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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.

Toxicology in vitro : an international journal published in association with BIBRA (2013-07-23)
Raju Y Prasad, John K McGee, Micaela G Killius, Danielle A Suarez, Carl F Blackman, David M DeMarini, Steven O Simmons
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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Citric acid, certified reference material, TraceCERT®
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Citric acid, meets analytical specification of Ph. Eur., BP, USP, E330, anhydrous, 99.5-100.5% (based on anhydrous substance)
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Cerium(IV) oxide, dispersion, nanoparticles, <25 nm particle size, 10 wt. % in H2O
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Polyvinylpyrrolidone, K 25, tested according to Ph. Eur.
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Cerium(IV) oxide, nanopowder, <50 nm particle size (BET), 99.95% trace rare earth metals basis
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Citric acid, Anhydrous, Pharmaceutical Secondary Standard; Certified Reference Material
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Polyvinylpyrrolidone, powder, average Mw ~29,000
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Cerium(IV) oxide, powder, <5 μm, 99.9% trace metals basis
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Cerium(IV) oxide, dispersion, 20 wt. % colloidal dispersion in 2.5% acetic acid, 30-50 nm avg. part. size
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Cerium, powder, −40 mesh, 99.9% trace rare earth metals basis
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Polyvinylpyrrolidone, powder, average Mw ~55,000
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Polyvinylpyrrolidone, K 30
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Cerium(IV) oxide, nanopowder, <25 nm particle size (BET)
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Cerium(IV) oxide, ≥99.0%
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Silver nitrate on silica gel, extent of labeling: ~10 wt. % loading, +230 mesh
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Cerium, chips
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Polyvinylpyrrolidone, average Mw ~1,300,000 by LS
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Citric acid, ACS reagent, ≥99.5%
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Citric acid, anhydrous, suitable for cell culture, suitable for plant cell culture
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Citric acid, 99%
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