title: Molecular Mechanism of Silver Nanoparticles-Induced Human Osteoblast Cell Death: Protective Effect of Inducible Nitric Oxide Synthase Inhibitor.
abstract: BACKGROUND
  Silver nanoparticles (AgNPs) show strong antibacterial properties, making them excellent candidates to be used in orthopaedic repair and regeneration. However, there are concerns regarding the cytotoxicity of AgNPs and molecular mechanisms underlying AgNPs-induced bone cells toxicity have not been elucidated. Therefore, the aim of our study was to explore mechanisms of AgNPs-induced osteoblast cell death with particular emphasis on the role of nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS).
  
  
  RESULTS
  Silver nanoparticles used in this study were 18.3±2.6 nm in size, uncoated, spherical, regular shape and their zeta potential was -29.1±2.4 mV as measured by transmission electron microscopy (TEM) and zetasizer. The release of silver (Ag) from AgNPs was measured in cell culture medium by atomic absorption spectroscopy (AAS). The exposure of human osteoblast cells (hFOB 1.19) to AgNPs at concentration of 30 or 60 μg/mL for 24 or 48 hours, respectively resulted in cellular uptake of AgNPs and changes in cell ultrastructure. These changes were associated with apoptosis and necrosis as shown by flow cytometry and lactate dehydrogenase (LDH) assay as well as increased levels of pro-apoptotic Bax and decreased levels of anti-apoptotic Bcl-2 mRNA and protein. Importantly, we have found that AgNPs elevated the levels of nitric oxide (NO) with concomitant upregulation of inducible nitric oxide synthase (iNOS) mRNA and protein. A significant positive correlation was observed between the concentration of AgNPs and iNOS at protein and mRNA level (r = 0.837, r = 0.721, respectively; p<0.001). Finally, preincubation of osteoblast cells with N-iminoethyl-l-lysine (L-NIL), a selective iNOS inhibitor, as well as treating cells with iNOS small interfering RNAs (siRNA) significantly attenuated AgNPs-induced apoptosis and necrosis. Moreover, we have found that AgNPs-induced cells death is not related to Ag dissolution is cell culture medium.
  
  
  CONCLUSIONS
  These results unambiguously demonstrate that increased expression of iNOS and generation of NO as well as NO-derived reactive species is involved in AgNPs-induced osteoblast cell death. Our findings may help in development of new strategies to protect bone from AgNPs-induced cytotoxicity and increase the safety of orthopaedic tissue repair.
authors.affiliation: Department of Medical Chemistry, Medical University of Gdansk, Gdansk, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Zielinska
authors.affiliation: Department of Electron Microscopy, Medical University of Gdansk, Gdansk, Poland.
authors.forename: Cecylia
authors.initials: C
authors.lastname: Tukaj
authors.affiliation: College of Medicine, University of Saskatchewan, Saskatoon, Canada.|Kardio-Med Silesia, Zabrze, Poland.
authors.forename: Marek Witold
authors.initials: MW
authors.lastname: Radomski
authors.affiliation: Department of Medical Chemistry, Medical University of Gdansk, Gdansk, Poland.
authors.forename: Iwona
authors.initials: I
authors.lastname: Inkielewicz-Stepniak
mesh_terms: D016923:Cell Death / Q000187:drug effects*; D002460:Cell Line; D006801:Humans; D053768:Metal Nanoparticles / Q000008:administration & dosage*; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors*; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D010316:Particle Size; D020011:Protective Agents / Q000008:administration & dosage*; D012333:RNA, Messenger / Q000378:metabolism; D012834:Silver / Q000008:administration & dosage*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D020011:Protective Agents; D012333:RNA, Messenger; D009569:Nitric Oxide; D012834:Silver; D052247:Nitric Oxide Synthase Type II