Unravelling immunomodulatory effects of bisphenol A substitutes on human macrophages, T and B lymphocytes using in vitro models

Unravelling immunomodulatory effects of bisphenol A substitutes on human macrophages, T and B lymphocytes using in vitro models Nina Franko , Tijana Markoviˇ c , Pia ˇ Ziˇ zek , Anja Kodila , Irena Mlinariˇ c Raˇ sˇ can, Marija Sollner Dolenc * University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia ARTICLE INFO Edited by Tao Zhang Keywords: Bisphenol A BPA substitutes Pergafast201 Immunotoxicity Cytokine Endocrine disruption Immunomodulation TLR4 MHC-I NF-kB/AP-1 ABSTRACT This study investigates the immunomodulatory effects of BPA substitutes using in silico and in vitro approaches. A comprehensive in silico screening with Endocrine Disruptome of BPA and its 25 substitutes revealed that BPG, BPS-MAE, BPS-MPE, BPPH and PF201 have the highest potential to bind nuclear receptors. Based on the in silico ranking, 12 compounds were selected and tested in vitro to investigate their effects on THP-1 derived macrophages, Jurkat T cells and LCLs. The metabolic activity tests showed that BPA has IC 50 values of 99–182 µM, while the values for BPG, BPPH and BPP are 5–10 times lower. Comparison of the IC 50 values with the logP of the tested compounds showed that the BPA analogues affect cell viability in proportion to their lipophilicity (R 2 = 0.9185). The results of the cytokine release assays showed that the BPA substitutes generally stimulated the proinflammatory response in THP-1 macrophages in a statistically significant manner when exposed to the environmentally relevant concentrations and suppressed it in the micromolar range in all cell lines tested. Further analysis using RamosBlue reporter cells indicated that BPAP, BPG, BPP and BPPH alter the NF-κB/AP-1 signalling pathway. Moreover, BPAP, BPG, BPP, BPPH, BPS-MAE, BPS-MPE, BTUM and PF201 were predicted in silico to be TLR4-MyD88 inhibitors. The overall results of this study identify BPG, BPP, BPPH, BPZ and TCBPA as BPA analogues with the highest immunomodulatory potential. In addition, the immunomodulatory potential of the BPA alternatives Pergafast201 and BTUM was confirmed in vitro for the first time. This study emphasises the need for cautious evaluation of BPA substitutes due to their potential immunomodulatory effect, which could significantly affect public health. 1. Introduction Bisphenol A (BPA) is a notorious building block of polycarbonate plastics that is also found in epoxy resins of faucets and can coatings, paints, thermal paper, dental sealants, and medical implants (Testai et al., 2016; Bisphenol A - EFSA). Because it is so widespread, it poses a major risk to human exposure – it can leach from plastic packaging into food and from taps into drinking water, as it can also penetrate through the skin from thermal paper (Zalko et al., 2011; Bisphenol A - EFSA). The European Food Safety Authority (EFSA) estimates that BPA intake via food is the highest, followed by transdermal exposure. In addition, house dust and toys are also a significant source of BPA, especially for children under the age of three (Bolognesi et al., 2015). Nevertheless, BPA is now widespread and is found in soil (Xu et al., 2021), surface water and seawater (ˇ Cesen et al., 2018; Han et al., 2023; Shimabuku et al., 2022; Wang et al., 2022), where it poses an ecotoxicological risk to the environment, animals and plants (Franko et al., 2024; Gomes et al., 2019; Wang et al., 2019; Zhang et al., 2021). Overall exposure to BPA is problematic due to its numerous harmful effects. First, it is a known endocrine disruptor, acting as an agonist of estrogen (ER) (Durcik et al., 2022) and antagonist of androgen (AR) and glucocorticoid receptors (GR) (Ma et al., 2022). Exposure to BPA has been negatively associated with markers of ovarian reserve, and some studies have found an association between BPA and polycystic ovary syndrome (Stavridis et al., 2022). BPA is also able to cross the placenta (Balakrishnan et al., 2010), and prenatal exposure has been associated with impaired male fertility and changes in white matter microstructure in the brains of preschool children (Grohs et al., 2019; Hart, 2020). In recent years, BPA has attracted more attention due to its immunotoxic effects, as it can affect various types of immune cells (Kodila et al., 2023). For example, it can downregulate the expression of CD1a and upregulate DC-SIGN in immature dendritic cells and reduce their * Corresponding author. E-mail address: [email protected] (M. Sollner Dolenc). Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv https://doi.org/10.1016/j.ecoenv.2025.118406 Received 1 February 2025; Received in revised form 20 May 2025; Accepted 21 May 2025 Ecotoxicology and Environmental Safety 300 (2025) 118406 Available online 30 May 2025 0147-6513/© 2025 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC license ( http://creativecommons.org/licenses/bync/4.0/ ). endocytotic capacity (ˇ Svajger et al., 2016), it disrupts the ratio between Th1 and Th2 cells and exerts the inflammatory effects by inducing the PI3K/Akt/mTOR signalling pathway (Gao et al., 2020). In monocytes, BPA reduces the release of IL-8 and TNF α by reducing the transcriptional activity of the RACK1 promoter (Buoso et al., 2021) and the expression of the surface markers CD54 and CD86 (ˇ Strukelj Pahovi´ c et al., 2023). The immune system’s pivotal role in maintaining homeostasis and defending against pathogens makes it a critical target for toxicological evaluation. Disruption of immune function can have profound and systemic health consequences, potentially leading to increased susceptibility to infections, autoimmune diseases, and other immune-related disorders. The finding that BPA increases Th17 levels in mice, leading to an increase in pro-inflammatory conditions, prompted EFSA to reduce the permitted daily intake by 20,000-fold, from 4 µg/kg to 0.2 ng/kg body weight in 2023 (Lambr´ e et al., 2023). This is one of the latest regulatory restrictions on the use of BPA – previously its use was banned in baby bottles and drinking cups (Bisphenol A in Batch 2 of the Challenge; 2011/8/EU) and its concentration in thermal paper was limited to <0.02 % by weight from 2020 (2016/2235 Annex XVII). At the end of 2024, the EFSA finally banned BPA materials that come into contact with food (Ban of Bisphenol A in food contact materials). To circumvent these restrictions, the industry has adapted by using BPA substitutes, which may be structural analogues of BPA (e.g., bisphenols S, F and AF) alternatives whose structure no longer resembles bisphenol (e.g., 4,4 ′ -bis (p-toluenesulfonylaminocarbonylamino)diphenylmethane (BTUM) and Pergafast201 (PF201)). As a result, new BPA analogues and alternatives are now found in consumer products, in environment and biological samples (Frederiksen et al., 2020; Huang et al., 2020; BPA being replaced by BPS in thermal paper). However, the studies have shown that they exhibit toxic effects as well (Kodila et al., 2023; Mokra et al., 2018; Shi et al., 2020; Winkler et al., 2022). For example, the human biomonitoring studies associated maternal exposure to bisphenol AP (BPAP) or AF (BPAF) with a higher risk of unexplained recurrent miscarriages (Ao et al., 2022), and adult exposures to bisphenol S (BPS) with obesity (Choi et al., 2022), to BPS or BPAF with an increased likelihood of developing polycystic ovary syndrome (Zhan et al., 2023) and to bisphenol F (BPF) with impaired thyroid function (Pei et al., 2025) There have already been proposals to restrict the use of bisphenols B, S, F and AF, although no legal action has yet been taken (ECHA: Germany proposes a restriction on bisphenol A and other bisphenols with endocrine disrupting properties for the environment). In 2015, the United States Environmental Protection Agency (EPA) compiled a list of possible BPA substitutes in thermal paper (EPA: Bisphenol A alternatives in thermal paper). This report points out that there is insufficient data available to assess the effects of the listed compounds on the human immune system. In 2021, the European Chemicals Agency (ECHA) categorised BPA substitutes into four groups based on available data: 1) compounds for which restriction is required; 2) compounds for which data are currently being generated; 3) compounds for which there is currently no need for regulatory risk management in the EU; and 4) compounds for which the hazards cannot be clarified due to lack of data (ECHA: Assessment of regulatory needs). Literature data indicate that the compounds mentioned in the above documents, including ECHA’s list of compounds for which there is insufficient data, are already in use and therefore pose a risk to human exposure (Franko et al., 2024). For example, bisphenol S 4-allyl ether (BPS-MAE), PF201 and BTUM are contained in thermal paper (Bj¨ ornsdotter et al., 2017; Eckardt and Simat, 2017; Goldinger et al., 2015), bisphenols PH (BPPH), G (BPG) and TMC (BP-TMC) in baby bottles (Siddique et al., 2021), bisphenol E (BPE) and Z (BPZ) in honey (ˇ Cesen et al., 2016) and BPZ also in infant formula (Karsauliya et al., 2021). Not surprisingly, these analogues are already detected in human serum. While exposure to BPA seems to be the highest in industrial areas, where the mean detected concentration was 42.1 ng/mL (equivalent to 184 nM), the detected concentrations of its substitutes are lower – the maximum serum concentrations detected in human biomonitoring studies were e.g., 0.828 ng/mL (4 nM) for BPE, 1.190 ng/mL (3.8 nM) for BPG, 0.917 ng/mL (2.5 nM) for bisphenol P (BPP) and 1.415 ng/mL (5.2 nM) for BPZ (Owczarek et al., 2018). Moreover, there is already evidence of their emergence. Frederiksen et al. showed that the exposure of Danish boys to BPG and BPE increased sharply between 2009 and 2017 (Frederiksen et al., 2020). Increasing exposure to BPAP, BPP and BPZ was observed in China, where the average serum concentrations of BPAP and BPZ have increased about 10-fold since 2015, while those of BPP doubled. These substances are now found in about 90 % of the population (Franko et al., 2024; H. Wang et al., 2023). While some studies have examined the endocrine effects of these substitutes, their immunomodulatory potential remains largely unexplored. Adverse outcome pathways (AOPs) describe a sequence of key events leading to an adverse outcome that occurs after the molecular initiating event. Several studies have recognised that immunotoxicity may be a consequence of endocrine disruption. For example, an AOP14 (as deposited in the AOP-WIKI (AOP-wiki, 2023a)) describes that activation of the glucocorticoid receptor leads to increased susceptibility to disease due to inhibition of NF-κB, suppression of inflammatory cytokines and reduced number of lymphocytes (AOP-wiki, 2023a). In addition, AOP314 finds that binding to ER α in immune cells leads to an exacerbation of systemic lupus erythematosus following the induction of GATA3 expression, an increase of Th2 cells producing IL-4 and an increase of anti-DNA antibodies from autoreactive B cells (AOP-wiki, 2023b). Furthermore, a 2021 systematic review proposed new AOPs leading to immunotoxicity based on the proven mechanisms of endocrine disrupting chemicals. The authors recognised that immunotoxicity could be the result of activation of the aryl hydrocarbon receptor (AhR), ER or peroxisome proliferator–activated receptor (PPAR), although the proposed key molecular and cellular events are cell-type specific. For example, activation of the ER is proposed to upregulate IL-6 and decrease antigen presentation, leading to an increase in inflammatory dendritic cells, whereas activation of PPAR upregulates Blimp-1 and impairs B cell differentiation, cell number and immunoglobulin production (Sabuz Vidal et al., 2021). Moreover, several in vitro studies have shown correlations between the activation/suppression of nuclear receptors and immunomodulatory effects (Table 1). In this study, we aimed to fill the knowledge gaps regarding the immunomodulatory functions of the new BPA substitutes by using cell lines as in vitro models for macrophages, T cells and B cells. Although the cell lines are subjected to genotypic and phenotypic changes and may therefore inadequately represent primary cells, they allow reproducible screening assays to be performed at low cost (Gennari et al., 2005; Kaur and Dufour, 2012). The selection of THP-1, Jurkat, and lymphoblastoid cell lines (LCL) for immunotoxicity evaluation of bisphenols was grounded in their established roles as effective models for assessing immunomodulation. Each cell line offers unique advantages that contribute to a comprehensive understanding of immunotoxic effects. THP-1 cells are human monocytic leukaemia cells that can differentiate into macrophageand dendritic cell-like cells, making them suitable for studying antigen-presenting cell activation (Chanput et al., 2014; Franko and Sollner Dolenc, 2024). Jurkat cells are a well-characterized human T-cell line, ideal for studying T-cell responses and signalling pathways related to immunotoxicity (OECD, 2023; Schmeits et al., 2015). LCL cells obtained from healthy individuals represent B-lymphocytes, providing a complementary perspective to T-cell and monocytic models in immunotoxicity studies (Markoviˇ c et al., 2015). To select the compounds to be tested, we first performed an in silico analysis of the endocrine effects of BPA and its 25 substitutes. We hypothesised that the more nuclear receptors are targeted by the compound, the greater the immunomodulatory potential. Therefore, on the basis of in silico screening with Endocrine Disruptome we selected 11 compounds (Table 2) that had the highest biding affinities to most nuclear receptors and investigated their effects on cellular metabolic activity and the release of cytokines from in vitro models representing human macrophages, T cells and B cells. Understanding the immunotoxic effects of N. Franko et al. Ecotoxicology and Environmental Safety 300 (2025) 118406 2 these chemicals is critical for assessing their potential risks and making regulatory decisions to protect public health. 2. Materials and methods 2.1. In silico screening In addition to BPA, a series of 25 BPA substitutes were selected on the basis of EPA’s list of BPA substitutes in thermal paper (EPA: Bisphenol A alternatives in thermal paper) and ECHA’s list of compounds whose hazard cannot be clarified due to the lack of data (ECHA: Assessment of regulatory needs) and analysed with Endocrine Disruptome (ED) (Kolˇ sek et al., 2014), available at http://endocrinedisruptome.ki.si/. Molecules of interest were prepared as SMILE strings and inserted into ED. The software is able to estimate the binding affinity of small molecules to 12 nuclear receptors (androgen receptor (AR), estrogen receptors α (ER α ) and β (ERβ), glucocorticoid receptor (GR), liver X receptors α (LXR α ) and β (LXRβ), mineralocorticoid receptor (MR), peroxisome proliferator-activated receptors α (PPAR α ), β (PPARβ) and γ (PPARγ), progesterone receptor (PR), retinoid X receptor α (RXR α ), thyroid receptors α (TR α ) and β (TRβ)). For the ERs, AR and GR, the binding affinities are predicted for both agonistic and antagonistic (an.) conformations. Docking results were displayed as a score of predicted binding energy (kcal x mol −1 ) and the output was colour coded based on sensitivity (derived from thresholds specific to each receptor), with red (sensitivity <0.25) indicating a high binding probability, orange (0.25 <sensitivity <0.5) a moderate binding probability, yellow (0.5 < sensitivity <0.75) a minor binding probability and green (sensitivity > 0.75) indicating a low binding probability. The threshold values for the binding energies for colour classification of the compounds were determined during model validation and are listed in Table S1. This in silico tool is suitable for the characterisation of molecules with a molecular weight of up to 600 g/mol, without multiple ionisation sites and without boron atoms. For each receptor (including agonistic and antagonistic modes for AR, GR and ERs), the compounds were ranked from 1 to 26, with the first rank representing the highest binding potential and thus the lowest predicted binding energy. If two or more compounds had the same binding energy, they were all assigned the same rank. A comprehensive ranking was then performed by calculating the sum of all ranks for each compound. The compounds with the lower sum have a higher potential for endocrine disruption (See Table 3 and Table S2). Predictions of compound binding to TLR4 were performed using AICpred, machine learning-based web application using eXtreme gradient boosting (XGBoost) model (Fry-Nartey et al., 2025). Molecules were prepared as SMILE strings obtained from PubChem database and copy-pasted into AICpred. Results are given as Active/Inactive. Predictions of compound binding to MHC-I (HLA class I histocompatibility antigen, A-2 alpha chain) were performed using 1-Click Docking (http s://mcule.com/apps/1-click-docking/). Molecules were prepared as SMILE strings obtained from PubChem database and copy-pasted into 1-Click Docking and docked into default binding centre of 2XHN. The software outputs are docking scores to four docking poses. The arithmetic means of all four poses were calculated, where the more negative number presents the higher binding affinity. 2.2. Chemicals Bisphenols BPA (99.9 %; CAS 80–05–7), BPE (99.9 %; CAS 2081–05–5), BPAP (99.9 %; CAS 1571–75–1), BPP (99.9 %; CAS 2167–51–3), BPZ (99.9 %; CAS 843–55–0), TCBPA (99.8 %; CAS 79–95–8) and BPS-MAE (99.9 %; CAS 97042–18–7) were obtained by Chiron AS, Norway. BPPH (98 %; CAS 24038–68–4), BPS-MPE (98 %; CAS 63134–33–8), BTUM (98 %; CAS 151882–81–4) and PF201 (98 %; CAS 232938–43–1) were obtained by Toronto Research Chemicals, Canada. BPG (≥98.0 %; CAS 127–54–8) and hydrocortisone (≥98 %; CAS 50–23–7) were obtained by Sigma-Aldrich, St. Louis, MO, USA. All compounds were solubilized as 50 mM stocks in dimethyl sulfoxide (DMSO; ACS grade, ≥99.9 %) by Sigma-Aldrich. Mirdametinib (>99 %; CAS 391210–10–9) was obtained by MedChemExpress as 10 mM stock. LogP and TPSA were calculated using Molinspration (Molinspriation). Other drug-likeness parameters (Table S3) were calculated using SwissADME (Daina et al., 2017) and cellular permeability data were obtained from ADMETlab 3.0 database (ADMETlab). 2.3. Cell lines, cell culture and macrophage differentiation The cell lines THP-1 and Jurkat (clone E6–1) (ATCC Jurkat; ATCC THP-1) were obtained from the American Type Culture Collection (ATCC, Manassas, USA). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich) supplemented with 10 % foetal bovine serum (Gibco, ThermoFisher Scientific, Waltham, MA, USA), 2 mM L-glutamine (Sigma-Aldrich) and 1 mM sodium pyruvate (Sigma-Aldrich). Human lymphoblastoid cell lines (LCLs) were acquired from the National Laboratory for the Genetics of Israeli Populations (NLGIP), a human diversity biobank based at Tel-Aviv University, Israel (Markoviˇ c et al., 2015). The cell lines were generated from fresh lymphocytes isolated from blood samples donated for this purpose by Table 1 Involvement of nuclear receptors in the processes of immunomodulation. nuclear receptor model pathway outcome compound reference ER THP−1 derived macrophages ER α /β/ERK/NFκB↑TNF α , IL−6↓IL−10, TGFβBPA (Liu et al., 2014) Macrophages IRF5 Promoted proinflammatory M1 phenotype BPA (Lu et al., 2019) Macrophages ER/JAK2/STAT3ER/ PI3K/AKT BPF (Kodila et al., 2023) Jurkat T cells GPER1/PI3K/Akt Proinflammatory effects TCBPA (Lu et al., 2024) Jurkat T cells, primary T cells ER/NFκB/IL−2↓IL−2 17-β-estradiol (McMurray et al., 2001) GR T cells GR/GILZ/ NFAT/AP−1/ IL−2 ↓IL−2 dexamethasone (Mittelstadt and Ashwell, 2001) THP−1 monocytes GR/RACK1 Decreased RACK1 expression BPA, BPAF (Buoso et al., 2021) LCL N/A ↑IL−10↓ TNF α , IL−2 hydrocortisone (Klopˇ ciˇ c et al., 2018) AR THP−1 monocytes AR/RACK1/NFκB Increased RACK1 expression BPAF (Buoso et al., 2021) LCL N/A ↓ TNF α , IL−2, IL−6 diclofenac (Klopˇ ciˇ c et al., 2018) PPAR Macrophages N/A Promoted anti-inflammatory M2 phenotype N/A (comparison with PPAR knockouts) (Christofides et al., 2021) T cells N/A Suppressed Th1 and TH17 differentiation rosiglitazone N/A: not applicable. LCL – lymphoblastoid cell lines. N. Franko et al. Ecotoxicology and Environmental Safety 300 (2025) 118406 3 consenting healthy adults. The experiments were performed with cell lines derived from three different donors. Cells were grown in RPMI 1640 medium (Sigma–Aldrich) supplemented with 10 % foetal bovine serum (Gibco, Thermo Scientific, Waltham, MA, USA), 4 mM L-glutamine (Sigma-Aldrich) and 1 % (v/v) penicillin/streptomycin (Sigma-Aldrich). Prior to the experiment, all cell lines were washed with phosphate buffer saline (PBS) and transferred to assay medium RPMI 1640 without phenol red (Sigma-Aldrich) supplemented with 5 % heat-inactivated charcoal-stripped foetal bovine serum (Gibco), 2 mM L-glutamine and 1 mM sodium pyruvate. Table 2 List of BPA analogues and alternatives, their MW and logP. LogP was calculated by Molinspiration. compound structure MW logP BPA 228.29 3.37 BPAP 290.36 4.58 BPE 214.26 2.92 BPG 312.45 6.24 BPP 346.46 5.57 BPPH 380.49 6.91 BPS-MAE 290.34 3.00 BPS-MPE 340.39 3.95 BPZ 268.35 4.53 TCBPA 366.07 6.31 BTUM 592.69 5.63 PF201 460.52 4.19 N. Franko et al. Ecotoxicology and Environmental Safety 300 (2025) 118406 4 Ramos Blue cells were purchased from InvivoGen (San Diego, CA, United States). The cells carry stable expression of an NF-κB/AP-1inducible reporter gene for secreted embryonic alkaline phosphatase (SEAP) (Gobec et al., 2015). The cell line was cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco, Thermo Scientific, Waltham, MA, USA) supplemented with 10 % heat-inactivated foetal bovine serum, 2 mM L-glutamine and 1 % (v/v) penicillin-streptomycin. For alternate passages, we performed a selection procedure with 100 µg/mL Zeocin (InvivoGen, San Diego, CA, USA). For differentiation into macrophages, THP-1 cells were prepared at a concentration of 5 x 10 5 cells/mL in assay medium and exposed to 80 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 72 h. Attached macrophages were then rested in the assay medium for 24 h before performing experiment. All cultures were maintained in humidified incubator with 5 % CO 2 atmosphere at 37 ◦C. 2.4. Metabolic activity assays For the evaluation of metabolic activity of cells after exposure to bisphenols, 5 x 10 4 cells/well of THP-1 derived macrophages or 3 x 10 4 cells/well of Jurkat or LCL cells were seeded in black 96-well plates. The stock solutions of the compounds were appropriately diluted in DMSO to reach 200-fold the final concentration, then diluted 20-fold in the test medium and then diluted 10-fold in the cell suspensions to reach the final concentration. The highest concentration of the compound tested was 250 µM. As control samples, the cells were exposed to the vehicle control (0.5 % DMSO). The final volume in the wells was 100 µL. After 24 h of incubation, 10 µL of 400 µM resazurin (Sigma-Aldrich), dissolved in PBS, was added to the cells and incubated for a further 3 h. Resorufin fluorescence was measured at λ ex /λ em =530/590 nm using an automated plate reader (Synergy 4 Hybrid; BioTek, Winooski, VT, USA). Three independent experiments were performed in technical duplicates. IC 50 values were calculated using GraphPad Prism 10.1.2 (San Diego, CA, USA). 2.5. Cell activations and cytokine measuerements 2.5.1. THP-1 derived macrophages THP-1 derived macrophages were prepared in 24-well plates at a concentration 5 x 10 5 cells/mL in 500 µL of the assay medium. Cells were pretreated for 2 h with increasing concentrations of the compounds or vehicle control (0.02 % DMSO) and then stimulated for 24 h with 10 ng/mL lipopolysaccharides of Escherichia coli O111:B4 (LPS; Sigma-Aldrich). Supernatants were collected by centrifugation and stored at –80 ◦C until analysis. IL-1β, IL-6, IL-8, IL-10, TNF α and IL12p70 were measured in the cell culture supernatants using the Human Inflammatory Cytokine Cytometric Bead Array (CBA) – I Kit (BD Biosciences, San Diego CA, USA) according to the manufacturer’s instructions (CBA Human Inflammatory kit). Standard curves were generated using recombinant cytokines from the kit. At least three independent experiments were performed. Results are expressed as arithmetic mean with standard error of the mean. The Shapiro – Wilk test showed that data used for the statistical analysis passed the normality test. The homogeneity of variances was tested using the Brown-Forsythe test. The statistical analysis was performed using Dunnett’s multiple comparison and if significant differences in the standard deviations were found in the Brown-Forsythe test, the Geisser-Greenhouse’s correction was applied. The statistical analysis was performed in GraphPad Prism 10.1.2. 2.5.2. Jurkat T-cells To evaluate the effects of the substances on the release of IL-2 from T lymphocytes, 500 µl Jurkat cells were seeded in 24-well plates at a concentration of 3 x 10 5 cells/mL in the assay medium. Cells were pretreated for 2 h with increasing concentrations of the compounds or an appropriate vehicle control and then stimulated for 24 h with 1 µM ionomycin (Sigma-Aldrich) and 50 nM PMA. The supernatants were collected by centrifugation and stored at –80 ◦C until analysis. IL-2, a marker for T cell activation, was measured in the culture supernatants by Enzyme-linked immunosorbent assay (ELISA) (Invitrogen, Waltham, Table 3 Results of in silico analysis of 26 compounds with Endocrine Disruptome. Colour coded fields represent high (red), moderate (orange), minor (yellow) and low (green) binding probabilities based on sensitivity (derived from thresholds specific to each receptor). Values within the colour coded fields represent ranks of each compound (1−26) for the binding to the receptor, where the lower number represents higher binding affinity comparing to the higher number for the receptor. Compounds (N =12) in bold were selected for the in vitro assays. N. Franko et al. Ecotoxicology and Environmental Safety 300 (2025) 118406 5 MA, USA) according to the manufacturer’s instructions (Human IL-2 Uncoated ELISA Kit). At least three independent experiments were performed in technical duplicates. IC 50 values were calculated using GraphPad Prism 10.1.2. The results are given as arithmetic mean with standard error. 2.5.3. Lymphoblastoid cell lines To evaluate the effects of the compounds on LCL cells, cytokine analysis was performed as previously described (Klopˇ ciˇ c et al., 2018; Markoviˇ c et al., 2015). In brief, cells were plated (1 x 10 6 cells/mL) were plated and pre-treated with the compounds for 1 h. They were then activated with 0.5 µM ionomycin and 3.33 ng/mL PMA and incubated for 24 h. The quiescent, untreated cells or the cells stimulated with ionomycin/PMA were used as controls. The cell-free supernatants were collected by centrifugation and stored at −80 ˚C until analysis. The cytokines released from the LCLs was measured using the BD cytometric Bead Array (CBA) – Human Th1/Th2/Th17 cytokine kit (BD Biosciences) in accordance to manufacturer’s instruction (CBA Human Th1/Th2/Th17). At least three independent experiments were performed. Results are expressed as arithmetic mean with standard error of the mean. Statistical analysis was performed as described in 2.5.1. 2.5.4. QUANTI-Blue™ Solution (InvivoGen) colorimetric enzyme assay Quanti-Blue assay was performed as described previously (Nabergoj et al., 2021). Briefly, before performing the experiment, the IMDM medium was inactivated at 56 ◦C to deactivate any residual SEAP activity that could interfere with the experiment. Cells were then harvested and seeded onto a 24-well plate at a concentration of 2 x 10 6 cells/mL and treated with appropriate concentrations of the tested compounds. After a one-hour incubation, the cells were stimulated with 10 ng/mL TNF α , transferred to a 96-well plate and incubated for a further 24 h. At the end of the incubation period, 40 μ l supernatant was collected and 160 μ l Quanti Blue reagent was added. After 30 min, the absorbance was measured at 640 nm using a microplate reader (Synergy 4 Hybrid; BioTek, Winooski, VT, USA). Data were normalised to the TNF α -activated control. At the end of the incubation period, cell viability was determined by tetrazolium MTS assay using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. 10 μ L of MTS reagent was added to 160 μ L of cell suspension. Assays were performed in at least two independent experiments in 96-well plates. Absorbance was measured after 3 h at 492 nm on an automated microplate reader (Synergy 4 Hybrid; BioTek, Winooski, VT, USA). Quanti Blue data were normalised to the relative metabolic activity of the cells. 3. Results and discussion 3.1. In silico screening To narrow down the set of compounds to be tested in immunomodulatory assays in vitro, we performed an in silico screening of BPA and its 25 substitutes with Endocrine Disruptome (ED), in which compounds were ranked based on their likelihood of binding to nuclear receptors. The predicted binding affinities are listed in Table S2 and the rankings including the comprehensive ranking are shown in Table 3. As shown in Table 3, the ED predicted that all compounds have a binding affinity to GR, a known immunomodulator (AOP-wiki, 2023a). Bisphenols were predicted to bind predominantly in agonistic mode to GR, albeit with minor binding probability (yellow class). However, BPPH has been found to be a potent GR agonist and antagonist and PF201, BPFL and BTUM are potent antagonists. The current in vitro studies show that the majority of BPA analogues act as GR antagonists in the micromolar range (Ma et al., 2022). However, consistent with ED predictions, BPE has been found to be a GR agonist (Ma et al., 2022), while there is evidence that BPPH can act as GR agonist (Chen et al., 2020) and antagonist (Grimaldi et al., 2019). To the best of our knowledge, there are no in vitro data available to support or omit the interaction of PF201 and BTUM with GR (Franko et al., 2024). The ED predicted that most of the tested bisphenols act as antagonists of AR, which is well supported by several in vitro studies (Grimaldi et al., 2019; Ma et al., 2022; Pelch et al., 2019). For the alternative PF201, however, the ED predicted a minor probability of binding to the AR, although no binding affinity was demonstrated in vitro (Keminer et al., 2020). The ER is a known target of bisphenols. BPA is able to agonise both ER α and ERβ (Durcik et al., 2022; Grimaldi et al., 2019), although its estrogenic potential is about 100,000 times weaker than that of 17-β-estradiol (E2) (Grignard et al., 2012). In our in silico screening, ED predicted that several compounds, including BPS-MAE, bisphenol S MPE (BPS-MPE), BPZ, BPAP and BTUM have a higher probability of binding to ER α as agonists or to ERβ as antagonists than BPA. The available in vitro data support the predictions that BPZ and BPAP are ER α and ERβ agonists. As shown in the assay using HeLa cells with ERE-luciferase plasmid (HELN ER) by Grimaldi et al., the estrogenic potency of BPAP is lower than that of BPA, while BPZ appears to be more potent (Grimaldi et al., 2019). In contrast to the predictions of the ED, the antiestrogenic potency of BPAP and BPZ has not been confirmed in vitro. Furthermore, although only two studies are available to date, no interaction of BPS-MAE and BPS-MPE with the ER has been demonstrated (Keminer et al., 2020; Pelch et al., 2019) and no data are available to support or exclude the estrogenic potency of BTUM. Several bisphenols were found in the ED as TR α /β binders, with BPPH standing out with a strong binding affinity to TRβ. Only limited data are available to support these results. While BPE showed no activity in the (anti)-TR α /βChemical-Activated Luciferase Gene Expression (CALUX) assay, it acted as a TR α agonist in the micromolar range in the yeast two-hybrid assay (Lei et al., 2017; ˇ Sauer et al., 2021). Currently, there is no data available to support the predictions that tetra-chloro-BPA (TCBPA) targets human TR α /β, although it may be a weak agonist of the rat isozyme (Mhaouty-Kodja et al., 2024). In agreement with Grimaldi et al., ED predicted that BPZ, BPAP, BPE and BPA interact with MR, while it missed the demonstrated interactions of BPBP and BPPH with this receptor (Grimaldi et al., 2019). There are also in vitro data confirming the interactions of BPAP and BPPH with PR, while BPBP, BPP and BPZ were missed in our in silico analysis (Grimaldi et al., 2019). There are currently no in vitro data available to assess the predicted reactivity of BPPH, PF201, BPP, HBPX-1 and D90 with RXR α . In terms of PPAR α /β/γ, the ED predicted that BPPH and PF201 react with all three isoforms. Currently, there is no evidence to support the predictions for BPPH, while PF201 showed no effect on PPARγ transcriptional activity in a recent study (Crosthwait et al., 2025). Although the ED did not predict the reactivity of BTUM, BPS-MAE and BPS-MPE with PPARs, they were identified as PPARγ activators in an in vitro model (Crosthwait et al., 2025). Moreover, unlike BPA, TCBPA is able to activate PPARγ (Riu et al., 2011b). To select the compounds to be tested in vitro, we performed a comprehensive ranking in which the ranks for each compound were summed for all receptors - therefore the lower value is attributed to the higher comprehensive rank and presumably leads to higher immunotoxicity (See Table 3). Of the total 26 compounds, BPA was ranked 15th. BPG, BPS-MPE, BPS-MAE, BPPH and PF201 were ranked as the 5 most toxic compounds based on their predicted interaction with nuclear receptors and were selected for analysis. In order to establish a preliminary structure-activity relationship between BPA substitutes and their immunomodulatory effects, we also selected BPZ, BPAP, BPP and BTUM with a higher predicted endocrine disrupting toxicity than BPA and BPE and TCBPA with a lower toxic potential for the in vitro assays. 3.2. Effects of BPA and its substitutes on metabolic activity of immune cells First, we investigated the effects of selected BPA substitutes on cell N. Franko et al. Ecotoxicology and Environmental Safety 300 (2025) 118406 6 viability using a resazurin-based assay to assess metabolic activity. The IC 50 values for each cell line are shown in Table 4. In general, the IC 50 values for each compound were comparable between the cell lines. Compared to BPA (IC 50 =99.4 – 182.8 µM), the analogues BPP, BPPH, BPG, BPAP, BPZ, BPS-MPE and TCBPA were more cytotoxic. BPG (IC 50 =19.5 – 28.7 µM), BPPH (IC 50 =17.5 – 30.6 µM) and BPP (IC 50 =11.1 – 28.6 µM) were the most cytotoxic compounds identified and had IC 50 values that were approximately five to ten times lower than those of BPA. PF201 (IC 50 =98.2 – 178.8 µM) and BPS-MAE (IC 50 =141.4 – 226.8 µM) showed comparable cytotoxicity to BPA, while BPE (IC 50 =208.2 – 220.2 µM) and BTUM (IC 50 >250 µM) affected the metabolic activity the least. Comparison of the IC 50 values with the logP of the compounds tested shows that the BPA analogues affect cell viability in proportion to their lipophilicity, with a higher logP correlating hyperbolically with a lower IC 50 (Fig. 1, panel A). The average IC 50 values of the analogues (excluding BTUM and PF201 with non-bisphenolic structure) of all cell lines listed in Table 4 were calculated (see Table S4) and when plotted against logP with a hyperbolic fit, an R 2 of 0.9185 was obtained (Fig. 1, panel B). The increased cytotoxicity due to the higher logP, which has a positive effect on membrane permeation, is consistent with previously published studies (Crump et al., 2021; Sharin et al., 2022; Wang et al., 2025). According to the Lipinski rule of 5, compounds with a logP ≤5 are classified as bioavailable when administered orally. However, the analogues BPG, BPP, BPPH and TCBPA (and the alternative BTUM) have a logP above 5 (see Table 2), which is advantageous for membrane permeation. It is also to be expected that such lipophilic compounds are more promiscuous towards several targets and could even accumulate in cell membranes. Therefore, the BPA analogues with the highest logP values could cause cytotoxicity via multiple mechanisms (Matsson et al., 2016). The IC 50 of TCBPA is slightly higher than expected for its logP, suggesting that the chlorine atoms affect either transmembrane transport or reactivity with intracellular targets. These observations are also consistent with the predicted compounds’ permeability in Caco-3 cells (Table S3), where TCBPA is predicted to have poor permeability in Caco-3 cells, while the other BPA analogues exhibit excellent permeability related parameters (ADMETlab). Moreover, TCBPA is also less soluble than the other bisphenols (e.g., BPA exhibits logS of −3.85 and TCBPA of −7.17) with the exception of BPP (logS −7.93). On the other hand, the structurally different alternatives BTUM and PF201 follow this correlation poorly. Both compounds have a higher logP value than BPA, but a lower or comparable cytotoxicity. This observation suggests that structurally different molecules with a larger total polar surface area and a higher molecular weight (Table S3) have lower effects on cellular metabolic activity and membrane permeation. Indeed, both alternatives are predicted to have low cellular permeability and solubility as judged by logS (logS <−7). 3.3. Effects of BPA and its substitutes on cytokine release from immune cells To investigate whether BPA substitutes affect the functions of the innate and adaptive immune system, we exposed THP-1 derived macrophages, Jurkat T cells and LCLs to the investigated compounds from the environmentally and toxicologically relevant nanomolar concentrations (Owczarek et al., 2018) up to the micromolar range. After cell exposure and activation, the cytokines released were measured in the cell culture supernatants. Prior to testing, metabolic activity assays confirmed that more than 80 % of the cells remained active at the highest concentration tested (Figures S1 – S4). The suitability of the models for the evaluation of the immunomodulatory effect was confirmed by testing cytokine release upon exposure to known immunomodulators, hydrocortisone and/or mirdametinib (Figure S5). 3.3.1. THP-1 derived macrophages The effects of BPA, a known ER α /β agonist, on cytokine release were previously thought to be mainly proinflammatory, although they are likely to be model and concentration dependent. Previous studies using non-activated THP-1 macrophages found that BPA at nanomolar concentrations increased the proinflammatory cytokines TNF α and IL-6 and decreased the anti-inflammatory IL-10 and TGFβ (Liu et al., 2014), while BPA at micromolar concentrations decreased TNF α in LPS-induced murine macrophages (Byun et al., 2005). The fact that the effect of bisphenols on cytokine secretion is dose-dependent was nicely demonstrated by Chen et al. (Chen et al., 2018) in U937 macrophages, where BPA, BPS and BPF generally suppressed cytokine release at 1 µM and increased their secretion at 10 µM. In the present study, the effects of BPA substitutes on the primary immune system were investigated by measuring the proinflammatory cytokines released by THP-1 macrophages after LPS activation. The macrophages secreted IL-1β, IL-6, IL-8 and TNF α (Fig. 2). IL-10 and IL-12p70 were below the detection limit. In our LPS-induced THP-1-derived macrophage model, IL-1β, the proinflammatory cytokine responsible for the activation of lymphocytes and neutrophils, was the most affected cytokine. BPA was only able to suppress it at 10 µM (fold induction 0.64 ±0.13). With the exception of BPAP, BPS-MAE, BPZ and BTUM, the other tested substitutes affected IL1β to a much greater extent than BPA. In general, the analogues promoted the IL-1β release in the nanomolar range (Fig. 2, panel A). Statistically significant increases were observed with 10 nM BPE (1.40 ±0.12) and TCBPA (1.47 ±0.13), with 100 nM BPPH (1.33 ±0.15), BPS-MPE (1.46 ±0.13) and TCBPA (1.70 ±0.13). On the other hand, BPG (0.26 ±0.14), BPP (0.61 ±0.13) and BPPH (0.44 ±0.13) suppressed the release of IL-1β at 10 µM, showing a biphasic effect, while TCBPA (1.71 ±0.13) and PF201 (1.38 ±0.13) stimulated it even at micromolar concentrations. IL-6 (Fig. 2, panel B), which stimulates the differentiation of B cells into plasma cells and of T lymphocytes into Th17, was not affected by BPAP, BPE, BPG, BPP and PF201 in THP-1 derived macrophages, while other analogues and alternatives showed a tendency towards its stimulated increase. Interestingly, BPPH again had a stimulatory effect at a concentration of 10 nM (1.41 ±0.05) and an inhibitory effect at 10 µM (0.44 ±0.11), while BPZ showed the opposite effect (0.77 ±0.03 at 10 nM and 1.69 ±0.11 at 10 µM). Statistically significant increases were observed at 100 nM BPS-MPE (2.16 ±0.21) and at 10 µM BPS-MAE (1.61 ±0.11), BPS-MPE (1.66 ±0.11), TCBPA (1.82 ±0.11) and BTUM (2.01 ±0.11). In comparison, BPA reduced IL-6 at a concentration of 10 nM (0.63 ±0.08), although this observation contrasts with the observed increase of IL-6 on nonactivated acute myeloid leukaemic cells HL-60 and U937 cells by 10 nM BPA (Zhang et al., 2020). TNF α and IL-8 (Fig. 2, panels C and D) were relatively unaffected by the bisphenols, only BPPH was potent enough to decrease TNF α (0.38 ±0.10) and TCBPA to increase IL-8 (1.25 ±0.07) at 10 µM concentration. A general overview of cytokine release from the macrophage model shows that selected BPA substitutes have predominantly Table 4 IC 50 values of tested compounds on cell metabolic activities. All results are expressed in µM with standard error. compound THP-1 macrophages Jurkat T cells LCLs BPA 122.5 ±3.2 99.4 ±4.6 182.8 ±16.1 BPAP 36.9 ±0.7 26.5 ±0.9 62.4 ±8.5 BPE 208.5 ±4.6 208.2 ±6.1 220.2 ±11.2 BPG 21.0 ±0.3 28.7 ±1.1 19.5 ±3.7 BPP 18.8 ±0.5 11.1 ±0.1 28.6 ±1.5 BPPH 17.5 ±0.4 20.8 ±0.4 30.6 ±2.4 BPS-MAE 145.2 ±8.0 141.4 ±6.5 226.8 ±34.5 BPS-MPE 58.7 ±2.0 87.7 ±6.7 107.4 ±19.4 BPZ 44.8 ±2.1 38.1 ±1.3 65.9 ±4.9 TCBPA 73.1 ±3.4 41.5 ±3.4 89.8 ±4.6 BTUM >250 >250 >250 PF201 98.2 ±5.6 148.1 ±7.5 178.8 ±0.7 N. Franko et al. Ecotoxicology and Environmental Safety 300 (2025) 118406 7 proinflammatory effects on human LPS-induced macrophages, with some concentration-dependent exceptions. At nanomolar concentrations similar to in vivo concentrations in blood, BPE, BPG, BPPH, BPSMPE, TCBPA and BTUM showed a statistically significant increase in the release of proinflammatory IL-1β and/or IL-6. Indeed, the observed fold inductions were relatively small but comparable to those observed in the previously published studies on LPS-activated THP-1 monocytes (24 h exposure) (Buoso et al., 2021) and U937 macrophages (48 h exposure) (Chen et al., 2018). On the other hand, Liu et. al (Liu et al., 2014) observed an approximately 5to 6-fold induction of IL-6 and TNF α in non-activated THP-1 macrophages after 24 h exposure to 100 nM BPA for 24 h. These discrepancies highlight the differences in the in vitro models used. In this study, the THP-1 monocytes were differentiated into macrophages using 80 nM PMA, while Liu et al. used 320 nM PMA (Liu et al., 2014). Using higher concentration of PMA leads to more complete differentiation resulting in different macrophage phenotype. In contrast to our study, where stimulation by LPS was needed to stimulate the IL-1β, IL-6 and TNF α release, the macrophages used by Liu et al. did not require LPS as a stimulant to release the cytokines. Although the activation of cells with LPS is an established practice in in vitro immunotoxicology (Chanput et al., 2014, 2013), their overstimulation might mask the effects of tested compounds. There is little data to support the pro-inflammatory effect of the new BPA substitutes. In mice fed BPP corn oil, an activated LPS/TLR4/NF-κB signalling pathway was observed in the intestinal cells, which was associated with increased gene expression of IL-1β and IL-6, leading to intestinal inflammation (Ma et al., 2023). TCBPA and BPS were found to increase intracellular ROS in human arterial smooth muscle cells and murine macrophages, leading to increased release of IL-1β, IL-6 and TNF α via activation of the NLRP3 inflammasome (Qiao et al., 2024; Xie et al., 2020). Other underlying mechanisms have not been explored, but immunomodulation could be an (in)direct consequence of either endocrine disruption (Durcik et al., 2022) or increased oxidative stress (L. Wang et al., 2023) . The fact that BPG and BPPH can greatly increase intracellular ROS, oxidative stress could be a contributing factor to the very pronounced effect on cytokine release and decreased metabolic activity (Jia et al., 2023). 3.3.2. Jurkat T cells The increasing evidence that BPA as well as some other bisphenols such as BPS and BPF (Kodila et al., 2023) disrupt the balance and function of T cells, prompted us to investigate whether the emerging substitutes affect the activation of Jurkat CD4 + cells. After activation with ionomycin and PMA, NF-κB and the NFAT/AP-1 are activated in Jurkat cells and IL-2 is released (Khalaf et al., 2010). IL-2 is a pleiotropic cytokine released by CD3 + CD4 + cells and is critical for the development, regulation, proliferation and maintenance of Tregs (Harris et al., 2023) and we speculate that its modulated secretion by BPA may contribute to the observed Treg/Th17 imbalance (Gao et al., 2020). Based on the reports of previous studies that BPA at nanomolar and micromolar concentrations does not significantly affect the release of IL2 from Jurkat cells (Ndebele et al., 2004), we exposed the cells to the highest concentrations of bisphenols at which they still retained >80 % of metabolic activity (all in the micromolar range). For the compounds that decreased the cytokine in a dose-dependent manner, the IC 50 values of decreased IL-2 secretion were calculated and are shown in Table 5. The behavioural changes of IL-2 by all tested compounds are shown in Figure S6. BPA showed an IC 50 of IL-2 secretion of 11.2 ±3.5 µM, which is more pronounced compared to the previously published studies (Ndebele et al., 2004). With the exception of TCBPA, all structural analogues of BPA also showed comparable IC 50 values in the low micromolar range, with BPP (IC 50 =4.02 ±2.8 µM) and BPPH (IC 50 =2.50 ±0.3 µM) being the most potent and BPE being slightly less potent (IC 50 =18.7 ±7.4 µM). As shown in Figure S6, BPA, BPAP, BPG, BPPH, BPS-MAE and BPS-MPE were able to completely inhibit the release of IL-2 when exposed to the concentrations at which the cells were still metabolically active. As described in Table 1, reduced IL-2 release from T cells can be achieved by GR agonists acting via the GR/GILZ/NFAT/AP-1/IL-2 pathway (Mittelstadt and Ashwell, 2001). However, Jurkat T cells suffer from a mutation in the GR that renders them resistant to glucocorticoids (Riml et al., 2004; Vacca et al., 1990), implying that the observed changes in IL-2 secretion in Table 5 and Fig. 3 are likely due to compounds that bind to other targets rather than the GR (Figure S5). Our group has previously shown that IL-2 secretion from Jurkat cells can be inhibited by inhibitors of mTOR, calcineurin phosphatase, myeloperoxidase and MAPK (Franko and Sollner Dolenc, 2024). In addition, IL-2 can also be downregulated by ER agonists (McMurray et al., 2001). Similar to the release of IL-1β and IL-6 from macrophages, a different pattern of behaviour compared to BPA and its structural analogues was observed with TCBPA, BTUM and PF201, with IL-2 secretion increasing at lower concentrations and decreasing at higher concentrations (Fig. 3). TCBPA increased T cell activation at concentrations up to 15 µM, BTUM at up to 50 µM and PF201 at up to 10 µM. TCBPA has previously been shown to exert proinflammatory effects on resting Jurkat T cells via modulation of estrogen signalling-related target genes GPER1/PI3K/Akt (Lu et al., 2024), while both proand anti-inflammatory cytokines, including IL-2, were increased in sera from mice exposed to TCBPA (Wang et al., 2021). 3.3.3. LCL cells The effect of BPA substitutes on B cell function was assessed by measuring cytokine release from ionomycin/PMA-activated LCL cells derived from three healthy, unrelated donors, two of them were obtained from male and one from female donor. The pro-inflammatory Fig. 1. Correlation of the IC 50 values with logP of the tested compounds. Panel A: All measured IC 50 values for each cell line, plotted against logP. Panel B: Average values of the IC 50 values determined on all cell lines, plotted against logP. BTUM and PF201 were removed and the plot was fitted to the hyperbolic equation with R 2 =0.9185. N. Franko et al. Ecotoxicology and Environmental Safety 300 (2025) 118406 8 cytokines IL-2, IL-6 and TNF α as well as the anti-inflammatory cytokine IL-10 were determined in three LCL cell lines derived from unrelated individuals (Fig. 4). As already observed in THP-1 derived macrophages, a trend towards non-monotonic behaviour was also observed in LCL cells, particularly in the release of pleiotropic IL-2 (Fig. 4, panel A). Statistically significant increase of IL-2 was observed upon exposure of cells to 10 nM BPAP (1.18 ±0.02), while BPZ caused small but significant IL-2 decrease at this concentration (0.91 ±0.01). At concentrations of 100 nM, BPE (1.29 ±0.02) and BPZ (1.28 ±0.04) significantly increased the release of IL-2. BPA and the analogues BPAP, BPG, BPP, BPPH and BPS-MPE showed a similar trend without statistical significance. On the other hand, BPA (0.67 ±0.11), BPG (0.20 ±0.13), BPP (0.08 ±0.13), BPPH (0.07 ±0.13), BPS-MAE (0.52 ±0.13), BPS-MPE (0.21 ±0.14), BPZ (0.40 ±0.14) and TCBPA (0.58 ±0.13) at 10 µM concentrations significantly suppressed IL-2 release from LCLs, which is consistent with the observed IL-2 behaviour on Jurkat T cells (Table 5). Unlike BPA, that significantly induced IL-6 release at 100 nM (1.32 ±0.10), the analogues did not alter its modulation at the environmentally relevant concentrations (Fig. 4, panel B). However, BPAP (0.75 ±0.10), BPE (0.57 ±0.11), BPG (0.50 ±0.11), BPP (0.13 ±0.11), BPPH (0.28 ±0.11), BPS-MAE (0.68 ±0.12), BPS-MPE (0.47 ±0.11) and BPZ (0.68 ±0.11) significantly suppressed the release of IL-6 at 10 µM concentrations, being equally or more potent than BPA (0.74 ±0.10). The decrease in IL-6 release from LCL cells contrasts with the stimulated release from THP-1 macrophages by BPS-MAE, BPS-MPE and BPZ (Fig. 2, panel B) and the pronounced release of IL-6 induced by 15 µM BPA and BPE from human primary lymphocytes (Zhang et al., 2024), which may be due to differences in both cell models and the activation mechanism (Franko and Sollner Dolenc, 2024). On the other hand, 10 µM TCBPA significantly increased the release of IL-6 (1.54 ±0.11), showing a similar effect as in THP-1 derived macrophages. Fig. 2. BPA and its substitutes modulate the release of proinflammatory cytokines from human THP-1 derived macrophages. Cells were exposed to the compounds at concentrations of 0.01 µM, 0.1 µM and 10 µM and activated with LPS for 24 h. The cytokines released were measured in the cell culture supernatants. The effects of the compounds on the individual cytokines are shown in separate panels. Panel A – IL-1β; Panel B – IL-6; Panel C – TNF α ; Panel D – IL-8. 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