Polystyrene nanoplastics elicit early mitochondria-associated phenotypic, metabolic, and functional responses in human hepatocytes.

Nanoplastics have recently been detected in human liver tissue, raising concerns about their potential impact on liver function. However, early hepatocyte responses associated with nanoplastics exposure remain poorly understood. Here, we combined high-throughput Cell Painting-based phenomics, untargeted metabolomics, and Seahorse mitochondrial functional assay to investigate the effects of 100 nm polystyrene nanoplastics on human HepaRG hepatocytes, a surrogate for primary human hepatocytes. At the tested concentrations (6.25-100 µg/mL), exposure did not induce overt cytotoxicity, enabling assessment of early sublethal cellular responses. Phenomics revealed widespread subcellular perturbations, with 16.4% of the measured phenotypic features significantly altered. Mitochondria-associated features represented the dominant altered phenotypic signature, showing pronounced changes in granularity, texture, and radial distribution, alongside alterations in endoplasmic reticulum- and cytoskeleton-associated features. Untargeted metabolomics of intracellular metabolites and the extracellular secretome revealed metabolic alterations, characterized by changes consistent with altered β-oxidation, lipid handling, membrane stress, and central carbon metabolism, including changes in the tricarboxylic acid (TCA) cycle and amino acid catabolism. Pathway analysis identified the TCA cycle as one of the most significantly affected pathways (FDR = 0.028). Integrated phenomic-metabolomic analysis revealed strong correlations between mitochondrial phenotypic features and metabolites involved in lipid and energy metabolism, indicating a coordinated structural-metabolic response to polystyrene nanoplastics exposure. Functional assessment using Seahorse assay showed reduced basal and maximal respiration and decreased ATP-linked O2 consumption. Together, these findings provide evidence that 100 nm polystyrene nanoplastics elicit early mitochondria-associated phenotypic, metabolic, and functional responses prior to overt cytotoxicity under the tested conditions. They also highlight the value of phenomic-metabolomic-functional integration for profiling sublethal nanotoxicological responses and guiding future targeted mechanistic studies.