The impact of toxic bisphenols on model human erythrocyte membranes

Journal Pre-proof The impact of toxic bisphenols on model human erythrocyte membranes Beata Wy˙zga, Karolina Połe´c, Karolina Olechowska, Katarzyna ˛ Hac-Wydro

PII:

S0927-7765(19)30814-8

DOI:

https://doi.org/10.1016/j.colsurfb.2019.110670

Reference:

COLSUB 110670

To appear in:

Colloids and Surfaces B: Biointerfaces

Received Date:

1 August 2019

Revised Date:

3 November 2019

Accepted Date:

23 November 2019

˛ Please cite this article as: Wy˙zga B, Połe´c K, Olechowska K, Hac-Wydro K, The impact of toxic bisphenols on model human erythrocyte membranes, Colloids and Surfaces B: Biointerfaces (2019), doi: https://doi.org/10.1016/j.colsurfb.2019.110670

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The impact of toxic bisphenols on model human erythrocyte membranes.

Beata Wyżga, Karolina Połeć, Karolina Olechowska, Katarzyna Hąc-Wydro* Department of Environmental Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387, Kraków, Poland

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Number of words: 7417 Number of figures: 6

Corresponding author (K. Hąc-Wydro)

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Number of tables: 0

Fax: +48 0-12-634-05-15 Phone: +48 0-12-686-25-72

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Graphical abstract

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e-mail: [email protected]

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Research highlights  Bisphenol A more strongly penetrates lipid monolayers than bisphenol S and F 

Bisphenols change condensation, morphology and interactions in erythrocyte membrane

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Bisphenol S and F interact with erythrocyte lipids less selectively than bisphenol A

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Bisphenol A and F are partially removed from cholesterol membrane

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bisphenols toxicity to erythrocytes may depend on the content of cholesterol in

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their membranes.

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Abstract Bisphenols are the environmental pollution of a highly harmful, but different in their magnitude, influence on the living organisms. Among various aspects of the toxicity of these compounds their effect on the red blood cells is intensively investigated. The aim of this work was to compare the effect of bisphenol A (BPA), bisphenol S (BPS) and bisphenol F (BPF) on model erythrocyte membranes and to get insight into the origin of the differences in the harmful effect of these substances on cells. Thus, the influence of bisphenols on multicomponent Langmuir

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films imitating the outer leaflet of erythrocyte membrane was thoroughly analyzed. An important step of the experiments were the studies on the effect of bisphenols on the films composed from particular erythrocyte membrane lipids. It was confirmed that both BPA and

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BPF affect model lipid systems more strongly than BPS, by changing their condensation, ordering, stability and morphology. However, the most essential conclusion was that BPA acts

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on the erythrocyte lipids more selectively than BPS and BPF and the influence exerted by this molecule is more strongly determined by the membrane composition. It was also suggested that

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cholesterol may act as the molecule of a decisive role from the point of view of the magnitude of the incorporation and the effect of BPA and BPF on membrane. Thus, the level of bisphenols

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toxicity to erythrocytes may depend on the concentration of cholesterol in their membranes.

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Keywords: bisphenols; erythrocyte membrane; lipid monolayer; Brewster angle microscopy

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Introduction Bisphenol A (BPA) is the compound used in the production of plastics, thermal paper, or dental materials. The thermal instability of this substance and its sensitivity to UV radiation, washing or alkaline conditions cause that BPA is systematically emitted from the products of everyday use and it is accumulated in the environment. In the consequence all the living organisms are continuously exposed to the toxic influence of bisphenol and its metabolites present in food, water as well as in various parts of the environment [1-7]. In fact bisphenol A is detected in the

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human urine or in the blood samples [8,9] and a strongly harmful influence of this compound on human health is well documented in literature. Bisphenol A belongs to the group of endocrine disrupting chemicals, which means that it is able to behave like hormones and to

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affect the functioning of the hormonal system and reproduction. However, the other toxic effects of BPA were also reported, for example this compound is associated with pathogenesis

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of breast or prostate cancer, as well as with the liver and cardiovascular diseases [6, 10,11].

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A strong toxicity of bisphenol A resulted in the replacement of this compound in the production of plastics or thermal papers by its analogues. Among them bisphenol S (BPS) and bisphenol F (BPF) seem to be the most frequently applied. However, today it is known that both BPS and

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BPF, similarly to BPA, are the xenoestrogenic substances and that the numerous toxic effects (cytotoxicity or genotoxicity), confirmed previously for BPA, were postulated also for BPS and

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BPF [6,12]. Moreover, similarly to BPA, also its analogues are present in the environment [12]

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and the living organisms are systematically exposed to their toxic influence by a dermal contact or via digestive system. It is worth noticing that BPS is chemically more stable than BPA, which may reduce its emission. On the other hand, it is produced in a large amounts and it is also more resistant to biodegradation than BPA. Moreover, it penetrates via skin more strongly than BPA [6]. In fact, the magnitude of the exposure to BPS and BPF as compared to BPA and its consequences are still intensively studied and they still need exploration.

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One of the area of the interest is the link between the exposure to bisphenols and the effect of these compounds on erythrocytes and cardiovascular system. For BPA the connection between the accumulation of this compound in the human body and the occurrence of the hypertension or the heart attack was suggested [13]. Moreover, the oxidative damage of erythrocytes induced by this compound and its metabolites was reported [14]. The number of the studies performed in this field for BPA substitutes (especially for BPF) is much lower than for BPA. However, for BPS its negative influence on the blood functions was found and the promotion of

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cardiovascular diseases in the living organisms was suggested [15]. Additionally, the comparative analysis of the toxic potential of the selected bisphenols to erythrocytes evidenced that BPS causes much weaker alterations in the morphology and functioning of the red cells

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than BPA and BPF [16]. An interesting aspect of these studies is the effect of bisphenols directly on cellular membrane, which is the barrier between the cell interior and the external

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environment. The in vitro experiments evidenced [17] that BPA is able to affect the erythrocyte membrane organization. As it was found, BPA alters hydrophobic region of membrane that is

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its penetrates the membrane deeply and disturbs its fluidity. These alterations made at the level of membrane may be the reason of the increase of the osmotic fragility and the internal viscosity

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of the red cells, occurring in the presence of BPA. In the same studies much weaker effect of BPS on the membrane properties was evidenced [17]. However, the influence of BPF on the

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membrane order parameter, the conformational alterations in membrane protein and the internal

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viscosity of human red blood cells was also significant [17]. The foregoing results motivated the experiments performed in this work. Namely, it is of great importance to get deeper insight into the origin of the differences in the effect of bisphenols on erythrocyte membrane, which can be connected with a different affinity of these harmful molecules to particular membrane lipids. A method, which is useful to perform this kind of investigations is based on the application of the model systems. One of them are Langmuir

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monolayers, that is the technique, which enables to construct separately the model of particular leaflets of the natural membranes. Moreover, the similarities in the behavior of the lipids in the monolayers and bilayers at the surface pressure range: 30-35 mN/m were proved [18], which allows one to link the results from the monolayer experiments to bilayer systems. The aim of this work was to compare the influence of BPA, BPS and BPF on the one component monolayers composed from the lipids, which are accumulated in the outer leaflet of the natural erythrocyte membrane (sphingomyelin, phosphatidylcholine, cholesterol) [19]. Then, the of

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bisphenols

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component

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(sphingomyelin/phosphatidylcholine/cholesterol) model erythrocyte membrane was studied. The results of the investigations on one component systems allowed one to deeply analyze the

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influence of BPA vs BPS vs BPF on elasticity and stability of more complex model membrane and to compare the effect of bisphenols on the interactions with the lipid molecules. Based on

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the collected data the selective affinity of bisphenols to particular erythrocyte membrane lipids was discussed and the role of the membrane cholesterol level in the influence of bisphenols was

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postulated.

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Experimental Materials The lipids used in the experiments, namely 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol (Chol) and egg yolk sphingomyelin (Sphingomyelin) were the compounds of high purity (≥99%) purchased from Avanti Polar Lipids Inc., USA. Bisphenol A (BPA) – 2,2bis(4-hydroxyphenyl)propane (99%), bisphenol S (BPS) – 4,4′-sulfonyldiphenol and bisphenol F bis(4-hydroxyphenyl)methane (BPF) (99%) were purchased from Sigma-Aldrich. The stock

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solutions of the lipids were made in chloroform/methanol (9:1 v/v) (HPLC grade, ≥99.9%, Aldrich) mixture. Bisphenol solutions for the application as the subphase were prepared in ultrapure water (their concentration in the subphase was equal to 0.1 mM). The stock solution

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of bisphenols used in the penetration experiments were prepared in ethanol and their final concentration in the subphase was equal to 0.1 or 0.01 mM. In all experiments ultrapure water

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of the resistivity 18.2 MΩ cm (produced with the application of the Merck-Millipore system) was used.

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Methods

The surface pressure (π) - area (A) isotherms were recorded for one component POPC, Chol

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and Sphingomyelin monolayers as well as for POPC/Chol/Sphingomyelin = 1:1:1 film treated as a model of erythrocyte membrane. To measure the isotherms the lipid solutions were

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deposited onto the subphase by using the Hamilton micro syringe (± 1.0 μL). As the subphase

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were used: pure water, BPA or BPS or BPF aqueous solutions, the latter in the concentration of 0.1 mM. After spreading of the lipid solution, the films were left for 5 min. and then the compression was started with the barrier speed 10 cm2/min. These experiments were performed on KSV-NIMA Langmuir trough (total area = 275 cm2) having two Delrin barriers enabling symmetrical compression of the monolayers. The trough was placed on an anti-vibration table. The surface pressure was measured (± 0.1 mN/m) with the Wilhelmy plate made of filter paper

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(ashless Whatman Chr1) connected to electrobalance. The experiments were done at 20 °C and the subphase temperature was controlled thermostatically (± 0.1°C) by a circulating water system. In all the experiments Ultra-pure Milli-Q water was used. The experiments were repeated at least twice to obtain consistent results. The penetration studies done for model erythrocyte membrane were based on the compression of the mixed lipid solution at the air/water interface to the target surface pressure. Then the monolayer was left for equilibration to the initial surface pressure πin (10; 20 and 30 mN/m).

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After equilibration the solution of particular bisphenols, prepared in ethanol, was injected into the subphase to final concentration of 0.1 or 0.01 mM. During experiments the subphase was continuously stirred. During experiments the changes in the surface pressure π (at a constant

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area) caused by penetration of bisphenols into the lipid film in time were recorded. In preliminary studies it was found that the injection of sole ethanol into the subphase (in the

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volume corresponding to the volume of ethanolic bisphenol solutions introduced during penetration experiments) does not modify the surface pressure. This ensures that the changes in

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the surface pressure observed in penetration studies were caused only by the presence of bisphenols.

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The another experiments done to gain some insight into the influence of bisphenols on the mixed monolayers stability were based on the measurements of the changes in the area per

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molecule values at constant surface pressure. To perform these experiments the monolayer

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spread on water or on the solutions of the respective bisphenol was compressed to a desirable surface pressure (30 mN/m). Then, the barriers were stopped at the area initial (A0) and the decrease in the area per molecule values (A) in time were monitored. To analyze the effect of bisphenols on the model membrane morphology Brewster angle microscopy studies were performed. In these experiments UltraBAM instrument (Accurion GmbH, Goettingen, Germany) equipped with a 50 mW laser emitting p-polarized light at a

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wavelength of 658 nm, a 10x magnification objective, polarizer, analyzer and a CCD camera was applied. The spatial resolution of the microscope was 2 μm. Both the microscope and the Langmuir trough were placed on the table (Standa Ltd. Vilnius, Lithuania) equipped with active vibration isolation system (antivibration system VarioBasic 40, Halcyonics, Göttingen, Germany). Calculations To obtain information on the effect of bisphenols on the monolayer elasticity the compressional

CS1   A (d / dA)

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modulus (CS-1) values were calculated based on the isotherms and according to eq. 1 [20]: (1)

wherein A is the mean area per molecule value at a given surface pressure π. The higher CS-1

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values the lower lateral elasticity of model membrane [21]:

Based on eq. 2 and 3 the excess area per molecule values (AExc) for the mixed films on water

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and on bisphenol solutions were calculated [22]:

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AExc = A - Aid Aid = ∑AiXi

(2) (3)

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Where: A is the mean area per molecule value at a given surface pressure, Aid is the ideal value of the mean molecular area, which is the linear combination of the areas of the respective

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components and their molar fractions in the mixtures, Ai is the mean area per molecule for the respective one component films and Xi is the mole fraction of the respective component in the

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mixed monolayer.

Additionally to compare the condensation of the model membrane on various subphases the percentage of condensation A% values were calculated (eq. 4) [23]: A% = [(Aid − A)/ Aid]×100%

(4)

From the results of penetration studies Δπ values were calculated (eq. 5): Δπ = π - πi

(5) 9

In the above equation π is the surface pressure at particular stages of monitoring, πi is the initial surface pressure (the surface pressure, at which bisphenol solution was injected into the subphase). The results of penetration experiments were presented in Δπ vs. time plots. Moreover, the equilibrium surface pressure values Δπeq were calculated according to eq. 6: Δπeq = πeq - πi

(6)

Where πeq is the value of the surface pressure achieved after stabilization during penetration experiments.

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Finally from the monolayer stability measurements the A/A0 values were calculated where A0 is the initial area at zero time and A is the area per molecules at different stages of monitoring. To compare the effect of the studied bisphenols on the mixed film stability the values of

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A/A0were presented as the percent of the value obtained for the film on water subphase.

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Results The surface pressure/area measurements The isotherms recorded for the model erythrocyte membrane as well as those for one component sphingomyelin and cholesterol films are shown in Fig. 1. In Fig. 2 the compressional modulus vs the surface pressure plots for these monolayers are presented. The studies on the effect of bisphenol A, S and F on one component POPC films were published previously [24], however,

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they are also included in the discussion performed in this work.

Fig. 1 The isotherms for model erythrocyte membrane and for one component lipid monolayers

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spread on water and on bisphenol solutions

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Fig. 2 The compressional modulus vs the surface pressure plots for model erythrocyte

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membrane and for one component lipid monolayers spread on water and on bisphenol solutions

The shape and the course of the curve for model membrane together with the maximal value of the compressional modulus for this film (CS-1max = 200 mN/m) indicate that the studied lipid mixture forms monolayer in a liquid condensed state. Among the studied lipids cholesterol molecules form the most ordered and the least elastic (CS-1max = 950 mN/m), while POPC the

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most fluid and the most elastic (CS-1max = 115 mN/m) [24] films. For sphingomyelin monolayer, during its compression, the phase transition between liquid expanded and liquid condensed state occurs. This is reflected in a kink in the course of the isotherm and in a minimum in the compressional modulus vs the surface pressure plot. The same lipid films were spread and compressed on the solutions containing bisphenol molecules. As it can be seen (Fig. 1) the presence of BPA, BPS and BPF in the subphase changes the shape and position of the isotherms in respect to the curves obtained on water. In general,

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bisphenols cause the shift of the isotherms to the larger areas and lowers its slope. This is accompanied by the decrease of the compressional modulus values (Fig. 2). Thus, bisphenols increase the fluidity and the lateral elasticity of the studied monolayers. However, the

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magnitude of these effects depends on the lipid forming the film as well on the kind of bisphenol added into the subphase. As indicate the collected results for all the lipids the effect of BPA is

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always visibly stronger than the effect of the remaining bisphenols. However, analysing the films of particular lipids, the additional differences in the effect of BPA, BPS and BPF can be

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noticed. As it is seen in Fig. 1 and 2 for cholesterol films the presence of BPA and BPF but not BPS, results in the kink in the course of the isotherm, which is visible as the minimum in the

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CS-1 vs π plots at ca. 28 and 30 mN/m for BPS and BPF, respectively. For the film on water this phenomenon does not occur, which indicates that the observed minimum evidences that BPA

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as well as BPF molecules are in part removed from the interface. Similar effect was found

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previously in the studies on the one-components plant sterol monolayers, and, less pronounced, for fungi sterol film [24]. The removal of BPA and BPF molecules is only partial since the isotherm for cholesterol film on the foregoing bisphenols, above the kink, does not cover with the curve registered on water. On the other hand, it covers well with the isotherms for the film on BPS. Thus, first of all, the content of BPA and BPF molecules in the film is sufficient to affect the film organisation. Secondly, the compression modulus values at π = 30-35 mN/m (in

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the region where the molecular packing of lipids in the monolayers is similar to that in bilayer) are lower for the film on BPA and BPF as compared to the film on BPS. Thus, BPA and BPF, even at lower concentrations than BPS, affect cholesterol film more strongly than BPS. The results obtained for sphingomyelin monolayer evidenced also that in the presence of bisphenols the transition surface pressure between an expanded and a more condensed state increases. Thus, the formation of the condensed phase in the presence of BPA, BPS and BPF is difficult, which additionally proves fluidizing effect of bisphenols.

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For POPC film [24] the presence of BPA in the subphase reflects not only in the shift of the isotherm to the larger areas but also in a strongly pronounced changes in the slope of the curve. In the consequence at π > 30 mN/m the mean molecular areas for the film on BPA solution are

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lower than those for the film on water. This effect indicates that the monolayer material is partially removed from the interface or in other word the lipids are extracted from the monolayer

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into the bulk phase. This is supported by a strong decrease of the compressional modulus values. As it can be observed for the mixed monolayer (Figs. 1 and 2) the presence of bisphenols leads

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to the fluidisation of the film (the shift of the isotherms and a decrease of the compressional modulus), however, the other effect is also observed. Namely, the studied bisphenols cause the

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decrease of the collapse surface pressure. The latter suggests that bisphenol molecules also destabilize the monolayer.

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To more deeply compare the effect of bisphenols on particular lipid films the changes in the

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position of the isotherm and in the value of the compressional modulus in respect to the values on water were calculated and expressed in percent (Fig. 3).

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Fig. 3 The shift of the isotherms (at π = 32.5 mN/m) and the drop of the compressional modulus

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values caused by bisphenols (the values are expressed as the percentage changes in respect for

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the monolayers on water).

Comparing the values of a percentage increase of the area per molecule it can be stated that for all the one component lipid films on BPS solution the effect is very similar in the error range. Also the changes in the compressional modulus are comparable for the majority of the studied films and only for sphingomyelin monolayer they are slightly stronger than for the remaining

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systems. On the other hand, for BPA both the differences in the isotherms position as well as the decrease of the compressional modulus for particular lipid films are more pronounced. Thus, in the case of BPA its effect depends more strongly on the lipid forming the monolayer as compared to BPS. In other words, BPS acts on the studied monolayers less selectively than BPA. Moreover, the effect of BPA on cholesterol film is stronger than the effect of BPS, despite the fact that BPA is partially removed from the interface. Interestingly, for BPF the alterations in the position of the isotherms were comparable, in the range of error, to those found for BPS.

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However, the effect of this compound on the values of compressional modulus is slightly more complicated. Namely, a decrease in this parameter value observed for cholesterol film is comparable to that induced by BPA and BPF. However, for the remaining lipid films the effect

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of BPF is intermediate between the influence of BPA and BPS. Moreover, based on the analysis of the parameters presented in Fig. 3, it can be concluded that BPF, similarly to BPS, acts on

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the studied one component films less selectively than BPA.

Based on the isotherms and according to the formulas 2 and 3 the excess area per molecule

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values for the mixed monolayer were calculated. The calculations were made at the surface pressure of 32.5 mN/m, which is in the surface pressure range important from the point of view

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of the natural membrane properties. The following values were obtained: -2.7; -2.5, -2.6 and 2.2 Å2/molecule (±0.2) for the film on water, on BPS, BPF and BPA solution, respectively. The

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values of this parameter are negative, which means a non ideal behaviour of the system and a

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favourable mixing between the molecules. The interactions between the mixed film components are more favourable than those between the particular lipids in their one component films. However, the presence of bisphenols changes these values to be less negative. This indicates that bisphenol molecules, incorporating into the monolayer, make the lipid molecules loosely packed. The lower lipid packing causes that the interactions between the lipids become weaker. This influence is stronger for BPA than for the remaining bisphenols. Unfortunatelly, the AExc

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values calculated for the films on bisphenol solutions cannot be directly compared with those obtained on water since the composition of the monolayer changes due to a possible penetration of bisphenols, However, the changes in the AExc values indicate that bisphenol molecules incorporate into the film and in this way they change intermolecular forces in the mixed system. oreover, the percent of condensation of the studied model membrane was calculated according to eq. 4. The comparison of the obtained results: 6.1; 5.8; 5.6 and; 4.5 % (±0.2) for the model system on water, BPS, BPF and BPA solution, evidences ones again that, among the studied

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bisphenols, BPA molecules affect the condensation of the model system the most strongly. Brewster angle microscopy

The effect of bisphenols on the morphology of the model system was investigated in Brewster

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angle microscopy experiments. The representative BAM images for the mixed monolayer on

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water subphase and in the presence of BPA, BPS and BPF are shown in Fig. 4.

Fig. 4. Bam images for model erythrocyte membrane on water and on bisphenol solutions 17

As it can be seen, even at low surface pressures the differences in the morphology of the monolayer spread on water and on the solutions of particular bisphenols appear. Namely, in the images taken at 0.5 mN/m for the mixture on water the coexistence of gaseous and more condensed homogenous phase together with a well detected nuclei of even more condensed domains appear. The images for the film on BPS solutions are very similar. However, in the film on BPA, the nuclei of the condensed phase are not formed. In the latter sense the images

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for the film on BPF solution are similar to those taken in the presence of BPA. Further compression of the film on water results in the formation of a large number of the condensed domains dispersed in a fluid matrix. These domains become larger and merge together and at

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the surface pressure > 10 mN/m the uniform condensed phase is visible in the images. For the film on BPS solution the domains are also formed, however, they are less pronounced and they

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do not enlarge with the film compression. This proves that BPS makes the formation of the condensed phase difficult. Analysing the pictures for the monolayers spread in the presence of

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BPA and BPF it can be conclude that these molecules prevent from the formation of the condensed domains and the monolayer, in the whole range of the surface pressure, is more fluid

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than the film on water. Summarizing, all the investigated bisphenols affect the morphology of model erythrocyte membrane, however, in a different degree. Namely the effect of BPS is the

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weakest, while BPF affects the model membrane morphology very similarly to BPA.

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Penetration studies

The results of penetration experiments are shown in Fig. 5. In Figs. 5a-b the ∆π vs time plots registered after injection of bisphenol solutions (0.1 mM) into the model membrane at different initial surface pressures (10; 20 and 30 mN/m) are shown. At the experimental conditions applied, after injection of bisphenol solutions the surface pressure increases and stabilizes in a short time. Thus, bisphenol molecules incorporate into the model membrane and stay at the

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interface, which is reflected in a positive values of ∆π.

Fig. 5 The penetration of bisphenols into model erythrocyte membrane at different initial surface pressures at the concetration of 0.1 mM (a, b); ∆πeq vs the initial surface pressure values

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after injection of particular bisphenols (c), the penetration of bisphenols into model erythrocyte membrane after injection of bisphenols at the concentration of 0.01 mN/m (d)

As it can be seen ∆π values are higher at lower initial surface pressures, which is the expected effect resulting from lower condensation of the monolayer at lower π value. To compare the results for all the bisphenols in Fig. 5c the equilibrium surface pressure values (∆πeq.) for a given initial surface pressure were presented. As it can be noticed independently on the surface

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pressure, ∆πeq. values are the highest after injection of BPA solution and the lowest in the presence of BPS. This proves stronger ability of BPA to penetrate model membrane as compared to BPF and BPS. The second highly important finding is that ∆πeq. are positive even

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at high surface pressures. The latter indicates that bisphenols are able to incorporate also into natural membranes, although the effect of BPS is much weaker as compared to the influence of

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the remaining bisphenols. To gain deeper insight into this issue the penetration experiments were performed also for the lower concentration of bisphenols (0.01 mM) at 30 mN/m. The

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results presented in Fig. 5d evidence that BPA is able to penetrate the monolayer even at lower concentration, which is in contrast to BPS. For the latter compound the surface pressure

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increases only slightly and in time the ∆πeq. values stabilize below 0 mN/m. This indicates that the injected BPS molecules practically do not incorporate into the model membrane and

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additionally, during experiments they escape from the film. The effect for BPF is intermediate

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between the effect of BPA and BPS, namely, initially the pronounced increase of the surface pressure occurs, however, in time the surface pressure decreases to zero values. Stability measurements To gain some insight into the stability of the studied model membrane the changes in the area per molecule values in time at constant surface pressure were monitored. To compare the effect of bisphenols, the percentage decrease of A/A0 values (A - the area occupied by the monolayers

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at time t, A0 - the initial area at t=0) in respect to the value on water at different stages of

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measurements was analyzed (Fig. 6).

Fig. 6 A decrease (in percent) of the A/A0 at selected stages of the monitoring of the decrease

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of the area in time.

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As it can be seen all the investigated bisphenols cause a decrease of the area per molecule values, which reflects in the drop of A/A0. Initially, the effect of all the compounds is very

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similar, however, in time, the stability of the model system decreases more strongly in the presence of BPA and BPF as compared to BPS. Moreover, the influence of BPA and BPF on

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the film stability is very similar. Discussion

As it is well known bisphenol A is a toxic compound emitted from many products of everyday use to the environment. One of the treatments of the reduction the exposure of the living organisms to this substance is to eliminate BPA from plastics production and use its analogues instead. The most promising and frequently used derivatives of BPA are BPS and BPF. 21

Although a lower toxicity of these compounds as compared to BPA is postulated, the investigations in this area are still performed. The aim of this work was to compare the influence of bisphenol A, bisphenol S and bisphenol F on the lipid monolayer imitating erythrocyte membranes, which are one of the targets for these molecules in the human body. The performed experiments evidenced that the influence of bisphenols on model erythrocyte membrane involves the decrease of the condensation and ordering of the system, as well as its destabilization. These effects reflect in the alterations in the position of the surface pressure

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area curves, the drop of the compressional modulus values as well as in the decrease of the collapse surface pressure and the mean molecular area with time. Moreover, Brewster angle microscopy experiments evidenced that bisphenols change the morphology of the model

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system, namely they make the formation of the condensed domains difficult. The results suggested that bisphenols make the lipid molecules loosely packed at the interface, which leads

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to the weakening of the interactions between the molecules in the monolayer. All the foregoing

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alterations result from the ability of bisphenols to incorporate into model lipid system, which was confirmed in our penetration experiments. The collected results evidenced also that the affinity of BPS to model lipid system is weaker as compared to BPA and BPF. Moreover, in

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general the effect of BPF on the studied films is intermediate between the effect of BPA and BPS. However, the changes in the film morphology verified based on BAM images (Fig. 4), as

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well as the stability of the monolayers (Fig. 6) is similarly affected by both BPF and BPA.

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The strongest effect of biphenol A on the mixed lipid film is an expected finding since BPA is more hydrophobic than the remaining bisphenols. This is indicated by the values of the logarithm of octanol/water partition coefficient (LogP) for these compounds (3.6, 2,9 and 1.5 for BPA, BPF and BPS respectively [25]. However, as prove our experiments, the logP value is not the only factor determining the behavior of both bisphenols in the lipid environment.

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A highly important result obtained in these studies is that BPS cannot be described as definitely safe for the cells. Despite weaker influence of this compound as compared to BPA on the monolayer properties at a given concentration, this substance is also able to affect the condensation, morphology and stability of the system as well as to alter the interactions between its components. On the other hand, its lower toxicity at a given concentration as compared to BPA seems to be well-founded also in our studies. The analysis of the obtained results together with a greater resistance of BPS than BPA to the effect of higher temperature and light [26] may

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lead to the conclusion that the risk due to exposure to bisphenol S is lower than to BPA. The collected results evidenced also that BPA is able to incorporate into model system at its lower concentration than BPS. This fact additionally confirms stronger affinity of this compound to

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model membrane and stronger risk resulting from the exposure to this compound. As far as BPF is concerned, this compounds similarly to BPS is considered as a less toxic alternative for BPA.

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However, our results evidence that also this substance is able to incorporate into lipid membrane and make the alterations in the organization of these structures. Moreover, both the penetration

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at higher surface pressures as well as the alterations caused in the film morphology and stability are very comparable to the effect observed for BPA.

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Probably the most important information resulting from our experiments and the results obtained previously [24] concern the affinity of the investigated bisphenols to particular lipids

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of model erythrocyte membranes. It can be concluded that BPS acts less selectively on the

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studied herein lipid monolayers than BPA. Namely, the modifications in the values of the analysed parameters caused by BPS are very similar for all the studied films. It should be however, stressed that the mentioned above low selectivity of BPS does not concern all type of lipids. Namely, our latter investigations evidenced pronounced differences in the magnitude of the effect of BPS on phosphatidylglycerol (POPG) vs phosphatidylethanol amine (POPE) and phosphatidylcholine monolayers (POPC) [24]. However, considering the lipids investigated

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herein, being typical of the external leaflet of human erythrocytes (sphingomyelin, phospahtidylcholines, cholesterol) our results prove very similar effect of BPS on the formed monolayers as well as on the model lipid membrane. Interestingly, the results collected for BPF, both herein and in the paper published previously [24], indicate that this compound acts comparably on all the lipids studied so far. Thus, the selectivity of BPF is low for a larger group of lipids as compared to the selectivity of BPS. For BPA the observed differences in the alterations in the values of the calculated parameters

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for the studied films were first of all more pronounced and secondly the effect of this bisphenol on particular lipids was more specific. Namely, the strongest decrease in the monolayer ordering (measured by the drop of the compressional modulus) was found for POPC, and for

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this system the changes in the isotherms position were negative. This means that BPA, by interacting with POPC, causes the extraction of the lipid molecules from the interface, leading

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to the strong decrease in the monolayer condensation. The latter phenomenon was not observed for BPS and BPF. Also the effect of BPA vs BPS on cholesterol films was different. Namely, it

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involves partial exclusion of BPA from the interface at higher surface pressures. The latter was not observed for BPS but it was found also for BPF, which additionally confirms some

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similarity in the membrane activity of BPA and BPF. The exclusion of the molecules from the model system leads to a decrease of BPA and BPF concentration in the lipid environment. Thus,

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cholesterol may act as the molecule preventing the membrane from the incorporation of

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bisphenol A and bisphenol F and weakening the harmful effect of their molecules on the system. Analysing the results published previously for ergosterol and β – sitosterol monolayers it can be concluded that BPS practically does not change the ordering of these two sterol films [24]. Its effect on the ordering of the cholesterol monolayer is slightly stronger, but it is also very weak. On the other hand, a significantly more pronounced are the differences in the effect of BPA and BPF on cholesterol vs the remaining sterols films. Namely, the influence of these two

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bisphenols on the ordering and condensation of the cholesterol monolayer is definitely stronger than on plant or fungi sterol films. This is reflected in the position of the isotherms and in the drop of the compressional modulus values [24]. The foregoing findings reveal also in the studies on multicomponent systems. Namely, at the same content of sterol in model system, the changes in the ordering of ergosterol – or β-sitosterol-containing multicomponent monolayers (measured by the drop in the compressional modulus at 30 mN/m) caused by BPA and BPF are lower [24] than those found for cholesterol-containing model erythrocyte membranes. Similar

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relationship can be found during the analysis of the penetration data. Namely, the penetration of BPA and BPF into cholesterol – containing model membrane (measured by the ∆πeq value at the surface pressure equal to 30 mN/ms) is slightly stronger than the penetration of these

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bisphenols into the plant and fungi membrane mimicking systems investigated previously. It is clear that the studied previously model plant and fungi membranes differ in their composition

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from the model studied herein, therefore the above mentioned analysis does not allow to draw definitive conclusions on the role of sterols in the activity of bisphenols. However, the data

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collected so far enable to suggest that from the point of view of the toxicity of BPA and BPF on erythrocyte membranes, the content of cholesterol may be important. That is, depending on the

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concentration of the sterol in membranes, both the incorporation of bisphenols as well as the magnitude of disturbances induced by their molecules may change.

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Summarizing all the collected results it can be concluded that both BPA, BPS and BPF can

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affect model erythrocyte membrane, but in a different degree. It was also found that the alterations caused in membranes by BPF are intermediate between those exerted by the remaining bisphenols. However, the effect of this molecule (BPF) on membrane organisation is closer to the effect of BPA than that of BPS. Thus, BPF molecule can be equally harmful for erythrocytes as BPA. Moreover, the influence of BPA is more strongly determined by the monolayer composition as compared to BPS and BPF. Thus, the reported in literature [16,17]

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differences in the alterations in the red cells caused by the presence of bisphenols may originate from a direct influence of these toxicants on membrane lipids. Considering the obtained results in relation to the natural membranes, they are in agreement with the reported findings that BPA and BPF are more toxic to the red cells than BPS [16,17]. However, our results allow one to additionally conclude that the toxicity of bisphenols can be modified by the composition of membrane. Namely, it is possible that the level of cholesterol in erythrocyte membrane may change the sensitivity of the cell to the effect of bisphenols. This is an important issue since the

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lipid composition of the erythrocyte membrane including the concentration of cholesterol therein, can change due to diseases, diet or even aging [27-30]. Therefore, it is highly important task to continue the investigations in this area.

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Conflict of Interest Statement: The authors declare that there are no conflicts of interest.

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