In vitro effects of cadmium and polystyrene microparticles on HepG2 cells

Cadmium (Cd) is a typical heavy metal that is ubiquitous in the environment. It represents a major public health concern due to its association with adverse health outcomes, including cardiovascular disease, renal toxicity, cancer, and congenital malformations. The liver plays an important role in the overall toxicity of Cd, as a large proportion of the body burden accumulates in this organ. Plastics microparticles (MPs) have been shown to either increase or decrease the exposure to environmental contaminants, including Cd, and this is an issue of growing concern for both the environment and human health. In this study, we aimed to evaluate the in vitro effect of Cd on the HepG2 liver cancer cell line in terms of cytotoxicity, lipid content and expression of marker genes and proteins related to liver metabolic disorders. We also investigated whether the presence of polystyrene microparticles (5 or 0.5 μm diameter) can modify the effects of Cd. The viability of HepG2 cells was evaluated by CellTiter-Glo® Luminescent Cell Viability Assay, based on the quantitation of ATP, which signals the presence of metabolically active cells. To evaluate lipid content, AdipoRed/NucBlue staining was performed; AdipoRed assay reagent quantifies intracellular triglycerides, while the DNA content was estimated by NucBlue staining; as a positive control for intracellular lipid accumulation, we induced hepatic steatosis on HepG2 cells incubated for 24h in serum-free MEM containing 0.5 mM FFA mixture. Our results show that 24-hour Cd exposure promotes HepG2 cell proliferation at low concentrations (below 10μM), while higher Cd concentrations induce a cytotoxic effect, with an IC50=80.27 μM. Cd toxicity was significantly increased in cells starved for 24 hours before Cd exposure. Cd at concentrations above 25 μM interfered also with lipid metabolism, reducing the lipid content per cell. Polystyrene microplastics (10 and 100 μg/ml) per se were not toxic to HepG2 cells, though some of them were internalized. Co-incubation of Cd and MPs was able to reduce Cd cytotoxicity (IC50=93.5 μM), however at Cd concentrations >100 μM, the presence of MPs was no longer able to rescue the cells. Ongoing experiments are determining the effects on MPs on lipid content and on the expression of marker genes and proteins of lipid metabolism, oxidative stress and pyroptosis. Our data confirm that Cd interferes with proper lipid metabolism and cell viability, and suggests that the presence of micro- or nanoplastics may modify its effects. These findings have important implications for understanding the health risks associated with exposure to Cd in the environment and the need for further research to develop strategies to mitigate its impact.