Impact of polystyrene nanoplastics on human hepatocytes in vitro

The inevitable exposure of humans to micro-nanoplastics (MNPs) has emerged as a pressing global environmental issue, raising growing concerns about their potential health impacts. Several aspects of MNPs are still unknown, including their direct effects on human health, their role as carriers of environmental pollutants and organic substances, and the consequences of prolonged exposure. All these aspects are being explored to enable more accurate risk assessments of their long-term impacts on both human and environmental health. This study considered, on an in vitro cell model, two aspects of NP exposure: the interaction between NPs and other pollutants or organic molecules, and prolonged exposure to concentrations comparable to those found in the environment. Although the NPs do not enter HepG2 cells, one-hour exposure to 100 µg/ml of NPs significantly increases ROS production (+25% compared to control), yet cell viability remains unaffected even at concentrations well above environmental levels. Interestingly, NPs significantly reduce Cd cytotoxicity at concentrations close to its LC50 (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 cellular lipid content after short-term exposure (24 hours); on the other hand, in presence of Cd, NPs reduce the cellular uptake of fatty acids added to the medium in a dose-dependent manner, probably decreasing their availability for cellular uptake. Dynamic Light Scattering and Scanning Electron Microscopy revealed an interaction between NPs, Cd, and free fatty acids. In the second part of the study, we focused on sub-chronic exposure: concentrations close to the environmental ones (104 and 106 NPs/ml), were not directly cytotoxic for HepG2 cells, since no significant effects on cell viability or morphology were observed following 28 days of exposure. On the other hand, a prolonged exposure to NPs significantly increased the cellular lipid content, first observed after 120 hours of exposure; a further rise of lipid content was observed by day 7, with a dose-dependent progressive increase, peaking at day 14 (120% ± 16 and 133% ± 12 respectively) which remained stable until day 21, before decreasing. Moreover, starting from day 14, a qualitative change in lipid content was observed through lipidomics analysis. This shift includes an increase in saturated fatty acids and a reduction in unsaturated fats in the cells exposed to NPs. Furthermore, on day 7, a significant increase in TNF-α is observed, which is involved in the inflammatory process. Until day 14 the expression of glutathione synthetase, an important antioxidant enzyme, is augmented, along with an increase in the expression of BiP, a master regulator of the unfolded protein response involved in endothelial reticulum stress. All these transient effects triggered by sub-chronic exposure to NPs appear to be restored after 28 days, suggesting a form of cellular adaptation. Despite this adaptation, lipid content continues to qualitatively change, which could eventually lead to lipotoxicity in the cell, potentially resulting in more severe consequences over time. While the direct toxicity of polystyrene NPs in our experimental model was minimal, our findings highlight the complexity of predicting NP effects due to their interactions with environmental pollutants and biological components. Notably, prolonged exposure to low NP concentrations alters the cellular lipid profile both qualitatively and quantitatively, likely as part of an inflammatory response.