ROS and NF-κB are involved in upregulation of IL-8 in A549 cells exposed to multi-walled carbon nanotubes

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Abstract

Carbon nanotubes (CNTs) have potential applications in biosensors, tissue engineering, and biomedical devices because of their unique physico-chemical, electronic and mechanical properties. However, there is limited literature data available concerning the biological properties and toxicity of CNTs. This study aimed to assess the toxicity exhibited by multi-walled CNTs (MWCNTs) and to elucidate possible molecular mechanisms underlying the biological effects of MWCNTs in A549 cells. Exposing A549 cells to MWCNTs led to cell death, changes in cell size and complexity, reactive oxygen species (ROS) production, interleukin-8 (IL-8) gene expression and nuclear factor (NF)-κB activation. Treatment of A549 cells with antioxidants prior to adding MWCNTs decreased ROS production and abrogated expression of IL-8 mRNA. Pretreatment of A549 cells with NF-κB inhibitors suppressed MWCNTs-induced IL-8 mRNA expression. These results indicate that MWCNTs are able to induce expression of IL-8 in A549 cells, at least in part, mediated by oxidative stress and NF-κB activation.

Section snippets

Materials and methods

Materials. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and F12 were products of Gibco BRL (Grand Island, NY, USA). NF-κB consensus oligonucleotide was purchased from Promega (Madison, WI, USA). γ-[32P]ATP was the product of NEN Life Science (Boston, MA, USA). Antibodies against p65, IκBa, PARP, β-actin, and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Escherichia coli 0111:B4 lipopolysaccharide (LPS) was obtained from Sigma

Cytotoxicity, cell size and complexity analysis

Among the widely used formazan-based cytotoxicity assays, WST-1 has been shown not to interact with CNTs, in contrast to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) [21]. Therefore, cytotoxicity induced by MWCNTs was assessed with the WST-1 assay. After 72 h incubation with A549 and BEAS-2A cells, MWCNTs elicited a concentration-dependent toxic effect on cell viability (Fig. 1A). The cell viability results were confirmed by a live–dead cell assay, which indicated that

Discussion

In the present study, we evaluated the toxicological potential of MWCNTs on pulmonary epithelial cells by studying their effects on cell viability, cell size and complexity, induction of oxidative stress, release of inflammatory mediators, and activation of NF-κB, which led to a number of interesting findings.

MWCNTs appeared to be water-miscible and dispersed at the concentration range used in the presence of the Pluronic surfactant as a dispersing agent, not toxic by itself (data not shown).

Acknowledgments

Project supported by the Science and Technology Innovation Project of Fujian Province for Young Scientific Researchers, China (2008F3097) and the Special Program for Key Basic Research of the Ministry of Science and Technology, China (2006CB93300).

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