title: 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.
abstract: 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.
authors.affiliation: Integrated Systems Toxicology Division, United States Environmental Protection Agency, Research Triangle Park, NC 27711, USA.
authors.forename: Raju Y
authors.initials: RY
authors.lastname: Prasad
authors.forename: John K
authors.initials: JK
authors.lastname: McGee
authors.forename: Micaela G
authors.initials: MG
authors.lastname: Killius
authors.forename: Danielle A
authors.initials: DA
authors.lastname: Suarez
authors.forename: Carl F
authors.initials: CF
authors.lastname: Blackman
authors.forename: David M
authors.initials: DM
authors.lastname: DeMarini
authors.forename: Steven O
authors.initials: SO
authors.lastname: Simmons
mesh_terms: D001692:Biological Transport; D002470:Cell Survival; D002563:Cerium / Q000633:toxicity; D019343:Citric Acid / Q000737:chemistry; D017930:Genes, Reporter; D005978:Glutathione / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D053768:Metal Nanoparticles / Q000737:chemistry / Q000633:toxicity*; D051267:NF-E2-Related Factor 2 / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics; D018384:Oxidative Stress / Q000187:drug effects; D010316:Particle Size; D011205:Povidone / Q000737:chemistry; D051818:Replication Protein C / Q000235:genetics; D012834:Silver / Q000737:chemistry / Q000633:toxicity*; D012835:Silver Nitrate / Q000633:toxicity
chemical_list: D051267:NF-E2-Related Factor 2; D016328:NF-kappa B; C495635:NFE2L2 protein, human; D019343:Citric Acid; D002563:Cerium; D012834:Silver; C030583:ceric oxide; D012835:Silver Nitrate; D008156:Luciferases; D051818:Replication Protein C; D011205:Povidone; D005978:Glutathione
keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 50% inhibitory concentration; AP1; ARE; Ag NPs; Ag(+); AgNO(3); BSO; CeO(2); DLS; EC(50); EC(max); FBS; GSH; HQ; IC(50); ICP-MS; IL-1b; MTT; NFκB; NRF2; Nanoparticles; OPD; Oxidative stress; PVP; PdI; ROS; Reporter genes; Silver; Stress response; activator protein 1; antioxidant response element; cerium oxide; dynamic light scattering; fetal bovine serum; glutathione; half maximal effective concentration; hydroquinone; inductively coupled plasma mass spectroscopy; interleukin 1b; l-buthionine (S,R)-sulfoximine; maximal effective concentration; nuclear factor (erythroid-derived 2)-like 2; nuclear factor kappa B; o-phenylenediamine; polydispersity index; polyvinylpyrrolidone; reactive oxygen species; silver ion; silver nanoparticles; silver nitrate