Artificial plasticenta: how polystyrene nanoplastics affect in-vitro cultured human trophoblast cells

Abstract

Background: In the human placenta, we have detected the MPs by Raman microspectroscopy analysis and, for the first time, with transmission electron microscopy. MPs fragments have been localized in different compartments of placental tissue, free in the cytoplasm and within organelles like lysosomes. Moreover, their presence has been correlated with ultrastructural alterations of some cell organelles, typical of metabolic stress, mainly dilated rough endoplasmic reticulum and numerous swollen electrodense mitochondria, as well as signs derived from involuting organelles. As a result, we have speculated that microplastics in the placenta could be responsible for pathological traits activation such as oxidative stress, apoptosis, and inflammation causing long-term effects on the health of the mother and child. To demonstrate the cytotoxicity of PS-NPs on the placenta and confirm the in vivo results, we performed in vitro experiments on a trophoblast human cell line, the HTR8/SVneo cells.

Materials and methods: HTR8/SVneo cells were treated, for 24 h and 48h, with increasing concentrations (10, 25, 50, 75, and 100 μg/mL) of 0.05 µm polystyrene (PS) and cellular viability was evaluated by Counting Kit-8. Fluorescent PS-NPs examined under fluorescence/confocal microscopy were used to investigate the internalization of plastics in the placenta cells. Transmission electron microscopy was used to evaluate possible PS-NPs-dependent ultrastructural alterations of cells and organelles.

Results: Our study shows that starting from 24 h exposure, PS-NPs treatment, at 50 μg/mL dose, has a cytotoxic effect on placental cells, causing the death of 40% of cells and affecting the morphology of the surviving cells. In addition, PS-NPs alter the ultrastructure of some organelles in the surviving cells, like those we have already described in vivo. We found that NPs enter the cells, affecting the endoplasmic reticulum and mitochondria morphology, accumulating as aggregates within lysosome-like organelles. Interestingly these aggregates become larger as the concentration of NPs increases. We speculated that the accumulation of NPs inside lysosome-like organelles could result from a prolonged and impossible attempt by the cell to remove and destroy PS. This would lead to ER and mitochondrial stress, impairing mitochondria/ER functions and oxidative stress, thus activating the apoptotic pathway and suggesting that PS-NPs could act as a cell stressor, leading to the death of cells. In support of our hypothesis, we also found NPs associated with morphological signs of cellular regression and degeneration, such as the presence of a highly vacuolized cytoplasm, dilatation, and vesiculation of ER, associated with the uncoupling/loss of associated mitochondria, cytoplasmic fragments, and free organelles deriving from cellular lysis.

Conclusion: Based on electron microscopy and immunofluorescence analysis and in vitro study, we demonstrate the cytotoxicity of PS-NPs in trophoblast cells together with ultrastructural alterations associated with cellular regression and degeneration typical of metabolic stress. An abnormal amount of NPs in the cells might determine a persistent cellular alarm CDR (cell danger response), the evolutionarily conserved metabolic response that protects the cells and hosts from harm triggered by chemical (as in the case of NPs/MPs), physical, or biological agents that exceed the cellular capacity for homeostasis. This in vitro study could further help to demonstrate that the inevitable exposure of MPs/NPs in the environment, which characterizes the modern world, might be partially responsible for the epidemic of non-transmissible disease.

Keywords: altered fetal programming; confocal and transmission electron microscopy; placenta; plastics; polystyrene (PS) nanoplastics; ultrastructure.

FIGURE 1

Schematic representation of NPs and MPs contamination during pregnancy (“Image Reprinted under terms of the CC-BY license (Ragusa et al., 2022c)”).

FIGURE 2

Schematic representation of study design. HTR-8 SVneo trophoblast cells were treated for 24–48 h with different concentrations of 0.05 µm PS-NPs and analyzed for cell viability and morphological alterations by light, confocal, and TEM microscopy.

FIGURE 3

PS-Ps treatment of trophoblast cells (A) Cell viability evaluation with CCK-8 assay. Cells were treated with increasing concentrations of 0.05 µm NPs (10, 25, 50 75, and 100 μg/mL). Reported data represent the mean ± SD of three experiments. The differences in values compared to the control are significant (p < 0.05 at 10 μg/mL; P < 0.01 at 25–100 μg/mL) (B) Representative contrast phase images of cells after 24 h of treatment. Cells treated for 24 h with 10, 50, and 100 μg/mL microspheres decrease their proliferation and undergo morphological changes becoming flatter and longer and losing contact with each other. Bar, 25 µm.

FIGURE 4

Uptake of PS-NPs by placental trophoblast cells detected with confocal microscopy. To investigate the intake of PS-NPs inside cells, trophoblast cells were treated with PS-YG Fluoresbrite NPs, for 24 h, at a 50 μg/mL dose (A, B) Representative microphotographs at a confocal microscope indicate the presence of aggregates of NPs (green) inside the cells. Cell nuclei were stained with DAPI (blue) (C) The cytoplasm of cells photographed in Differential Interference Contrast (DIC, grey) at a confocal microscope (D) Representative image, at fluorescence microscope, of an NPs aggregate (green), in trophoblast cells stained with DAPI. Bar, 25 µm (see attached Figure 5) (E) Coverslips with the Fluoresbrite^® YG Microspheres alone at 100 μg/mL concentration were used as positive controls. Bar, 50 µm (A–C). Bar, 25 µm (D, E).

FIGURE 5

Microphotography of ultrathin sections of human control trophoblast cells by TEM (A, B) Low magnification of HTR-8 SVneo cells morphology. The cells are irregularly rounded, with oval large nuclei (N) and less abundant cytoplasm. Numerous blebs and microvilli (mv) are present on the surface. Bar, 2 µm (C, D) Ultrastructural details at higher magnification of mitochondria (m) in association with tubular vesicles of ER, clathrin vesicles (cL) under the plasma membrane, vacuoles (V), Golgi membranes (G), and polyribosomes (r). Bar, 500 nm.

FIGURE 6

NPs in human trophoblast cells (A, B) NPs (50 μg/mL) were able to enter the cells, probably via endocytosis and (C, E) accumulate in structures constituted by a double membrane (arrows) resembling lysosomes (D) PS-NPs in cell medium (100 μg/mL) observed by TEM were used as a positive control, confirming the commercial particle size of 0.05 µm. Bar, 200nm. CL, clathrin vesicles; m, mitochondria; mv, microvilli. Bar, 500 µm (A, B). Bar = 200 µm (C–E).

FIGURE 7

Microphotographs of trophoblast cells treated with 50 μg/mL NPs (A–C) NPs aggregates induce morphological changes in ER and mitochondria. The ER appears dilated, with sparse ribosomes on the outer surface. Degranulation and disaggregation of polyribosomes (r) free in the cytoplasm also occur (D) Microphotographs of placental cells treated with 100 μg/mL NPs in which swollen electrondense mitochondria (m) appear in the cytoplasm. Note that the size of aggregates is enhanced by increasing NP concentration. Bar, 500 nm (A, B); Bar, 200 nm (C, D). N, nucleus.

FIGURE 8

Ultrastructural changes in trophoblast cells treated with 100 μg/mL PS-NPs (A) PS NPs, encapsulated in a lysosome-like double membrane structure occur (arrows) in the cytoplasm together with numerous smaller, pycnotic, and electrodense mitochondria (m_p) with prominent electron-dense granules (G), swollen mitochondria (m_s) and numerous highly dilated ER vesicles. Bar, 500 nm (B–D) Morphological details at higher magnification correlating the presence of NPs with ultrastructural changes of ER and mitochondria suggest a strong turnout in support of the hypothesis of an NPs-induced possible metabolic stress. Bar, 200 nm. Golgi membranes (G).

FIGURE 9

Regressing and degenerating trophoblast cells treated with 100 μg/mL PS-NPs (A) vacuoles (V) in the cytoplasm (B–D) highly vacuolized cytoplasm derived from swelling and coalescence of ER vesicles and swelling mitochondria (m_s) with prominent electron-dense granule (G) (E, F) Cytoplasmic fragments and matrix-free organelles deriving from cellular lysis also occur. Bar, 500 nm (A–D); Bar, 2 µm (E, F).