Polystyrene nanoplastics disrupt iron homeostasis by promoting FPN1 ubiquitination in GC-2spd(ts) cells

Nanoplastics are emerging environmental pollutants ubiquitously found in natural ecosystems. Although studies have shown that nanoplastics can accumulate in the testes of mice and affect spermatogenic cells, the specific toxicological mechanisms remain unclear. To investigate the specific mechanism by which polystyrene nanoplastics induce ferroptosis in mouse spermatocyte-derived GC-2spd(ts) cells and subsequently lead to male reproductive toxicity, this study exposed mouse germ cell lines (GC-1 spg and GC-2spd(ts)) to PS-NPs of two sizes (50 nm and 90 nm). Cell viability assays indicated that GC-2spd(ts) cells were more sensitive to PS-NPs exposure. Transcriptomic and proteomic analyses revealed that PS-NPs exposure induced intracellular reactive oxygen species (ROS) accumulation and significant alterations in related pathways, specifically activating the ferroptosis signaling pathway. Further mechanistic studies demonstrated that PS-NPs disrupted intracellular iron homeostasis, leading to the accumulation of labile Fe , enhanced lipid peroxidation, depletion of the antioxidant glutathione, and mitochondrial dysfunction. At the molecular level, PS-NPs upregulated the expression of iron uptake-related proteins and significantly downregulated the iron exporter protein ferroportin1 (FPN1). In-depth investigation revealed that PS-NPs did not affect the transcriptional level of FPN1 but promoted FPN1 protein degradation by enhancing its ubiquitination modification, subsequently via the proteasome-dependent pathway. This process resulted in blocked cellular iron efflux, iron ion accumulation, and ultimately triggered ferroptosis. This study elucidates the molecular mechanism by which PS-NPs regulate FPN1 degradation through the ubiquitin-proteasome pathway, disrupt iron metabolic homeostasis, and thereby induce ferroptosis in germ cells, providing novel experimental evidence for assessing the male reproductive toxicity of nanoplastics.