents/2021-10/pfas-natl-test-strategy.pdf

Facciotti, F., Larghi, P., Bosotti, R. et al. (2020). Evidence for a pathogenic role of extrafollicular, IL-10-producing CCR6+B helper T cells in systemic lupus erythematosus. Proc Natl Acad Sci USA 117, 7305-7316. doi:10.1073/pnas.1917834117

Filer, D. L., Kothiya, P., Setzer, R. W. et al. (2017). tcpl: The ToxCast pipeline for high-throughput screening data. Bioinformatics 33, 618-620. doi:10.1093/bioinformatics/btw680

Fink, E. E., Mannava, S., Bagati, A. et al. (2016). Mitochondrial thioredoxin reductase regulates major cytotoxicity pathways of proteasome inhibitors in multiple myeloma cells. Leukemia 30, 104-111. doi:10.1038/leu.2015.190

Fu, J., Gaetani, S., Oveisi, F. et al. (2003). Oleylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-alpha. Nature 425, 90-93. doi:10.1038/nature01921

Gaballah, S., Swank, A., Sobus, J. R. et al. (2020). Evaluation of developmental toxicity, developmental neurotoxicity, and tissue dose in zebrafish exposed to GenX and other PFAS. Environ Health Perspect 128, 47005. doi:10.1289/ehp5843

Gerçel-Taylor, C., Ackermann, M. A. and Taylor, D. D. (2001). Evaluation of cell proliferation and cell death based assays in chemosensitivity testing. Anticancer Res 21, 2761-2768.

Griffith, E. C., Su, Z., Niwayama, S. et al. (1998). Molecular recognition of angiogenesis inhibitors fumagillin and ovalicin by methionine aminopeptidase 2. Proc Natl Acad Sci USA 95, 15183-15188. doi:10.1073/pnas.95.26.15183

Grulke, C. M., Williams, A. J., Thillainadarajah, I. et al. (2019). EPA’s DSSTox database: History of development of a curated chemistry resource supporting computation toxicology research. Comput Toxicol 12, 100096. doi:10.1016/j.comtox.2019.100096

Halloran, P. F., Helms, L. M., Kung, L. et al. (1999). The temporal profile of calcineurin inhibition by cyclosporine in vivo. Transplantation 68, 1356-1361. doi:10.1097/00007890-199911150-00023

Hammitzsch, A., Tallant, C., Fedorov, O. et al. (2015). CBP30, a selective CBP/p300 bromodomain inhibitor, suppresses human Th17 responses. Proc Natl Acad Sci USA 112, 10768-10773. doi:10.1073/pnas.1501956112

Houck, K. A., Dix, D. J., Judson, R. S. et al. (2009). Profiling bioactivity of the ToxCast chemical library using BioMAP primary human cell systems. J Biomol Screen 14, 1054-1066. doi:10.1177/1087057109345525

Hungria, V. T. M., Crusoé, E. Q., Bittencourt, R. I. et al. (2019). New proteasome inhibitors in the treatment of multiple myeloma. Hematol Transfus Cell Ther 41, 76-83. doi:10.1016/j.htct.2018.07.003

Itoh, K., Inoue, T., Ito, K. et al. (1994). The interplay of interleukin-10 (IL-10) and interleukin-2 (IL-2) in humoral immune responses: IL-10 synergizes with IL-2 to enhance responses of human B lymphocytes in a mechanism which is different from upregulation of CD25 expression. Cell Immunol 157, 478-488. doi:10.1006/cimm.1994.1243

Ji, C. H. and Kwon, Y. T. (2017). Crosstalk and interplay between the ubiquitin-proteasome system and autophagy. Mol Cells 40, 441-449. doi:10.14348/molcells.2017.0115

Kapetanovic, M. C., Nagel, J., Nordström, I. et al. (2017). Methotrexate reduces vaccine-specific immunoglobulin levels but not numbers of circulating antibody-producing B cells in rheumatoid arthritis after vaccination with a conjugate pneumococcal vaccine. Vaccine 35, 903-908. doi:10.1016/j.vaccine.2016.12.068

Khalesi, N., Korani, S., Korani, M. et al. (2021). Bortezomib: A proteasome inhibitor for the treatment of autoimmune diseases. Inflammopharmacology 29, 1291-1306. doi:10.1007/s10787-021-00863-2

Kisselev, A. F. and Goldberg, A. L. (2001). Proteasome inhibitors: From research tools to drug candidates. Chem Biol 8, 739-758. doi:10.1016/s1074-5521(01)00056-4

Kleinstreuer, N. C., Yang, J., Berg, E. L. et al. (2014). Phenotypic screening of the ToxCast chemical library to classify toxic and therapeutic mechanisms. Nat Biotechnol 32, 583-591. doi:10.1038/nbt.2914

Knight, S. D., Adams, N. D., Burgess, J. L. et al. (2010). Discovery of GSK2126458, a highly potent inhibitor of PI3K and the mammalian target of rapamycin. ACS Med Chem Lett 1, 39-43. doi:10.1021/ml900028r

Kumon, Y., Suehiro, T., Hashimoto, K. et al. (2001). Dexamethasone, but not IL-1 alone, upregulates acute-phase serum amyloid A gene expression and production by cultured human aortic smooth muscle cells. Scand J Immunol 53, 7-12. doi:10.1046/j.1365-3083.2001.00829.x

Kunkel, E. J., Dea, M., Ebens, A. et al. (2004a). An integrative biology approach for analysis of drug action in models of human vascular inflammation. FASEB J 18, 1279-1281. doi:10.1096/fj.04-1538fje

Kunkel, E. J., Plavec, I., Nguyen, D. et al. (2004b). Rapid structure-activity and selectivity analysis of kinase inhibitors by BioMAP analysis in complex human primary cell-based models. Assay Drug Dev Technol 2, 431-441. doi:10.1089/adt.2004.2.431

Lau, C., Anitole, K., Hodes, C. et al. (2007). Perfluoroalkyl acids: A review of monitoring and toxicological findings. Toxicol Sci 99, 366-394. doi:10.1093/toxsci/kfm128

Lee, C. R., Chun, J. N., Kim, S.-Y. et al. (2012). Cyclosporin A suppresses prostate cancer cell growth through CaMKKβ/AMPK-mediated inhibition of mTORC1 signaling. Bioch Pharmacol 84, 425-431. doi:10.1016/j.bcp.2012.05.009

Levitt, D. and Liss, A. (1986). Toxicity of perfluorinated fatty acids for human and murine B cell lines. Toxicol Appl Pharmacol 86, 1-11. doi:10.1016/0041-008x(86)90394-7

Levy, J., Barnett, E. V., MacDonald, N. S. et al. (1972). The effect of azathioprine on gammaglobulin synthesis in man. J Clin Invest 51, 2233-2238. doi:10.1172/jci107031

Liberatore, H. K., Jackson, S. R., Strynar, M. J. et al. (2020). Solvent suitability for HFPO-DA (“GenX” Parent Acid) in toxicological studies. Environ Sci Technol Lett 7, 477-481. doi:10.1021/acs.estlett.0c00323

Loveless, S. E., Hoban, D., Sykes, G. et al. (2008). Evaluation of the immune system in rats and mice administered linear ammonium perfluorooctanoate. Toxicol Sci 105, 86-96. doi:10.1093/toxsci/kfn113

Matsuda, S. and Koyasu, S. (2000). Mechanisms of action of cyclosporine. Immunopharmacology 47, 119-125. doi:10.1016/s0162-3109(00)00192-2

Melton, A. C., Melrose, J., Alajoki, L. et al. (2013). Regulation of IL-17A production is distinct from IL-17F in a primary human cell co-culture model of T cell-mediated B cell activation. PLoS One 8, e58966. doi:10.1371/journal.pone.0058966

NTP (2016). NTP Monograph: Immunotoxicity Associated with Exposure to Perfluorooctanoic Acid or Perfluorooctane Sulfonate. https://ntp.niehs.nih.gov/ntp/ohat/pfoa_pfos/pfoa_pfosmonograph_508.pdf

O’Mahony, A., John, M. R., Cho, H. et al. (2018). Discriminating phenotypic signatures identified for tocilizumab, adalimumab, and tofacitinib monotherapy and their combinations with methotrexate. J Transl Med 16, 156. doi:10.1186/s12967-018-1532-5

OECD (2015). Risk Reduction Approaches for PFASs – A Cross-Country Analysis. Series on Risk Management, No. 29. OECD Environment, Health and Safety Publications, Paris. https://bit.ly/3lSEy3z

OECD (2018). Toward a new comprehensive global database of per- and polyfluoroalkyl substances (PFASs): Summary report on updating the OECD 2007 list of per- and polyfluoroalkyl substances (PFASs). Series on Risk Management, No. 39. OECD Environment Health and Safety Publications. https://bit.ly/3klctBG

OECD (2021). Reconciling Terminology of the Universe of Per- and Polyfluoroalkyl Substances: Recommendations and Practical Guidance. Series on Risk Management, No. 61. OECD Environment, Health and Safety Publications, Paris. https://bit.ly/3m03xBX

Ojo, A. F., Peng, C. and Ng, J. C. (2020). Assessing the human health risks of per- and polyfluoroalkyl substances: A need for greater focus on their interactions as mixtures. J Hazard Mater 407, 124863. doi:10.1016/j.jhazmat.2020.124863

Patlewicz, G., Richard, A. M., Williams, A. J. et al. (2019). A chemical category-based prioritization approach for selecting 75 per- and polyfluoroalkyl substances (PFAS) for tiered toxicity and toxicokinetic testing. Environ Health Perspect 127, 14501. doi:10.1289/ehp4555

Peden-Adams, M. M., Keller, J. M., Eudaly, J. G. et al. (2008). Suppression of humoral immunity in mice following exposure to perfluorooctane sulfonate. Toxicol Sci 104, 144-154. doi:10.1093/toxsci/kfn059

Perkins, R. G., Butenhoff, J. L., Kennedy, G. L. et al. (2004). 13‐week dietary toxicity study of ammonium perfluorooctanoate (APFO) in male rats. Drug Chem Toxicol 27, 361-378. doi:10.1081/dct-200039773

Popa-Burke, I. G., Issakova, O., Arroway, J. D. et al. (2004). Streamlined system for purifying and quantifying a diverse library of compounds and the effect of compound concentration measurements on the accurate interpretation of biological assay results. Anal Chem 76, 7278-7287. doi:10.1021/ac0491859

Rackham, O. J., Sills, J. A. and Davidson, J. E. (2002). Immunoglobulin levels in methotrexate treated paediatric rheumatology patients. Arch Dis Child 87, 147-148. doi:10.1136/adc.87.2.147

Sakamoto, J., Kimura, H., Moriyama, S. et al. (2000). Activation of human peroxisome proliferator-activated receptor (PPAR) subtypes by pioglitazone. Biochem Biophys Res Comm 278, 704-711. doi:10.1006/bbrc.2000.3868

Scepanovic, P., Alanio, C., Hammer, C. et al. (2018). Human genetic variants and age are the strongest predictors of humoral immune responses to common pathogens and vaccines. Genome Med 10, 59. doi:10.1186/s13073-018-0568-8

Shah, F., Stepan, A. F., O’Mahony, A. et al. (2017). Mechanisms of skin toxicity associated with metabotropic glutamate receptor 5 negative allosteric modulators. Cell Chem Biol 24, 858-869.e5. doi:10.1016/j.chembiol.2017.06.003

Simms, L., Mason, E., Berg, E. L. et al. (2021). Use of a rapid human primary cell-based disease screening model, to compare next generation products to combustible cigarettes. Curr Res Toxicol 2, 309-321. doi:10.1016/j.crtox.2021.08.003

Singer, J. W., Al-Fayoumi, S., Taylor, J. et al. (2019). Comparative phenotypic profiling of the JAK2 inhibitors ruxolitinib, fedratinib, momelotinib, and pacritinib reveals distinct mechanistic signatures. PLoS One 14, e0222944. doi:10.1371/journal.pone.0222944

Smeltz, M. G., Clifton, M. S., Henderson, W. M. et al. (in preparation). An analytical framework to evaluate per- and poly-fluoroalkyl substances (PFAS) stock quality for in vitro high-throughput toxicity testing.

Stahn, C., Löwenberg, M., Hommes, D. W. et al. (2007). Molecular mechanisms of glucocorticoid action and selective glucocorticoid receptor agonists. Mol Cell Endocrinol 275, 71-78. doi:10.1016/j.mce.2007.05.019

Steenland, K., Tinker, S., Frisbee, S. et al. (2009). Association of perfluorooctanoic acid and perfluorooctane sulfonate with serum lipids among adults living near a chemical plant. Am J Epidemiol 170, 1268-1278. doi:10.1093/aje/kwp279

UN – United Nations (2020). Stockholm Convention on Persistent Organic Pollutants. Annex A and B. http://www.pops.int/Portals/0/download.aspx?d=UNEP-POPS-COP-CONVTEXT-2021.English.pdf

Wambaugh, J. F., Wetmore, B. A., Ring, C. L. et al. (2019). Assessing toxicokinetic uncertainty and variability in risk prioritization. Toxicol Sci 172, 235-251. doi:10.1093/toxsci/kfz205

Wang, Z., DeWitt, J. C., Higgins, C. P. et al. (2017). A never-ending story of per- and polyfluoroalkyl substances (PFASs)? Environ Sci Technol 51, 2508-2518. doi:10.1021/acs.est.6b04806

Williams, A. J., Gaines, L. G. T., Grulke, C. M. et al. (2022). Assembly and curation of lists of per- and polyfluoroalkyl substances (PFAS) to support environmental science research. Front Environ Sci 10, 1-13. doi:10.3389/fenvs.2022.850019

Yang, C., Tarkhov, A., Marusczyk, J. et al. (2015). New publicly available chemical query language, CSRML, to support chemotype representations for application to data mining and modeling. J Chem Inf Model 55, 510-528. doi:10.1021/ci500667v

Yang, D., Han, J., Hall, D. R. et al. (2020). Nontarget screening of per- and polyfluoroalkyl substances binding to human liver fatty acid binding protein. Environ Sci Technol 54, 5676-5686. doi:10.1021/acs.est.0c00049

Young, P. W., Buckle, D. R., Cantello, B. C. et al. (1998). Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma. J Pharmacol Exp Ther 284, 751-759.

Yu, S. and Reddy, J. K. (2007). Transcription coactivators for peroxisome proliferator-activated receptors. Biochim Biophys Acta 1771, 936-951. doi:10.1016/j.bbalip.2007.01.008

Zhang, C., McElroy, A. C., Liberatore, H. K. et al. (2021). Stability of per- and polyfluoroalkyl substances in solvents relevant to environmental and toxicological analysis. Environ Sci Technol 56, 6103-6112. doi:10.1021/acs.est.1c03979

Zhang, W., Pang, S., Lin, Z. et al. (2020). Biotransformation of perfluoroalkyl acid precursors from various environmental systems: Advances and perspectives. Environ Pollut 272, 115908. doi:10.1016/j.envpol.2020.115908

Most read articles by the same author(s)