Polystyrene nanoplastics enhance lead-induced ferroptosis in HT22 cells via clathrin-mediated endocytosis

The co-occurrence of nanoplastics and heavy metals like lead (Pb) in the environment presents a significant neurotoxic risk, yet the mechanistic basis for their combined effects remains poorly defined. In this study, we investigated the combined neurotoxic effects of 50 nm polystyrene nanoplastics (PS-NPs) and Pb in mouse hippocampal HT22 cells. Co-exposure to PS-NPs (50 μg/mL) and Pb (10 μg/mL)-concentrations commonly used in in vitro mechanistic studies-for 24 h, but not individual treatments, enhanced cytotoxicity and induced ferroptosis-related features, including iron metabolism dysregulation, lipid peroxidation, antioxidant defense impairment, and mitochondrial damage. Furthermore, the ferroptosis inhibitor Ferrostatin-1 (Fer-1) attenuated cell death, supporting ferroptosis as an important contributor to the observed cytotoxicity. Transcriptomic analysis revealed significant alterations in ferroptosis-related pathways, with selected differentially expressed genes validated by qRT-PCR. Physicochemical characterization (DLS/zeta potential) revealed changes in the hydrodynamic size and zeta potential of PS-NPs following incubation with Pb, while ICP-OES analysis of the supernatant provided evidence for the association of Pb with PS-NPs. This enhanced toxicity was associated with increased intracellular Pb accumulation facilitated by PS-NPs, involving the clathrin-mediated endocytosis (CME) pathway, as indicated by upregulation of key CME-related proteins (CHC, AP2M1, and Dynamin 2) and enhanced CHC-AP2M1 co-localization. Pharmacological inhibition of CME using chlorpromazine (CPZ) effectively reduced Pb uptake and attenuated ferroptosis-related cellular damage. Our findings provide insights into a proposed CME-associated Trojan horse-like mechanism whereby PS-NPs enhance ferroptosis-related neuronal responses to Pb exposure, highlighting the potential implications of nanoplastic-heavy metal co-exposure for environmental health.