Pregnant Women and Endocrine Disruptors: Role of P2X7 Receptor and Mitochondrial Alterations in Placental Cell Disorders

In pregnant women, the lungs, skin and placenta are exposed daily to endocrine-disrupting chemicals (EDCs). EDCs induce multiple adverse effects, not only on endocrine organs, but also on non-endocrine organs, with the P2X7 cell death receptor being potentially the common key element. Our objective was first to investigate mechanisms of EDCs toxicity in both endocrine and non-endocrine cells through P2X7 receptor activation, and second, to compare the level of activation in lung, skin and placental cells. In addition, apoptosis in placental cells was studied because the placenta is the most exposed organ to EDCs and has essential endocrine functions. A total of nine EDCs were evaluated on three human cell models. We observed that the P2X7 receptor was not activated by EDCs in lung non-endocrine cells but was activated in skin and placenta cells, with the highest activation in placenta cells. P2X7 receptor activation and apoptosis are pathways shared by all tested EDCs in endocrine placental cells. P2X7 receptor activation along with apoptosis induction could be key elements in understanding endocrine placental and skin disorders induced by EDCs.


Introduction
Endocrine-disrupting chemicals (EDCs) are defined by the World Health Organization as exogenous substances or mixtures that alter function(s) of the endocrine system and consequently cause adverse health effects in an intact organism, or its progeny, or (sub)populations [1]. EDCs are mostly found in personal care products, food contaminants, metals, additives and plastics and even some medications. Pregnant women and children are the most vulnerable populations to be affected by EDCs exposure, and the effects of exposure to EDCs may not become evident until later in life. Adverse pregnancy outcomes induced by EDCs can be harmful for both the mother and the baby.
Chronic exposure to EDCs can occur through breathed air, food and daily life products such as cosmetics; therefore, inhalation, ingestion and skin contact are the main routes of exposure. In pregnant women, the lungs, skin and placenta (through blood circulation) are then continuously exposed to EDCs that have been reported to induce multiple adverse effects including asthma [2], urticaria, allergic contact dermatitis and skin aging [3], preterm birth and the worst case scenario of preeclampsia [4][5][6][7][8][9][10]. It is then obvious that EDCs act not only in endocrine organs but also in non-endocrine organs, and exert pleiotropic effects.
Whether EDCs share a common mechanism of action on both endocrine and nonendocrine organs remains unclear. The above-cited pathologies that EDCs can induce are different in terms of clinical features, morbidity and consequences for health, but we observed in the literature that the P2X7 receptor seems to be implicated in their development [11][12][13][14][15][16]. P2X7 receptor activation is reported to be involved in multiple pathologies from immune disorders to degenerative diseases [17][18][19][20]. The P2X7 receptor is a ubiquitous membrane receptor that induces many intracellular signaling pathways after alterations of the ion permeability or after formation of a large pore, depending on the duration of the stimulus. Pore formation after prolonged activation of the P2X7 receptor leads to apoptosis via multiple mechanisms including caspase-8 and caspase-9 activation, ROS production, mitochondrial dysfunction and caspase-3/7 activation [21][22][23][24]. In human placental cells, we previously showed that P2X7 receptor activation plays a pivotal role in toxicity induced by both known EDCs such as bisphenols (bisphenol A, bisphenol F and bisphenol S) [25] and suspected EDCs such as benzo[a]pyrene [26].
The question that we raised is then: do EDCs share P2X7 receptor activation as a common cellular mechanism of toxicity in pregnant women organs? Reported alterations of the placenta upon EDCs exposure (preterm birth, preeclampsia) are more dangerous for both the mother and her fetus than reported alterations of the lungs and skin (asthma and dermatitis). Another question that can therefore be asked is: should the level of P2X7 receptor activation after EDCs exposure be the same in the lungs, skin and placenta? Lungs being a non-endocrine organ, skin being closely related to the endocrine system and referred to as steroidogenic tissue [27] and placenta being an endocrine organ, we hypothesize that the level of P2X7 receptor activation induced by EDCs can be classified as follows: higher in placenta than in skin and higher in skin than in lungs. To provide preliminary in vitro answers, we studied P2X7 receptor activation after incubation with EDCs in human cells that express P2X7 receptor: human lung A549 cells [28], human keratinocytes HaCaT cells [29] and human placental JEG-Tox cells [25]. A total of nine EDCs belonging to different chemical families were selected for their susceptibility to be either inhaled, directly applied to the skin and/or ingested (Figure 1). In each case, EDCs can pass into the blood circulation after diffusion through skin and pulmonary barriers and digestion, ultimately reaching the placenta where they can accumulate [30]. The placenta being the most exposed organ, further investigations were performed to study apoptosis through the measurements of caspases-3, -8 and -9 activity, mitochondrial potential and chromatin condensation. The EDCs we tested in lung cells were selected because of their pulmonary exposition route: bisphenol A and benzyl butyl phthalate are plasticizers which can be contained in air or dust. The same rationale was used in skin cells: propylparaben is a commonly used preservative in cosmetics and 3-benzylidene camphor serves as an ultraviolet (UV) filter in sunscreen products. The EDCs tested in placental cells were selected because they are the most abundant EDCs in pregnant women fluids and placentas [8,9,[31][32][33]: bisphenol A, 4-heptylphenol (additive found in lubricants and greases), 4-tert-amylphenol (germicide and fumigant), phthalates (benzyl butyl phthalate and di(2-ethylhexyl) phthalate, DEHP), propylparaben, 3-benzylidene camphor and triclosan (biocide used in cosmetics). Diethylstilbestrol, a well-known EDC, was also tested because it was prescribed to pregnant women between 1940 and 1970 to prevent miscarriage, premature labor and related complications of pregnancy. For the record, the EDCs detected in placentas are listed because of their endocrine properties either as substances of very high concern (SVHC) under REACH legislation, or restricted to people aged under 3 years in cosmetic products by the European Commission (regulation 358/2014), or banned by the U.S. Food and Drug Administration.
All chemicals were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Di(2-ethylhexyl)phthalate was dissolved in culture medium. Benzyl butyl phthalate and propylparaben were dissolved in absolute ethanol. Bisphenol A, diethylstilbestrol, 4-tert-amylphenol, 4-heptylphenol, triclosan and 3-benzylidene camphor were dissolved in dimethylsulfoxyde (DMSO). Stock solutions were stored at -20 • C and work solutions were obtained after a 1/1 000 dilution in culture medium. The final concentration of absolute ethanol and DMSO on cells was less than or equal to 0.1%.
Cell viability: Neutral Red assay. The Neutral Red solution at 0.4% (m/v in water) was diluted in cell culture medium to obtain a working concentration of 50 µg/mL. Neutral Red working solution was distributed in the plates for a 3 h incubation time at 37 • C. The cells were then rinsed with PBS and lysed with a solution of ethanolwater-acetic acid (50.6/48.4/1, v/v/v). After homogenization, the fluorescence signal was scanned (λ ex = 540 nm, λ em = 600 nm) using a Spark ® microplate reader (Tecan, Männedorf, Switzerland).
Cell death P2X7 receptor activation: YO-PRO-1 ® assay. P2X7 cell death receptor activation was evaluated using the YO-PRO-1 ® assay [38]. The YO-PRO-1 ® probe only enters into cells after pore opening induced by P2X7 receptor activation and binds to DNA, emitting fluorescence. A 1 mM YO-PRO-1 stock solution was diluted at 1/500 in PBS just before being used and distributed in the wells of the microplate. After a 10 min incubation time at room temperature, the fluorescence signal was read (λ ex = 485 nm, λ em = 531 nm) using the Spark ® microplate reader.
Caspase 3 activity: CellEvent TM Caspase-3/7 Green Detection Reagent. Caspase-3 activity was evaluated using the CellEvent TM Caspase3/7 Green Detection Reagent. Cell Event TM Caspase-3/7 Green Detection reagent was diluted in PBS with 2.5% FBS to a final concentration of 8µM. The cells were incubated with the reagent for 30 min and then rinsed with PBS. The cells were observed under fluorescence microscopy and pictures were captured under the same acquisition parameters by Evos FL fluorescence microscope (Thermo Fisher Scientific).
Mitochondrial membrane potential: To determine mitochondrial potential we used the membrane potential-sensitive probe JC-1, which forms J-aggregates (with red color) at higher potential and JC-1 monomers (with green color) at low membrane potential, and the ratio between the red and green signals is a measure of mitochondrial potential. The dye at 6.5µg/mL of PBS was added to living adherent cells. The microplate was incubated at 37 • C for 15 min and then read at λ ex = 485 nm and λ em = 600 nm for the red fluorescence and λ ex = 485 nm and λ em = 520 nm for the green fluorescence. Carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma-Aldrich) was used as a positive control for mitochondrial depolarization.
Chromatin condensation: Hoechst 33342 assay. Chromatin condensation was evaluated using the Hoechst 33342 assay. The Hoechst 33342 fluorescent probe enters and intercalates into DNA in living and apoptotic cells. The fluorescent signal is proportional to chromatin condensation. A 0.5µg/mL Hoechst 33342 solution was distributed in the wells of the microplate. The fluorescence signal was read after a 30 min incubation time at room temperature (λ ex = 350 nm, λ em = 450 nm) using a Spark ® microplate reader.
Results exploitation and statistical analysis: Results are expressed in percentage or fold change compared with control cells and presented as means of at least three independent experiments ± standard errors of the mean. Statistical analysis was performed using Prism software (version 8, GraphPad software, La Jolla, CA, USA). The normal distribution of the data was confirmed by D'Agostino-Pearson test. Then, a one-way analysis of variance for repeated measures followed by a Dunnett's test with risk α set at 5% was performed to compare EDCs incubation with control (p-value expressed as follows: *) and a t-test was used to compare results in the presence of BBG with results in its absence (p-value expressed as follows: #).

Cell Viability
We investigated A549, HaCaT and JEG-Tox cells viability after incubation with EDCs, using the neutral red assay. Any concentration inducing a loss of cell viability greater than or equal to 30% was considered as cytotoxic (ISO 10993-5:2009).
Bisphenol A and benzyl butyl phthalate, 3-benzylidene camphor and propylparaben had no cytotoxic effects at the tested concentrations in A549 cells (Figure 2a

P2X7 Receptor Activation
P2X7 pore opening, reflecting P2X7 receptor activation, was assessed using the fluorescent YO-PRO-1 ® assay. There is no effect of bisphenol A and benzyl butyl phthalate on P2X7 receptor in A549 cells (Figure 3a,c).
In HaCaT cells, 3 benzylidene camphor induced a high fold change in P2X7 receptor activation (×1.35 at 10 µM and ×2.07 at 50 µM compared with the control, Figure 3g). The activation induced at 50µM was significantly inhibited by the P2X7 receptor antagonist BBG. Propylparaben had no effect on P2X7 receptor in HaCaT cells (Figure 3e).

EDCs Effects on Apoptosis in JEG-Tox Cells
The P2X7 receptor was activated by all the tested EDCs except one (4-heptylphenol) in JEG-Tox cells. As the P2X7 cell death receptor is known to trigger apoptosis, we studied apoptosis in JEG-Tox cells through the assessment of caspase-8, caspase-9 and caspase-3 activities, mitochondrial membrane potential and chromatin condensation.  Figure 4a) and benzyl butyl phthalate (×1.15 without BBG versus x0.96 with BBG, Figure 4a) but had no effects on capase-8 activation induced by 4-heptylphenol. Bisphenol A, 4-tert-amylphenol, triclosan, DEHP and 3-benzylidene camphor had no effect on caspase-8 activity ( Figure  4a).

EDCs Effects on Apoptosis in JEG-Tox Cells
The P2X7 receptor was activated by all the tested EDCs except one (4-heptylphenol) in JEG-Tox cells. As the P2X7 cell death receptor is known to trigger apoptosis, we studied apoptosis in JEG-Tox cells through the assessment of caspase-8, caspase-9 and caspase-3 activities, mitochondrial membrane potential and chromatin condensation.

Caspase-8, Caspase-9 and Caspase-3 Activity
The activity of initiator caspases (8 and 9) and the activity of executioner caspase-3 are reported in Figures 4 and 5.

Mitochondrial Membrane Potential
Caspase-9 activation being associated with mitochondrial disruption during apoptosis, we analysed mitochondrial membrane potential with a JC-1 assay. A positive control, CCCP, known to trigger mitochondrial depolarization, was used to ensure that JEG-Tox possessed functional mitochondria. CCCP led to low mitochondrial membrane potential, as expected (x0.65 compared with the control, Figure 6) corresponding to low mitochondrial activity. BBG induced an unexpected weak fluorescence signal in control cells (x0.47 compared with the control without BBG), which could be attributed to an artifact; BBG seems therefore not suitable to study the relationship between mitochondrial membrane potential and the P2X7 receptor.

Discussion
The objective of the present study was to explore the ability of different EDCs to induce toxicity in different cells by a common cellular mechanism, in particular P2X7 receptor activation. We compared the level of P2X7 receptor activation in human epithelial pulmonary cells, and keratinocytes and placental cells after incubation with EDCs that can be inhaled, directly applied to the skin and/or ingested. The placenta being a crucial organ during pregnancy and the most exposed organ to EDCs, further investigations were performed to study apoptosis, one of the major cell death pathways [39] that can be induced by P2X7 receptor activation [21,40]. Apoptosis is involved in pregnancy disorders [41][42][43].
The three cell lines were selected for their different endocrine properties and because they all express functional P2X7 receptors [13,26,34]. Human epithelial pulmonary A549 cells share similar ultrastructural characteristics and cytochromes expression to in situ type II pneumocytes, the most abundant cells in lungs. Human keratinocyte HaCaT cells have been extensively used to study epidermal homeostasis and its physiopathology. Furthermore, they are metabolically active since they have cytochromes from 1, 2, 3 and 4 families [44]. Human villous trophoblastic placental JEG-3 cells provide an appropriate model to detect placental toxicity [13,25,26]. They are also able to synthesize and secrete hormones.
The present study is the first to compare EDCs effects on P2X7 receptor activation in three organs present in pregnant woman and possessing different endocrine properties. We highlighted that the P2X7 receptor in lung cells is not sensitive to EDCs and that the P2X7 receptors in skin cells are less sensitive to EDCs than in placental cells where they induce apoptosis.
Bisphenol A and benzyl butyl phthalate did not activate the P2X7 receptor in lung cells, while 3-benzylidene camphor activated the P2X7 receptor in keratinocytes, unlike propylparaben. All of the tested EDCs in human placental cells activated the P2X7 receptor (except 4-heptylphenol). These differences may be explained by the endocrine properties of the different cells. The placenta is considered the most important endocrine organ during pregnancy. Villous trophoblast cells have a lot of steroid or polypeptide hormone receptors and the ability to synthesize, secrete and control different maternal and fetus hormones. Skin, in addition to its main protective function, can also be classified as an endocrine organ endowed with local steroidogenic activities [47,48]. HaCaT keratinocytes possess different steroid receptors [49][50][51] and metabolize progesterone to deoxycorticosterone, cortisone, aldosterone and cortisol [47]. On the contrary, pulmonary epithelial cells do not possess any hormonal receptors and are not able to produce hormones. The lack of P2X7 receptor activation by EDCs in A549 cells did not allow us to arrive at a conclusion on the potential existence of a common mechanism of EDCs in pregnant women organs, but it suggests that P2X7 receptor activation could be a common mechanism in endocrine organs and its activation would be linked to hormonal dysregulation. To confirm this statement, it would be interesting to evaluate P2X7 receptor activation induced by the same EDCs in other endocrine cells, such as adrenal H295 cells, used for the OECD Steroidogenesis Assay (OECD Guidelines for the Testing of Chemicals, Section 4 Test No. 456).
P2X7 receptor activation is known to trigger pregnancy disorders such as preeclampsia and preterm birth [14,16]. In this study, we have shown that EDCs, known in the literature to induce the same disorders, activate the P2X7 receptor. Our results suggest that EDCs could trigger pregnancy disorders through P2X7 receptor activation. P2X7 receptor activation could be a key element in the understanding of placental disorders induced by EDCs.
Prolonged activation of the P2X7 receptor has been linked to apoptosis [21,40]. Depending on the cleaved caspase, apoptosis can be initiated through two major pathways [52]: the extrinsic receptor mediated pathway through caspase-8 activation [53,54] or the intrinsicmediated pathway, resulting in caspase-9 activation [21,55]. Mitochondrial damages result in cytochrome c release and formation of the apoptosome, a multimeric protein complex containing Apaf-1, cytochrome c and caspase-9, resulting in caspase-9 activation [21,55]. Extrinsic and intrinsic-mediated pathways lead to caspase-3 activation [56], followed by chromatin condensation [57]. Most tested EDCs, despite different chemical structures, triggered apoptosis in placental cells, through different pathways depending on the studied EDC (Table 1). Bisphenol A and 3-benzylidene camphor induced P2X7 receptor activation and mitochondrial membrane potential disturbance. Diethylstilbestrol induced the activation of all the assessed apoptosis markers, meaning that both extrinsic and intrinsicmediated apoptosis are triggered through P2X7 receptor activation. Propylparaben also induced both extrinsic and intrinsic-mediated apoptosis through P2X7 receptor activation, but contrary to diethylstilbestrol did not induce mitochondrial membrane potential disturbance. Benzyl butyl phthalate induced both extrinsic and intrinsic-mediated apoptosis, but only the intrinsic-mediated apoptosis was associated with P2X7 receptor activation. 4-heptylphenol induced extrinsic and intrinsic-mediated apoptosis, but it was independent from P2X7 receptor activation. 4-tert-amylphenol and DEHP induced P2X7 receptor-dependent intrinsic-mediated apoptosis with mitochondrial membrane potential disturbance. Triclosan induced P2X7 receptor-dependent intrinsic-mediated apoptosis without mitochondrial membrane potential disturbance. Our study highlights that most of the tested EDCs, belonging to different chemical families, induced mitochondrial alterations through either intrinsic-mediated apoptosis or membrane potential disturbance or both. We were unfortunately unable to link P2X7 receptor activation with mitochondrial membrane potential alteration due to a technical artifact with BBG in the JC-1 assay. The P2X7 receptor and mitochondria have a close connection to ATP. ATP, produced by mitochondria, is the natural ligand of the P2X7 receptor. In syncytiotrophoblast, the majority of ATP is utilized for cholesterol transport and steroidogenesis [58]. Placental mitochondria play a key role in steroidogenesis with huge production of progesterone necessary for maintaining pregnancy [58]. Furthermore, progesterone increases mitochondrial membrane potential [59,60]. As mitochondria are required for steroidogenesis, mitochondrial disorders may contribute to preeclampsia [61,62] and other endocrine diseases such as diabetes mellitus and obesity. All these data suggest that the P2X7 receptor along with mitochondria are specific targets for EDCs to exert their effects on hormones alteration.

Conclusions
In conclusion, the results of our study suggest that the P2X7 receptor would be a common cellular mechanism of EDCs toxicity in endocrine pregnant women cells. In placental cells, EDCs induce P2X7 receptor activation and mitochondrial alterations, reported to trigger preeclampsia and preterm birth in clinics. P2X7 receptor activation and mitochondrial alterations could be key elements in understanding placental disorders induced by EDCs.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.