Apoptotic responses stimulated by the trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine depend on cell differentiation state in BeWo human trophoblast cells

Abstract

During pregnancy, the placental villous cytotrophoblasts differentiate via cell fusion and multinucleation to create syncytiotrophoblasts, a cell type at the maternal-fetal interface. Apoptosis of syncytiotrophoblasts is associated with adverse pregnancy outcomes. The human trophoblast BeWo cell line has been used as an in vitro model for this differentiation process, also known as syncytialization. In the current study, we exposed unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells to S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a metabolite of the industrial chemical trichloroethylene (TCE). DCVC exposure at 50 μM for 48 h decreased cell viability, increased cytotoxicity, increased caspase 3/7 activity, and increased nuclear condensation or fragmentation in BeWo cells regardless of their differentiation status. Investigating mechanisms of apoptosis, DCVC increased H₂O₂ abundance and decreased PRDX2 mRNA in all three BeWo cell models. DCVC decreased tumor necrosis factor-receptor 1 (TNF-R1) concentration in media and decreased NFKB1 and PRDX1 mRNA expression in syncytialized BeWo cells only. DCVC decreased BCL2 mRNA expression in syncytializing BeWo cells and in syncytialized BeWo cells only. Decreased LGALS3 mRNA was seen in unsyncytialized BeWo cells only. Together, these data suggest roles for oxidative stress and pro-inflammatory mechanisms underlying apoptosis in BeWo cells with differences depending on differentiation state.

Keywords: Apoptosis; Placenta; S-(1,2-dichlorovinyl)-L-cysteine (DCVC); Syncytialization; Trichloroethylene (TCE); Villous trophoblasts.

Fig. 1.

Concentration-dependent effects of forskolin on syncytialization with and without DCVC co-exposure. (A) Visualization of BeWo cell syncytialization. The green stain is a phalloidin-FITC membrane stain, and the blue stain corresponds to a 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) nuclear stain. White scale bar represents 200 μm. (B) Quantification of staining assessment of syncytialization using the fusion index and percentage of nuclei in syncytia. N = 4 independent experiments. (C) DCVC effects on forskolin-stimulated syncytialization. N = 5 independent experiments. Controls were treated with 0.1% DMSO (vehicle control for forskolin) and are indicated as Vehicle (Fig. A), treatment with 0 μM forskolin alone (Figs. B1 and B2), or 0 μM DCVC +0 μM forskolin (Figs. C1-C4). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc multiple comparisons of means. Statistical significance is indicated by non-overlapping letters. Error bars represent mean ± SEM.

Fig. 2.

Concentration-dependent effects of DCVC on cell viability and cytotoxicity. (A) Unsyncytialized BeWo cells exposed to DCVC for 48 h. (B) Unsyncytialized BeWo cells exposed to DCVC for 72 h. (C) BeWo cells co-exposed to DCVC while undergoing forskolin-stimulated syncytialization for 48 h. (D) Syncytialized BeWo cells exposed to DCVC for 48 h. For all graphs, the control group is indicated as treatment with 0 μM DCVC alone or 0 μM DCVC +0 μM forskolin. In experiments that included forskolin co-treatment, cells in the 0 μM DCVC +0 μM forskolin control group were treated with 0.1% DMSO (vehicle control for forskolin). Statistical analysis was performed using mixed models ANOVA followed by Tukey’s post-hoc comparison of means. Statistical significance is denoted by non-overlapping letters. N = 3 independent experiments with triplicate wells for each experiment. Error bars represent mean ± SEM. Cell viability was measured as relative fluorescence units (RFU) and cytotoxicity was measured as relative luminescence units (RLU)/mg protein using the Multitox-Glo Multiplex Cytotoxicity Assay as described in Materials and Methods. Camptothecin (CPT) was included as a positive control.

Fig. 3.

Effects of DCVC on caspase 3/7 activity. (A) Unsyncytialized BeWo cells exposed to DCVC for 48 h. (B) Unsyncytialized BeWo cells exposed to DCVC for 72 h. (C) BeWo cells co-exposed to DCVC while undergoing forskolin-stimulated syncytialization for 48 h. (D) Syncytialized BeWo cells exposed to DCVC for 48 h. For all graphs, the control group is indicated as treatment with 0 μM DCVC alone or 0 μM DCVC +0 μM forskolin. In experiments that included forskolin co-treatment, cells in the 0 μM DCVC +0 μM forskolin control group were treated with 0.1% DMSO (vehicle control for forskolin). Statistical analysis was performed using mixed models ANOVA followed by Tukey’s post-hoc comparison of means. Statistical significance is denoted by non-overlapping letters. N = 4 independent experiments for (A) and N = 3 independent experiments for (B—D), performed in triplicate for each experiment. Error bars represent mean ± SEM. Camptothecin (CPT) was included as a positive control.

Fig. 4.

Effects of DCVC on nuclear condensation or fragmentation in BeWo cells. (A) Representative images of Hoechst 33342 staining to visualize nuclear condensation or fragmentation in unsyncytialized BeWo cells following 48-h exposure to DCVC. White scale bar represents 100 μm. (B) Quantification of DCVC effects on nuclear condensation or fragmentation in (B1) unsyncytialized BeWo cells exposed to DCVC for 24 h, (B2) unsyncytialized BeWo cells exposed to DCVC for 48 h, (B3) BeWo cells undergoing syncytialization for 48 h, (B4) syncytialized BeWo cells exposed to DCVC for 24 h, and (B5) syncytialized BeWo cells exposed to DCVC for 48 h. For all graphs, the control group is indicated as treatment with 0 μM DCVC alone or 0 μM DCVC +0 μM forskolin. In experiments that included forskolin co-treatment, cells in the 0 μM DCVC +0 μM forskolin control group were treated with 0.1% DMSO (vehicle control for forskolin). The percentage data were converted to fractions and arcsine transformed prior to statistical analysis. Statistical analysis was performed using one-way ANOVA. Statistically significant differences are indicated by non-overlapping letters. N = 4–5 independent experiments. Error bars represent mean ± SEM.

Fig. 5.

DCVC effects on H₂O₂ abundance, PRDX1 mRNA expression, and PRDX2 mRNA expression as biomarkers of oxidative stress. (A) Unsyncytialized BeWo cells exposed to DCVC for 48 h. (B) BeWo cells co-treated with DCVC while undergoing forskolin-stimulated syncytialization. (C) Syncytialized BeWo cells exposed to DCVC for 24 h. (D) Syncytialized BeWo cells exposed to DCVC for 48 h. For all graphs, the control group is indicated as treatment with 0 μM DCVC alone or 0 μM DCVC +0 μM forskolin. In experiments that included forskolin co-treatment, cells in the 0 μM DCVC +0 μM forskolin control group were treated with 0.1% DMSO (vehicle control for forskolin). For the mRNA expression data, statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc comparison of means, with N = 4–5 independent experiments. For the H₂O₂ abundance data, statistical analysis was performed using mixed models ANOVA followed by Tukey’s post-hoc comparison of means, with N = 3 independent experiments performed in triplicate. Statistically significant differences are indicated by non-overlapping letters. Error bars for all graphs represent mean ± SEM. Menadione (MD) was included as a positive control for the experiments involving measurement of H₂O₂.

Fig. 6.

DCVC effects on NFKB1 mRNA expression and cell media concentrations of TNF-R1 as biomarkers of pro-inflammatory response. (A) Unsyncytialized BeWo cells exposed to DCVC for 48 h. (B) BeWo cells co-treated with DCVC while undergoing forskolin-stimulated syncytialization for 48 h. (C1) Syncytialized BeWo cells exposed to DCVC for 24 h. (C2-C3) Syncytialized BeWo cells exposed to DCVC for 48 h. For all graphs, the control group is indicated as treatment with 0 μM DCVC alone or 0 μM DCVC +0 μM forskolin. In experiments that included forskolin co-treatment, cells in the 0 μM DCVC +0 μM forskolin control group were treated with 0.1% DMSO (vehicle control for forskolin). Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc comparison of means. Statistically significant differences are indicated by non-overlapping letters. For the TNF-R1 data, N = 5, 5, and 3 independent experiments for unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells, respectively. For the NFKB1 mRNA expression data, N = 5 independent experiments for all except N = 3 for exposures on syncytialized cells for 48-h duration. Error bars represent mean ± SEM.

Fig. 7.

Effects of DCVC on apoptotic pathway genes BCL2 and LGALS3. (A) Unsyncytialized BeWo cells exposed to DCVC for 48 h. (B) BeWo cells co-treated with DCVC while undergoing forskolin-stimulated syncytialization for 48 h. (C) Syncytialized BeWo cells exposed to DCVC for 24 h. (D) Syncytialized BeWo cells exposed to DCVC for 48 h. For all graphs, the control group is indicated as treatment with 0 μM DCVC alone or 0 μM DCVC +0 μM forskolin. In experiments that included forskolin co-treatment, cells in the 0 μM DCVC +0 μM forskolin control group were treated with 0.1% DMSO (vehicle control for forskolin). Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc comparison of means. Statistical significance is indicated by non-overlapping letters. Error bars represent mean ± SEM. N = 5 independent experiments for the experiments in (A) and (B). N = 4 independent experiments for the experiments in (C). N = 3 and N = 4 independent experiments for the BCL2 and LGALS3 outcomes, respectively, in Panel (D).

Fig. 8.

Summarized mechanism of apoptosis stimulated by DCVC in unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells. Whereas hydrogen peroxide increase and PRDX2 mRNA decrease were observed in all BeWo models, a decrease of PRDX1 mRNA, TNF-R1, and NFKB1 mRNA were only observed in syncytialized BeWo cells. A decrease of BCL2 mRNA was observed for BeWo cells undergoing syncytialization and syncytialized BeWo cells. A decrease of LGALS3 mRNA was observed only for unsyncytialized BeWo cells. Together, this suggests that mechanisms towards apoptosis differ depending on the BeWo model.