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Title: Multi-walled carbon nanotubes induce COX-2 and iNOS expression via MAP kinase-dependent and -independent mechanisms in mouse RAW264.7 macrophages.

Authors: Lee, Jong Kwon; Sayers, Brian C; Chun, Kyung-Soo; Lao, Huei-Chen; Shipley-Phillips, Jeanette K; Bonner, James C; Langenbach, Robert

Published In Part Fibre Toxicol, (2012)

Abstract: 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).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.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.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.

PubMed ID: 22571318 Exiting the NIEHS site

MeSH Terms: Animals; Cell Line; Cyclooxygenase 2/biosynthesis*; Dinoprostone/metabolism; Dose-Response Relationship, Drug; Enzyme Induction/drug effects*; Extracellular Signal-Regulated MAP Kinases/metabolism*; Inflammation Mediators/metabolism; MAP Kinase Signaling System/physiology; Macrophages/drug effects*; Macrophages/enzymology; Macrophages/ultrastructure; Mice; Nanotubes, Carbon/toxicity*; Nitric Oxide Synthase Type II/biosynthesis*; Nitric Oxide/metabolism; Soot/toxicity

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