Chemicals and reagents: Minimum essential Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Fœtal Bovine Serum (FBS), 2 mM glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin, trypsin-EDTA 0.05% and Phosphate Buffer Saline (PBS) were provided by Gibco (Paisley, UK) and cell culture plastics such as flasks and microplates by Corning (Schiphol-Rijk, The Netherlands). Cell EventTM Caspase-3/7 green ReadyProbesTM, YO-PRO-1®, JC-1 and Hoechst 33342 probes were obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA) and Caspase-Glo® 8 Assay and Caspase-Glo® 9 Assay from Promega (Madison, WI, USA).
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%.
Cells culture: Human placental JEG-3 cell line (ATCC HTB-36), human lung A549 cell line (ATCC CCL-185) and human keratinocytes HaCaT cell line (Cell lines service-CLS-Germany) were cultured under standard conditions (37 °C, saturated humidity and 5% CO2), in Minimum Essential Medium (MEM) for JEG-3 cells and Dulbecco’s Modified Eagle Medium (DMEM) for HaCaT and A549 cells, supplemented with 10% FBS, 1% L-glutamine, 0.5% penicillin and streptomycin in 75 cm2 polystyrene flasks. Confluent cells were detached by trypsin-EDTA incubation. Cells were seeded into 96-well culture microplates at a density of 80,000 cells/mL (200µL/wells for JEG-3 and HaCaT, 100µL/wells for A549) for analysis. Cultures were kept at 37 °C for 24 h.
Cell incubation: To study the P2X7 receptor and its relationship between apoptosis, the cells were preincubated with either PBS or P2X7 antagonist Brilliant Blue G (BBG) at 25 µM for 15 min [34 (link)]. After removal of PBS and BBG, the cells were incubated for 72 h with bisphenol A (5, 10 and 20 µM), diethylstilbestrol (3.75, 7.5 and 15 µM), 4-tert-amylphenol (1, 10 and 50 µM), 4-heptylphenol (1, 10 and 50 µM), triclosan (0.1, 1 and 10 µM), propylparaben (20, 50 and 100 µM), benzyl butyl phthalate (1, 10 and 50 µM), DEHP (1, 10 and 50 µM) and 3-benzylidene camphor (1, 10 and 50 µM) in MEM with 2.5% FBS according to Olivier et al.’s protocol that describes the JEG-Tox model [35 (link)] or DMEM with 2.5% FBS for the HaCaT and A549. Concentrations tested in placental cells were selected according to the literature and the same concentrations were used to study the lung and skin cells [6 (link),7 (link),9 (link),32 (link),36 (link),37 (link)].
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 ethanol–water–acetic acid (50.6/48.4/1, v/v/v). After homogenization, the fluorescence signal was scanned (λex = 540 nm, λem = 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 (link)]. 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 (λex = 485 nm, λem = 531 nm) using the Spark® microplate reader.
Caspase-8, -9 activity: Caspase-Glo® Assays. Caspase-8 and -9 activities were evaluated using the Caspase-Glo® 8 and 9 assay kits, respectively. The assay was performed according to the manufacturer’s instructions. Luminescence was quantified using a Spark® microplate reader.
Caspase 3 activity: CellEventTM Caspase-3/7 Green Detection Reagent. Caspase-3 activity was evaluated using the CellEventTM Caspase3/7 Green Detection Reagent. Cell EventTM 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 λex = 485 nm and λem = 600 nm for the red fluorescence and λex = 485 nm and λem = 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 (λex = 350 nm, λem = 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: #).
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%.
Cells culture: Human placental JEG-3 cell line (ATCC HTB-36), human lung A549 cell line (ATCC CCL-185) and human keratinocytes HaCaT cell line (Cell lines service-CLS-Germany) were cultured under standard conditions (37 °C, saturated humidity and 5% CO2), in Minimum Essential Medium (MEM) for JEG-3 cells and Dulbecco’s Modified Eagle Medium (DMEM) for HaCaT and A549 cells, supplemented with 10% FBS, 1% L-glutamine, 0.5% penicillin and streptomycin in 75 cm2 polystyrene flasks. Confluent cells were detached by trypsin-EDTA incubation. Cells were seeded into 96-well culture microplates at a density of 80,000 cells/mL (200µL/wells for JEG-3 and HaCaT, 100µL/wells for A549) for analysis. Cultures were kept at 37 °C for 24 h.
Cell incubation: To study the P2X7 receptor and its relationship between apoptosis, the cells were preincubated with either PBS or P2X7 antagonist Brilliant Blue G (BBG) at 25 µM for 15 min [34 (link)]. After removal of PBS and BBG, the cells were incubated for 72 h with bisphenol A (5, 10 and 20 µM), diethylstilbestrol (3.75, 7.5 and 15 µM), 4-tert-amylphenol (1, 10 and 50 µM), 4-heptylphenol (1, 10 and 50 µM), triclosan (0.1, 1 and 10 µM), propylparaben (20, 50 and 100 µM), benzyl butyl phthalate (1, 10 and 50 µM), DEHP (1, 10 and 50 µM) and 3-benzylidene camphor (1, 10 and 50 µM) in MEM with 2.5% FBS according to Olivier et al.’s protocol that describes the JEG-Tox model [35 (link)] or DMEM with 2.5% FBS for the HaCaT and A549. Concentrations tested in placental cells were selected according to the literature and the same concentrations were used to study the lung and skin cells [6 (link),7 (link),9 (link),32 (link),36 (link),37 (link)].
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 ethanol–water–acetic acid (50.6/48.4/1, v/v/v). After homogenization, the fluorescence signal was scanned (λex = 540 nm, λem = 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 (link)]. 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 (λex = 485 nm, λem = 531 nm) using the Spark® microplate reader.
Caspase-8, -9 activity: Caspase-Glo® Assays. Caspase-8 and -9 activities were evaluated using the Caspase-Glo® 8 and 9 assay kits, respectively. The assay was performed according to the manufacturer’s instructions. Luminescence was quantified using a Spark® microplate reader.
Caspase 3 activity: CellEventTM Caspase-3/7 Green Detection Reagent. Caspase-3 activity was evaluated using the CellEventTM Caspase3/7 Green Detection Reagent. Cell EventTM 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 λex = 485 nm and λem = 600 nm for the red fluorescence and λex = 485 nm and λem = 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 (λex = 350 nm, λem = 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: #).
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