Synopsis
To identify the respective contributions of released zinc and solid particles on the cytotoxicity of zinc oxide nanoparticles (ZnO-NPs), we exposed A549 cells to ZnO-NP suspensions, their extractions collected after centrifugation, and medium containing zinc chloride (ZnCl₂). We then assayed the cytotoxicity of these samples using water-soluble tetrazolium salts (WSTs) and intracellular reactive oxygen species (ROS) with 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA) as outputs. Only the ZnO-NP suspension caused cytotoxicity and increased intracellular ROS; the extractions and ZnCl₂ did not cause cytotoxicity or oxidative stress. Global gene expression analysis revealed that both ZnCl₂-containing medium and the ZnO-NP suspension caused upregulation of the "cadmium binding" gene functional category. This category consisted of metallothioneins (MTs), important zinc-homeostasis proteins. To further investigate the role of MTs in ZnO-NP-dependent cytotoxicity, we inhibited the overexpression of MTs with corresponding siRNA and found that released zinc contributed to ZnO-NP-dependent cytotoxicity. We conclude that both solid particles and released zinc contributed to ZnO-NP-dependent cytotoxicity. Additionally, we propose a syn-ergic relationship between ZnO-NPs and MTs.

Key words: zinc oxide nanoparticles, synergic cytotoxicity, released zinc, metallothioneins, intracellular reactive oxygen species

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