Figure 6

ZnONPs induce excessive ROS production resulting from the accumulation of damaged mitochondria. (a) A549 cells were pretreated with 1, 3 or 10 mM NAC, followed by 30 or 50 μg/ml ZnONPs treatment. MTS analysis was performed to detect the cell viability at 24 h after treatment. Data are representative of three independent experiments (n=6 for each group) and values are expressed in mean±S.E.M. *P<0.05, **P<0.01. (b) A549 cells were pretreated with NAC (10 mM) or DTPA (1 mM), followed by treatment with 30 μg/ml ZnONPs. FACS analysis of cells stained with 2'7'-dichlorofluorescin diacetate was performed at 24 h after treatment. Images are representative of three independent experiments. (c) TEM images of untreated or 30 μg/ml ZnONPs-treated A549 cells. Note that aberrant mitochondria accumulated in ZnONPs-treated cells. Images are representative of three independent experiments. (d) FACS analysis of cells stained with MitoTracker-Green FM. The effects of vehicle or ZnONPs (30 μg/ml) on the mass of mitochondria were analyzed 24 h after treatment. Images are representative of three independent experiments. (e) A549 cells were transfected with 75 nM siRNA targeted to LAMP-1, LAMP-2 or control siRNA for 48 h. Thereafter, cells were treated with 30 μg/ml ZnONPs, and FACS analysis of cells stained with TMRE was performed to detect mitochondrial membrane potential at 24 h after treatment. Images are representative of three independent experiments. **P<0.01, N.S., not significant. (f) A549 cells were transfected with 75 nM siRNA targeted to LAMP-1, LAMP-2 or control siRNA for 48 h. Then, cells were treated with 30 μg/ml ZnONPs and FACS analysis of cells stained with 2'7'-dichlorofluorescin diacetate was performed to detect intracellular ROS at 24 h after treatment. Images are representative of three independent experiments. (g) Schematic of the mechanism of ZnONPs-induced cytotoxicity. ZnONPs are delivered into lysosomes via autophagy, and subsequently dissolved in lysosomes to release zinc ions, which is the crucial factor triggering ZnONPs cytotoxicity. Furthermore, zinc ions (intracellular or extracellular) can damage mitochondria and lysosomes; thereafter, they further disrupt the negative feedback mechanisms of ROS and mitophagy, leading to the accumulation of damaged mitochondria, excessive ROS and finally cell death. Aberrant LAMP-2 expression is probably involved in this process. Inhibition of autophagy, chelating of zinc ions or treatment with antioxidant NAC effectively mitigates ZnONPs-induced cytotoxicity