Absrtact

Recent studies have proved that zinc oxide (ZnO) nanoparticles can cause toxicity in different cell lines, oxidative stress is often hypothesized to be an important factor in cytotoxicity of ZnO nanoparticles. However, the mechanisms are incompletely understood. The present study aimed to investigate the role of oxidative stress in toxicity and possible involvement of mitochondria in the production of reactive oxygen species (ROS) upon exposure of retinal ganglion cells (RGC-5) to ZnO nanoparticles. In this study, the effects of ZnO nanoparticles on mitochondrial membrane potential and ROS levels involved in hydrogenperoxide and hydroxyl radical production were investigated via inverted fluorescence microscope and hydrogen peroxide and hydroxyl radical assay kits, respectively. Furthermore, the mRNA of caspase-12 and the protein secreted into culture supernatant were also determined by means of real-time quantitative PCR and ELISA techniques. Our studies indicate that ZnO nanoparticles could apparently decrease the mitochondrial membrane potential, increase the production of ROS and lead to the overexpression of caspase- 12 in RGC-5 cells, suggesting that ZnO nanoparticle-induced toxicity via ROS overproduction will trigger endoplasmic reticulum stress, lead to the RGC-5 cell damage and finally induce apoptosis/necrosis,the overexpression of caspase-12 may be involved in cell death in RGC-5 cells.

Materials and methods

2.9. Determination of extracellular caspase-12
After incubation with different concentrations of ZnO nanoparticles, the amounts of caspase-12 proteins secreted into culture medium were determined using ELISA kit (Rat Caspase 12 ELISA Kit, Wuhan ColorfulGene biological technology Co., Ltd, China), respectively. The determination was according to the manufacturer’s instructions. Briefly, RGC-5 cells were seeded in a 6-well plate at a density of 4 105 cells/well in 2 mL volume and grown overnight, then replaced with different concentrations (i.e., 0, 2.5, 5.0, 10.0 lg/mL) of ZnO nanoparticles (2 mL per well) for 24 h, subsequently 100 lL aliquots of the supernatant were centrifuged at 2000g for 20 min to remove RGC-5 cells. After which 10 lL of the supernatant was added to 10 lL of regent on the 96-well ELISA plate. The absorbance of samples was measured at 450 nm using a 96-well microplate reader (Elx800, BioTek Instruments, USA). Caspase-12 was quantified from the relevant standard curve, respectively. The data were expressed as ng/mL. All samples were measured in triplicate and the experiment was repeated three times independently.