ZnO, Ag and ZnO-Ag nanoparticles exhibit differential modes of toxic and oxidative action in hemocytes of mussel Mytilus galloprovincialis

Highlights: • ZnO, Ag and ZnO-Ag NPs were synthesized with Flame Spray Pyrolysis. • PXRD and DLS measurements were conducted in MQ water and hemolymph serum (HS). • Cytotoxic and oxidative effects of NPs were assessed in mussel hemocytes. • All NPs exhibited cytotoxic and oxidative potential. • Each NP demonstrated a different mechanism of action. The present study investigates the cytotoxic and oxidative effects of custom-made nanoparticles (NPs) on hemocytes of Mytilus galloprovincialis, utilizing hemolymph serum (HS) as exposure medium. Specifically, hemocyte lysosomal membrane destabilization (in terms of neutral red retention time assay/NRRT), superoxide anion (O₂⁻), nitric oxide (NO, in terms of nitrites) and lipid peroxidation content (in terms of malondialdehyde/MDA equivalents) were determined in cells treated for 1 h with different concentrations (0.1–50 μg mL⁻¹) of ZnO NPs, Ag NPs and ZnO-Ag NPs, as well as AgNO₃ and/or ZnCl₂ (bulk ions, respectively). According to the results, Ag NPs were more cytotoxic than ZnO-Ag NPs and/or ZnO NPs, while NRRT values observed in AgNO₃ treated cells were lower than those of ZnCl₂. Furthermore, high levels of both O₂⁻ and MDA were detected in cells treated with Ag NPs, ZnO-Ag NPs, and AgNO₃ at concentrations lower than 5 μg mL⁻¹, while high NO generation was observed only in cells treated with 5–25 μg mL⁻¹ of ZnO NPs or ZnCl₂. Despite the absence of data, regarding the formation of NP-serum protein corona complexes that could mediate NP surface energy and uptake efficiency, the current study firstly revealed that ZnO NPs, probably via their surface charge, particle agglomeration, and NP Zn⁺ release could promote an immune-related generation of O₂⁻ and NO via the respiratory burst stimulation, a process that is questioned in the case of Ag NPs and/or ZnO-Ag NPs. Moreover, ZnO-Ag NP interaction with biological membranes and their oxidative mode of action seemed to be regulated by the release and the antagonistic/synergistic response of its ionic counterparts (ZnO⁺ and Ag⁺), but further studies are needed to elucidate the oxidative mode of action of NP metal ions in complex NP mixtures.