title: Multi-walled carbon nanotubes induce COX-2 and iNOS expression via MAP kinase-dependent and -independent mechanisms in mouse RAW264.7 macrophages.
abstract: BACKGROUND
  Carbon nanotubes (CNTs) are engineered graphene cylinders with numerous applications in engineering, electronics and medicine. However, CNTs cause inflammation and fibrosis in the rodent lung, suggesting a potential human health risk. We hypothesized that multi-walled CNTs (MWCNTs) induce two key inflammatory enzymes in macrophages, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), through activation of extracellular signal-regulated kinases (ERK1,2).
  
  
  METHODS
  RAW264.7 macrophages were exposed to MWCNTs or carbon black nanoparticles (CBNPs) over a range of doses and time course. Uptake and subcellular localization of MWCNTs was visualized by transmission electron microscopy (TEM). Protein levels of COX-2, iNOS, and ERK1,2 (total ERK and phosphorylated ERK) were measured by Western blot analysis. Prostaglandin-E(2) (PGE(2)) and nitric oxide (NO) levels in cell supernatants were measured by ELISA and Greiss assay, respectively.
  
  
  RESULTS
  MWCNTs, but not CBNPs, induced COX-2 and iNOS in a time- and dose-dependent manner. COX-2 and iNOS induction by MWCNTs correlated with increased PGE(2) and NO production, respectively. MWCNTs caused ERK1,2 activation and inhibition of ERK1,2 (U0126) blocked MWCNT induction of COX-2 and PGE2 production, but did not reduce the induction of iNOS. Inhibition of iNOS (L-NAME) did not affect ERK1,2 activation, nor did L-NAME significantly decrease COX-2 induction by MWCNT. Nickel nanoparticles (NiNPs), which are present in MWCNTs as a residual catalyst, also induced COX-2 via ERK-1,2. However, a comparison of COX-2 induction by MWCNTs containing 4.5 and 1.8% Ni did not show a significant difference in ability to induce COX-2, indicating that characteristics of MWCNTs in addition to Ni content contribute to COX-2 induction.
  
  
  CONCLUSIONS
  This study identifies COX-2 and subsequent PGE(2) production, along with iNOS induction and NO production, as inflammatory mediators involved in the macrophage response to MWCNTs. Furthermore, our work demonstrates that COX-2 induction by MWCNTs in RAW264.7 macrophages is ERK1,2-dependent, while iNOS induction by MWCNTs is ERK1,2-independent. Our data also suggest contributory physicochemical factors other than residual Ni catalyst play a role in COX-2 induction to MWCNT.
authors.affiliation: Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, Durham, NC 27709, USA.
authors.forename: Jong Kwon
authors.initials: JK
authors.lastname: Lee
authors.forename: Brian C
authors.initials: BC
authors.lastname: Sayers
authors.forename: Kyung-Soo
authors.initials: KS
authors.lastname: Chun
authors.forename: Huei-Chen
authors.initials: HC
authors.lastname: Lao
authors.forename: Jeanette K
authors.initials: JK
authors.lastname: Shipley-Phillips
authors.forename: James C
authors.initials: JC
authors.lastname: Bonner
authors.forename: Robert
authors.initials: R
authors.lastname: Langenbach
mesh_terms: D000818:Animals; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000096:biosynthesis*; D015232:Dinoprostone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction / Q000187:drug effects*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D018836:Inflammation Mediators / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D051379:Mice; D037742:Nanotubes, Carbon / Q000633:toxicity*; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis*; D053260:Soot / Q000633:toxicity
chemical_list: D018836:Inflammation Mediators; D037742:Nanotubes, Carbon; D053260:Soot; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D048049:Extracellular Signal-Regulated MAP Kinases; D015232:Dinoprostone