title: Evaluation of cytotoxic, genotoxic and inflammatory responses of nanoparticles from photocopiers in three human cell lines.
abstract: BACKGROUND
  Photocopiers emit nanoparticles with complex chemical composition. Short-term exposures to modest nanoparticle concentrations triggered upper airway inflammation and oxidative stress in healthy human volunteers in a recent study. To further understand the toxicological properties of copier-emitted nanoparticles, we studied in-vitro their ability to induce cytotoxicity, pro-inflammatory cytokine release, DNA damage, and apoptosis in relevant human cell lines.
  
  
  METHODS
  Three cell types were used: THP-1, primary human nasal- and small airway epithelial cells. Following collection in a large volume photocopy center, nanoparticles were extracted, dispersed and characterized in the cell culture medium. Cells were doped at 30, 100 and 300 μg/mL administered doses for up to 24 hrs. Estimated dose delivered to cells, was ~10% and 22% of the administered dose at 6 and 24 hrs, respectively. Gene expression analysis of key biomarkers was performed using real time quantitative PCR (RT-qPCR) in THP-1 cells at 5 μg nanoparticles/mL for 6-hr exposure for confirmation purposes.
  
  
  RESULTS
  Multiple cytokines, GM-CSF, IL-1β, IL-6, IL-8, IFNγ, MCP-1, TNF-α and VEGF, were significantly elevated in THP-1 cells in a dose-dependent manner. Gene expression analysis confirmed up-regulation of the TNF-α gene in THP-1 cells, consistent with cytokine findings. In both primary epithelial cells, cytokines IL-8, VEGF, EGF, IL-1α, TNF-α, IL-6 and GM-CSF were significantly elevated. Apoptosis was induced in all cell lines in a dose-dependent manner, consistent with the significant up-regulation of key apoptosis-regulating genes P53 and Casp8 in THP-1 cells. No significant DNA damage was found at any concentration with the comet assay. Up-regulation of key DNA damage and repair genes, Ku70 and Rad51, were also observed in THP-1 cells, albeit not statistically significant. Significant up-regulation of the key gene HO1 for oxidative stress, implicates oxidative stress induced by nanoparticles.
  
  
  CONCLUSIONS
  Copier-emitted nanoparticles induced the release of pro-inflammatory cytokines, apoptosis and modest cytotoxicity but no DNA damage in all three-human cell lines. Taken together with gene expression data in THP-1 cells, we conclude that these nanoparticles are directly responsible for inflammation observed in human volunteers. Further toxicological evaluations of these nanoparticles, including across different toner formulations, are warranted.
authors: Department of Work Environment, One University Avenue, University of Massachusetts Lowell, Lowell, MA 0185, USA. m2khatri@gmail.com.
authors: Madhu
authors: M
authors: Khatri
authors: Dhimiter
authors: D
authors: Bello
authors: Anoop K
authors: AK
authors: Pal
authors: Joel M
authors: JM
authors: Cohen
authors: Susan
authors: S
authors: Woskie
authors: Thomas
authors: T
authors: Gassert
authors: Jiaqi
authors: J
authors: Lan
authors: April Z
authors: AZ
authors: Gu
authors: Philip
authors: P
authors: Demokritou
authors: Peter
authors: P
authors: Gaines
mesh_terms: D000393:Air Pollutants / Q000737:chemistry / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D018929:Cell Culture Techniques; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D020552:Comet Assay; D003308:Copying Processes; D016207:Cytokines / Q000276:immunology*; D004249:DNA Damage; D004847:Epithelial Cells / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles / Q000737:chemistry / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects; D010316:Particle Size; D020545:Respiratory Mucosa / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D012995:Solubility; D013499:Surface Properties
chemical_list: D000393:Air Pollutants; D016207:Cytokines
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