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

Pregnant women are exposed to complex chemical mixtures, many of which reach the placenta. Some of these chemicals interfere with epidermal growth factor receptor (EGFR) activation, a receptor tyrosine kinase that modulates several placenta cell functions. We hypothesized that a mixture of chemicals (Chem-Mix) known to reduce EGFR activation (polychlorinated biphenyl (PCB)-126, PCB-153, atrazine, trans-nonachlor, niclosamide, and bisphenol S) would interfere with EGFR-mediated trophoblast cell functions. To test this, we determined the chemicals' EGFR binding ability, EGFR and downstream effectors activation, and trophoblast functions (proliferation, invasion, and endovascular differentiation) known to be regulated by EGFR in extravillous trophoblasts (EVTs). The Chem-Mix competed with EGF for EGFR binding, however only PCB-153, niclosamide, trans-nonachlor, and BPS competed for binding as single chemicals. The effects of the Chem-Mix on EGFR phosphorylation were tested by exposing the placental EVT cell line, HTR-8/SVneo to control (0.1% DMSO), Chem-Mix (1, 10, or 100 ng/ml), EGF (30 ng/ml), or Chem-Mix + EGF. The Chem-Mix - but not the individual chemicals - reduced EGF-mediated EGFR phosphorylation in a dose dependent manner, while no effect was observed in its downstream effectors (AKT and STAT3). None of the individual chemicals affected EVT cell invasion, but the Chem-Mix reduced EVT cell invasion independent of EGF. In support of previous studies that have explored chemicals targeting a specific pathway (estrogen/androgen receptor), current findings indicate that exposure to a chemical mixture that targets the EGFR pathway can result in a greater impact compared to individual chemicals in the context of placental cell functions.

Binding sites in the epidermal growth factor receptor are responsible for bisphenol S effects on trophoblast cell invasion.

Bisphenol S (BPS) is an endocrine disrupting chemical and the second most abundant bisphenol detected in humans. We have recently demonstrated that in utero exposure to BPS reduces human placenta cell fusion by interfering with epidermal growth factor (EGF)-dependent EGF receptor (EGFR) activation. Our previous work suggests that this occurs via binding of BPS to the extracellular domain of EGFR. However, whether BPS directly binds to EGFR has not been confirmed. We evaluated the binding ability of BPA, BPF and BPS to EGFR to determine whether EGFR binding is a unique attribute of BPS. To test these hypotheses, we first exposed HTR-8/SVneo cells to BPS, BPA, or BPF, with or without EGF. When co-exposed to EGF, BPS, but not BPA nor BPF, reduced EGFR phosphorylation by ∼60%, demonstrating that only BPS can interfere with EGF-dependent EGFR activation. As this indicates that BPS binding to the extracellular domain is responsible for its effect, we performed a computational search for putative binding sites on the EGFR extracellular domain, and performed ligand docking of BPS, BPA, and BPF at these sites. We identified three sites where polar interactions between positively charged residues and the sulfonyl group of BPS could lead binding selectivity over BPA and BPF. To test whether EGFR mutations at the predicted BPS binding sites (Arg255, Lys454, and Arg297) could prevent BPS's interference on EGFR activation, mutations for each EGFR target amino acids (R255A, R297A, and K454A) were introduced. For variants with R297A or K454A mutations, BPS did not affect EGF-mediated EGFR phosphorylation or EGFR-mediated cell invasion, suggesting that these residues are needed for the BPS antagonism effect on EGFR. In conclusion, BPS, but not BPA or BPF, interferes with EGFR-mediated trophoblast cell functions through binding at Arg297 and Lys454 amino acid residues in the extracellular domain of EGFR.

The organotin triphenyltin disrupts cholesterol signaling in mammalian ovarian steroidogenic cells through a combination of LXR and RXR modulation.

Organotins, a chemical family with over 30 congeners to which humans are directly exposed to through food consumption, are a chemical class widely used as stabilizers in polyvinyl chloride, and biocides in antifouling products. Aside from tributyltin (TBT), toxicological information on other organotin congeners, such as triphenyltin (TPT), remains scarce. Our previous work has demonstrated that TBT can interfere with cholesterol trafficking in steroidogenic cells. Given their structural similarities, we hypothesized that TPT, similar to TBT, disrupts intracellular cholesterol transport and impairs steroidogenesis in ovarian theca cells. To test this, human and ovine primary ovarian theca cells were isolated, purified and exposed to TPT at environmentally relevant doses (1 or 10 ng/ml) in pre-luteinized (48 h exposure) or luteinizing cells (72 h exposure). Intracellular cholesterol levels, progesterone, and testosterone secretion and gene expression of nuclear receptors, cholesterol transporters, and steroidogenic enzymes were evaluated. In ovine cells, TPT upregulated StAR, ABCA1, and SREBF1 mRNA and ABCA1 protein in both pre-luteinized and luteinized stages. TPT did not alter intracellular cholesterol or testosterone synthesis, but upregulated progesterone production. Inhibitor and shRNA knockdown approaches were then used to evaluate the role of retinoid X receptor (RXR) and liver X receptor (LXR) on TPT's effects. TPT upregulated ABCA1 and StAR expression was blocked by both LXR and RXR antagonists. TPT's effect on ABCA1 expression was reduced in LXRß and RXRß knockdown theca cells. Similar findings were obtained with primary human theca cells. No synergistic effect of TBT and TPT was observed. In conclusion, at an environmentally relevant dose, TPT upregulates theca cell cholesterol transporter ABCA1 expression via RXR and LXR pathways. Similar effects of TPT on human and sheep theca cells supports its conserved mechanism across mammalian theca cells.

Bisphenol S Impairs Invasion and Proliferation of Extravillous Trophoblasts Cells by Interfering with Epidermal Growth Factor Receptor Signaling.

The placenta supports fetal growth and is vulnerable to exogenous chemical exposures. We have previously demonstrated that exposure to the emerging chemical bisphenol S (BPS) can alter placental endocrine function. Mechanistically, we have demonstrated that BPS interferes with epidermal growth factor receptor (EGFR) signaling, reducing placenta cell fusion. Extravillous trophoblasts (EVTs), a placenta cell type that aids with vascular remodeling, require EGF to invade into the maternal endometrium. We hypothesized that BPS would impair EGF-mediated invasion and proliferation in EVTs. Using human EVTs (HTR-8/SVneo cells), we tested whether BPS could inhibit the EGF response by blocking EGFR activation. We also evaluated functional endpoints of EGFR signaling, including EGF endocytosis, cell invasion and proliferation, and endovascular differentiation. We demonstrated that BPS blocked EGF-induced phosphorylation of EGFR by acting as a competitive antagonist to EGFR. Transwell assay and a three-dimensional microfluidic chip invasion assay revealed that BPS exposure can block EGF-mediated cell invasion. BPS also blocked EGF-mediated proliferation and endovascular differentiation. In conclusion, BPS can prevent EGF-mediated EVT proliferation and invasion through EGFR antagonism. Given the role of EGFR in trophoblast proliferation and differentiation during placental development, our findings suggest that maternal exposure to BPS may contribute to placental dysfunction via EGFR-mediated mechanisms.

Bisphenol S and Epidermal Growth Factor Receptor Signaling in Human Placental Cytotrophoblasts.

BACKGROUND: Bisphenol S (BPS) is an endocrine-disrupting chemical and the second most abundant bisphenol detected in humans. In vivo BPS exposure leads to reduced binucleate cell number in the ovine placenta. Binucleate cells form by cellular fusion, similar to the human placental syncytiotrophoblast layer. Given that human placental syncytialization can be stimulated through epidermal growth factor (EGF), we hypothesized that BPS would reduce human cytotrophoblast syncytialization through disruption of EGF receptor (EGFR) signaling. OBJECTIVE: We tested whether BPS interferes EGFR signaling and disrupts human cytotrophoblast syncytialization. METHODS: We first tested BPS competition for EGFR using an EGF/EGFR AlphaLISA assay. Using human primary term cytotrophoblast cells (hCTBs) and MDA-MD-231 cells, a breast cancer cell line with high EGFR expression, we evaluated EGFR downstream signaling and tested whether BPS could inhibit the EGF response by blocking EGFR activation. We also evaluated functional end points of EGFR signaling, including EGF endocytosis, cell proliferation, and syncytialization. RESULTS: BPS blocked EGF binding in a dose-dependent manner and reduced EGF-mediated phosphorylated EGFR in both cell types. We further confirmed that BPS acted as an EGFR antagonist as shown by a reduction in EGF internalization in both hCTBs and MDA-MD-231 cells. Finally, we demonstrated that BPS interfered with EGF-mediated cell processes, such as cell proliferation in MDA-MD-231 cells and syncytialization in hCTBs. EGF-mediated, but not spontaneous, hCTB syncytialization was fully blocked by BPS (200 ng/mL), a dose within urinary BPS concentrations detected in humans. CONCLUSIONS: Given the role of EGFR in trophoblast proliferation and differentiation during placental development, this study suggests that exposures to BPS at environmentally relevant concentrations may result in placenta dysfunction, affecting fetal growth and development. https://doi.org/10.1289/EHP7297.

Diferentes ofertas de forragem e a produção de leite em vacas mestiças Holandês x Gir / Different herbage allowances and milk yield in crossbred Holstein x Gir

Objetivou-se avaliar o efeito de diferentes ofertas de forragem em pastagem de estrela africana (Cynodon nlemfuensis Vanderyst var. nlemfuensis), sobre a taxa de desaparecimento de forragem (TDF) e a produção de leite de vacas mestiças Holandês x Gir. Trinta animais foram submetidos a três ofertas de forragem (OF) distintas, sendo 10,0 12,5 e 15,0% do peso corporal. Houve influência da OF sobre a TDF (P<0,001). Para cada unidade de acréscimo da OF, a TDF aumentou 140,0kg ha-1 dia-1. Houve efeito da relação folha:colmo sobre a produção de leite (P<0,05). O aumento da oferta não refletiu em incremento da produção de leite em função do manejo empregado para obtenção das ofertas e estádio de crescimento das plantas.

The objective of this study was to evaluate different herbage allowances in stargrass (Cynodon nlemfuensis Vanderyst var. nlemfuensis), on the herbage disappearance rate (HDR) and milk yield in crossbred Holstein x Gir cows. Thirty animals were assigned to three different herbage allowances (HA), ranging from 10.0, 12.5 and 15.0% BW. There was effect of HA on the HDR (P<0.001). Increasing the HA in one unit had effect on the HDR increasing by 140.0kg ha-1 day-1. There was effect of leaf:stem ratio on milk yield (P<0.05). The increasing in supplying herbage allowances did not resulted in increased milk yield because the management for herbage allowance and herbage growth.