Benzo(a)pyrene and Cerium Dioxide Nanoparticles in Co-Exposure Impair Human Trophoblast Cell Stress Signaling

Abstract

Human placenta is a multifunctional interface between maternal and fetal blood. Studying the impact of pollutants on this organ is crucial because many xenobiotics in maternal blood can accumulate in placental cells or pass into the fetal circulation. Benzo(a)pyrene (BaP) and cerium dioxide nanoparticles (CeO₂ NP), which share the same emission sources, are found in ambient air pollution and also in maternal blood. The aim of the study was to depict the main signaling pathways modulated after exposure to BaP or CeO₂ NP vs. co-exposure on both chorionic villi explants and villous cytotrophoblasts isolated from human term placenta. At nontoxic doses of pollutants, BaP is bioactivated by AhR xenobiotic metabolizing enzymes, leading to DNA damage with an increase in γ-H2AX, the stabilization of stress transcription factor p53, and the induction of its target p21. These effects are reproduced in co-exposure with CeO₂ NP, except for the increase in γ-H2AX, which suggests a modulation of the genotoxic effect of BaP by CeO₂ NP. Moreover, CeO₂ NP in individual and co-exposure lead to a decrease in Prx-SO₃, suggesting an antioxidant effect. This study is the first to identify the signaling pathways modulated after co-exposure to these two pollutants, which are common in the environment.

Keywords: AhR; CYP1A1; benzo(a)pyrene; cerium dioxide nanoparticles; chorionic villi; human placenta; p21; p53; villous cytotrophoblasts; γ-H2AX.

Figure 1

Effect of increasing concentrations of benzo(a)pyrene on human primary trophoblast metabolic activity and AhR pathway activation. Villous cytotrophoblasts (VCT) purified from term placentas were plated overnight. They were then either untreated (Ctrl) or incubated with dimethyl sulfoxide (DMSO, solvent control), benzo(a)pyrene (BaP), or cerium dioxide nanoparticles (CeO₂ NP) at the indicated concentrations for 30 min, 24 h, 48 h, or 72 h. (A) WST-1 assay showing metabolic activity of VCT as mean ± SEM (n = 3) relative to DMSO control for each time of treatment. * p < 0.05, ** p < 0.01 vs. DMSO. (B) VCT were also incubated with 0.5 µM of staurosporine (STS) for 2 h or 6 h. Total protein extracts were subjected to SDS-PAGE under reducing conditions and membranes were immunoblotted with anti-PARP-1, anti-AhR, anti-CYP1A1, and anti-vinculin antibodies (the latter was used as loading control).

Figure 2

Effect of benzo(a)pyrene and cerium dioxide nanoparticles on the AhR pathway and metabolic activity of human primary trophoblasts. VCT purified from term placentas were plated overnight. They were then incubated either with DMSO or with BaP and/or CeO₂ NP at the indicated concentrations for 30 min, 4 h, 24 h, or 72 h, or with 0.5 µM of STS for 4 h. (A) Total protein extracts were subjected to SDS-PAGE under reducing conditions and membranes were immunoblotted with anti-AhR, anti-CYP1A1, anti-PARP-1, and anti-vinculin antibodies (the latter was used as loading control). (B) WST-1 assay showing metabolic activity of VCT as mean ± SEM (n = 3) relative to DMSO after 72 h of treatment. * p < 0.05, ** p < 0.01 vs. DMSO. (C) The human chorionic gonadotropin (hCG) assay was performed by enzyme-linked immunosorbent assay in VCT supernatants. VCT were incubated for 48 h with 1 µM of BaP and/or 10 μg/cm² of CeO₂ NP, or 500 µM of TBHP. The hCG assays were normalized to µg of protein per condition. Results represent the mean ± SEM (n = 3) relative to control (Ctrl). **** p < 0.0001 vs. Ctrl.

Figure 2

Effect of benzo(a)pyrene and cerium dioxide nanoparticles on the AhR pathway and metabolic activity of human primary trophoblasts. VCT purified from term placentas were plated overnight. They were then incubated either with DMSO or with BaP and/or CeO₂ NP at the indicated concentrations for 30 min, 4 h, 24 h, or 72 h, or with 0.5 µM of STS for 4 h. (A) Total protein extracts were subjected to SDS-PAGE under reducing conditions and membranes were immunoblotted with anti-AhR, anti-CYP1A1, anti-PARP-1, and anti-vinculin antibodies (the latter was used as loading control). (B) WST-1 assay showing metabolic activity of VCT as mean ± SEM (n = 3) relative to DMSO after 72 h of treatment. * p < 0.05, ** p < 0.01 vs. DMSO. (C) The human chorionic gonadotropin (hCG) assay was performed by enzyme-linked immunosorbent assay in VCT supernatants. VCT were incubated for 48 h with 1 µM of BaP and/or 10 μg/cm² of CeO₂ NP, or 500 µM of TBHP. The hCG assays were normalized to µg of protein per condition. Results represent the mean ± SEM (n = 3) relative to control (Ctrl). **** p < 0.0001 vs. Ctrl.

Figure 3

Homogeneity of the cellular response to benzo(a)pyrene and cerium dioxide nanoparticles in independent VCT cultures from 5 placentas. VCT purified from 5 different term placentas were plated overnight in culture and then were incubated either with DMSO or with BaP (0.6 µM) or CeO₂ NP (6.3 µg/cm²), and in BaP and CeO₂ NP co-exposure for 24 h. Total protein extracts (30 µg) were subjected to SDS-PAGE under reducing conditions and membranes were immunoblotted with anti-CYP1A1 and anti-vinculin (loading control) antibodies. Immunoblots were quantified with an Odyssey System Imager and results in the bar scale graph represent the mean ± SEM after normalization to vinculin (n = 5). * p < 0.05 vs. DMSO.

Figure 4

Cell Stress Array heatmap of the relative variations in protein expression after exposure of human primary trophoblasts to benzo(a)pyrene and cerium dioxide nanoparticles. VCT purified from term placentas were plated overnight and were either untreated or incubated with BaP (0.6 µM) or CeO₂ NP (6.3 µg/cm²) alone or together for 24 h. Total protein extracts were pooled (n = 5) and protein expression was measured by the Cell Stress Array. Immunoblots were quantified with an Odyssey System Imager and the results are shown in the heatmap as variation relative to the DMSO-treated cells.

Figure 5

Intracellular oxidative stress after trophoblast exposure to benzo(a)pyrene and/or cerium dioxide nanoparticles. VCT purified from term placentas were plated overnight and were untreated (control) or incubated with DMSO, BaP (0.6 µM), or CeO₂ NP (6.3 µg/cm²) and in co-exposure for 24 h. Tert-butyl hydroperoxide (TBHP, 100 μM, 4 h) and STS (0.5 μM, 3 h) were used as positive controls for oxidative stress and apoptosis, respectively. Total protein extracts were subjected to SDS-PAGE under reducing conditions and membranes were immunoblotted with anti-Prx-SO₃, anti-NFκB p65, anti-HO-1, and anti-vinculin antibodies (loading control). Immunoblots were quantified with an Odyssey System Imager and the results represent the mean ± SEM after normalization to vinculin (n = 5). * p < 0.05 vs. DMSO.

Figure 6

p53 signaling and DNA damage after trophoblast exposure to benzo(a)pyrene and/or cerium dioxide nanoparticles. VCT purified from term placentas were plated overnight and were untreated (control) or incubated with DMSO, BaP (1 µM), and/or CeO₂ NP (10 µg/cm²) for 24 h. (A) Total protein extracts were subjected to SDS-PAGE under reducing conditions and membranes were immunoblotted with anti-CYP1A1, anti-p53, anti-p21, anti-γH2AX, and anti-vinculin antibodies (loading control). Immunoblots were quantified with an Odyssey System Imager and the results represent the mean ± SEM after normalization to vinculin (n = 6). * p < 0.05 vs. DMSO. (B) Total mRNA was extracted from VCT, followed by reverse transcription. Real-time quantitative PCR (RT-qPCR) was performed for the genes of interest CYP1A1, p53, and p21. The results, expressed as fold change, represent the mean ± SEM after normalization to the geometric mean of the reference genes HPRT, RPLO, and SDHA (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO.

Figure 7

Protein expression and mRNA levels of p53 signaling and the AhR pathway after trophoblast exposure to benzo(a)pyrene. (A–C) VCT purified from term placentas were plated overnight and were untreated (control), incubated with DMSO or BaP (1 µM), preincubated for 1 h with AhR antagonist (3 µM), preincubated for 1 h with pifithrin-α (20 µM), incubated with BaP (1 µM) after preincubation for 1 h with AhR antagonist for 24 h, or incubated with BaP (1 µM) after preincubation for 1 h with pifithrin-α for 24 h. Total protein extracts were subjected to SDS-PAGE under reducing conditions and membranes were immunoblotted with anti-CYP1A1, anti-p53, anti-p21, anti-γH2AX, and anti-vinculin antibodies (loading control). Immunoblots were quantified with an Odyssey System Imager and the results represent the mean ± SEM after normalization to vinculin (n = 6). * p < 0.05 vs. DMSO, # p < 0.05 vs. BaP, ## p < 0.01 vs. BaP. (D) VCT purified from term placentas were plated overnight and incubated with DMSO or BaP (1 µM), preincubated for 1 h with AhR antagonist (3 µM) or pifithrin-α (20 µM), or incubated with BaP (1 µM) after preincubation for 1 h with AhR antagonist or pifithrin-α for 24 h. Total mRNA was extracted from VCT, followed by reverse transcription. RT-qPCR was performed for the genes of interest, i.e., p53 and p21. The results, expressed as fold change, represent the mean ± SEM after normalization to the geometric mean of the reference genes HPRT, RPLO, and SDHA (n = 5). * p < 0.05 vs. DMSO, # p < 0.05 vs. BaP, ## p < 0.01 vs. BaP.

Figure 8

Immunohistochemistry of HIF, p53, and p21 after chorionic villi exposure to benzo(a)pyrene or/and cerium dioxide nanoparticles. Representative images of immunohistochemical staining of HIF-1α, HIF-2α, p53, p21, and negative controls (rabbit IgG and mouse IgG2b) from placental explants incubated with DMSO, BaP (1 µM), and/or CeO₂ NP (10 µg/cm²) for 24 h (n = 3). Quantitative analysis of HIF-1α, HIF-2α, p53, and p21 expression was performed using the ImageJ plugin. Values represent means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. DMSO.