In vitro estrogenic activity of binary and multicomponent mixtures with bisphenol A

Journal Pre-proofs In vitro estrogenic activity of binary and multicomponent mixtures with bisphenol A Darja Gramec Skledar, Lucija Peterlin Mašič PII: DOI: Reference:

S0048-9697(19)35203-9 https://doi.org/10.1016/j.scitotenv.2019.135211 STOTEN 135211

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Science of the Total Environment

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18 July 2019 22 October 2019 24 October 2019

Please cite this article as: D.G. Skledar, L.P. Mašič, In vitro estrogenic activity of binary and multicomponent mixtures with bisphenol A, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv. 2019.135211

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In vitro estrogenic activity of binary and multicomponent mixtures with bisphenol A

Darja Gramec Skledar, Lucija Peterlin Mašič*

Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia

*Corresponding author: Lucija Peterlin Mašič Faculty of Pharmacy University of Ljubljana Aškerčeva 7 1000 Ljubljana, Slovenia Tel: +386-1-4769635 E-mail: [email protected]

1

Abstract Bisphenol A and its analogs are environmental contaminants with well known estrogenic and anti-androgenic activities. In studies of human biomonitoring, simultaneous exposure to multiple bisphenols was shown in different biological samples, at picomolar to low nanomolar concentrations. Evaluation of their combined toxicities will therefore be a more realistic and reliable predictor for estimation of health risks than evaluation of only the single chemicals. In the present study, estrogenic activities of individual bisphenols were evaluated, along with their binary and multicomponent mixtures including three- and four-component mixtures, using the Organisation for Economic Co-operation and Development validated transactivation assay with the hERα-Hela9903 cell line. Concentration-dependent estrogenic activity was confirmed for all of the tested bisphenols, in the nanomolar to micromolar range. Estrogenic activities of binary and multicomponent mixtures followed a concentration addition model. Although exposure to individual bisphenols remains below their effective doses, we demonstrate that as a mixture, they can contribute additively to toxicity. This study thus emphasizes the importance of mixture toxicity evaluation for risk assessment of compounds that act like the bisphenols.

Keywords: bisphenols, mixtures, estrogenic activity, concentration addition

Abbreviations BPA, bisphenol A; BPAF, bisphenol AF; BPC, bisphenol C; BPF, bisphenol F; BPS, bisphenol S; BPZ, bisphenol Z; CA, concentration addition; E2, estradiol; ER, estrogen receptor; LDH, lactate dehydrogenase; NOEC, no observed effects concentration; OECD, Organisation for Economic Co-operation and Development. 2

1. Introduction

Bisphenol A (BPA) is a well-known endocrine disruptor with estrogenic and antiandrogenic activities (Fic et al., 2014; Skledar et al., 2016; Skledar et al., 2019). Use of BPA is linked to various diseases of modern times, such as metabolic syndrome, cardiovascular diseases and cancer (Michałowicz, 2014). BPA is therefore being gradually replaced with its analogs, like bisphenol S (BPS), bisphenol F (BPF), bisphenol AF (BPAF), bisphenol C (BPC), bisphenol Z (BPZ), and a number of others (Figure 1). Increasing use of BPA analogs has resulted in their detection in different biological and environmental samples, such as sewage, sediments, indoor dust and surface water (Caballero-Casero et al., 2016). Different bisphenols were detected in sediments collected from industrialized areas of United states, Korea and Japan in concentrations from below the limit of quantification to 25.3 µg/g dry weight (mean value of 0.2 µg/g dry weight) (Liao et al., 2012b), while the concentrations of BPA in surface water ranged from 0.12-12 µg/L (Flint et al., 2012). Various bisphenols have been simultaneously determined in urine, blood and other biological samples (Yang et al., 2014; Ye et al., 2015; Kolatorova Sosvorova et al., 2017). The concentrations of BPA and its analogs in urine (total concentration of the parent bisphenol with metabolites) are in the micrograms per liter range (Table 1), with BPA still the most frequently detected bisphenol (in >90% of biological samples), followed by BPS and BPF (Table 1).

Table 1: Concentrations of BPA and its analogs found in human urine.

It is thus certain that people are simultaneously exposed to a variety of endocrine-disrupting chemicals found in different everyday products, such as food and 3

beverages, cosmetics, pharmaceuticals, and plastics. Combined exposure to complex chemical mixtures can result in elevated toxicity. In different studies, the so-called ‘something from nothing’ effect was observed for chemical mixtures (Thrupp et al., 2018); namely, significant mixture effects were observed even when the chemicals were combined together at very low doses, with each below its ‘no observed effects concentration’ (NOEC) (Silva et al., 2002; Orton et al., 2014). Using a yeast estrogen screen, Silva and coworkers reported significant estrogenic activity for a mixture of eight environmental estrogens, with each present at a very low concentration, below the NOEC (Silva et al., 2002). The same has been reported also in vivo by Tinwell and Ashby (2004), who observed strong uterotropic effects for a combined mixture of seven estrogens, with each at sub-effective doses (i.e., doses that show negative uterotropic responses when tested alone) (Tinwell and Ashby, 2004). There are different principles to predict combined effects of chemicals in such mixtures. Mixture effects of noninteractive chemicals that share the same mode of action can be adequately described by the concentration addition (CA) concept, which was first described by Loewe and Muischek in 1926 (Loewe and Muischnek, 1926). Howdeshell and coworkers (2008) showed, for example, that a mixture of five phthalates with the same mechanism of action suppressed testosterone synthesis in fetal rats in a CA manner (Howdeshell et al., 2008), and Ermler and coworkers confirmed the suitability of a CA model for in vitro effects of 17 structurally diverse androgen receptor antagonists (Ermler et al., 2011). In contrast, for noninteractive chemicals that act through different modes of action, the independent action concept should be applied. However, Rider et al (2010) investigated mixture effects of seven chemicals with different mechanisms of action (antagonists of androgen receptors; inhibitors of androgen synthesis); these were precisely predicted by the CA concept, and not by the 4

independent action concept (Rider et al., 2010). In practice, it is often difficult to indisputably distinguish between these two concepts, and in such cases, the CA concept is usually used as a more conservative approach. While the estrogenic activities of individual bisphenols have been well established in numerous in vitro and in vivo assays (Fic et al., 2014; Gramec Skledar and Peterlin Mašič, 2016; Skledar et al., 2016; Skledar et al., 2019), their mixtures are still particularly underexplored. Conley and coworkers used an in vitro T47D-KBluc transactivation assay and an in vivo uterotropic assay for in vitro to in vivo extrapolation of the estrogenic activities of individual compounds, as well as for binary mixtures composed of BPAF and methoxychlor and BPS and methoxychlor, and multicomponent mixtures composed of BPAF, BPC, BPS, ethinyl estradiol, and   methoxychlor (Conley et al., 2016). In vivo estrogenic activities of BPAF, BPC, and BPA were accurately predicted from in vitro data, while in vivo predictions of estrogenic activity were overestimated for estradiol (E2) and underestimated for methoxychlor and BPS. The estrogenic activities of the mixtures were accurately predicted using a CA model with the individual chemical in vivo data, although there were large deviations when they use in vitro data (Conley et al., 2016). Recently, in vitro genotoxic activity of bisphenols mixtures in HepG2 cells was studied (Hercog et al., 2019). Binary and multicomponent mixtures of bisphenols did not exert genotoxic activity at concentrations relevant for human exposure. However, their mixtures additively increased gene expression of key metabolic enzymes CYP1A1 and UGT1A1 (Hercog et al., 2019). The purpose of the present study was to determine the estrogenic activities of binary and multicomponent mixtures of bisphenols using the Organisation for Economic Co-operation

and

Development

(OECD)

validated

in

vitro

hERα-Hela9903

transactivation assay, and to compare these in vitro data with predictions performed 5

with the CA model. Additionally, we wanted to determine whether complex mixtures of bisphenols can produce measurable estrogenic activity with the current in vitro assay, even when each bisphenol is present in the mixture at a concentration below its individual NOEC. Experiments will show how multicomponent mixtures of bisphenols act together at the receptor level and we hypothesized that CA is the appropriate prediction model.

Figure 1: Chemical structures of bisphenols tested in mixtures.

2. Materials and methods 2.1. Materials Bisphenol AF (BPAF; 97%; CAS 1478-61-1), bisphenol A (BPA; >99%; CAS 80-05-7), bisphenol F (BPF; 98%; CAS 620-92-8), bisphenol S (BPS; 98%; CAS 80-09-1), bisphenol C (BPC; 98%; CAS 79-97-0), bisphenol Z (BPZ; 98%; CAS 843-55-0) and dimethyl sulfoxide were all from Sigma-Aldrich (St. Louis, MO, USA). 17-Estradiol (E2; ≥98%; CAS 50-28-2), 17-estradiol (≥98%; CAS 57-91-0), corticosterone (≥98.5%; CAS 50-226), and kanamycin solution from Streptomyces kanamyceticus (10 mg/mL; CAS 2538994-0) were from Sigma-Aldrich, Germany. Methyltestosterone (>98%; CAS 58-18-4) was from Tokyo Chemical Industry (TCI Europe, Belgium).

2.2. Transactivation assay using ERα-HeLa-9903 cells The ERα-Hela 9903 cell line was from the Japanese Collection of Research Bioresources Cell Bank (JCRB1318; Osaka, Japan). Estrogenic activities were determined according to protocol 455 of the OECD (OECD, 2016). Briefly, the cells were maintained in phenolred-free minimal essential medium (Gibco), supplemented with 10% charcoal stripped 6

fetal bovine serum (Sigma), 2 mM glutamine (Sigma), and 60 mg/L-kanamycin (Sigma) at 37 °C and under 5% CO2. The cells were first seeded into white 96-well luminometer plates (Greiner, Bio one) at 1 ×105 cells/mL (100 μL/well). After 3 h, they were treated with the selected bisphenols or the vehicle control (0.1% dimethyl sulfoxide) and positive control (1 nM E2). After a 24-h incubation, the metabolic activity of the cells was determined using resazurin reduction assays, and luciferase levels used the One-glo Luciferase assay system (Promega, Madison, WI, USA), according to the manufacturer instructions (Luciferase assay system, instructions for use, 12/11; Promega). Luminescence and fluorescence were measured with an automatic microplate reader (Synergy 4 Hybrid; BioTek, Winooski, VT, USA).

Testing of the bisphenols mixtures For mixture effects we tested an equimolar mixtures of two, three, four or six bisphenols (Table 2). Additionally, we evaluated combined estrogenic effects of bisphenols at fixed mixture ratio, proportional to individual bisphenol EC50 values. Compositions of tested mixtures are listed in the Table 2.

Table 2: Compositions of testing mixtures.

2.3. Lactate dehydrogenase assay The lactate dehydrogenase (LDH) viability assay was performed using LDH cytotoxicity assay kits (CyQUANT; Thermo Fisher Scientific, Massachusetts, USA), according to the manufacturer instructions. Briefly, the cells were seeded into 96-well microtiter plates (1 ×105 cells/mL) and allowed to attach overnight. The cells were then treated with selected compounds and incubated for 24 h at 37 °C and under 5% CO2. Then 10 µL of 7

10× lysis buffer was added to the maximal LDH activity control wells, and the plates were incubated for an additional 45 min. Then 50 µL of the cell culture supernatant was transferred to a new 96-well microtiter plate and mixed with 50 µL of the reaction mixture. After 30 min at room temperature, the reactions were stopped with 50 µL stop solution. Absorbance was measured at 490 nm and 680 nm with an automatic microplate reader (Synergy 4 Hybrid microplate reader; BioTek, Winooski, VT, USA). Cytotoxicity was determined according to Equation (1):

(1).

2.4. Data analysis GraphPad Prism 5.04 software for Windows (GraphPad Software Inc., San Diego, CA, USA) and Microsoft Excel were used to analyze the data obtained in the in vitro assays, according to OECD protocol 455. The data collected from at least two independent experiments performed in triplicate were fitted using the GraphPad Prism software, and the EC50 values were calculated. Bisphenols were considered as agonists of the estrogen receptor (ER) pathway if their maximal response was at least 10% of the maximum response induced by the positive control (i.e., 1 nM E2). The endocrine-disrupting activities toward the ER were analyzed using two-sample Student’s t-tests, where *p <0.05, **p <0.01 and ***p <0.001 were considered statistically significant.

2.5. Concentration addition prediction model The CA model can be used for the prediction of mixture effects for chemicals that share the same mechanism of action (i.e., one chemical is a dilution of the other). The model can be described according to Equation (2): 8

(2),

where ci is the individual concentration of a chemical in the mixture that produces an effect x, and ECx is the effect concentration of a single chemical that alone would produce the same effect x as the mixture. Ci can be further replaced with proportions of the total concentration and described according to Equation (3), which enables construction of concentration response curves for mixtures, and comparison with experimental data (Thorpe et al., 2006).

,

(3).

Where ECxi represents the effect concentration of the compound in the mixture that independently elicits same effect as mixture and pi represents the molar fraction of the component in the mixture. To evaluate accuracy of the model, model deviation ratio (MDR) was used, which is defined as:

Where expected activity is the effective concentration activity for the mixture predicted by CA model and observed activity is effective concentration activity for the mixture obtained with transactivation assay on Hela9903 cell line. Mixtures with MDR values lower than 0.5 and higher than 2 have high probability of synergistic (MDR < 0.5) and antagonistic interactions (MDR > 2), while MDR values within 0.50–0.71 and 1.40–2.00 indicate, respectively, under- and overestimation of calculated models. 3. Results 9

3.1. Cytotoxicities of the bisphenols determined with the resazurin and LDH assays The noncytotoxic concentrations of tested bisphenols and six component bisphenols mixture were determined with two different assays: the resazurin assay, which measures the metabolic activity of the cells (i.e., metabolically active cells can reduce resazurin to highly fluorescent resofurin); and the LDH assay, which measures the integrity of the plasma membrane. According to OECD protocol 455, the concentrations of the tested bisphenols or mixtures that reduced cell viability by >20% were considered as cytotoxic and were not included in the further evaluation of estrogenic activity. LDHbased cytotoxicity was observed for BPAF at 50 µM (Figure 2b). None of the tested individual bisphenols were considered cytotoxic at 25 µM, neither with the resazurin assay nor the LDH assay (Figure 2a, b). Multicomponent equimolar mixture of all of these tested bisphenols showed LDH-based cytotoxicity at 50 µM and 25 µM of individual bisphenol in the mixture, but not at 10 µM (54%, 49%, 10% cytotoxicity, respectively) (Figure 2b). Similarly, using the resazurin assay, the cell viability with the multicomponent mixture tested at 25 µM was reduced by 47% (Figure 2). Therefore, the highest tested concentration for all the multicomponent mixtures was set to 10 µM.

Figure 2

3.2. Estrogenic activities of the individual bisphenols The estrogenic activities of the individual bisphenols were determined with the hERαHela9903 transactivation assay according to OECD protocol 455. The estrogenic activities of BPA and BPAF determined with OECD protocol 455 assay have been reported previously (Skledar et al., 2019), while the remaining bisphenols were evaluated here for the first time. The responsiveness of the assay was initially evaluated 10

with the reference substances (i.e., 17-E2 as a strong agonist; 17α-E2 as a weak agonist; 17α-methyltestosterone as a very weak agonist; and corticosterone as inactive), and the values obtained fell within the specified ranges (Figure 3, Table 3). All of these bisphenols showed concentration-dependent hERα agonistic activities with EC50 values in the nanomolar or low micromolar range. BPAF showed the highest estrogenic activity (EC50, 0.13 µM), followed by BPC, BPA, BPZ, BPS, and BPF (EC50, 1.01, 1.14, 1.15, 3.90, 4.39 µM, respectively) (Figure 3, Table 3).

Figure 3

3.3. Estrogenic activities of the mixtures Binary and multicomponent mixtures were evaluated for estrogenic activities, with the experimental data further compared with the CA prediction model (Figures 4-7; Table 3). We evaluated estrogenic activity of all binary mixtures with BPA (Figure 4), selected three-component (Figure 5) and four-component mixtures (Figure 6), and the multicomponent mixture of all six bisphenols (Figure 7). At the beginning we tested equimolar mixtures of bisphenols and then we also tested selected binary and multicomponent mixtures at fixed equi-effective ratios; proportional to the EC50 value of each bisphenol to ensure a balanced contribution of each single bisphenol to the overall mixture effect. In addition, we also tested a four-component mixture of the most commonly identified bisphenols BPA, BPS, BPF and BPAF in biological samples based on ratios in the urine samples determined in the study of Yang and coworkers (Yang et al., 2014). Here, the in vitro data were generally well predicted by the CA model, with MDR values between 0.77-1.35. The highest difference between the experimental and the predicted data was seen for the six component mixtures, however the deviation from the 11

model can be explained with larger standard deviations between biological replicates and does not represent significant deviation from additivity.

Figure 4 Figure 5 Figure 6 Figure 7

Table 3: Summary of the experimental and predicted (mixtures: concentration addition model) estrogenic activities of the individual bisphenols and their mixtures, as determined in vitro with the transactivation assay using the hERα-Hela9903 cell line. Data are means ±standard deviation of at least two independent experiments, each carried out in triplicate.

3.4. Estrogenic activities of bisphenols and their mixture at no observed effect concentrations The in vitro estrogenic activities were also determined for the individual bisphenols at their NOEC (as initially determined in dose-response assays with the individual bisphenols; i.e., BPA, BPS, BPF, BPC, BPZ, 0.1 µM; BPAF, 0.01 µM) and for their mixture. Estrogenic activities of these individual bisphenols were absent (i.e., BPAF, BPS, BPF) or very low (i.e., BPA, BPC, BPZ), and were well below the limit defined for estrogenic agonists in OECD protocol 455 (i.e., 10% estrogenic activity of the positive control). Nevertheless, the mixture of all six bisphenols showed significant estrogenic activity (i.e., 30% of maximal estrogenic activity induced by 1 nM E2), which was higher than was predicted by the summation of the effects of the individual bisphenols (Figure 8). 12

The estrogenic activity of the bisphenols mixture predicted with the CA model was a little higher than that observed experimentally (40% of maximal estrogenic activity); however, this CA prediction was closer to the experimental data than the simple summation of the effects of each of the individual bisphenols (Figure 8).

Figure 8

4. Discussion Bisphenol A is a well-known endocrine disruptor that can be found frequently in the environment as well as in humans. Prohibitions on the use of BPA in different products have resulted in increased use of its analogs in the manufacture of plastics, and consequently to higher human exposure to these chemicals. Indeed, BPA analogs have been found in biological samples at concentrations from picomolar to low nanomolar. Reported geometric mean concentrations of BPA in urine, for example, have ranged from 0.36 µg/L to 5.0 µg/L (Table 1), which corresponds to 1.6 nM to 21.9 nM. This is lower than the concentrations needed to provoke estrogenic effects through the nuclear ERs (Caballero-Casero et al., 2016). However, people can be simultaneously exposed to complex mixtures of different bisphenols and to many other environmental estrogens, which can contribute to mixture toxicity. The in vitro estrogenic activities of these bisphenols determined with the OECD validated transactivation assay using the Hela9903 cell line were in the low micromolar range, which are in agreement with previous in vitro studies (Kitamura et al., 2005). Considering human exposure to those chemicals (i.e., in the picomolar to low nanomolar range) and their rapid metabolism to inactive glucuronides and sulfates, it can be concluded that these chemicals do not pose a risk to human health through their ER13

mediated activities. However, it was shown previously in in vitro and in vivo studies, even if they are individually at sub-effective concentrations, mixtures of these chemicals can evoke estrogenic effects (Silva et al., 2002; Tinwell and Ashby, 2004). We have here confirmed this so-called ‘something from nothing’ phenomenon also for this mixture of the six bisphenols that are most commonly detected in biological samples. Although individually at the tested concentrations none of these bisphenols showed agonistic estrogenic activities, their mixture was significantly estrogenic (30% of the maximal estrogenic effect). Moreover, we are continuously exposed to low concentrations of various other ER agonists (e.g., flavonoids, urolithins from food; parabens from cosmetics; phthalates from plastics) that might accumulate and result in toxic outcomes. Long-term exposure to low doses of complex mixtures can therefore be particularly problematic, especially for the most vulnerable groups, such as children. Herein, we have confirmed that low ineffective concentrations of these individual environmental estrogens does not automatically mean that there is no risk for human health. In current in vitro experiment we demonstrated realistic exposure to complex mixture of similar acting chemicals, tested at low concentrations. The effects observed in vitro can be expected also in vivo, as reported previously by Tinwell and Ashby (2004). They used uterotropic assay and confirmed that environmental chemicals tested at low, inactive doses, gave a positive response when tested as complex mixture (Tinwell and Ashby, 2004). Mixture effects of bisphenols determined in vitro with reporter gene assay gave us valuable mechanistic information and potencies at the receptor level. However, in vitro assays do not consider toxicokinetics parameters. For example, rapid metabolism, mainly to inactive glucuronide was reported for some bisphenols in vivo (Gramec Skledar and Peterlin Mašič, 2016). In vitro assays therefore do not necessarily correctly predict in vivo effects, at concentration relevant for human exposure. 14

All of these tested bisphenols have the same mode of action (i.e., ER activation), and therefore the CA model prediction was expected for their mixtures. The CA model has accurately predicted the mixture effects of noninteractive compounds acting through the same mechanism in various in vitro and in vivo studies (Houtman et al., 2006; Correia et al., 2007; Ramirez et al., 2014; Seeger et al., 2016; Yu et al., 2019). In a recent in vitro study conducted by Yu et al. (2019), the combined estrogenic activity of six environmental estrogens that work through a similar mechanism (i.e., as here; ER activation) was better predicted with the CA model than with the response addition model (Yu et al., 2019). The predictive power of CA is very good and is influenced by the number of components in the mixture, the mixture ratio and the slope of the concentration response curve of individual components. Nevertheless, the CA often overestimates mixture toxicity and is therefore used as a conservative approach in the risk assessment. The CA model is a good predictor of the overall activity of bisphenols mixtures, with MDR values within 0.77-1.35. The CA model generally under-predicted the estrogenic activity for these bisphenols mixtures, although the predicted combined EC50 was maximally 1.35-fold higher (i.e., binary mixture with BPA and BPS and sixcomponent mixture), which does not represent a significant deviation from additivity. Overall, our findings are in concordance with previous studies which showed that CA is a good prediction model for mixtures of compounds with the same mechanism of action.

5. Conclusions

Exposure to bisphenols is widespread, and we have demonstrated in the present study that as a mixture they can cause toxic effects even at concentrations that are low enough to be considered safe for exposure to the individual bisphenols. These combined effects 15

of bisphenols can be accurately predicted with the CA model. Evaluation of this mixture toxicity for these bisphenols represents a more realistic and reliable approach than the traditional risk assessment approach that is based on the effects of the individual bisphenols, and can thus underestimate the real threat that bisphenols present to human health.

Acknowledgements The authors acknowledge the financial support of the Slovenian Research Agency (Grant No. P1-0208).

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spectrometry. Talanta 154, 511-519. Rocha, B.A., de Oliveira, A.R.M., Barbosa, F., 2018. A fast and simple air-assisted liquidliquid microextraction procedure for the simultaneous determination of bisphenols, parabens, benzophenones, triclosan, and triclocarban in human urine by liquid chromatography-tandem mass spectrometry. Talanta 183, 94-101. Seeger, B., Klawonn, F., Nguema Bekale, B., Steinberg, P., 2016. Mixture effects of estrogenic pesticides at the human estrogen receptor α and β. PLoS One 11, e0147490. Silva, E., Rajapakse, N., Kortenkamp, A., 2002. Something from "nothing" - eight weak estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects. Environ. Sci. Technol. 36, 1751-1756. Skledar, D.G., Carino, A., Trontelj, J., Troberg, J., Distrutti, E., Marchianò, S., Tomašič, T., Zega, A., Finel, M., Fiorucci, S., Mašič, L.P., 2019. Endocrine activities and adipogenic effects of bisphenol AF and its main metabolite. Chemosphere 215, 870-880.

20

Skledar, D.G., Schmidt, J., Fic, A., Klopcic, I., Trontelj, J., Dolenc, M.S., Finel, M., Masic, L.P., 2016. Influence of metabolism on endocrine activities of bisphenol S. Chemosphere 157, 152-159. Thorpe, K.L., Gross-Sorokin, M., Johnson, I., Brighty, G., Tyler, C.R., 2006. An assessment of the model of concentration addition for predicting the estrogenic activity of chemical mixtures in wastewater treatment works effluents. Environ. Health Perspect. 114 (1), 90-97. Thrupp, T.J., Runnalls, T.J., Scholze, M., Kugathas, S., Kortenkamp, A., Sumpter, J.P., 2018. The consequences of exposure to mixtures of chemicals: something from 'nothing' and 'a lot from a little' when fish are exposed to steroid hormones. Sci. Total Environ. 619-620, 1482-1492. Tinwell, H., Ashby, J., 2004. Sensitivity of the immature rat uterotrophic assay to mixtures of estrogens. Environ. Health Perspect. 112, 575-582. Yang, Y., Guan, J., Yin, J., Shao, B., Li, H., 2014. Urinary levels of bisphenol analogues in residents living near a manufacturing plant in south China. Chemosphere 112, 481486. Ye, X., Wong, L.Y., Kramer, J., Zhou, X., Jia, T., Calafat, A.M., 2015. Urinary concentrations of bisphenol A and three other bisphenols in convenience samples of U.S. adults during 2000-2014. Environ. Sci. Technol. 49, 11834-11839. Yu, H., Caldwell, D.J., Suri, R.P., 2019. In-vitro estrogenic activity of representative endocrine

disrupting

chemicals

mixtures

concentrations. Chemosphere 215, 396-403.

21

at

environmentally

relevant

Table’s legend Table 1: Concentrations of BPA and its analogs found in human urine. Table 2: Compositions of the testing mixtures. Table 3: Summary of the experimental and predicted (mixtures: concentration addition model) estrogenic activities of the individual bisphenols and their mixtures, as determined in vitro with the transactivation assay using the hERα-Hela9903 cell line. Data are means ±standard deviation of at least two independent experiments, each carried out in triplicate.

Figures legend: Figure 1: Chemical structures of bisphenols tested in mixtures. Figure 2: Metabolic activities (a) and cytotoxicities (b) of the selected individual bisphenols and equimolar six-component mixture determined with the resazurin reduction assay (a) and the LDH assay (b). Data are means ±standard errors of two independent experiments, each carried out in triplicate. Significant differences between treated cells and the solvent control is indicated by ***, p  <  0.001 (Two-tailed Student's t-test). Figure 3: Estrogenic activities of the individual bisphenols determined with the hERαHela9903 transactivational assay. Figure 4: Estrogenic activities of the individual equimolar (Mix 1, 3, 5, 6, 7) and equieffective (Mix 2, 4) binary bisphenol mixtures determined with the in vitro hERαHela9903 transactivation assay and as predicted with the concentration addition (CA) model.

22

Figure 5: Estrogenic activities of the equimolar (Mix 8, 10, 12) and equi-effective (Mix 9, 11, 13) trinary bisphenols mixtures determined with the in vitro hERα-Hela9903 transactivation assay and as predicted with the concentration addition (CA) model. Figure 6: Estrogenic activities of the equimolar (Mix 14, 17, 18) and equi-effective (Mix 15) four component mixture and four component BPA, BPAF, BPF and BPS mixture based on bisphenols ratio in urine samples (Mix 16) as determined with the in vitro hERα-Hela9903 transactivation assay and as predicted with the concentration addition (CA) model. Figure 7: Estrogenic activities of the equimolar (Mix 19) and equi-effective (Mix 20) six component bisphenol mixture determined with the in vitro hERα-Hela9903 transactivation assay and as predicted with the concentration addition (CA) model. Figure 8: Estrogenic activities of the individual bisphenols at their no observed effects concentration (NOEC; i.e., 0.1 µM for bisphenols A, S, F, C and Z and 0.01 µM for BPAF). Mix: Bisphenols mixture, containing all six bisphenols, each at its NOEC. Mix 10x: Bisphenols mixture, containing all six bisphenols at a concentration 10-fold their NOEC. CA, concentration addition prediction for mixture effect; EA, effect addition, as estrogenic response obtained as summation of estrogenic effects of individual bisphenols. Data are means ±standard error of two biological replicates, each carried out in triplicate. Lower dashed lined (0.1-fold control) represents lower limit of estrogenic activity as determined in OECD protocol 455, and upper dashed line (1-fold control) represents estrogenic activity of positive control (i.e., 1 nM E2). Significant differences between treated cells and the solvent control is indicated by **, p <0.01, ***, p <0.001 and ****, p <0.0001 (Two-tailed Student's t-test).

23

Table 1: Concentrations of BPA and its analogs found in human urine. Compound Bisphenol A

Bisphenol S

Bisphenol F

Bisphenol AF

Geometric mean

Frequency of

concentration (µg/L)

detection (%)

2.55

100

(Koppen et al., 2019)

1.24

95.7

(Lehmler et al., 2018)

0.36–2.07

74–99

1.9

92

(Rocha et al., 2016)

1.2

96

(Rocha et al., 2018)

5.0

100

(Heffernan et al., 2016)

0.89

100

(Yang et al., 2014)

0.37

89.4

(Lehmler et al., 2018)

<0.1−0.25

19–74

0.168

81

(Liao et al., 2012)

0.52

14

(Rocha et al., 2018)

0.029

40.4

(Yang et al., 2014)

0.35

66.5

(Lehmler et al., 2018)

0.15−0.54

42–88

1.27

24

(Rocha et al., 2018)

0.23

<30

(Yang et al., 2014)

0.018

<30

(Yang et al., 2014)

24

Reference

(Ye et al., 2015)

(Ye et al., 2015)

(Ye et al., 2015)

Table 2: Compositions of the testing mixtures.

Mix 1 Mix 2 Mix 3 Mix 4 Mix 5 Mix 6 Mix 7 Mix 8 Mix 9 Mix 10 Mix 11 Mix 12 Mix 13 Mix 14 Mix 15 Mix 16 Mix 17 Mix 18 Mix 19 Mix 20

BPA 50% 90% 50% 23% 50% 50% 50% 33.3% 22% 33.3% 20% 33.3% 12% 25% 12% 75% 25% 25% 15.6% 9.7%

BPAF 50% 10%

BPS

BPF

BPC

BPZ

50% 78% 50% 50% 50% 33.3% 2.4% 33.3% 2%

25% 1% 1% 25% 15.6% 1%

33.3% 75.6%

33.3% 41% 25% 41% 2% 25% 15.6% 33.3%

25

33.3% 78% 33.3% 47% 25% 46% 22% 25% 25% 15.6% 34.9%

25% 25% 15.6% 8.5%

15.6% 9.7%

Table 3: Summary of the experimental and predicted (mixtures: concentration addition model) estrogenic activities of the individual bisphenols and their mixtures, as determined in vitro with the transactivation assay using the hERα-Hela9903 cell line. Data are means ±standard deviation of at least two independent experiments, each carried out in triplicate. Condition

E2 (strong agonist)

EC50 (µM)

2.14 ×10-5

Predicted

MDR

EC50 CA

(Expected/o

model (µM)

bserved)

NA

±0.32 ×10-5 17-α E2 (weak agonist)

0.0028

NA

±0.0013 Methyltestosterone (very weak agonist)

ND

NA

Inactive

NA

BPA

1.14 ±0.16

NA

BPF

4.39 ±1.01

NA

BPAF

0.13 ±0.05

NA

BPS

3.90 ±0.94

NA

BPC

1.01 ±0.20

NA

BPZ

1.16 ±0.31

NA

Mix 1 (BPA + BPAF)

0.23 ±0.002

0.23

1

Mix 2 (BPA + BPAF)

0.52 ±0.05

0.63

1.19

Mix 3 (BPA + BPS)

1.30 ±0.68

1.76

1.35

Mix 4 (BPA + BPS)

2.33 ±0.78

2.63

1.12

Mix 5 (BPA + BPF)

1.72 ±0.42

1.81

1.05

Mix 6 (BPA + BPC)

0.83 ±0.31

1.06

1.28

Mix 7 (BPA + BPZ)

0.98 ±0.23

1.14

1.16

Mix 8 (BPA + BPAF+BPS)

0.33 ±0.002

0.34

1.03

Mix 9 (BPA + BPAF+BPS)

1.94 ±0.09

1.78

0.92

Mix 10 (BPA + BPAF+BPF)

0.37 ±0.03

0.33

0.89

Mix 11 (BPA + BPAF+BPF)

1.51 ±0.30

1.98

1.31

Mix 12 (BPA + BPF + BPS)

1.86 ±0.16

2.21

1.19

Mix 13 (BPA + BPF + BPS)

2.52 ±0.06

3.16

1.25

Corticosterone (negative control)

26

Mix 14 (BPA + BPAF + BPF + BPS)

0.56 ±0.04

0.44

0.79

Mix 15 (BPA + BPAF + BPF + BPS)

2.18 ±0.04

2.47

1.13

Mix 16 (BPA + BPAF + BPF + BPS)

0.99 ±0.15

1.29

1.30

Mix 17 (BPA + BPAF + BPF + BPC)

0.43 ±0.02

0.40

0.93

Mix 18 (BPA + BPF + BPS + BPC)

1.35 ±0.32

1.78

1.32

Mix 19 (BPA + BPAF + BPF + BPS + BPC +

0.70 ±0.21

0.54

0.77

1.46 ±0.54

1.97

1.35

BPZ) Mix 20 (BPA + BPAF + BPF + BPS + BPC + BPZ) NA; not applicable; ND, not determined

27

28

a)

b) 1.5

BPA BPAF BPF BPS BPC BPZ

1.0

0.5

Cell viability (n fold over control)

Cell viability (n fold over control)

1.5

0.0 5

10

0.5

0.0

25

5

[Bisphenols], µM

10

25

[Bisphenols], µM

c) 30

BPA BPAF BPF BPS BPC BPZ

20 10

% Cytotoxicity

d)

40

% Cytotoxicity

BPA+BPAF BPA+BPF BPA+BPS BPA+BPC BPA+BPZ All bisphenols

1.0

80 60

BPA+BPAF BPA+BPF BPA+BPS BPA+BPC BPA+BPZ All bisphenols

40 20 0

0 0

20

40

[Bisphenols], µM

60

0

20

40

[Bisphenols], µM

29

60

2.0

BPA BPAF BPS E2

1.0 0.5 0.0 10 -8 -0.5

10 -6

10 -4

10 -2

[Bisphenols], µM

10 0

10 2

Estrogen activity (Normalized to E2)

Estrogen activity (Normalized to E2)

1.5

1.5 1.0

BPF BPC BPZ E2

0.5 0.0 10 -8 -0.5

10 -6

10 -4

10 -2

[Bisphenols], µM

30

10 0

10 2

0.0 10 -5

Normalized to E2

1.5 1.0

Mix 3

0.0

1.0

10 0

10 5

c, µM

Mix 5

0.0 10

1.5 1.0

10 -8

1.0

10 -6

10 -4

10

0

10

5

c, µM

10 -2

10 0

10 2

10 -2

10 0

10 2

0.0 10 -8

1.0

10 -6

10 -4

c, µM

Mix 6 BPA+BPC CA E2

0.5 0.0 10 -8 -0.5

10 -6

10 -4

c, µM

Mix 7

0.0 10 0

10 2

Mix 4

BPA+BPZ CA E2

10 -5

10 0

BPA+BPS CA E2

0.5

-0.5

10 -2

c, µM

0.5

1.5

0.5

-0.5

0.0

-0.5

BPA+BPF CA E2

-5

0.5

1.5

BPA+BPS CA E2

10 -5

1.0

BPA+BPAF CA E2

-0.5

0.5

1.5

Normalized to E2

10 5

c, µM

-0.5

Normalized to E2

10 0

Normalized to E2

0.5

-0.5

Mix 2

1.5

Normalized to E2

1.0

Mix 1 BPA+BPAF CA E2

Normalized to E2

Normalized to E2

1.5

10 5

c, µM

31

Mix 8

Mix 9 1.5

BPA+BPAF+BPS CA

1.0

E2

0.5 0.0 10 -5

10 0

10 5

Normalized to E2

Normalized to E2

1.5

c, µM

-0.5

1.0

BPA+BPAF+BPS CA E2

0.5 0.0 10 -8

10 -6

Mix 10 BPA+BPAF+BPF

1.0

E2

0.5 0.0 10 -5

10 0

10 5

c, µM

-0.5

1.5 1.0

1.0

CA E2

0.5 0.0 10 -5 -0.5

10 0

c, µM

10 5

10 -2

10 0

10 2

10 -2

10 0

10 2

0.0 10 -8

10 -6

10 -4

c, µM

Mix 13

2.0

Normalized to E2

Normalized to E2

1.5

10 2

0.5

-0.5

BPA+BPF+BPS

10 0

BPA+BPAF+BPF CA E2

Mix 12 2.0

10 -2

Mix 11

2.0

CA

Normalized to E2

Normalized to E2

1.5

10 -4

c, µM

-0.5

1.5 1.0

BPA+BPS+BPF CA E2

0.5 0.0 10 -8

10 -6

10 -4

c, µM

-0.5

32

Mix 15

BPA+BPAF+BPS+BPF

1.0

CA E2

2.0

0.5 0.0 10

-5

10

0

10

5

Normalized to E2

Normalized to E2

Mix 14 1.5

c, µM

-0.5

1.5

BPA+BPS+BPF+BPAF CA E2

1.0 0.5 0.0 10 -8

10 -6

1.5

BPA+BPAF+BPF+BPC

Real urine CA E2

0.0 10 -8

10 -6

10 -4

10 -2

10 0

10 2

c, µM

-0.5

10 -8

10 -6

Normalized to E2

E2

0.0 10 -6

10 -4

10 -2

10 0

10 -4

c, µM

-0.5

BPA+BPF+BPS+BPC

10 -8

10 2

0.0

0.5

-0.5

10 0

E2

0.5

CA 1.0

10 2

CA 1.0

Mix 18 1.5

10 0

Mix 17 Normalized to E2

Normalized to E2

0.5

10 -2

c, µM

Mix 16 1.5 1.0

10 -4

-0.5

10 2

c, µM

33

10 -2

Mix 19

1.0

Mix 20

BPA+BPS+BPF+BPAF+BPC+BPZ CA E2

0.5 0.0 10 -8 -0.5

10 -6

10 -4

c, µM

10 -2

10 0

10 2

2.0

Normalized to E2

Normalized to E2

1.5

1.5

BPA+BPS+BPF+BPAF+BPC+BPZ CA E2

1.0 0.5 0.0 10 -8 -0.5

10 -6

10 -4

c, µM

34

10 -2

10 0

10 2

35

Highlights    

Bisphenols exhibited estrogenic activities in OECD validated transactivation assay Estrogenic activities of mixtures with BPA followed a concentration addition model ‘Something from nothing’ phenomenon was confirmed for mixture of the six bisphenols Mixture effects should be considered in the risk assessment of chemicals

36

c, µM

-0.5

1.5

BPA+BPS CA model E2

0.5 0.0 10 0

10 -5

10 5

Normalized to E2

Normalized to E2

1.5 1.0

c, µM

-0.5

c, µM

-0.5

BPA+BPC CA model E2

1.0 0.5 0.0

10 -4

10 -6

10 -8

10 -2

10 0

10 2

10 -2

10 0

10 2

c, µM

-0.5

In vitro toxicity testing 1.5

10 0

10 -5 -0.5

c, µM

Concentration addition model

10 5

bisphenols CA model E2

1.0 0.5 0.0

10 -4

10 -6

10 -8

c, µM

-0.5

Estrogen activity (Normalized to E2)

Normalized to E2

Combined exposure

0.0

Normalized to E2

1.5

BPA+BPZ OECD validated CA model 1.0 transactivation assay E2 on Hela9903 cell line 0.5

1.0

0.5

1nM E2

EA

Mix 10x

CA

Mix

BPZ

BPF

BPC

BPS

BPA

Environmental pollutants and endocrine disruptors

BPAF

0.0