Bioaccumulation Rate of Non-Biodegradable Polystyrene Microplastics in Human Epithelial Cell Lines

Abstract

Environment plastic accumulation has been attracting the attention of both political and scientific communities, who wish to reduce global pollution. Plastic items have been detected everywhere, from oceans to the air, raising concerns about the fate of plastics within organisms. Leaked plastics are ingested by animals, entering the food chain and eventually reaching humans. Although a lot of studies focused on the evaluation of plastic particles in the environment and living organisms have already been published, the behavior of plastic at the cellular level is still missing. Here, we analyzed the bioaccumulation and extrusion trend of two differently sized plastic particles (1 and 2 µm), testing them on three human epithelial cell lines (liver, lung, and gut) that represent epithelial sites mainly exposed to plastic. A different behavior was detected, and the major plastic uptake was shown by liver cells, where the 1 µm beads accumulated with a dose-dependent profile. Moreover, a 60% reduction in the content of 1 µm particles in cells was evaluated after plastic removal. Finally, the viability and proliferation of the three human cell lines were not significantly affected by both the 1 and 2 µm beads, suggesting that cells might have a defense mechanism against plastic exposure risk.

Keywords: bioaccumulation; cell survival; microplastic; polystyrene; release.

Figure 1

PS-MPs microscopy characterization. (A) SEM images of 1 and 2 µm ulPS-MPs, also representative of the corresponding flPS-MPs (scale bar: 1 µm); (B) fluorescent microscopy images of 1 and 2 µm flPS-MPs excited at 488 nm (scale bar: 10 µm).

Figure 2

Confocal microscopy of Mahlavu cells after 24 h of exposure to 1 µm PS beads (5000 beads/mm²). (A) Five consecutive optical sections, Z-step: 0.3 µm; (B) Z-stack projection deriving from the superimposition of 29 optical sections taken 0.3 µm apart; (C) 3D views of the Z-stack along the x-axis (upper image) and the z-axis (lower image). Cytoskeleton: red (Phalloidin Alexa Fluor-555 conjugated); PS-MPs: green/yellow. Scale bar: 10 µm.

Figure 3

Fluorescent microscopy of bioaccumulation and subcellular localization of 1 µm flPS-MPs in Mahlavu cells after 48 h of exposure time (20,000 beads/mm²). The image in (A) was acquired with a Plan-Apochromat 60×/1.45 in oil objective, whereas the one in (B) was acquired with a Plan-Apochromat 100×/1.45 in oil objective, to zoom in and obtain a more detailed visualization; localization of the PS-MPs within cells is denoted by white arrows. Nucleus: blue (DAPI); cytoskeleton: red (Phalloidin Alexa Fluor-555 conjugated); PS-MPs: green/yellow. Scale bar: 10 µm.

Figure 4

(A) Bioaccumulation of 1 µm PS-MPs by Mahlavu cells after 24 h and 48 h of exposure to beads at different densities (5000–10,000–20,000 beads/mm²). Fluorescent microscopy representative images of PS-MPs-treated cells. Nucleus: blue (DAPI); cytoskeleton: red (Phalloidin Alexa Fluor-555 conjugated); PS-MPs: green/yellow. Scale bar: 10 µm. (B) Percentage of positive cells for PS beads internalization. Error bars: 95% CI. (C) number of 1 µm PS-MPs per single cell. * p < 0.05; ** p < 0.01; circle sign for data normalization.

Figure 5

Extrusion process of 1 µm PS-MPs in Mahlavu cells exposed to 20,000 beads/mm² for 24 or 48 h. (A) Percentage of positive cells for PS beads internalization. Error bars: 95% CI. (B) Number of beads per cell in the different tested conditions. * p < 0.05.

Figure 6

Confocal microscopy of Mahlavu cells after 24 h of exposure to 2 µm PS beads (5000 beads/mm²). (A) Five consecutive optical sections, Z-step: 0.3 µm; (B) Z-stack projection deriving from the superimposition of 26 optical sections with a 0.3 µm Z-step; (C) three-dimensional views of the Z-stack along the x-axis (upper image) and the z-axis (lower image). Cytoskeleton: red (Phalloidin Alexa Fluor-555 conjugated); PS-MPs: green/yellow. Scale bar: 10 µm.

Figure 7

Bioaccumulation and localization pattern of 2 µm PS-MPs in Mahlavu cells, after 24 h and 48 h of exposure to beads at different densities (5000–10,000–20,000 beads/mm²). (A) Fluorescent microscopy representative images of PS-MPs-treated cells. Nucleus: blue (DAPI); cytoskeleton: red (Phalloidin Alexa Fluor-555 conjugated); PS-MPs: green/yellow. Scale bar: 10 µm. (B) Percentage of positive cells for PS beads internalization. Error bars: 95% CI. (C) Number of beads per single cell. * p < 0.05; ** p < 0.01; circle sign for data normalization.

Figure 8

Extrusion of 2 µm PS-MPs. (A) Percentage of positive cells for PS beads internalization. Error bars: 95% CI. (B) Number of beads per single cell. ** p < 0.01.

Figure 9

Internalization of 1 and 2 µm flPS-MPs by human cell lines (20,000 beads/mm²; exposure time: 48 h). Nucleus: blue (DAPI); cytoskeleton: red (Phalloidin Alexa Fluor-555 conjugated); PS-MPs: green/yellow. Scale bar: 10 µm.

Figure 10

Effects of 1 and 2 µm PS-MPs on cell viability and proliferation of Mahlavu, HCT-116, and A549 cell lines exposed to 20,000 beads/mm². (A) Percentage of cell proliferation in cells exposed to PS-MPs for 24 or 48 h. Doxorubicin was used as positive control; (B) percentage of cell viability in cells exposed to PS-MPs for 24 or 48 h. Doxorubicin was used as positive control. * p < 0.05; ** p < 0.01; **** p < 0.0001.