JEG-3 placental cells in toxicology studies: a promising tool to reveal pregnancy disorders.

Placental alterations are responsible for adverse pregnancy outcomes like preeclampsia and intrauterine growth restriction. And yet, placenta toxicology has not become a fully-fledged toxicology field. Because placenta is very often seen only as a barrier between the mother and the fetus, there is a lack and therefore a need for an experimental human model with technical recommendations to study placenta toxicology. In vitro approaches are recommended in experimental toxicology as they focus on a specific biological process and yield high-throughput screening methods. In the present study, we first established incubation conditions to preserve signatures of the human JEG-3 cell line identity while enabling toxicity detection. JEG-3 cells prepared in our incubation conditions were renamed JEG-Tox cells. As placental alterations are mainly triggered by uncontrolled apoptosis, we second used known apoptotic agents pregnant women are exposed to, to check that JEG-Tox cells can trigger apoptosis. Ethanol, bisphenol F, quinalphos, 4,4'-DDT, benzalkonium chloride, phenoxyethanol, propylparaben, and perfluorooctanic acid all induced chromatin condensation in JEG-Tox cells. Our incubation conditions allow JEG-Tox cells to keep placental cell identity and to respond to toxic chemicals. JEG-Tox cells are a pertinent model for placenta toxicology and could be used to better understand pregnancy alterations.

Expression, Localization, and Activity of the Aryl Hydrocarbon Receptor in the Human Placenta.

The human placenta is an organ between the blood of the mother and the fetus, which is essential for fetal development. It also plays a role as a selective barrier against environmental pollutants that may bypass epithelial barriers and reach the placenta, with implications for the outcome of pregnancy. The aryl hydrocarbon receptor (AhR) is one of the most important environmental-sensor transcription factors and mediates the metabolism of a wide variety of xenobiotics. Nevertheless, the identification of dietary and endogenous ligands of AhR suggest that it may also fulfil physiological functions with which pollutants may interfere. Placental AhR expression and activity is largely unknown. We established the cartography of AhR expression at transcript and protein levels, its cellular distribution, and its transcriptional activity toward the expression of its main target genes. We studied the profile of AhR expression and activity during different pregnancy periods, during trophoblasts differentiation in vitro, and in a trophoblast cell line. Using diverse methods, such as cell fractionation and immunofluorescence microscopy, we found a constitutive nuclear localization of AhR in every placental model, in the absence of any voluntarily-added exogenous activator. Our data suggest an intrinsic activation of AhR due to the presence of endogenous placental ligands.

Lipidome-wide disturbances of human placental JEG-3 cells by the presence of MEHP.

During pregnancy, exposure to environmental contaminants can lead to adverse effects on fetal growth and development, especially by targeting the placenta. Di(2-ethylhexyl)phthalate (DEHP), the most abundant chemical used in plastic materials, is known to induce toxicity on animals reproductive system and is suspected to give rise to similar effect in humans. Toxicity of DEHP is due to its main metabolite, MEHP, which is also known to disturb lipid synthesis in several organs. Moreover, mono-(2-ethylhexyl)phtalate (MEHP) is a high affinity ligand of the peroxisome proliferator-activated receptor PPARγ which is essential for placental development and lipid metabolism. In order to investigate possible lipid disruptions induced by MEHP, in the JEG-3 human trophoblast cell line, a differential lipidomic analysis was carried out by UPLC-MS on both exposed and control cells. Our results showed that MEHP induced an important change of JEG-3 cells lipidome, especially in glycerolipids and glycerophospholipids, with a marked accumulation of triacylglycerols. For the first time, our results highlighted adverse effects of MEHP on human placental cells lipidome and thus, its potential effect on placental physiology.

A fast and reproducible cell- and 96-well plate-based method for the evaluation of P2X7 receptor activation using YO-PRO-1 fluorescent dye.

The YO-PRO-1 assay provides a quantitative estimation of P2X7 receptor activation. P2X7 receptor is associated to pathological conditions including infectious, inflammatory, neurological, musculoskeletal disorders, pain and cancer. Most primary cells and cell lines from diverse origin may be used thanks to the ubiquitous distribution of P2X7 receptor. To study the activation of P2X7 receptor by chemicals or biological agents, we established a microplate-based cytometry protocol to accurately and rapidly quantify the activation of P2X7 receptor that leads to the formation of large pores in cell membranes. The YO-PRO-1 assay is based on the ability of cells to incorporate and bind YO-PRO-1 dye to DNA after activation of P2X7 receptor through pore formation. Cells are seeded in 96-well plates and incubated with the compound being tested for the appropriate time. The microplate is then incubated for 10 min with YO-PRO-1 staining solution. After the 10 min staining time, fluorescence signal is read using a microplate reader in 1 min. This procedure is easier and requires less handling steps than flow cytometry. 96-well plate based YO-PRO-1 assay is a reproducible and fast method to study both P2X7 receptor activation by toxic agents at subnecrotic concentrations and P2X7 receptor inhibition by antagonists.

The Role of the P2X7 Receptor in Ocular Stresses: A Potential Therapeutic Target.

The P2X7 receptor is expressed in both anterior and posterior segments of the eyeball. In the ocular surface, the P2X7 receptor is activated in case of external aggressions: preservatives and surfactants induce the activation of P2X7 receptors, leading to either apoptosis, inflammation, or cell proliferation. In the retina, the key endogenous actors of age-related macular degeneration, diabetic retinopathy, and glaucoma act through P2X7 receptors' activation and/or upregulation of P2X7 receptors' expression. Different therapeutic strategies aimed at the P2X7 receptor exist. P2X7 receptor antagonists, such as divalent cations and Brilliant Blue G (BBG) could be used to target either the ocular surface or the retina, as long as polyunsaturated fatty acids may exert their effects through the disruption of plasma membrane lipid rafts or saffron that reduces the response evoked by P2X7 receptor stimulation. Treatments against P2X7 receptor activation are proposed by using either eye drops or food supplements.

Amyloid ß Peptide Induces Apoptosis Through P2X7 Cell Death Receptor in Retinal Cells: Modulation by Marine Omega-3 Fatty Acid DHA and EPA.

Retinal Müller glial cells have already been implicated in age-related macular degeneration (AMD). AMD is characterized by accumulation of toxic amyloid-ß peptide (Aß); the question we raise is as follows: is P2X7 receptor, known to play an important role in several degenerative diseases, involved in Aß toxicity on Müller cells? Retinal Müller glial cells were incubated with Aß for 48 h. Cell viability was assessed using the alamarBlue assay and cytotoxicity using the lactate dehydrogenase (LDH) release assay. P2X7 receptor expression was highlighted by immunolabeling observed on confocal microscopy and its activation was evaluated by YO-PRO-1 assay. Hoechst 33342 was used to evaluate chromatin condensation, and caspases 8 and 3 activation was assessed using AMC assays. Lipid formulation rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) used in Age-Related Eye Disease Study 2 was incubated on cells for 15 min prior to Aß incubation. For the first time, we showed that Aß induced caspase-independent apoptosis through P2X7 receptor activation on our retinal model. DHA and EPA are polyunsaturated fatty acids recommended in food supplement to prevent AMD. We therefore modulated Aß cytotoxicity using a lipid formulation rich in DHA and EPA to have a better understanding of the results observed in clinical studies. We showed that fish oil rich in EPA and DHA, in combination with a potent P2X7 receptor antagonist, represents an efficient modulator of Aß toxicity and that P2X7 could be an interesting therapeutic target to prevent AMD.

New in vitro biomarkers to detect toxicity in human placental cells: The example of benzo[A]pyrene.

Our aim was to study the toxicity of benzo(a)pyrene (BaP), an environmental pollutant that can reach placenta, on two human placental models in order to propose biomarkers in risk assessment for pregnancy. Ex vivo human placental cells isolated from term placenta and JEG-3 cancer cell line were incubated with BaP at 0.1-10 µM for 48 h or 72 h. BaP induced neither loss of cell viability nor apoptosis in ex vivo placental cells. To go further, we performed experiments on JEG-3 cell line that provides near-unlimited cells. The results we obtained in JEG-3 cells confirmed that BaP, in our experimental conditions, is neither necrotic nor apoptotic for placental cells. BaP toxicity on placental cells resulted in cell cycle arrest (G2/M phase) associated with inhibition of cell proliferation. Besides, we observed that BaP remodeled the protein content of membrane microdomains via increased expression of ZO-1, caveolin-1 and P2X7 cell degenerescence receptor. In conclusion, we identified nuclear and membrane potential biomarkers of risks for placenta and then pregnancy. These potential biomarkers detected on placental cell lines could represent useful tools for toxicological studies.