Polystyrene Nanoplastics Induce Time-Dependent Cytotoxic, Cytoskeletal and Oxidative Stress-Associated Responses in Rainbow Trout Gill Epithelial Cells

Nanoplastics are emerging aquatic contaminants whose cellular effects induced in freshwater fish remain incompletely understood. In this study, RTgill-W1 epithelial cells of rainbow trout (Oncorhynchus mykiss) gills were incubated with polystyrene nanoplastics (PS-NPLs) at concentrations of 1–500 µg/mL for 24 and 72 h. Cytotoxicity was evaluated by Live/Dead staining and lactate dehydrogenase (LDH) release, while cytoskeletal alterations were assessed by F-actin staining. Oxidative stress-associated responses were investigated by measuring reduced glutathione (GSH), malondialdehyde (MDA), and the expressions of Hsp60 and Hsp70 proteins. PS-NPLs induced cellular responses that varied with concentration and exposure duration, with the most pronounced cytotoxic effects observed at 500 µg/mL. At this concentration, reduced cell density, altered cellular morphology, changes in F-actin organization, and increased LDH release were observed, particularly after 72 h. GSH and MDA levels increased relative to controls, with the strongest responses occurring after 24 h at 500 µg/mL and attenuating after prolonged exposure. Hsp60 and Hsp70 protein expression was elevated after 24 h, whereas after 72 h, Hsp70 expression was no longer significantly different from the control at either concentration, and Hsp60 expression remained elevated at 50 µg/mL but was not significantly different from the control at 500 µg/mL. These findings indicate that PS-NPLs exhibit dynamic responses in fish gill epithelial cells, involving membrane damage, cytoskeletal remodelling, lipid peroxidation, antioxidant responses, and modulation of stress-related proteins. The results support the use of RTgill-W1 cells as a mechanistically informative in vitro model for investigating cellular responses to nanoplastic exposure in fish.