# Inhibition of human organic anion transporter 4 (OAT4) activity by bisphenol analogues

Hélène Le Mentec, David Malnoë, Marc Le Vée, Olivier Fardel

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Hélène Le Mentec, David Malnoë, Marc Le Vée, Olivier Fardel. Inhibition of human organic anion transporter 4 (OAT4) activity by bisphenol analogues. Environmental Pollution, 2026, 390, pp.127540. 10.1016/j.envpol.2025.127540. hal-05446443

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# Inhibition of human organic anion transporter 4 (OAT4) activity by bisphenol analogues*

Hélène Le Mentec<sup>a</sup>, David Malnoë<sup>b, c</sup>, Marc Le Vée<sup>a</sup>, Olivier Fardel<sup>b, * d</sup>

## ARTICLE INFO

**Keywords:**

Bisphenol analogues

In vitro to in vivo extrapolation

Organic anion transporter

Pergafast® 201

Membrane transport

## ABSTRACT

Organic anion transporter (OAT) 4 is a kidney and placental membrane protein implicated in the transport of xenobiotics, including pollutants. The present study was designed to determine whether environmental bisphenol (BP) analogues, including emerging bisphenol A substitutes, can interact with OAT4. 6-carboxyfluorescein-related OAT4 activity displayed by OAT4-overexpressing HEK-293 cells was inhibited by 7/23 BP analogues used at 10 µM, especially by the thermal paper-contained color developer Pergafast® 201 (PF201) and the flame retardants tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), acting in a concentration-dependent manner (with IC₅₀ values in the 0.95–1.48 µM range). Most of these BP analogues moreover decreased membrane transport of estrone-3-sulfate, a hormonal endogenous substrate for OAT4. Some of them, especially PF201, additionally blocked activity of the renal transporters OAT1 and OAT3. Interaction of BP analogues with OAT4 was further supported by docking simulation studies, indicating that PF201 binds to the Arg473-containing electropositive pocket of OAT4, presumed to play a key role in inhibition of OAT4-mediated transport. BP analogues however failed to trans-stimulate OAT4 activity and OAT4-overexpressing cells exhibited no or marginally increased sensitivity to PF201, TBBPA and TCBPA, thus suggesting that these BP analogues are not or poorly transported by OAT4. In vitro to in vivo extrapolation finally demonstrated that occupational exposure to PF201 is predicted to result in human OAT4 inhibition. Overall, OAT4 was demonstrated to be a target for BP analogues, including BPA substitutes, with possible deleterious consequences in terms of OAT4-mediated transport of endogenous compounds or drugs, notably for workers exposed to PF201.

## 1. Introduction

Organic anion transporter (OAT) 4 (SLC22A11) is a solute carrier acting as a bidirectional plasma membrane transporter of anionic drugs, such as antibiotics, nonsteroidal anti-inflammatory drugs, diuretics and antiviral drugs (Burckhardt, 2012; Takeda et al., 2002). It also transports endogenous compounds, including metabolites like urate, or hormones like estrone-3-sulfate (E3S) and thyroxine (Chen et al., 2023; Hagos et al., 2012). OAT4 is expressed at the apical pole of renal proximal tubular cells (Ekaratanawong et al., 2004), where it can assume either reabsorption or secretion of its substrates from or into urine, according to its influx or efflux mode of activity. OAT4 is additionally present at the basolateral membrane of syncytiotrophoblasts (Ugele et al., 2003), where it may mediate elimination of anionic drugs and endogenous metabolites from the fetus (Cha et al., 2000); efflux of drugs via OAT4

from syncytiotrophoblasts to the fetal circulation might also be considered, as proposed for olmesartan (Noguchi et al., 2015).

Besides drugs and endogenous compounds, chemical pollutants have to be considered for putative interactions with OAT4, as already highlighted for other drug transporters (Michel et al., 2023; Tastet et al., 2023). In this context, it is noteworthy that some per- and polyfluoroalkyl substances (PFAS), such as the legacy PFAS perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) and the emerging PFAS 6:2 fluorotelomer sulfonate (6:2 FTS), are substrates for human OAT4 (Louisse et al., 2023; Nakagawa et al., 2009; Ryu et al., 2024). The transport by OAT4 of these toxic environmental chemicals may slow their renal clearance through facilitating their tubular reabsorption and thus increase their accumulation into humans (Louisse et al., 2023). It may also decrease the fetal exposure to these PFAS (Kummu et al., 2015; Lu et al., 2021). PFOA and PFOS additionally exert

<footnote><sup>a</sup> Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail), UMR_S 1085, F-35000, Rennes, France</footnote>

<footnote><sup>b</sup> Univ Rennes, CHU Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail), UMR_S 1085, F-35000, Rennes, France</footnote>

<footnote>* This paper has been recommended for acceptance by Mingliang Fang.</footnote>

<footnote>* Corresponding author. Irset, 9 Avenue du Pr Léon Bernard, 35000, Rennes, France.</footnote>

<footnote>E-mail address: olivier.fardel@univ-rennes.fr (O. Fardel).</footnote>
potent inhibitory effects towards OAT4-mediated transport of thyroxine, which may contribute to their endocrine disruptive effects (Wagenaars et al., 2024). In contrast to PFAS, other pollutants remain rather poorly characterized with respect to putative interactions with OAT4. This is notably the case for bisphenol A (BPA) and its structural and/or functional derivatives, called bisphenol (BP) analogues and constituting a major class of plastics-derived hazardous pollutants, to which humans are rather highly exposed. Indeed, even if the halogenated BP analogue tetrabromobisphenol A (TBBPA), in contrast to the BP class leader BPA, was very recently shown to potently inhibit OAT4-mediated transport of thyroxine (Wagenaars et al., 2024), interactions of BP analogues with OAT4 activity are poorly studied. The present study was therefore designed to characterize the possible interactions of OAT4 with a wide range of BP analogues/derivatives. These BP analogues include many substitutes to BPA, whose use in various products, including epoxy resins, polycarbonate plastic, canned foods, and beverage containers, is now regulatory restricted due to its potent human endocrine-disruptive effects (Tarafdar et al., 2022). Our data indicate that the chlorinated flame retardant tetrachlorobisphenol A (TCBPA) and the thermal paper-contained color developer Pergafast® 201 (PF201, also known as 3-(3-tosylureido)phenyl 4-methylbenzenesulfonate), in addition to TBBPA, act as potent inhibitors of OAT4, thus fully supporting the hypothesis that OAT4 interacts with some BP analogues.

## 2. Material and methods

### 2.1. Chemicals

BPA and twenty-two structural and/or functional BP analogues/derivatives were included in the study. Their chemical structure is indicated in Fig. S1. They correspond to (i) BPA structural analogues, i.e., chemicals that share a common structure of two hydroxyphenol functionalities (BPA, bisphenol AF (BPAF), 4,4'-((1-phenylethylidene) bisphenol (BPAP), bisphenol B (BPB), bisphenol E (BPE), bisphenol F (BPF), bisphenol G (BPG), bisphenol P (BPP), bisphenol PH (BPPH), bisphenol S (BPS), bisphenol T (BPT), bisphenol Z (BPZ), TBBPA and TCBPA), (ii) compounds with one phenol (bisphenol S 4-isopropyl ether (BPS-IP), bisphenol S 4-allyl ether (BPS-MAE), bisphenol S 4-benzyl ether (BPS-MPE), 2,4'-dihydroxydiphenyl sulfone (2,4-BPS) and benzyl 4-hydroxybenzoate (BzP, also known as benzyl paraben)), (iii) compounds with non-phenolic aromatic rings (diglycidyl ether (BADGE), 4,4'-bis(p-tolylsulfonylureido)diphenylmethane (BTUM) and PF201) and (iv) compounds with non-aromatic ring (2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCB)). BP analogues were provided by Sigma-Aldrich (St. Louis, MO, USA), LGC Standards (Molsheim, France) or Santa Cruz Biotechnology (Dallas, TX, USA). Stock solutions of BPA and BP analogues were prepared in 100% dimethyl sulfoxide (DMSO); final solvent concentrations amounted to 0.1% (vol/vol) for both cell viability assays (performed with DMEM medium (Thermo Fischer Scientific, Villebon sur Yvette, France), supplemented with 5% (vol/vol) fetal bovine serum, as described below) and OAT-related transport assays (performed with Hanks' balanced salt solution (HBSS)). Probencid, bromosulfophthalein (BSP) and 6-carboxyfluorescein (6-CF) were obtained from Sigma-Aldrich and [6,7-³H(N)]-E3S (specific activity = 51.8 Ci/mmol) from PerkinElmer (Villepinte, France).

### 2.2. Cell culture

Human HEK-293 cells stably overexpressing human OATs, i.e., OAT1 (SLC22A6) (HEK-OAT1 cells), OAT3 (SLC22A8) (HEK-OAT3 cells) or OAT4 (HEK-OAT4 cells) were generated by transduction of HEK-293 cells by the lentiviral pLV-EF1-hOAT1-hPGK-GFP, pLV-EF1-hOAT3-hPGK-GFP or pLV-EF1-hOAT4-hPGK-GFP vectors. Control HEK-MOCK cells were obtained in parallel by transduction of HEK-293 cells with an empty lentiviral pLV-EF1-hPGK-GFP vector. Construction of the lentiviral vectors, production of lentivirus supernatants, transduction of

HEK-293 cells, cloning and initial characterization of HEK-OAT1, HEK-OAT3, HEK-OAT4 and HEK-MOCK cells were performed by Vectalys (Labège, France). The functionality of HEK-OAT1 cells, HEK-OAT3 and HEK-OAT4 cells has previously been verified by studying the uptake of reference OAT substrates in the absence or presence of known OAT inhibitors (Bruyère et al., 2021; Chedik et al., 2017; Le Vée et al., 2019). Cells were cultured in DMEM medium containing 4.5 g/L D-glucose and supplemented with 5% (vol/vol) fetal bovine serum, 20 IU/mL penicillin, 20 µg/mL streptomycin, 1% (vol/vol) MEM non-essential amino acids solution (Life Technologies, Carlsbad, CA, USA) and 1 µg/mL bovine insulin (Sigma Aldrich).

### 2.3. Transporter activity inhibition assays

The effects of BP analogues on OAT1, OAT3 and OAT4 activities were investigated by analysing the intracellular accumulation of the reference OAT substrates 6-CF or E3S in HEK-OATs cells, in the absence or presence of reference OAT inhibitors or BP analogues, as previously described (Bruyère et al., 2021). Briefly, HEK-OAT cells were incubated for 5 min at 37 °C in HBSS supplemented with 10 µM 6-CF (HEK-OAT1, -OAT3 and -OAT4 cells) or 3.5 nM [³H]-E3S (HEK-OAT4 cells), in the absence or presence of 2 mM probenecid (OAT1/3 inhibitor) (Hagos et al., 2017), 100 µM BSP (OAT4 inhibitor) (Wagenaars et al., 2024) or BP analogues (used at 10 µM for initial screening assays); cells were consequently simultaneously exposed to OAT substrates and BP analogues or OAT reference inhibitors for 5 min. After washing with phosphate-buffered saline (PBS), cells were lysed in distilled water by performing several aspiration/reflux cycles within the wells. Intracellular accumulation of 6-CF was determined by spectrofluorimetry using a Biotek Synergy H1 spectrofluorometer (Agilent, Les Ulis, France); excitation and emission wavelengths were 492 nm and 517 nm, respectively. Intracellular accumulation of [³H]-E3S was measured by scintillation counting with a Beckman LS6500 counter (Beckman Coulter, Fullerton, CA, USA). Values of substrate accumulation were finally normalized to total protein content, determined by the Bradford method (Bradford, 1976).

Data were routinely expressed as percentages of OAT activity found in control cells not exposed to reference inhibitors or BP analogues, arbitrarily set at 100%, according to the following equation:

$$ \text{OAT activity (\%)} = \frac{[\text{Substrate BP}] - [\text{Substrate Reference inhibitor}]}{[\text{Substrate Control}] - [\text{Substrate Reference inhibitor}]} \times 100 \quad (1) $$

with [Substrate BP] = cellular concentration of substrate in the presence of BP analogue, [Substrate Reference inhibitor] = cellular concentration of substrate in the presence of the reference inhibitor and [Substrate Control] = cellular concentration of substrate in control cells.

The percentage of OAT activity inhibition by BP analogues was next calculated according to equation (2):

$$ \text{OAT activity inhibition (\%)} = 100\% - \text{OAT activity (\%)} \quad (2) $$

with OAT activity (%) determined as described in equation (1).

BP analogues (10 µM) significantly inhibiting 6-CF-related OAT activity were routinely ranked as potent inhibitors (OAT inhibition by more than 80%), moderate inhibitors (OAT inhibition between 50% and 80%) or weak inhibitors (OAT inhibition by less than 50%).

The half maximal inhibitory concentration (IC₅₀) values of BP analogues towards OAT activities were determined from nonlinear regression of concentration-response data based on the four-parameter logistic function. They were calculated using the following equation:

$$ A = \frac{100}{1 + 10^{[\text{Log}(IC_{50})] \times \text{Hill slope}}} \quad (3) $$

with A = percentage of transporter activity for a given concentration of
BP analogue, determined as described in equation (1), [I] = the BP analogue concentration in the medium, and Hill slope = a coefficient describing the steepness of the curve.

## 2.4. OAT4 trans-stimulation assays

Trans-stimulation assays were performed in HEK-OAT4 cells using the OAT4 substrate E3S as a reference tracer substrate and glutarate as a reference trans-stimulating agent (Fokina et al., 2022; Hagos et al., 2012). HEK-OAT4 cells were first incubated with 1 mM glutarate or 10 µM BP analogues for 15 min at 37 °C. After washing with PBS, cells were next re-incubated with 3.5 nM [³H]-E3S for 5 min at 37 °C in HBSS as described above. Intracellular accumulation of E3S was finally determined by scintillation counting. Data were expressed as percentages of E3S accumulation found in control cells not exposed to glutarate or BP analogues.

## 2.5. Cell viability assay

The effects of BP analogues on cellular viability/proliferation of HEK-OAT4 cells and HEK-MOCK cells were determined using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Carmichael et al., 1987). Briefly, cells were seeded at 15000 cells/well in 96 wells plates. After 24 h seeding, cells were treated either with the control solvent DMSO or with various concentrations of BP analogues for 72 h. Cells were next incubated with 0.5 mg/mL MTT for 3 h, allowing the reduction of MTT into formazan dye, which was quantified by its absorbance at 570 nm. Data for BP analogues-exposed cells were expressed as percentages of cell viability comparatively to control cells, arbitrarily set at 100 %, according to the following equation:

$$ \text{Cell viability (\%)} = \frac{\text{Absorbance}_{\text{BP}}}{\text{Absorbance}_{\text{Control}}} \times 100 \quad (4) $$

with Absorbance BP = MTT-related absorbance of cells exposed to BP analogues and Absorbance Control = MTT-related absorbance of control cells.

IC50 values of BP analogues towards cell viability/proliferation were calculated as described in equation (3), with A = percentage of cell viability for a given concentration of BP analogue determined as described in equation (4).

The sensitization factor to BP analogue toxicity due to OAT4 expression was next calculated as reported in equation (5):

$$ \text{Sensitization factor} = \frac{\text{HEK-MOCK cell IC}_{50}}{\text{HEK-OAT4 cell IC}_{50}} \quad (5) $$

with HEK-MOCK cell IC50 = IC50 of BP analogue towards HEK-MOCK cells and HEK-OAT4 cell IC50 = IC50 of BP analogue towards HEK-OAT4 cells.

## 2.6. BP analogue accumulation assays

HEK-OAT4 and HEK-MOCK cells were incubated for 5 min at 37 °C with 10 µM PF201 or 10 µM TCBPA in HBSS medium. Cells were next washed twice with ice-cold PBS and lysed in distilled water. Cellular proteins were precipitated using 50 % (vol/vol) acetonitrile. After 1 h 45 min centrifugation at 4500g and 4 °C, supernatants were collected, diluted in an aqueous solution also supplemented with BPA, used here as an internal standard. The samples were finally analyzed using a liquid chromatography-tandem mass spectroscopy (LC-MS/MS) system. This system corresponds to a high-performance LC 1260 Infinity III system (Agilent Technologies, Les Ulis, France) equipped with a Poroshell 120 EC-C18 column (2.7 µm, 4.6 × 100 mm) and coupled to an API 4000 triple quadrupole mass spectrometer (AB Sciex, Les Ulis, France), with an electrospray ionization source operating in negative mode. The

mobile phase for LC separation consisted of a gradient of water (solvent A) and acetonitrile (solvent B), each containing 2 mM ammonium formate, with a flow rate of 250 µL/min. MS/MS analysis was performed in multiple reaction monitoring (MRM) mode. LC-MS/MS parameters, including retention times, monitored ion transitions and source ionization parameters for PF201, TCBPA and BPA, are indicated in Table S1. Cellular accumulations of PF201 and TCBPA were finally normalized to protein content.

## 2.7. Molecular descriptor generation

Ligand structures were obtained in SMILES format from the Center for Computational Toxicology and Exposure (CCTE) (US Environment Protection Agency (EPA)), via an application programming interface (API) (https://api-ccte.epa.gov/docs/index.html), and using CAS numbers for identification of BP analogues. Fourteen common physicochemical molecular descriptors/properties (listed in Table S2) were in silico simulated using the online websites SwissADME (http://www.swissadme.ch/) (Daina et al., 2017), Admetlab3.0 (https://admetlab3.scbdd.com/) (Fu et al., 2024) and Deep-PK (https://biosig.lab.uq.edu.au/deeppk/) (Myung et al., 2024), or were additionally obtained from ACD/Labs (Toronto, ON, Canada) (via the CCTE-API) or using ADMET Predictor 12.0 (SimulationsPlus, Lancaster, Ca, USA). When available from multiple sources, parameters were summarized as means of the different simulated data. Values of pKa were in silico predicted by the Chemaxon Playground version 1.6.2 online platform (https://playground.calculators.cxn.io/) and ionized fractions at physiological pH (i. e., pH = 7.4) were calculated using the High-Throughput Toxicokinetics (HTTK) R Package from EPA (https://www.epa.gov/comptox-tools/high-throughput-toxicokinetics-httk-r-package) (Pearce et al., 2017).

## 2.8. Molecular docking

Predicting binding of BP analogues and of the reference OAT4 inhibitor BSP to OAT4 protein was performed by dynamic docking using the DynamicBind version 1.0 model (Lu et al., 2024). The cryogenic electron microscopy (cryo-EM) structure of OAT4 (He et al., 2024) was retrieved from the Protein Data Bank (PDB; https://www.rcsb.org/). PyMOL version 3.1 (https://www.pymol.org/) was used to remove the chloride ion present within the cryo-EM structure. The protein structure, saved as a PDB file, along with ligand structures in SMILES format, was submitted to the DynamicBind version 1.0 model through the Neurosnap online platform (https://neurosnap.ai/service/DynamicBind), which requires only ligand SMILES and protein structure to predict complex structures. Visualization of the docking complexes was conducted using Biovia Discovery Studio 2024 (https://www.3ds.com/products/biovia/discovery-studio). The level of the simulated binding to OAT4 was assessed using the maximal affinity score, corresponding to the highest binding score returned by DynamicBind.

## 2.9. In vitro to in vivo extrapolation (IVIVE)

In vivo prediction of OAT activity inhibition by BP analogues was performed from in vitro data using the criteria defined by the International Transporter Consortium (ITC) (Giacomini et al., 2010) and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) M12 drug-drug interaction guideline (https://www.ema.europa.eu/en/ich-m12-drug-interaction-on-studies-scientific-guideline). Briefly, drug transporters present in the kidney such as OATs can be inhibited in vivo by plasma concentrations of xenobiotics if the ratio of the “maximum unbound plasma concentration of chemicals (Imax,u)/IC50 towards activity” ≥ 0.1. As data on the human pharmacokinetics of BP analogues are not or very poorly available, Imax,u values for BP analogues were determined for maximal daily doses, considered as possibly occurring in an occupational and/or environmental/dietary context, according to environmental/health
authorities such as the US EPA, the European Food Safety Authority (EFSA) and the Department of Health from the Australian Government; these doses are indicated in Table 1. $I_{max,u}$ values related to BP analogue doses were calculated according to equation (6) assuming a one-compartment model with a bioavailability of 1. The volume of distribution ($V_d$) and unbound plasma fraction ($f_u$) of each BP analogues were predicted in silico using the online tool Admetlab3.0 (Fu et al., 2024).

$$I_{max,u} = \frac{\text{Dose} \times f_u}{V_d} \qquad (6)$$

with dose and $V_d$ expressed in mole/kg and L/kg, respectively.

## 2.10. Statistical analysis

Data were usually expressed as means ± SD from at least three independent experiments, each being performed in technical triplicate. They were statistically analyzed using Prism 10.5.0 software (GraphPad software, Boston, MA, USA) through analysis of variance (ANOVA) followed by Dunnett's post-hoc test, Mann-Whitney U test or F test. Correlation between molecular descriptors (as continuous variables) and percentages of OAT4 activity inhibition by 10 µM BP analogues, as well as those between OAT1, OAT3 and OAT4 activity percentages and also between 6-CF- and E3S-related OAT4 activity percentages in the presence of BP analogues were done through Pearson correlations. The criterion of significance for statistical tests was $p < 0.05$. The level of correlation was evaluated according to the Pearson r correlation coefficient value: the correlation was considered to be strong when $r \ge 0.7$ and moderate when $0.7 > r \ge 0.4$, as previously described (Akoglu, 2018).

# 3. Results

## 3.1. Inhibition of OAT4 activity by BP analogues

Twenty-three BP analogues (10 µM), including BPA, were tested against OAT4-mediated transport of 6-CF. 7/23 BP analogues significantly reduced 6-CF-related OAT4 activity (Fig. 1A). Four of them, i.e., BPS-IP, BPS-MAE, BPS-MPE and BTUM, decreased OAT4 activity in a rather moderate manner (inhibition by 50–80 %) whereas three of them, i.e., TBBPA, TCBPA and PF201, reduced it in a more potent manner (inhibition by more than 80 %). Transport of the endogenous compound E3S by OAT4 was also significantly reduced by TBBPA, TCBPA, PF201, BPS-IP, BPS-MAE and BTUM, whereas the other BP analogues failed to inhibit it or even stimulated it (for BADGE and BPP) (Fig. 1B). 6-CF- and

E3S-related OAT4 activities in the presence of BP analogues were next demonstrated to be significantly strongly correlated ($r = 0.75, p < 0.0001$) (Fig. S2), suggesting a similar profile of OAT4 activity regulation by BP analogues, whatever the used OAT4 substrate. The inhibitory effects of the most inhibitory chemicals towards 6-CF-related OAT4 activity, i.e., TBBPA, TCBPA and PF201, were finally found to be concentrations-dependent, with $IC_{50}$ values ranging from 0.95 µM (for PF201) to 1.48 µM (for TCBPA) (Fig. 2).

## 3.2. Comparison of OAT1, OAT3 and OAT4 activity inhibition by BP analogues

We compared the effects of BP analogues against OAT4 activity to those against OAT1 and OAT3 activities, because OAT1 and OAT3, expressed at the basolateral pole of proximal tubular cells, are presumed to share some substrates and inhibitors with OAT4 (Burckhardt, 2012). This was performed using 6-CF as a common substrate for these OATs. As indicated in Fig. 3A, only 4/23 BP analogues (10 µM) significantly reduced 6-CF-related OAT1 activity. TCBPA exerted moderate inhibition (by 59.5 %) and PF201 potent inhibition (by 81.3 %) of OAT1 activity, whereas BzP and TBBPA appeared to be weak inhibitors (OAT1 inhibition by less than 50 %). With respect to 6-CF-related OAT3 activity, it was significantly inhibited by 10/23 BP analogues, with BPA, BPS-IP, BPS-MAE and BPT acting as weak inhibitors (inhibition by less than 50 %), BPB, BzP and BTUM as moderate inhibitors (inhibition between 50 and 80 %) and PF201, TBBPA and TCBPA as potent inhibitors (inhibition by more than 80 %) (Fig. 3B). These strong inhibitory effects of PF201, TBBPA and TCBPA towards OAT3 activity, as well as that of PF201 towards OAT1 activity, were concentration-dependent, with $IC_{50}$ values ranging from 0.21 µM (inhibition of OAT3 by TBBPA) to 2.18 µM (inhibition of OAT1 by PF201) (Fig. S3). As shown in Fig. 4, OAT1 and OAT3 activities in the presence of BP analogues were significantly correlated to that of OAT4. According to the proposed concordance between the Pearson r correlation coefficient values and the level of correlation (Akoglu, 2018), the correlation was considered to be strong for OAT4-OAT3 inhibition ($r = 0.73$) and moderate for OAT4-OAT1 inhibition ($r = 0.55$).

## 3.3. BP analogue molecular descriptors associated with OAT4 inhibition

Various basic molecular descriptors have been previously associated with OAT1, OAT3 and/or OAT4 inhibition by xenobiotics, including molecular weight (MW), number of H-bond acceptors or donors, number of aromatic or non-aromatic rings, total polar surface area (TPSA) or lipophilicity (LogP) (Duan et al., 2012; Liu et al., 2016;

<table><caption>**Table 1**

Prediction of *in vivo* inhibition of OAT4 activity by TBBPA, TCBPA or PF201.</caption><thead><tr><th>Chemical</th><th>Type of exposure</th><th>Dose</th><th>V<sub>d</sub><sup>a</sup> (L/kg)</th><th>F<sub>u</sub><sup>f</sup> (%)</th><th>I<sub>max, u</sub><sup>g</sup> (nM)</th><th>IC<sub>50</sub><sup>h</sup></th><th>Ratio I<sub>max, u</sub>/IC<sub>50</sub></th><th>In vivo prediction<sup>i</sup></th></tr></thead><tbody><tr><td rowspan="2">TBBPA</td><td>Occupational</td><td>387 µg/kg BW<sup>a</sup></td><td rowspan="2">2.129</td><td rowspan="2">1.3</td><td rowspan="2">4.38</td><td rowspan="2">1.39 µM</td><td>0.0032</td><td>No inhibition</td></tr><tr><td>Environmental/dietary</td><td>0.7 µg/kg BW<sup>b</sup></td><td>5.78x10<sup>-6</sup></td><td>No inhibition</td></tr><tr><td>TCBPA</td><td>Environmental/dietary</td><td>0.38 µg/kg BW<sup>c</sup></td><td>0.882</td><td>0.5</td><td>0.0059</td><td>1.48 µM</td><td>4.00x10<sup>-6</sup></td><td>No inhibition</td></tr><tr><td rowspan="2">PF201</td><td>Occupational/Workers in contact with PF201 powder</td><td>1512 µg/kg BW<sup>d</sup></td><td>0.221</td><td>1</td><td>148.7</td><td>0.95 µM</td><td>0.16</td><td>Inhibition</td></tr></tbody></table>

<footnote><sup>a</sup> According to the National Industrial Chemicals Notification and Assessment Scheme, Department of Health, Australian Government (https://www.industrialchemicals.gov.au/sites/default/files/2021-06/PEC42-Tetrabromobisphenol-A-TBBPA_0.pdf); BW = body weight.</footnote>

<footnote><sup>b</sup> According to the EFSA Panel on Contaminants in the Food Chain (CONTAM) (Panel on Contaminants in the Food Chain CONTAM et al., 2024).</footnote>

<footnote><sup>c</sup> According to exposure prediction from the EPA CompTox Chemicals Dashboard (https://comptox.epa.gov/dashboard/).</footnote>

<footnote><sup>d</sup> According to the National Industrial Chemicals Notification and Assessment Scheme, Department of Health, Australian Government (https://www.industrialchemicals.gov.au/sites/default/files/STD1058/Public/Report/PDF.pdf).</footnote>

<footnote><sup>e</sup> Defined as the volume of distribution and *in silico* predicted using ADMETlab 3.0.</footnote>

<footnote><sup>f</sup> Defined as the unbound plasma fraction and *in silico* predicted using ADMETlab 3.0.</footnote>

<footnote><sup>g</sup> Defined as the maximum unbound plasma concentration.</footnote>

<footnote><sup>h</sup> $IC_{50}$ values against OAT4 activity, as reported in Fig. 2.</footnote>

<footnote><sup>i</sup> *In vivo* inhibition of OAT4 activity was predicted if the ratio $I_{max, u}/IC_{50} \ge 0.1$.</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Inhibition of OAT4 activity by BP analogues. HEK-OAT4 cells were incubated at 37 °C for 5 min with OAT4 substrates, i.e., (A) 10 µM 6-CF or (B) 3.5 nM [³H]-E3S, in the absence (control) or presence of the reference OAT4 inhibitor BSP (100 µM) or BP analogues (10 µM). After washing, intracellular uptake of substrates was determined by (A) spectrofluorimetry (6-CF) or (B) scintillation counting (E3S). Data are expressed as percentages of OAT4 activity compared to untreated control, arbitrarily set at 100 % and indicated by a dotted line on the graphs; they correspond to means ± SD of four independent experiments. **, p < 0.01 and ***, p < 0.001, when compared to control untreated cells.</figcaption></figure>

Martinez-Guerrero et al., 2025). To determine whether such molecular descriptors can also be associated with OAT4 activity inhibition by BP analogues, we investigated whether percentages of OAT4 inhibition in response to BP analogues were correlated with molecular descriptor indexes of the chemicals. As shown in Table 2, OAT4 inhibition percentages were positively strongly correlated with ionized (anionic) fraction at pH = 7.4, the number of heteroatoms and the molecular weight (p < 0.001). TPSA, the number of H-bond-acceptors and the number of heavy atoms were additionally positively correlated with OAT4 inhibition percentages, whereas the Caco2 permeability and the solubility (LogS) were significantly negatively correlated with BP analogues-mediated OAT4 inhibition percentages (Table 2). Correlation graphs for these molecular descriptors are shown in Fig. S4. Other parameters, including the lipophilicity (LogP) and the fraction of Sp3 carbon atoms (Fsp3), characterizing the complexity of the spatial structure of the molecules (Lovering et al., 2009), were not significantly correlated with OAT4 inhibition percentages (Table 2). With respect to OAT3 inhibition percentages due to BP analogues, they were significantly correlated with ionized (anionic) fraction at pH = 7.4, the number of heteroatoms, the molecular weight and the Caco2 permeability (Table 2).

### 3.4. Putative transport of BP analogues by OAT4

To investigate whether BP analogues may be substrates for OAT4, we first performed *trans*-stimulation assays because *trans*-stimulating agents are thought to be substrates for SLC transporters, including OAT4 (Hagos et al., 2012). As shown in Fig. 5, pre-incubation with BP analogues acting as OAT4 inhibitors did not enhance accumulation of the OAT4 substrate E3S. OAT4 activity was even *trans*-inhibited by TBBPA and PF201, i.e., accumulation of E3S was significantly reduced by pre-incubation with these compounds (Fig. 5). By contrast, the OAT4 substrate glutarate was able to *trans*-stimulate OAT4 activity by a 3.4-fold factor (Fig. 5).

Cellular overexpression of OAT4 likely results in enhanced cellular accumulation of OAT4 substrates, which may consequently lead to reinforced toxicity of these chemicals. A chemical exerting increased toxicity in HEK-OAT4 cells versus HEK-MOCK cells may consequently be hypothesized to be substrate for OAT4. Toxicities of TBBPA, TCBPA and PF201, that represent the most interacting compounds with OAT4 according to activity inhibition data, were consequently compared in HEK-OAT4 cells and HEK-MOCK cells, using the MTT cell viability assay. As indicated in Fig. 6, HEK-OAT4 cells exhibited similar sensitivities to
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Concentration-dependent inhibition of OAT4 activity by PF201, TBBPA and TCBPA. HEK-OAT4 cells were incubated at 37 °C for 5 min with 10 µM 6-CF in the absence or presence of the reference OAT4 inhibitor BSP (100 µM) or various concentrations of PF201, TBBPA and TCBPA. After washing, intracellular uptake of 6-CF was determined by spectrofluorimetry. Data are expressed as percentages of transporter activity found in control untreated cells, arbitrarily set at 100%; they are the means ± SD of at least four independent assays. IC₅₀ values as well as 95 % confidence intervals (in brackets) are indicated at the top of graphs.</figcaption></figure>

TBBPA and TCBPA as control HEK-MOCK cells, whereas they were marginally, but significantly, more sensitive to PF201 than their control counterpart. The sensitization factors to BP analogue toxicities due to OAT4 overexpression were consequently low, i.e., less than 1.3, even for PF201 (Table S3).

Accumulations of PF201 and TCBPA in HEK-OAT4 and HEK-MOCK cells were finally investigated by LC-MS/MS. As indicated in Fig. S5, uptakes of PF201 and TCBPA in HEK-OAT4 cells did not significantly differ from those found in HEK-MOCK cells.

### 3.5. *In silico* simulation of BP analogue binding to OAT4

Binding of BP analogues to OAT4 protein was in silico predicted with the DynamicBind version 1.0 model. Maximal affinity scores for BP analogue docking are shown in Fig. S6; the scores for 7/23 BP analogues inhibiting 6-CF-related OAT4 activity were significantly higher than those for 16/23 non-inhibitory counterparts (Fig. S6). The mean of maximal affinity scores for BP analogues blocking activity (mean ± SD = 6.48 ± 0.52, n = 7) was moreover close to the maximal affinity score of the reference OAT4 inhibitor BSP (maximal affinity score for BSP = 7.12). 3D-visualization of the docking was next performed for PF201, TBBPA and TCBPA, that represent the most inhibitory compounds. As shown in Fig. 7, these three BP analogues were predicted to interact with the Arg473-containing electropositive pocket of OAT4, presumed to play a key role in inhibition of OAT4-mediated transport of organic anions (He et al., 2024), and containing both hydrophobic and hydrophilic regions. Indeed, PF201 was simulated to interact with OAT4 hydrophobic regions via alkyl and π-alkyl interactions with Arg473, as well as with Phe211 via π-π stacking (Fig. S7). Additionally, PF201 was presumed to engage with OAT4 hydrophilic regions through van der Waals interactions, hydrogen bonding, and π-ion interactions involving Asp385 and Arg389 (Fig. S7). Similarly, TBBPA and TCBPA exhibited comparable profiles of interaction with OAT4, with some variations in the residues involved and the types of chemical bonds formed (Fig. S7). It is noteworthy that OAT4 residues that have been recently demonstrated to bind to the OAT4 substrate dehydroepiandrosterone sulfate, such as Phe238, Tyr360, Tyr361 and Leu445 (Zhang et al., 2025), were also predicted to interact with PF201, TBBPA and/or TCBPA (Fig. S7).

### 3.6. Prediction of *in vivo* OAT4 inhibition by BP analogues

Prediction of *in vivo* inhibition of OAT4 activity in humans occupationally or environmentally exposed to PF201, TBBPA or TCBPA was performed by applying the regulatory criteria for OAT1/OAT3 inhibition to OAT4 inhibition. As indicated in Table 1, environmental/dietary exposure to TBBPA or TCBPA as well as occupational exposure to TBBPA were not predicted to result in *in vivo* inhibition of OAT4 activity by an IVIVE approach. By contrast, occupational exposure to PF201 was predicted to *in vivo* block OAT4 activity (Table 1). Workers exposed to PF201 may additionally exhibit inhibition of OAT3 activity, in contrast to TBBPA or TCBPA (Table S4). It is noteworthy that environmental/dietary exposure of humans to PF201 remains yet uncharacterized, which precludes to predict its effects towards OAT3/OAT4 via an IVIVE approach.

## 4. Discussion

OAT4 is a plasma membrane drug transporter playing a major role in the renal elimination and the feto-placental passage of various drugs and endogenous compounds. OAT4 consequently deserves growing interest (Martinez-Guerrero et al., 2025). Recently, OAT4 has been highlighted for its interactions with environmental pollutants, notably PFAS, with putative consequences for toxicokinetics and toxicity of these chemicals (Louisse et al., 2023; Ryu et al., 2024; Wagenaars et al., 2024). The present study adds some BP analogues as potent OAT4-interacting chemicals, thus fully confirming that OAT4 is a membrane transporter
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Inhibition of OAT1 and OAT3 activity by BP analogues. (A) HEK-OAT1 and (B) HEK-OAT3 cells were incubated at 37 °C for 5 min with 10 µM 6-CF in the absence (control) or presence of the reference OAT inhibitor probenecid (2 mM) or of BP analogues (10 µM). After washing, intracellular uptake of 6-CF was determined by spectrofluorimetry. Data are expressed as percentages of (A) OAT1 and (B) OAT3 activity compared to untreated control, arbitrarily set at 100 % and indicated by a dotted line on graphs; they correspond to means ± SD of at least three independent experiments. **, p < 0.01; ***, p < 0.001 when compared to control untreated cells.</figcaption></figure>

worthy of interest for pollutants.

The strong interaction of BP analogues with OAT4 is supported by the potent inhibition of OAT4 activity in response to TBBPA, TCBPA and PF201, with IC₅₀ values in the 0.9–1.5 µM range against 6-CF transport (Fig. 2). These IC₅₀ values are among the lowest ones reported for drugs inhibiting OAT4. Indeed, the 30 best inhibitors of OAT4 function among 1436 drugs have IC₅₀ values in the 0.6–8.6 µM range against OAT4-mediated 6-CF transport (Martinez-Guerrero et al., 2025). This highlights the potent inhibitory potential of TBBPA, TCBPA and PF201 towards OAT4. Other BP analogues such as BPS-IP, BPS-MAE, BPS-MPE and BTUM also decreased 6-CF-related OAT4 activity, but in a weaker manner than TBBPA, TCBPA and PF201 (inhibition by 50–80 % versus inhibition by more than 80 %, when tested compounds are used at 10 µM). Transport of 6-CF by OAT4 in the presence of BP analogues was

correlated to that of E3S, thus likely discarding a substrate-restricted regulation of OAT4 activity, described for other SLC transporters like organic cation transporter (OCT) 2 (SLC22A2) (Hacker et al., 2015). The exact mechanism by which BP analogues inhibit OAT4 remains to be determined. Interaction with the drug binding site on OAT4 is however likely. Indeed, dynamic docking studies have predicted that BP analogues acting as potent OAT4 inhibitors, such as PF201, TBBPA and TCBPA, interact with the hydrophobic Arg473-containing electropositive drug pocket of OAT4 (Fig. 7 and Fig. S7). The simulation also indicated binding to hydrophilic regions of OAT4, thus supporting a membrane-embedded binding pocket formed by a combination of hydrophobic and hydrophilic domains, thought as suitable for interactions with organic compounds (Sriramulu and Lee, 2020). Inhibition of drug transporter activity via direct/competitive binding to drug binding sites
r = 0.55; p = 0.007

<figure><img src="image_9.png" /></figure>

r = 0.73; p < 0.001

<figure><img src="image_10.png" /><figcaption>**Fig. 4.** Correlation between OAT4 and OAT1 or OAT3 activities in the presence of BP analogues. Pearson correlations were done between 6-CF-related OAT4 activities and OAT1 or OAT3 activities performed in the presence of 10 µM BP analogues and reported in Figs. 1A and 3. Data correspond to the means of OAT activities. Pearson correlation coefficients (r) and p values are provided on the top of each correlation graph.</figcaption></figure>

has already been presumed for other pollutants, including for the inhibition of OAT3 by BPA (Bruyere et al., 2017).

It is noteworthy that not all tested BP analogues behave as OAT4 inhibitors. Thus, 16/23 BP analogues failed to reduce 6-CF-related OAT4 activity when used at 10 µM (Fig. 1A). Notably, the BP class leader BPA did not significantly alter OAT4-mediated transport of 6-CF, which agrees with its recently reported lack of effects towards OAT4-mediated transport of thyroxine (Wagenaars et al., 2024). Specific molecular descriptors among BP analogues may consequently be implicated in OAT4 inhibition. Among them, the ionized (anionic) fraction at pH = 7.4, the number of heteroatoms, the molecular weight, TPSA, the number of H-bond-acceptors (HBA), the number of heavy atoms, the solubility and the Caco2 permeability are correlated with OAT4 inhibition (Table 2). Some of these molecular descriptors, i.e., the molecular weight, TPSA and the number of H-bond-acceptors, have been recently associated with OAT4 inhibition among 1436 drugs (Martinez-Guerrero et al., 2025); these descriptors may consequently constitute robust parameters

<caption>**Table 2**</caption>

Correlations of BP analogue molecular descriptor indexes with percentages of OAT4 and OAT3 inhibition.

<table><thead><tr><th rowspan="2">Molecular descriptor</th><th colspan="2">Correlation with OAT4 inhibition %</th><th colspan="2">Correlation with OAT3 inhibition %</th></tr><tr><th>Pearson coefficient (r)</th><th>Significance (p value)</th><th>Pearson coefficient (r)</th><th>Significance (p value)</th></tr></thead><tbody><tr><td>Ionized (anionic) fraction at pH = 7.4</td><td>0.85</td><td>p &lt; 0.0001</td><td>0.63</td><td>p = 0.0012</td></tr><tr><td>Number of heteroatoms</td><td>0.71</td><td>p = 0.0002</td><td>0.57</td><td>p = 0.005</td></tr><tr><td>Molecular weight (MW)</td><td>0.66</td><td>p = 0.0006</td><td>0.52</td><td>p = 0.020</td></tr><tr><td>Total polar surface area (TPSA)</td><td>0.53</td><td>p = 0.001</td><td>0.38</td><td>p = 0.074</td></tr><tr><td>Caco2 permeability</td><td>-0.50</td><td>p = 0.014</td><td>-0.48</td><td>p = 0.020</td></tr><tr><td>Number of H-bond acceptors</td><td>0.48</td><td>p = 0.020</td><td>0.35</td><td>p = 0.10</td></tr><tr><td>Solubility (LogS)</td><td>-0.42</td><td>p = 0.045</td><td>-0.33</td><td>p = 0.13</td></tr><tr><td>Number of heavy atoms</td><td>0.42</td><td>p = 0.045</td><td>0.29</td><td>p = 0.17</td></tr><tr><td>Number of rotatable bonds</td><td>0.36</td><td>p = 0.093</td><td>0.35</td><td>p = 0.10</td></tr><tr><td>Number of non-aromatic rings</td><td>-0.32</td><td>p = 0.13</td><td>-0.36</td><td>p = 0.088</td></tr><tr><td>Fraction of Sp3 carbon atoms (Fsp3)</td><td>-0.31</td><td>p = 0.15</td><td>-0.28</td><td>p = 0.20</td></tr><tr><td>Lipophilicity (LogP)</td><td>0.30</td><td>p = 0.17</td><td>0.31</td><td>p = 0.15</td></tr><tr><td>Number of aromatic rings</td><td>0.27</td><td>p = 0.20</td><td>0.15</td><td>p = 0.50</td></tr><tr><td>Number of H-bond donors</td><td>0.11</td><td>p = 0.61</td><td>0.20</td><td>p = 0.37</td></tr></tbody></table>

correlated with OAT4 inhibition. The molecular weight, as well as the ionized fraction at pH = 7.4, the Caco2 permeability and the number of heteroatoms, were also correlated with inhibition of OAT3 activity by BP analogues, suggesting that these descriptors may be linked to inhibition of both OAT3 and OAT4. Interestingly, higher solubility and greater Caco-2 permeability were significantly associated with lower OAT4 inhibition. This supports the idea that compounds belonging to the Biopharmaceutics Classification System (BCS) class I, i.e., chemicals/drugs characterized by high solubility and high permeability (Amidon et al., 1995), likely exhibit reduced inhibitory potential toward OAT4. This agrees with the conclusion that such BCS class I chemicals are generally considered to have minimal interactions with transporters (Wu and Benet, 2005).

In addition to OAT4, OAT1 and OAT3 were also blocked by TBBPA, TCBPA and PF201, indicating that these BP analogues may act as pancreatic OAT inhibitors. Moreover, the profile of OAT4 activity in the presence of BP analogues was found to be significantly correlated to those of OAT1 (moderate correlation) and OAT3 (high correlation) (Fig. 4). This clearly supports the conclusion that OAT1, OAT3 and OAT4 share inhibitors, as already claimed (Burckhardt, 2012). 6-CF-related OAT3 activity was however significantly inhibited by more BP analogues (10/23) than those of OAT4 (7/23) or OAT1 (4/23). This may be explained by differences in the structural selectivity of the different OATs. For example, OAT3 has a more permissive binding site than OAT1, capable of accommodating a greater diversity of chemical structures (Astorga et al., 2011). Nevertheless, some BP analogues like
<figure><img src="image_11.png" /><figcaption>Fig. 5. Lack of *trans*-stimulation of OAT4 activity by BP analogues. HEK-OAT4 cells were first incubated in the absence (control) or presence of 10 µM BP analogues or 1 mM glutarate (used here as a positive *trans*-stimulating agent) for 15 min at 37 °C. After washing, cells were next incubated with 3.5 nM [³H]-E3S for 5 min at 37 °C. Accumulation of E3S was finally determined by scintillation counting. Data are expressed as percentages of E3S accumulation in control cells, arbitrarily set at 100 % and indicated by a dotted line on the graph. They are the means ± SD of three independent experiments. *, p < 0.05 when compared to control.</figcaption></figure>

BPE and BPF showed no effect on OATs transporters (Figs. 1 and 3). This confirms that interactions with transporters are not identical for all substances in the same chemical or pharmacological class, as has already been shown for various pesticides (Chedik et al., 2017; Kerhoas et al., 2024).

Substrates of SLC transporters, including OATs, are thought to *trans*-stimulate their activity (Hagos et al., 2012). BP analogues inhibiting OAT4 activity however failed to *trans*-stimulate it, and even *trans*-inhibited it (for TBBPA and PF201) (Fig. 5), thus indirectly suggesting that they are not or only poorly transported by OAT4. This hypothesis is also supported by the fact that HEK-OAT4 cells were not, or only marginally more sensitive to toxic effects of the potent OAT4-interacting compounds TBBPA, TCBPA and PF201 (Fig. 6), thus likely discarding any marked preferential accumulation of these chemicals in HEK-OAT4 cells. The fact that HEK-OAT4 cells failed to exhibit significant increased accumulation of PF201 and TCBPA when compared to HEK-MOCK cells, as demonstrated by LC-MS/MS assays, also argues in favour of this hypothesis.

The possible clinical consequences of OAT4 inhibition due to BP analogues, especially those of the potent OAT4 inhibitors TBBPA, TCBPA and PF201, remain to be clarified. IVIVE using criteria from the FDA nevertheless reported that environmental human exposure to TBBPA and TCBPA is not predicted to result in *in vivo* OAT4 inhibition (Table 1). By contrast, occupational exposure to PF201, unlike that to TBBPA, is predicted to *in vivo* block OAT4 activity (Table 1), which is likely of concern for workers exposed to this alternative to BPA, largely used as the main color developer in thermal papers (Demierre et al., 2024). Workers in contact with PF201 may also exhibit inhibition of OAT3 activity, as revealed by IVIVE (Table S4). This IVIVE approach has inherent limitations, notably because the *in vitro* systems used, i.e., HEK-OAT cell clones, may not fully recapitulate the *in vivo* functional characteristics of OATs physiologically expressed in human tissues and organs. In addition, the concentrations of BP analogues that may actually occur in human plasma and tissues following environmental or occupational exposure remain to be formally established. Nevertheless, the predicted *in vivo* OAT3/OAT4 blockage by PF201 is likely to impair OAT3/OAT4-mediated drug transport at the renal tubule, thus possibly

<figure><img src="image_12.png" /><figcaption>Fig. 6. Comparative effects of PF201, TBBPA and TCBPA on viability of HEK-OAT4 and HEK-MOCK cells. HEK-MOCK and HEK-OAT4 cells were exposed to various concentrations of PF201, TBBPA and TCBPA for 72 h. Cell viability was next determined by the MTT assay. Data are expressed as percentages of cellular viability found in control cells, arbitrarily set at 100 %, and are the means ± SD from 4 independent assays. IC₅₀ values as well as 95 % confidence intervals (in brackets) are indicated at the top of graphs. **, p < 0.01 and ns, not statistically significant, when compared to IC₅₀ values in control HEK-MOCK cells.</figcaption></figure>

resulting in transporter-related pollutant-drug interactions. *In vivo* inhibition of OAT4 may also result in altered transport of the endogenous substrates of this SLC at the renal and placental barrier. This may particularly concern hormones handled by OAT4, including steroid hormones and thyroid hormones (Tomi et al., 2015; Wagenaars et al., 2024). The fact that BP analogues, including PF201, decreased E3S uptake in HEK-OAT4 cells argues in favour of this hypothesis. Inhibition of OAT4 by BP analogues may thus contribute to the endocrine
<figure><img src="image_13.png" /><figcaption>**Fig. 7.** 3D representation of PF201, TBBPA and TCBPA docking within the Arg473-contained drug-binding pocket of OAT4. Hydrophobic and hydrophilic regions of the OAT4 binding pocket are colored in brown and blue, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

disruptive properties of these environmental contaminants (Pellerin et al., 2021). In addition to the kidney and placenta, several other organs and tissues, including the brain, retina, choroid plexus and testes, express OAT1, OAT3, and/or OAT4 (Nigam et al., 2015). Consequently, inhibition of these transporters by BP analogues, including PF201, may induce toxic effects at these sites of OAT expression, notably through altered transport of drugs, pollutants and endogenous molecules across the blood-testis and the blood-cerebrospinal fluid barriers.

Finally, the potent inhibition of OAT4 by PF201, TBBPA and TCBPA likely deserves special attention with respect to the overall use and safety issues for these chemicals. Such compounds are presently rather poorly characterized with respect to possible adverse effects towards human health (Mhaouty-Kodja et al., 2024). The fact that such BP analogues can strongly interact with human proteins such as OAT4 may

question the postulated safety of these chemicals, initially thought to not exert the established pleiotropic toxicity of the BP leader class BPA. This suggests to more extensively investigate the human health risk caused by BPA alternatives. The fact that the poorly studied PF201 has been very recently predicted to interact with various human molecular targets in addition to OAT4, including immune and non-immune ones (Franko et al., 2025; Montes-Grajales et al., 2021), supports this conclusion.

In summary, various BPs analogues, especially PF201, TBBPA and TCBPA, were demonstrated to act as potent inhibitors of OAT4 activity, thus fully confirming that OAT4 is a transporter of interest for pollutants. This moreover highlights the fact that BP analogues, initially proposed as safe substitutes for BPA, can interact with human proteins, including drug transporters. Caution may therefore be considered with respect to their use as non-toxic surrogates for BPA.
## CRediT authorship contribution statement

Hélène Le Mentec: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. David Malnoë: Writing – review & editing, Methodology, Investigation, Formal analysis. Marc Le Vée: Methodology, Investigation. Olivier Fardel: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

This study has received funding from the European Union's Horizon Europe research and innovation program under Grant Agreement N° 101057014 (European Partnership for the Assessment of Risks from Chemicals).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2025.127540.

## Data availability

Data will be made available on request.

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