Nanostructured zinc carbonate hydroxide microflakes: assessing the toxicity against erythrocytes and L929 cells in vitro

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Nanostructured materials have been suggested to be used as a source of dietary zinc for livestock animals. In this study, we assessed the cytotoxicity of newly synthesized nanostructured zinc carbonate hydroxide (ZnCH) Zn5(CO3)(OH)6microflakes. Cytotoxicity of the microflakes was assessed against murine L929 cell line and rat mature erythrocytes. Viability, motility, cell death pathways, implication of Ca2+, reactive oxygen species and reactive nitrogen species (RNS) signaling, caspases, and alterations of cell membranes following exposure of L929 cells to the microflakes were assessed. To assess hemocompatibility of the Zn-containing microflakes, osmotic fragility and hemolysis assays were performed, as well as multiple eryptosis parameters were evaluated. Our findings indicate a dose-response cytotoxicity of ZnCH microflakes against L929 cells with no toxicity observed for low concentrations (10 mg l-1and below). At high concentrations (25 mg l-1and above), ZnCH microflakes promoted nitrosyl stress, Ca2+- and caspase-dependent apoptosis, and altered lipid order of cell membranes in a dose-dependent manner, evidenced by up to 7-fold elevation of RNS-dependent fluorescence, 2.9-fold enhancement of Fura 2-dependent fluorescence, over 20-fold elevation of caspases-dependent fluorescence (caspase-3, caspase-8, and caspase-9), and up to 4.4-fold increase in the ratiometric index of the NR12S probe. Surprisingly, toxicity to enucleated mature erythrocytes was found to be lower compared to L929 cells. ZnCH microflakes induced eryptosis associated with oxidative stress, nitrosyl stress, Ca2+signaling and recruitment of caspases at 25-50-100 mg l-1. Eryptosis assays were found to be more sensitive than evaluation of hemolysis. Zn5(CO3)(OH)6microflakes show no cytotoxicity at low concentrations indicating their potential as a source of zinc for livestock animals.