Objective  To investigate whether exposure pathways influence the distribution pattern and toxicity of polystyrene nanoplastics (PSNPs) in hepatic cells.

Methods  Male C57BL/6J wild-type healthy mice aged 6 to 8 weeks old and weighed 18 to 22 g were administered with PSNPs via gavage or tail vein injection. Then, we tracked PSNPs distribution in the major organs of mice via an in vivo imaging system (IVIS). After that, we analyzed the cellular accumulation patterns in hepatic cell subpopulations (hepatocytes and Kupffer cells) using immunofluorescence and transmission electron microscopy (TEM). 300 nm PSNPs were administered via gastric gavage or tail vein injection, and 70 nm PSNPs were injected via the portal vein. The cellular localization of PSNPs in the liver was analyzed using immunofluorescence. Subsequently, using AML-12 cells, a normal mouse liver cell line, as the parenchymal hepatocyte model, the uptake of PSNPs in AML-12 cells was analyzed by confocal laser scanning microscope (CLSM). Flow cytometry was performed to observe and quantify PSNPs uptake, and to analyze the underlying endocytosis mechanisms. IVIS was used to analyze PSNPs uptake features in vivo. Finally, using mouse macrophage line RAW264.7 as a Kupffer cell model and AML-12 cells as a parenchymal hepatocyte model, the cell-type-specific toxic effects induced by 100 μg/ml PSNPs were examined through transcriptomics and metabolomics analyses.

Results IVIS revealed predominant hepatic accumulation of PSNPs regardless of exposure pathways via intragastric gavage or tail vein injection. Immunofluorescence/TEM demonstrated exposure pathway-dependent cellular distribution: intragastric PSNPs were localized mainly in hepatocytes, while intravenous PSNPs were accumulated in Kupffer cells. Changes in particle size (300 nm vs. 70 nm) did not alter the cellular distribution pattern, while 70 nm PSNPs injected via the portal vein accumulated in Kupffer cells, which suggested that the cell-type-specific distribution of PSNPs in the liver was independent of PSNPs size and might be related to the transport of PSNPs in the gastrointestinal tract. Flow cytometry showed that PSNPs uptake by AML-12 was time-dependent and that the underlying endocytosis mechanism involved pathways mediated by clathrin (P < 0.0001), macropinocytosis (P = 0.0026), and lipid rafts (P < 0.0001). Findings on PSNPs distribution in blood revealed that the uptake of PSNPs by hepatocytes exhibited a rate saturation phenomenon. Multi-omics analysis identified distinct toxicity patterns: PSNPs disrupted lipid metabolism and neurotransmitter homeostasis in AML-12 cells and induced inflammation and oxidative stress in Kupffer cells.

Conclusion Exposure pathways mediate the hepatic cell-type-specific distribution of PSNPs, thereby altering the downstream toxicological consequences induced by exposure to PSNPs.