title: Silver Nanoparticle-Induced Autophagic-Lysosomal Disruption and NLRP3-Inflammasome Activation in HepG2 Cells Is Size-Dependent.
abstract: Silver nanoparticles (AgNPs) are incorporated into medical and consumer products to exploit their excellent antimicrobial properties; however, potential mechanisms of toxicity of AgNPs in mammalian cells are not fully understood. The objective of this study was to determine the mechanism of size- and concentration-dependent cytotoxicity of AgNPs in human liver-derived hepatoma (HepG2) cells. Mechanisms of toxicity were explored at subcytotoxic concentrations (≤10 µg/ml AgNPs) and autophagy induction, lysosomal activity, inflammasome-dependent caspase-1 activation, and apoptosis were examined. Using enhanced dark-field light microscopy, hyperspectral imaging, electron microscopy, and energy dispersive X-ray spectroscopy, AgNPs were shown to rapidly accumulate in cytoplasmic vesicles for up to 24 h and 10-nm AgNPs exhibited the highest uptake and accumulation. Autophagy and enhanced lysosomal activity were induced at noncytotoxic concentrations (1 µg/ml; primary particle size:10 > 50 >100 nm), whereas increased caspase-3 activity (associated with apoptosis) was observed at cytotoxic concentrations (10, 25, and 50 µg/ml). Subcytotoxic concentrations of AgNPs enhanced expression of LC3B, a pro-autophagic protein, and CHOP, an apoptosis inducing ER-stress protein, and activation of NLRP3-inflammasome (caspase-1, IL-1β). Disrupting the autophagy-lysosomal pathway through chloroquine or ATG5-siRNA exacerbated AgNPs-induced caspase-1 activation and lactate dehydrogenase release, suggesting that NLRP3-inflammasome plays an important role in AgNPs-induced cytotoxicity. Overall, 10-nm AgNPs showed the highest cellular responses compared with 50- and 100-nm AgNPs based on equal mass dosimetry. The results indicate the potential of vesicle-engulfed 10-nm AgNPs to induce cytotoxicity by a mechanism involving perturbations in the autophagy-lysosomal system and inflammasome activation.
authors.affiliation: Division of Biology, Chemistry, and Materials Science, Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Anurag R
authors.initials: AR
authors.lastname: Mishra
authors.affiliation: Division of Biology, Chemistry, and Materials Science, Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Jiwen
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Division of Biology, Chemistry, and Materials Science, Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Xing
authors.initials: X
authors.lastname: Tang
authors.affiliation: Division of Biology, Chemistry, and Materials Science, Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, Maryland, USA Peter.Goering@fda.hhs.gov.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Goering
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects*; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D056945:Hep G2 Cells; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism*; D008247:Lysosomes / Q000187:drug effects* / Q000648:ultrastructure; D053768:Metal Nanoparticles / Q000737:chemistry / Q000633:toxicity*; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein / Q000378:metabolism*; D010316:Particle Size; D012834:Silver / Q000737:chemistry / Q000633:toxicity*
chemical_list: D058847:Inflammasomes; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D012834:Silver
keywords: apoptosis; autophagy; caspase-1.; inflammasome; nanomaterials; silver nanoparticle