Chemical mixture that targets the epidermal growth factor pathway impairs human trophoblast cell functions.

To evaluate individual chemicals and Chem-Mix’s ability to block EGF from binding to EGFR, an AlphaLISA EGF/EGFR binding assay was used, and results shown in Figure 1 . The positive control cetuximab significantly reduced EGF binding by 99.2% ( p < 0.05 ). The Chem-Mix at 0.1 and 1 μg/ml significantly blocked EGF binding compared to the control ( p < 0.05 ) with a 56.5 % decrease in alpha signal at 1 μg/ml ( Figure 1 ). Of the individual chemicals, PCB-153, niclosamide, and trans -nonachlor reduced the alpha signal by 55.56 %, 22.96 % and 68.15 %, respectively at 1 μg/ml ( p < 0.05 ). BPS significantly blocked EGF binding ( p < 0.05 ) at 0.01, 0.1 and 1 μg/ml. In contrast, PCB-126 and atrazine did not compete with EGF for EGFR binding ( Figure 1 ). To test the effects of the Chem-Mix on EGFR, AKT, and STAT3 activation, their protein abundance were evaluated in HTR-8/SVneo cells ( Figure 2 ). After 15 min of exposure, the Chem-Mix did not affect EGFR phosphorylation at any of the doses tested. Similarly, none of the individual chemicals was able to induce EGFR phosphorylation (not shown). However, in the presence of EGF, the Chem-Mix reduced the pEGFR/EGFR ratio compared to EGF alone starting at the lowest dose ( p < 0.05 ; Figure 2 ). After 15 min exposure, EGF significantly increased AKT phosphorylation in HTR-8/SVneo cells ( Supplemental Figure S3B ). Neither Chem-Mix doses in the presence or absence of EGF affected total AKT abundance or its phosphorylation ( Figure 2 ). EGF did not affect abundance of total or phosphorylated STAT3. The Chem-Mix did not affect total and phosphorylated STAT3 even in the presence of EGF ( Figure 2 ). After 15 min exposure, niclosamide, trans -nonachlor, and BPS of the individual chemicals (at 100 ng/ml) plus EGF (3 ng/ml) significantly reduced EGFR phosphorylation compared to EGF ( p < 0.05 ; Supplemental Figure S2B ). Individual chemicals (100 ng/ml) in combination with 30 ng/ml of EGF did not affect EGFR phosphorylation when compared to EGF alone ( Figure 3 ). The effect of the Chem-Mix on EVT cell proliferation is shown in Figure 4 . EGF significantly increased cell proliferation ( p < 0.05 ) at 36 and 100 h ( Figure 4 ). At 36 and 100 h, doses of 1 and 10 ng/ml of the Chem-Mix plus EGF (30 ng/ml) did not affect proliferation compared to EGF. However, in the absence of EGF, 1 ng/ml significantly increased cell proliferation compared to control ( p < 0.05 ). By 36 h, 100 ng/ml of the Chem-Mix plus EGF (30 ng/ml) significantly decreased cell proliferation compared to EGF ( p < 0.05 ). At 100 h, 100 ng/ml of the Chem-Mix significantly decreased EVT cell proliferation regardless of the presence of EGF ( p < 0.05 ). The effect of the Chem-Mix and the individual chemicals on EVT cell invasion are shown in Figure 5 . Exposure to EGF significantly increased HTR-8/SVneo invasion compared to the control ( p < 0.05 ; Figure 5 ). The Chem-Mix alone (100 ng/ml) significantly attenuated cell invasion by 29.62% compared to the control group ( p < 0.05 ; Figure 5 ). Similarly, the Chem-Mix + EGF also significantly reduced HTR-8/SVneo cells’ invasion compared to EGF ( Figure 5 ). None of the individual chemicals affected EVT cell invasion compared to the control group ( Figure 5 ). A tube forming assay was used to evaluate the effects of the Chem-Mix on HTR-8/SVneo cells’ endovascular differentiation ( Figure 6 ). After 16 h, no significant changes in the number of nodes, junctions, or segments, mean mesh size, total length, and the segment length were observed in any of the groups tested compared to the control group.

The chemical mixture (Chem-Mix) used in this study included the combination of the following six chemicals: PCB-126, PCB-153, atrazine, niclosamide, trans -nonachlor, and BPS; all reported to interfere with EGFR activation ( Hardesty et al., 2017 ; Sauer et al., 2017 ; Hardesty et al., 2018 ; Ticiani et al., 2021 ; Ticiani et al., 2022 ). Chemical source details and purity are listed in Table 1 and human exposure sources and ranges are detailed in Table 2 ( Dearth and Hites, 1991 ; Newsome and Ryan, 1999 ; Perry et al., 2000 ; Rylander et al., 2005 ; Costopoulou et al., 2006 ; Diamanti-Kandarakis et al., 2009 ; Zhu et al., 2009 ; Ritter et al., 2011 ; Mendas et al., 2012 ; Zoeller et al., 2012 ; 2014; National Academy of Medicine, 2014 ; Megersa, 2015 ; Faroon and Ruiz, 2016 ; Namulanda et al., 2017 ; Schweizer et al., 2018 ; Wang et al., 2018 ). All chemicals were dissolved in dimethyl sulfoxide (DMSO, Cat# BP231-100, Thermo-Fisher, Rockford, IL, USA) to a final concentration of 0.1%, which was used as the vehicle control in all experiments. The Chem-Mix consisted of the same concentration for each individual chemical dissolved in DMSO. An AlphaLISA EGF/EGFR binding assay (Cat# AL366HV, PerkinElmer, Waltham, MA, USA) was used to evaluate the chemicals’ ability to block EGF from binding to EGFR as previously described ( Ticiani et al., 2021 ). Each of the six individual chemicals (PCB-126, PCB-153, atrazine, niclosamide, trans -nonachlor, and BPS at 0.01, 0.1, or 1 μg/ml) or the Chem-Mix (a mixture made of a range of doses from 0.001, 0.1, or 1 μg/ml of each of the six chemicals) were tested. In brief, anti-human IgG Fc-specific AlphaLISA acceptor beads and EGFR-Fc were mixed, incubated, and added to each well containing each of the respective chemical doses (listed above) and biotinylated EGF (n = 3 replicates/treatment group). Cetuximab, an antibody inhibitor of EGFR (0.1 μg/ml; Cat# A2000, Selleck Chemicals LLC, Houston, TX, USA) was used as negative control. Streptavidin-coated donor beads were added to each well and binding signal was measured in alpha units (A.U.) at 615 nm using the AlphaLISA mode on a multimode microplate reader and imager (Cytation 1 Cell Imaging Multimode Reader; BioTek, Santa Clara, CA, USA). Results were expressed as relative light units (RLU). The HTR-8/SVneo cell line derived from human first trimester extravillous trophoblasts was used in this study as it expresses EGFR and proliferates and invades in response to EGF ( Denys et al., 2008 ; Bolnick et al., 2011 ). Cells were maintained in basic cell culture medium consisting of Dulbecco’s modified Eagle’s medium/F12 medium (Cat# 124000-024, Millipore Sigma, Saint Louis, MO, USA), supplemented with 10% of fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cells were incubated at 37°C and 5% CO 2 . Cell viability testing was performed using concentration ranges found in human samples as described in Table 2 . Cytotoxicity of each of the six individual chemicals and the Chem-Mix was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously reported ( Ticiani et al., 2021 ). In brief, HTR-8/SVneo cells were cultured in basic cell culture medium in 96-well plates and exposed to either control (0.1% DMSO), each of the six individual chemicals (0.01, 0.1, 1, 10, 100, 1,000, or 2,000 ng/ml) or the Chem-Mix (1, 10, or 100 ng/ml) for 24 h. Likewise, HTR-8/SVneo cells were also exposed to control (0.1% DMSO), EGF (30 ng/ml), the Chem-Mix at 1, 10, or 100 ng/ml ± EGF (30 ng/ml) for 36 or 100 h. Thereafter, the exposure medium was replaced with 100 μl of phenol red-free MTT working solution (50 μg/ml) and incubated for 4 h. The MTT working solution was discarded and replaced with 100 μl of 0.1% DMSO per well. Plates were vortexed and absorbances of each treatment (n = 6 replicates/treatment group) were quantified at 570 nm in a multimode microplate reader (Cytation 1 Cell Imaging Multimode Reader; BioTek, Santa Clara, CA, USA). The effect of each of the six chemicals on EGFR’s pathway activation were evaluated in HTR-8/SVneo cells as previously described ( Ticiani et al., 2022 ). Cells were cultured in 6-well plates overnight in basic cell culture medium to reach 80% confluency. Rapid single chemical effects were tested by exposing cells for 15 min to control (0.1% DMSO), EGF (3 or 30 ng/ml) or each of the six individual chemicals + EGF (100 ng/ml individual chemical + 3 or 30 ng/ml EGF). Chem-Mix effects were tested by exposing cells to control (0.1% DMSO), EGF (30 ng/ml), Chem-Mix (1, 10, or 100 ng/ml), or the Chem-Mix + EGF for 15 min for EGFR and serine/threonine kinase (AKT) expression based on prior EGF exposure time-course results ( Ticiani et al., 2022 ) and for 120 min for the signal transducer and activator of transcription 3 (STAT3) expression. After exposure, protein abundance and phosphorylation of EGFR, AKT, and STAT3 were evaluated by western blotting. After exposure, cells were harvested, and protein was extracted as previously described ( Ticiani et al., 2021 ). Protein extraction was performed using RIPA lysis buffer (Cat# N653; VWR Life Science, San Francisco, CA, USA) containing 1 mM sodium orthovanadate 20%, 1 M sodium fluoride, and 1% protease inhibitor cocktail (Cat# M221; VWR Life Science, San Francisco, CA, USA). Protein concentration was determined using a Pierce bicinchoninic acid protein assay kit (Cat# 23225; Thermo-Fisher, Rockford, IL, USA) and 20 μg of protein per sample from cell lysates were subjected to electrophoresis on a 10% SDS-polyacrylamide gel (70 V for 120 min). Proteins were then transferred from the gel onto a nitrocellulose membrane (200 mA for 90 min) and subjected to Western blotting. Membranes were blocked with 5% nonfat dry milk in TBS containing 0.03% tween-20 (TBST) (block solution) and incubated with primary antibodies diluted in block solution overnight at 4 °C. Primary antibodies used were: anti-EGFR, anti-phospho-EGFR (Tyr 1068), anti-STAT3, anti-phospho-STAT3 (Tyr 705), anti-AKT, anti-phospho-AKT (Tyr 204), and anti-β-actin ( Supplemental Table S1 ). The membranes were washed 3 times with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies goat anti-mouse HRP-conjugated and goat anti-rabbit HRP-conjugated (Cat#115-005-003 and 111-005-003, respectively; Jackson Immunoresearch, West Grove, PA, USA) diluted 1:5,000 in block for 1 hour at room temperature in the dark. Western Bright ECL (Cat# K12045; Advansta, Menlo Park, CA, USA) was used for enhanced chemiluminescence and visualized on ChemiDoc Imaging System (Image Lab Touch Software; Biorad, Hercules, CA, USA). Quantification of band intensities was performed using ImageJ software ( Schneider et al., 2012 ). Differences in protein loading were accounted for by normalizing the target protein band by the control β-actin band for each sample. All original western blots images are presented in Supplemental Figures S2A to S4 ). The effect of the Chem-Mix on trophoblast cell proliferation was tested using a Sartorius IncuCyte system (A Sartorius Company, Ann Arbor, MI, USA). Green fluorescent protein tagged HTR-8/SVneo cells (as previously described in ( Pu et al., 2021 ); ~ 2,000 cells/well) were cultured in basic cell culture medium with the control (0.1% DMSO), EGF (30 ng/ml), Chem-Mix (1, 10, or 100 ng/ml), or the Chem-Mix + EGF in 96-well plates for 4 days. Cells were incubated at 37 °C and 5% CO 2 and monitored on the IncuCyte Live-Cell Analysis Systems. Cells were supplemented with basic cell culture medium consisting of the above chemical treatments every 48 h after initial seeding (n = 6 replicates/treatment group). Green fluorescent protein-tagged cells were imaged with phase contrast every 4 h. The number of live cells were analyzed using the Incucyte Basic Analysis Software (version 2022A Rev1). Results per time point were averaged, normalized to 0 h, and expressed as phase object count (per image). The effects of the six individual chemicals and the Chem-Mix on extravillous trophoblast cell invasion were tested in HTR-8/SVneo cells using a Transwell cell culture system as previously reported ( Ticiani et al., 2022 ). Each Transwell cell culture insert pre-coated with Matrigel (24-wells, 8 μm pore size; Corning, Tewksbury, MA, USA) was rehydrated for 2 h in 24-well plates. Cells (~50,000) were suspended in 250 μl of serum free DMEM, seeded in each insert, and exposed to either one of the following treatments: control (0.1% DMSO), EGF (30 ng/ml), the Chem-Mix (100 ng/ml), or the Chem-Mix + EGF (Chem-Mix 100 ng/ml + 30 ng/ml EGF). In another 24-well plate, HTR-8/SVneo cells were exposed to control (0.1% DMSO), EGF (30 ng/ml), or each of the 6 individual chemicals (100 ng/ml each). Basic cell culture medium supplemented with above treatments were added to each well below the inserts and incubated in 37°C and 5% of CO 2 for 16 h. Cells that did not invade were removed with a cotton swab from the upper side of the insert. Invasive cells were then fixed with 10% neutral-buffered formalin for 30 min at room temperature and the cell nuclei were stained with DAPI. With 5 technical replicates, three random images were taken of inserts at 10x magnification with a Nikon inverted microscope with NIS-Element Imaging Software (version 5.41.02). The number of DAPI stained cells per image were counted using CellProfiler software ( Adomshick et al., 2020 ; Pu et al., 2021 ; Ticiani et al., 2022 ), then averaged and normalized to the control group. Results were expressed as number of cells per field. The effects of the Chem-Mix on endovascular differentiation were evaluated as previously described ( Ticiani et al., 2022 ). HTR-8/SVneo cells were seeded at in 60 mm plate and kept in basic cell culture medium. Twenty-four-well plates were coated in 400 μl of Matrigel (Cat# 356234; Corning, Woodland, CA, USA) and allowed to polymerize at 37 °C and 5% CO 2 for 30 min. Cells were trypsinized, harvested, and resultant pellet diluted in basic cell culture medium containing control (0.1% DMSO), EGF (30 ng/ml), the Chem-Mix (100 ng/ml), the Chem-Mix + EGF (Chem-Mix 100 ng/ml + 30 ng/ml EGF). HTR-8/SVneo cells (80,000 cells/well) in 400 μl of exposure medium were added to each well and incubated overnight at 37 °C and 5% of CO 2 for 16 h. Thereafter, formation of cell networks (n = 3 replicates per treatment group) was imaged on a Nikon inverted microscope with NIS-Element Imaging Software (version 5.41.02). Evaluation of the cell networks was performed using ImageJ software ( Schneider et al., 2012 ). All data comprising invasive cells, protein abundance, fluorescent signal, alpha signal, cell density, and tube formation parameters were analyzed by comparing treatment groups using a generalized linear model (MIXED procedure) that allows adjustment of means while considering an effect from exposure time. The model used included treatment groups and time as fixed effects. LSMEANS was used to adjust the means and to compare treatments. Significance was set at p < 0.05 and outliers excluded after outlier testing. All data were analyzed using SAS software (version 9.4; SAS Institute Inc.) and plotted on GraphPad Prism 8 (GraphPad Software, Inc.).

As the mediator of many of placenta’s critical functions, EGFR is required for proper placental development ( Clemente and Bird, 2023 ). Because epidemiological studies evidence that exposures to chemical mixtures occur in pregnant women ( Woodruff et al., 2011 ; Vilahur et al., 2014 ; Zota et al., 2014 ; Kalloo et al., 2018 ; Montrose et al., 2018 ; Goodrich et al., 2019 ; Kelley et al., 2019 ; Kalloo et al., 2020 ; Banker et al., 2021 ; Tung et al., 2022 ) resulting in a “ mixed body burden ” ( Ribeiro et al., 2017 ), in this study, a chemical mixture that includes chemicals previously reported as interfering with EGFR activation ( Medicine, 2014 ; Hardesty et al., 2017 ; Sauer et al., 2017 ; Hardesty et al., 2018 ; Ticiani et al., 2021 ; Ticiani et al., 2022 ) was investigated for its effects on EGFR-dependent placental cell function. We have demonstrated that at the highest dose tested, the chemical mixture competed with EGF for EGFR binding, reduced EGFR phosphorylation, and independently of EGF, attenuated EVT cell proliferation and invasion. We also provide evidence that these effects were larger compared to those observed in single chemical exposures. These results are significant because 1) pregnant women are concomitantly exposed to complex chemical mixtures, 2) EGFR is highly expressed in the placenta, and 3) EGFR mediates critical placental cell functions (cell invasion and proliferation). These results add to the body of literature that have previously reported that when in mixture, chemicals that target a specific pathway (androgen, estrogen, and PPARγ receptor) ( Silva et al., 2002 ; Rajapakse et al., 2004 ; Orton et al., 2012 ; Watt et al., 2016 ; Schlezinger et al., 2020 ; Nielsen et al., 2022 ) can elicit larger responses when in combination compared to single chemicals. In an EGF competitive binding assay, PCB-153, niclosamide, trans -nonachlor, and BPS displaced EGF to bind to the extracellular domain of EGFR. In agreement with these findings, we and others have previously demonstrated in docking simulations (and site EGFR specific mutagenesis for BPS) that several of these chemicals interfere with EGF for EGFR binding. Specifically, BPS binds to EGFR subdomains II and III, trans -nonachlor between subdomains II and IV, PCB-126 within subdomains III and II, and PCB-153 between subdomains II and IV ( Hardesty et al., 2018 ; Ticiani et al., 2023 ). Of note, even though EGF naturally binds to EGFR at subdomains I and III ( Ferguson et al., 2003 ; Ferguson, 2008 ), other ligands such as growth factors, or chemicals can bind to the other extracellular subdomains and sterically block EGF’s high affinity for EGFR binding, thus preventing EGFR’s dimerization and activation ( Schmiedel et al., 2008 ). On the other hand, atrazine’s inability to compete with EGF is supported by docking simulations reporting that atrazine likely binds within the ATP pocket of the tyrosine kinase domain of EGFR as opposed to its extracellular domains ( Hardesty et al., 2018 ). Considering that the PCB-153, PCB-126, niclosamide, and trans -nonachlor at high doses and BPS at all doses competed with EGF, it is not surprising that the two highest doses of the chemical mixture blocked EGF binding to EGFR. Out of the 5 chemicals predicted bind to EGFR, only BPS’ binding site has been proven by us through site EGFR specific mutagenesis in EVT cells to block ( Ticiani et al., 2023 ). Similar to previous studies, mutagenesis studies are necessary to verify the binding sites of the individual chemicals as well as the chemical mixture to determine the effects of the chemical mixture on EGF interference with EGFR activation. Since the dose response binding effects are derived from a non-cell-based assay, we next investigated the Chem-Mix effect on EGFR activation using the EVT cell line HTR-8/SVneo, a human derived first trimester cell line that retains the ability to invade in vitro and routinely used in placental studies ( Graham et al., 1993 ; Chakraborty et al., 2002 ; Ticiani et al., 2022 ). At environmentally relevant doses, the Chem-Mix reduced EGF mediated total and phosphorylated EGFR. In determining which specific chemicals were contributing to the mixture effect, we observed that despite PCB-153, niclosamide, trans -nonachlor, and BPS binding to EGFR (binding assay results), none of these chemicals affected EGFR phosphorylation individually. While results using 30 ng/ml of EGF contrasts with previous studies where PCB-126, PCB-153, trans-nonachlor, atrazine and niclosamide have been reported to reduce EGFR phosphorylation ( Sauer et al., 2017 ; Hardesty et al., 2018 ), our results using a lower EGF concentration (3 ng/ml) indicates that 30 ng/ml of EGF oversaturates EGFR ( Supplemental Figure S2A ) and does not allow the detection of individual chemical effects, at least on EGFR phosphorylation ( Supplemental Figure S2B ). Our mixture chemical results are further strengthened by the detection of an effect at a saturated concentration of EGF (30 ng/ml). Upon phosphorylation, EGFR activates downstream effectors such as AKT and STAT3, which further elicit trophoblast cell growth, proliferation, migration, invasion, angiogenesis, and survival ( Straszewski-Chavez et al., 2010 ; Johnston and Grandis, 2011 ; Clemente and Bird, 2023 ). Neither AKT or STAT3 protein abundance or phosphorylation were affected by the Chem-Mix. While the lack of effect of the Chem-Mix on STAT3 is not surprising, the lack of effect of the Chem-Mix on AKT phosphorylation is rather paradoxical, especially since the Chem-Mix, at the same dose, reduced EVT invasion ( Figure 5 ). While we could have missed the window of AKT activation, the selection of timing for AKT activation evaluation was based on our previous work (AKT activation occurs between 15 min upon EGF exposure) ( Ticiani et al., 2022 ). Further studies on additional AKT phosphorylation sites are needed to further support these findings. Functionally, we first confirmed that EGF induces EVT cell invasion ( Bass et al., 1994 ). We have demonstrated that the Chem-Mix blocked EVT invasion at 100 ng/ml, in the presence of EGF. These findings support the Chem-Mix’s interference with EGFR and align with both, EGFR binding and EGFR phosphorylation results. Yet, in the absence of EGF, the Chem-Mix also reduced cell invasion compared to the control, suggesting that the Chem-Mix is likely affecting other mediators of EVT cell invasion, in addition to EGFR. This is supported by evidence in cancer cell lines in which some of the individual chemicals of the Chem-Mix (PCB-126, PCB-153, atrazine, niclosamide, trans -nonachlor, and BPS) can alter cell invasion through matrix metalloproteinases (MMP)-9, MMP2, Rho-associated kinase, and estrogen receptor pathways ( Liu et al., 2010 ; Pestana et al., 2015 ; Ali et al., 2016 ; Chen et al., 2018 ; Koual et al., 2020 ; Lee et al., 2020 ; Chen et al., 2021 ; Liu et al., 2021 ). Future studies should explore if any of these pathways are involved in the effect induced by the Chem-Mix in the context of trophoblast cell invasion. The reduction in cell invasion by the Chem-Mix, but none of the individual chemicals also supports the synergistic effect of the Chem-Mix and that this synergism may occur through non-EGFR dependent pathways. Of clinical significance, the attenuation of EVT invasion by the Chem-Mix can have pathophysiological implications for human health because: 1) some chemicals in the Chem-Mix have been detected in circulation of pregnant women and human placentas ( Park et al., 2008 ; Kezios et al., 2012 ; Trabert et al., 2012 ; Liu et al., 2017 ; Gingrich et al., 2020 ; Ouidir et al., 2020 ; Carles et al., 2021 ), 2) the Chem-Mix invasion effects occur at environmentally relevant doses for the chemicals included in the mixture (see Table 2 ), 3) defects in EVT invasion can contributes to pathological pregnancies such as placenta accreta, preeclampsia, intrauterine growth restriction, and miscarriages ( Pollheimer et al., 2018 ), and 4) and certain chemicals within the Chem-Mix (PCBs, ATZ, and BPS) are associated with miscarriage risk and can results in birth defects, small for gestational age birth weight, and fetal growth abnormalities ( Goodman et al., 2014 ; Sol et al., 2021 ; Montano et al., 2022 ). Proliferation, a partially EGF-controlled function in EVT trophoblast cells ( Li and Zhuang, 1997 ) was reduced only at the highest dose of the Chem-Mix in the presence of EGF after 36 and 100 h of exposure. Whether this reduction is driven by an EGFR-dependent mechanism and fully independent of potential cytotoxicity (observed at 100 h of exposure; Supplemental Figure S1 ), remains to be further explored. Pathways that may be involved as they crosstalk with EGFR in the context of differentiation, proliferation, and survival include STATs ( Ospina-Prieto et al., 2015 ), ESR1 ( Giuliano et al., 2013 ), Notch, Wnt Beta-catenin ( Haider et al., 2014 ; Dietrich et al., 2022 ), KISS1 ( Fang et al., 2021 ), VEGF ( Hastie et al., 2019 ), and PGF ( Whigham et al., 2021 ). In conclusion, some of the individual chemicals tested do not affect EGFR activation, nor EGFR-mediated EVT cell functions. Yet, we have demonstrated a synergistic effect of the chemical mixture on EGFR activation as well as attenuations in EGF-mediated trophoblast proliferation and invasion. To our knowledge this is among the first studies to combine different chemical classes targeting the EGFR pathway to investigate their synergistic effects on placenta cell function. Given the abundance and importance of EGFR in the placenta understanding how chemical exposures that target the EGFR pathway should be prioritized in epidemiological studies of pregnancy cohorts.

With over 85,000 man-made chemicals and over 40,000 in commerce ( Medicine, 2014 ; Organization, April 1, 2022 ), human exposure to complex chemical mixtures is virtually unavoidable ( LaKind et al., 2012 ; Zota et al., 2017 ; Naidu et al., 2021 ). Epidemiological evidence demonstrates chemical mixture exposure in pregnant women ( Woodruff et al., 2011 ; Vilahur et al., 2014 ; Zota et al., 2014 ; Kalloo et al., 2018 ; Montrose et al., 2018 ; Goodrich et al., 2019 ; Kelley et al., 2019 ; Kalloo et al., 2020 ; Banker et al., 2021 ; Tung et al., 2022 ) with most chemicals being detectable in maternal blood, urine, umbilical cord blood, breast milk, and the placenta ( Vandenberg et al., 2007 ; Kolatorova et al., 2018 ; Bjorvang et al., 2021 ). This is of significance since the placenta is responsible for pregnancy maintenance and is itself vulnerable to environmental chemical exposures. Specifically, chemicals can disrupt placenta cell functions, including proliferation, differentiation, fusion, invasion, materno-fetal nutrient and gas exchange, and hormone synthesis ( Gingrich et al., 2020 ; Padmanabhan et al., 2021 ). Notably, chemical exposures have been associated with pregnancy complications such as preeclampsia, gestational hypertension, miscarriage, and intrauterine growth restriction ( Snijder et al., 2012 ; Zhao et al., 2014 ; Cantonwine et al., 2016 ; Krieg et al., 2016 ; Shaffer et al., 2019 ; Welch et al., 2022 ; Hirke et al., 2023 ). The placenta highly expresses the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase that regulates several key placenta cell functions, including proliferation, fusion, and invasion. Highly expressed in almost every placenta cell type, EGFR is expressed in both the extravillous trophoblasts (EVTs) that invade into the maternal decidua as well as the cytotrophoblasts (CTBs) that layer the placental villi ( Chegini and Rao, 1985 ; Rao et al., 1985 ; Bulmer et al., 1989 ; Muhlhauser et al., 1993 ; Clemente and Bird, 2023 ). Importantly, EGFR modulates cytotrophoblast cell proliferation and differentiation ( Ohlsson, 1989 ), while in extravillous trophoblasts, EGFR activation induces EVT cell invasion into the maternal decidua ( Fang et al., 2021 ). In our previous work, we have demonstrated that the emerging bisphenol, bisphenol S (BPS) competes with the epidermal growth factor (EGF) for EGFR binding, blunts EGF-mediated EGFR phosphorylation, EGF endocytosis, and cell fusion of human primary cytotrophoblast cells ( Ticiani et al., 2021 ). In addition, we have recently reported that BPS impairs EGF-mediated extravillous trophoblast cell proliferation and invasion ( Ticiani et al., 2022 ) by interfering with specific binding sites of the extracellular domain of EGFR ( Ticiani et al., 2023 ). Altogether, these studies with BPS, underscore the importance of understanding whether other chemicals known to interfere with EGFR, are also placental toxicants. Other studies have identified additional chemicals that modify EGFR activation ( Medicine, 2014 ; Pogrmic-Majkic et al., 2016 ; Hardesty et al., 2017 ; Hardesty et al., 2018 ), but their effects on EGFR expression in placenta cells have not been investigated. Specifically, one study identified that the persistent chemicals trans -nonachlor, polychlorinated biphenyl (PCB)-126, PCB-153, and atrazine blocked EGF endocytosis and reduced EGF-mediated EGFR phosphorylation in human epidermoid carcinoma and hepatoma cells ( Hardesty et al., 2018 ). In addition, atrazine exposure in Leydig cells enhanced rapid human chorionic gonadotropin-stimulated androgen synthesis through EGFR signaling ( Pogrmic-Majkic et al., 2016 ). To our knowledge, aside from these three studies, the effects of PCB-126, PCB-153, atrazine, or trans -nonachlor on EGFR signaling remain virtually unexplored. While many of these (PCBs, atrazine, trans -nonachlor, BPS) are environmental chemicals, niclosamide an anthelminthic drug that has been recently repurposed as a therapeutic for lung cancer, rheumatoid arthritis, type 2 diabetes mellitus, and endometriosis ( Tao et al., 2014 ; Liang et al., 2015 ; Prather et al., 2016 ; Lee et al., 2020 ), can also reduce EGFR activation, proliferation, and colony growth in estrogen receptor-negative breast cancer cells ( Sauer et al., 2017 ). Given EGFR’s high expression and prominent role in the placenta ( Clemente and Bird, 2023 ), we investigated whether chemicals reported to interfere with EGFR in carcinoma cell lines disrupt EGFR-mediated function in placenta cells. Chemical mixture studies have used multiple approaches, including testing of complex chemical mixtures (of unknown specific composition) ( Carter et al., 1997 ; Hetland et al., 2004 ; Panas et al., 2014 ; Lampron et al., 2023 ), multiple exposures based on human epidemiological studies ( Langie et al., 2015 ; Hendryx and Luo, 2018 ), as well as chemical mixtures that target a specific pathway ( Hohenbichler et al., 2020 ; Lampron et al., 2023 ). With regards to the latter case, several studies have demonstrated that the combination of chemicals targeting the same pathway can result in an exacerbated effects ( Silva et al., 2002 ; Fang et al., 2003 ; Rajapakse et al., 2004 ; Orton et al., 2012 ; Watt et al., 2016 ; O'Driscoll et al., 2018 ; Schlezinger et al., 2020 ; Nielsen et al., 2022 ). For example, exposure to 11 weak xenoestrogens led to enhanced 17β-estradiol action in recombinant yeast cells compared to single exposure ( Rajapakse et al., 2002 ). Similar findings have been observed in rats, that a mixture of 18 thyroid-disrupting chemicals had an additive effect at low doses and synergy at high doses effects on thyroid hormone concentrations ( Crofton et al., 2005 ). In the context of placental toxicology, only a handful of studies have explored the effects of chemical mixtures of the same or similar chemical classes ( Bonfanti et al., 2009 ; Kalkunte et al., 2017 ; Speidel et al., 2018 ; Drwal et al., 2019 ; Andersen et al., 2021 ), while most have investigated single chemical exposures ( Gingrich et al., 2020 ; Padmanabhan et al., 2021 ). To fill the gap on the effects of chemical mixtures on placental function, we have used a chemical mixture paradigm that specifically targets the EGFR pathway, while including compounds of different classes, half-lives, and from different sources. We aimed to evaluate the effect of the chemicals individually and as a mixture (PCB-126, PCB-153, atrazine, trans -nonachlor, niclosamide, and BPS) on EGFR signaling and EGFR-mediated trophoblast cell functions. To test this, we combined approaches such as EGF competitive binding and EGFR activation, along with functional assays such as trophoblast cell viability, proliferation, invasion, and endovascular differentiation.