Polystyrene nanoplastics potentiate cadmium-induced pyroptosis and apoptosis in primary duck hepatocytes: differential responses to N-acetylcysteine

Abstract

Nanoplastics (NPs) can act as carriers for heavy metals, and co-exposure to NPs and co-pollutants may produce greater toxicity than single exposure. However, the combined toxicity of nanoplastics and heavy metals in poultry liver cells remains poorly understood. Primary duck embryonic hepatocytes (PDEH) were used as an in vitro model. Cells were exposed to polystyrene nanoplastics (PS-NPs, 80 nm, 10 μg/mL) and/or cadmium chloride (CdCl2, 4 μg/mL) for 12 h. Pyroptosis- and apoptosis-associated endpoints were assessed by brightfield microscopy, PI staining, SEM, flow cytometry, qRT-PCR, and Western blotting. The antioxidant N-acetylcysteine (NAC, 5 μg/mL) was used to evaluate the responses of these pathways. Co-exposure to PS-NPs and Cd potentiated cadmium-induced pyroptosis-associated morphological changes, including cell swelling, rounding, membrane blebbing, and surface pore formation. Mechanistically, co-exposure activated the NLRP3/Caspase-1/GSDMD signaling axis, as indicated by increased NLRP3, cleaved Caspase-1 (P20/P10), GSDMD-NT, and intracellular IL-1β maturation. Co-exposure also exacerbated apoptosis-associated changes, including an increased Bax/Bcl-2 ratio and a higher Annexin V-positive cell population. NAC effectively reversed the apoptosis-associated endpoints but did not significantly alter the selected pyroptosis-associated markers under the present in vitro exposure conditions. These findings suggest that PS-NPs potentiate Cd-induced hepatotoxicity in primary duck hepatocytes, with concurrent activation of pyroptosis- and apoptosis-associated signaling. The differential responses to NAC indicate that antioxidant intervention may preferentially attenuate apoptosis-associated injury under the present in vitro exposure conditions, while pyroptosis-associated signaling may require alternative targeted interventions. These results provide new insights into the combined toxicity of nanoplastics and heavy metals in avian hepatocytes.