Interaction of Polystyrene Nanoplastics with Biomolecules and Environmental Pollutants: Effects on Human Hepatocytes

Abstract

The inevitable exposure of humans to micro/nanoplastics has become a pressing global environmental issue, with growing concerns regarding their impact on health. While the direct effects of micro/nanoplastics on human health remain largely unknown, increasing attention is being given to their potential role as carriers of environmental pollutants and organic substances. This study investigates the direct toxicity of 500 nm polystyrene nanoplastics (NPs) on human hepatocytes (HepG2) in vitro, both alone and in combination with cadmium (Cd), a hazardous heavy metal and a prevalent environmental pollutant. One-hour exposure to 100 µg/mL of NPs causes a significant increase in ROS production (+25% compared to control) but cell viability remains unaffected even at concentrations much higher than environmental levels. Interestingly, NPs significantly reduce Cd cytotoxicity at LC₅₀ concentrations (cell viability compared to control: 55.4% for 50 µM Cd, 66.9% for 50 µM Cd + 10 µg/mL NPs, 68.4% for 50 µM Cd + 100 µg/mL NPs). Additionally, NPs do not alter the cellular lipid content after short-term exposure (24 h). However, when Cd and fatty acids are added to the medium, NPs appear to sequester fatty acids, reducing their availability and impairing their uptake by cells in a dose-dependent manner. We confirmed by Dynamic Light Scattering and Scanning Electron Microscopy the interaction between NPs, Cd and free fatty acids. Although polystyrene NPs exhibited minimal cytotoxicity in our experimental model, collectively our findings suggest that predicting the effects of cell exposure to NPs is extremely challenging, due to the potential interaction between NPs, environmental pollutants and specific components of the biological matrix.

Keywords: cadmium; hepatocyte viability; lipid sequestration; nanoplastics; oxidative stress.

Figure 1

The effects of increasing concentrations of nanoparticles (NPs) on cell viability, as measured by the CellTiter-Glo^® luminescent Cell Viability Assay after (a,b) 24 or 48 hours of exposure to NPs. Cell viability is expressed as luminescence intensity, which is directly proportional to the number of viable cells. Each graph represents the mean of three independent experiments (n = 3) ± standard deviation (SD).

Figure 2

The effects of cadmium (Cd) alone (panel a) and in combination with two different concentrations of nanoparticles (NPs) (panel b) on cell viability, as determined by the CellTiter-Glo^® luminescent Cell Viability Assay. Cell viability is quantified based on luminescence intensity, which is proportional to the number of living cells. The LC₅₀ (lethal concentration 50) of Cd is indicated in panel (a). Each graph depicts the mean results of three independent experiments (n = 3) with error bars representing the standard deviation (SD). * p < 0.05.

Figure 3

Production of reactive oxygen species (ROS) after exposing cells to 20 µM of MEN or different concentrations of nanoparticles (NPs) for 1 h. After exposure, cells were loaded with the CellROX^® Green probe, which fluoresces upon oxidation by ROS, allowing for quantification of ROS production. Fluorescence was recorded using a microplate reader. The data, expressed as a percentage relative to the control condition, represent the mean ± standard deviation (SD) of three independent experiments (n = 3) * p < 0.05; **** p < 0.001.

Figure 4

Lipid content quantification in HepG2 cells following exposure to NPs, Cd, or a combination of both. (a) Cells were exposed for 24 h to NPs, Cd, or NPs + Cd in standard culture conditions. (b) Following a 24 h starvation period, cells were grown in a medium containing a mixture of fatty acids (FFA, 0.5 mM) for 24 h. After this period, FFA were removed, and cells were then exposed to NPs, Cd, or NPs + Cd. (c) After 24 h starvation, cells were exposed for 24 h to FFA together with NPs, Cd, or NPs + Cd. At the end of each experiment, the AdipoRed/NucBlue (A/N) assay was performed for lipid quantification. The data displayed in all three panels are expressed as percentages of the control condition and are represented as mean ± standard deviation (SD). Statistical significance is indicated by asterisks: * p < 0.05.

Figure 5

Hydrodynamic diameters of nanoparticles (NPs) measured using Dynamic Light Scattering (DLS). NPs were dispersed in Minimum Essential Medium with or without cadmium (Cd) and free fatty acids (FFA), following the same procedure used for the in vitro tests. For each sample, triplicate measurements were performed, with each measurement averaging at least 10 runs. The data shown represent the mean hydrodynamic diameters ± standard deviation (SD).

Figure 6

Scanning Electron Microscopy images acquired with secondary electrons (SE, first row) and backscattered electrons (BSE, second row) of (A) NPs, (B) NPs + Cd, (C) NPs + FFA, (D) NPs + Cd + FFA. Energy-Dispersive X-ray Spectroscopy line analysis of (E) NPs, (F) NPs + Cd, (G) NPs + FFA, (H) NPs + Cd + FFA. Schematics illustrate the interaction between NPs, Cd, and FFA for each condition, visually representing their combined effects.