Cytotoxic Effects of Carboxyl-Modified Polystyrene Microplastics: Differential Effects on Hepatoma Cells and Immortalized Hepatocytes

Abstract

Carboxyl-modified polystyrene microplastics (cPS-MPs) may accumulate in the liver and interfere with cellular metabolism; however, their effects may differ substantially between cancerous and non-cancerous hepatocytes. In this study, we compared the responses of HUH-7 hepatocellular carcinoma cells and immortalized human hepatocytes (IHHs) to two cPS-MP size fractions, M1 (2.1 µm) and M2 (0.49 µm), at concentrations of 10–1000 µg/mL for 24, 48 and 72 h. Cell viability was evaluated by the MTT assay, while mitochondrial morphology and membrane potential (ΔΨm) were assessed using MitoTracker fluorescence microscopy and fluorescence measurements. HUH-7 cells exhibited concentration- and time-dependent loss of viability, with the strongest cytotoxicity observed after 72 h exposure to M2 particles, when viability decreased to approximately 48% at concentrations ≥750 µg/mL. In contrast, IHH cells showed no significant cytotoxicity and exhibited metabolic stimulation under several M1 exposure conditions. Brightfield microscopy further revealed pronounced accumulation of cPS-MPs in association with HUH-7 cells, whereas IHH cells showed substantially less apparent particle accumulation. In HUH-7 cells, prolonged exposure to high cPS-MP concentrations caused extensive mitochondrial network rearrangement, loss of nuclear integrity and heterogeneous changes in ΔΨm, including pronounced hyperpolarization in individual cells and depolarization that predominated at the population level. IHH cells largely maintained mitochondrial network integrity and stable ΔΨm. Based on the experimental dataset, HepatoMP, an open-source web-based Predictive Modelling Tool, was developed as a proof-of-concept framework integrating concentration-, particle size- and time-dependent responses for interactive data visualization and extrapolation. Overall, the findings demonstrate markedly different responses of hepatoma and immortalized hepatocytes to cPS-MPs and identify particle accumulation and mitochondrial dysfunction as prominent features associated with cPS-MP cytotoxicity in HUH-7 cells. HepatoMP provides a complementary hypothesis-generating framework for guiding future experimental investigation of hepatic microplastic toxicity.