Investigating oxidative stress and inflammatory responses elicited by silver nanoparticles using high-throughput reporter genes in HepG2 cells: Effect of size,surface coating,and intracellular uptake

Silver nanoparticles (Ag NP) have been shown to generate reactive oxygen species; however, the association between physicochemical characteristics of nanoparticles and cellular stress responses elicited by exposure has not been elucidated. Here, we examined three key stress-responsive pathways activated by Nrf-2/ARE, NFκB, and AP1 during exposure to Ag NP of two distinct sizes (10 and 75 nm) and coatings (citrate and polyvinylpyrrolidone), as well as silver nitrate (AgNO₃), and CeO₂ nanoparticles. The in vitro assays assessed the cellular response in a battery of stable luciferase-reporter HepG2 cell lines. We further assessed the impact of Ag NP and AgNO₃ exposure on cellular redox status by measuring glutathione depletion. Lastly, we determined intracellular Ag concentration by inductively coupled plasma mass spectroscopy (ICP-MS) and re-analyzed reporter-gene data using these values to estimate the relative potencies of the Ag NPs and AgNO₃. Our results show activation of all three stress response pathways, with Nrf-2/ARE displaying the strongest response elicited by each Ag NP and AgNO₃ evaluated here. The smaller (10-nm) Ag NPs were more potent than the larger (75-nm) Ag NPs in each stress-response pathway, and citrate-coated Ag NPs had higher intracellular silver concentrations compared with both PVP-coated Ag NP and AgNO₃. The cellular stress response profiles after Ag NP exposure were similar to that of AgNO₃, suggesting that the oxidative stress and inflammatory effects of Ag NP are likely due to the cytotoxicity of silver ions.