Systematic Analysis of Multiwalled Carbon Nanotube-Induced Cellular Signaling and Gene Expression in Human Small Airway Epithelial Cells

Abstract

Multiwalled carbon nanotubes (MWCNT) are one of the most commonly produced nanomaterials, and pulmonary exposure during production, use, and disposal is a concern for the developing nanotechnology field. The airway epithelium is the first line of defense against inhaled particles. In a mouse model, MWCNT were reported to reach the alveolar space of the lung after in vivo exposure, penetrate the epithelial lining, and result in inflammation and progressive fibrosis. This study sought to determine the cellular and gene expression changes in small airway epithelial cells (SAEC) after in vitro exposure to MWCNT in an effort to elucidate potential toxicity mechanisms and signaling pathways. A direct interaction between SAEC and MWCNT was confirmed by both internalization of MWCNT and interaction at the cell periphery. Following exposure, SAEC showed time-dependent increases in reactive oxygen species production, total protein phosphotyrosine and phosphothreonine levels, and migratory behavior. Analysis of gene and protein expression suggested altered regulation of multiple biomarkers of lung damage, carcinogenesis, and tumor progression, as well as genes involved in related signaling pathways. These results demonstrate that MWCNT exposure resulted in the activation of SAEC. Gene expression data derived from MWCNT exposure provide information that may be used to elucidate the underlying mode of action of MWCNT in the small airway and suggest potential prognostic gene signatures for risk assessment.

Monocyte adhesion induced by multi-walled carbon nanotubes and palmitic acid in endothelial cells and alveolar–endothelial co-cultures

“…A model consisting of macrophages and A549 cells in the upper chamber and dendritic cells on the basal side of the membrane in the lower chamber showed substantial inflammatory signaling induced by MWCNT exposure (Gasser et al, 2012). It has also been shown that 1.2 mg/ml MWCNT exposure to small airway epithelial cells resulted in elevated expression of genes related to inflammation and release of inflammatory cytokines (Snyder-Talkington et al, 2013b) and also on activated human microvascular endothelial cells in the lower chamber of a co-culture system (Snyder-Talkington et al, 2013a). Therefore, small airway epithelial cells could be more capable of releasing inflammatory mediators than A549 cells, although the latter do respond with increased IL-6 and IL8 transcription at very high exposure concentrations of, e.g., diesel exhaust particles (Dybdahl et al, 2004).…”

Multi-walled carbon nanotube-induced gene expression in vitro: Concordance with in vivo studies

“…Six biological replicates of each SAEC and HMVEC monoculture exposure condition were collected for microarray analysis. Both SAEC and HMVEC in monoculture have been shown to interact with MWCNT (Pacurari et al 2012; Snyder-Talkington et al 2013b). …”

Evaluating the mechanistic evidence and key data gaps in assessing the potential carcinogenicity of carbon nanotubes and nanofibers in humans

“…These findings show that MWCNT-7 exposure engages cells to control their DNA integrity and growth, and that cell growth may be activated by the overexpression of genes that activate growth and the underexpression of genes which normally exert a negative control. Snyder-Talkington et al (2013b) reported alterations of genes associated to cellular growth and proliferation in telomerase immortalized-human small airway epithelial cells (SAEC) after in vitro exposure to MWCNT-7. Several of these genes, were upregulated such as V-Akt murine thymoma viral oncogene homolog 1 ( AKT1 ), which is activated by growth factors; vascular endothelial growth factor A ( VEGFA ); smoothened, frizzled class receptor ( SMO ) and Sonic Hedgehog ( SHH ), involved in hedgehog signaling and carcinogenesis; as well as downregulation of B-cell CLL/lymphoma 2 ( BCL2 ), an apoptosis inhibitor.…”

“…This may suggest a growth advantage of MWCNTs-exposed SAEC. Moreover, these genes have a role in lung adenocarcinoma (Snyder-Talkington et al (2013b). Early passage immortalized cells are used in these studies to analyze genetic changes in a cell population in response to a toxic agent; these results are reliable because early passage immortalized cells have a stable genotype.…”