Genotoxicity of Particles From Grinded Plastic Items in Caco-2 and HepG2 Cells

Abstract

Large plastic litters degrade in the environment to micro- and nanoplastics, which may then enter the food chain and lead to human exposure by ingestion. The present study explored ways to obtain nanoplastic particles from real-life food containers. The first set of experiments gave rise to polypropylene nanoplastic suspensions with a hydrodynamic particle size range between 100 and 600 nm, whereas the same grinding process of polyethylene terephthalate (PET) produced suspensions of particles with a primary size between 100 and 300 nm. The exposure did not cause cytotoxicity measured by the lactate dehydrogenase (LDH) and water soluble tetrazolium 1 (WST-1) assays in Caco-2 and HepG2 cells. Nanoplastics of transparent PET food containers produced a modest concentration-dependent increase in DNA strand breaks, measured by the alkaline comet assay [net induction of 0.28 lesions/10⁶ bp at the highest concentration (95% CI: 0.04; 0.51 lesions/10⁶ base pair)]. The exposure to nanoplastics from transparent polypropylene food containers was also positively associated with DNA strand breaks [i.e., net induction of 0.10 lesions/10⁶ base pair (95% CI: -0.04; 0.23 lesions/10⁶ base pair)] at the highest concentration. Nanoplastics from grinding of black colored PET food containers demonstrated no effect on HepG2 and Caco-2 cells in terms of cytotoxicity, reactive oxygen species production or changes in cell cycle distribution. The net induction of DNA strand breaks was 0.43 lesions/10⁶ bp (95% CI: 0.09; 0.78 lesions/10⁶ bp) at the highest concentration of nanoplastics from black PET food containers. Collectively, the results indicate that exposure to nanoplastics from real-life consumer products can cause genotoxicity in cell cultures.

Keywords: DNA damage; comet assay; microplastic; nanoparticles; oxidative stress.

Figure 1

Preparation of polypropylene (PP) and polyethylene terephthalate (PET) suspensions for the pilot study. The images demonstrate from left to right the original food containers, pieces of containers, grinding process, primary slurry of PP and PET plastics particles.

Figure 2

Preparation of PET particles for the main study. Top from left to right: the PET food container was first sterilized using 96% ethanol. The plastic was then cut into 5 cm long strips. The pieces were mixed with ethanol for 10 min at room temperature. The plastic-ethanol suspension was left to sediment for 5 min and 400 ml of the suspension extracted using a plastic syringe and filtered through a paper filter using a vacuum pump. The filtrate was further filtered through a 0.45 disk filter. The final suspension was left to evaporate at 65°C in a heating block.

Figure 3

Particle size distribution of final exposure suspensions from transparent polypropylene (A,B) and polyethylene terephthalate plastics (C,D) in the pilot study, and suspensions from black polyethylene terephthalate plastics in the main (E,F) study. The suspensions were analyzed in filtered water for injection. The mean particle size distribution (B,D,F) has been obtained from five consecutive size distribution measurements (A,C,E).

Figure 4

Cytotoxicity in Caco-2 and HepG2 cells after 24 h exposure to polypropylene (PP) and polyethylene terephthalate (PET). The results are reported as fold-difference compared to the positive control (LDH assay) and unexposed (WST-1 assay). Symbol and error bars are means and SEM from three independent experiments.

Figure 5

Levels of DNA strand breaks in Caco-2 and HepG2 cells after 3 h exposure to grinded particles of polypropylene (PP) and polyethylene terephthalate (PET) food containers. The high concentration is 63 ng/ml, 175 ng/ml, and 100 μM of PET, PP, and H₂O₂, respectively. The medium and low concentrations correspond to sequential two-fold dilutions. Each bar is the mean and SEM of three independent experiments, except H₂O₂ in Caco-2 cells (n = 2). *P < 0.05, linear mixed effect model.

Figure 6

Metabolic activity (WST-1 assay) and cell membrane leakage (LDH assay) after 24 h exposure to nanoplastics from black PET food containers (main study). The results are mean and SEM from three independent experiments.

Figure 7

Cell cycle distribution HepG2 and Caco-2 cells after 24 h exposure to PET nanoplastics from black food containers. The results are means of 2-3 independent experiments (mean and standard deviation). The exposure to nanoplastics is not associated with changes in the cell cycle distribution (P > 0.05), whereas culture of cells in serum free medium (SFM) shifted the cell cycle to G0/G1 phase from DNA synthesis phase (S). *P < 0.05, linear mixed effects model.

Figure 8

Levels of DNA strand breaks in Caco-2 and HepG2 cells after 3 h exposure to particles from black polyethylene terephthalate (PET) food containers. The correlation coefficient refers to the concentration response relationship in linear mixed effect model. Symbols are individual experiments. The positive control (100 μM H₂O₂) is 2.49 lesions/10⁶ bp (standard error of the mean = 0.06 lesions/10⁶ bp).