Effects of a silver nanomaterial on cellular organelles and time course of oxidative stress in a fish cell line (PLHC-1)

Among the nanomaterials currently in commercial products, those based on silver are the most used, and so there is a high probability that silver nanoparticles (AgNPs) will be released into aquatic environments where they could adversely affect aquatic organisms, including fish. Taking this into account, the aim of the present work was to characterize in depth the mechanisms underlying the toxic action of AgNPs using fish cell lines, determining specifically the contribution of alterations in cellular structures and oxidative stress time course to the cytotoxicity of AgNPs. Since liver plays a key role in detoxification, the hepatoma cell line PLHC-1 was used. Exposure to AgNPs (NM-300K, obtained from the Joint Research Centre Repository) caused alterations at the lysosomal and mitochondrial levels at lower concentrations than those that disrupted plasma membrane (evaluated by means of neutral red, alamarBlue, and 5-carboxyfluorescein diacetate, acetoxymethyl ester assays respectively). AgNO3, used as a control Ag⁺ ion source, produced similar cytotoxic effects but at lower concentrations than AgNPs. Both silver forms caused oxidative disruption but the initial response was delayed in AgNPs until 6h of exposure. Transmission electron microscopy analysis also evidenced the disruption of mitochondrial structures in cells exposed to cytotoxic concentrations of both forms of silver. At non-cytotoxic concentrations, AgNPs were detected inside the nucleoli and mitochondria, thereby pointing to long-term effects. The present work evidences the mutual interaction between the induction of oxidative stress and the alterations of cellular structures, particularly mitochondria, as cytotoxicity mechanisms not exclusively associated to NPs.