Transcriptomic responses to repeated exposure of human C3A liver spheroids to polystyrene nanoplastics

Increasing evidence of nanoplastics (NPs) accumulation in the human liver has raised concerns regarding their potential hepatotoxicity. However, current knowledge is largely derived from animal studies or simplistic in vitro models, often with unrealistic exposure scenarios, thereby constraining their relevance to human health. Human liver spheroids have emerged as relevant tools for long-term mechanistic toxicological studies, yet their application to the hepatotoxicity assessment of NPs remains limited. This study aimed to establish and characterize a human liver C3A spheroid model to explore the effects of repeated exposure to polystyrene (PS) NPs for up to 21 days at concentrations informed by real-life human intake and internal exposure estimates, namely 0.0068, 0.068, and 1.6 µg/mL. Throughout the 21-day culture period, the human liver spheroids preserved stable morphology, overall viability, low levels of cell death, and stable expression of hepatic-associated genes. PS NPs accumulated within the human liver spheroids in a concentration-dependent and spatially heterogeneous manner, but did not induce detectable changes in spheroid size, viability, or cell death across all concentrations and time points measured. Transcriptomic profiling revealed subtle, concentration-dependent gene expression changes, which were most pronounced at day 3 and attenuated over time. Although limited in magnitude, these changes involved genes and pathways related to liver lipid and bile acid homeostasis, stress signaling, and detoxification. Collectively, these findings indicate that human-relevant concentrations of PS NPs do not induce overt hepatotoxic effects, while potentially modulating molecular pathways implicated in cellular processes essential for normal liver function.