Toxicoproteomic analysis of pulmonary carbon nanotube exposure using LC-MS/MS

Abstract

Toxicoproteomics is a developing field that utilizes global proteomic methodologies to investigate the physiological response as a result of adverse toxicant exposure. The aim of this study was to compare the protein secretion profile in lung bronchoalveolar lavage fluid (BALF) from mice exposed to non-functionalized multi-walled carbon nanotubes (U-MWCNTs) or MWCNTs functionalized by nanoscale Al2O3 coatings (A-MWCNT) formed using atomic layer deposition (ALD). Proteins were identified using liquid chromatography tandem mass spectrometry (LC-MS/MS), and quantified using a combination of two label-free proteomic methods: spectral counting and MS1 peak area analysis. On average 465 protein groups were identified per sample and proteins were first screened using spectral counting and the Fisher’s exact test to determine differentially regulated species. Significant proteins by Fisher’s exact test (p<0.05) were then verified by integrating the intensity under the extracted ion chromatogram from a single unique peptide for each protein across all runs. A two sample t-test based on integrated peak intensities discovered differences in 27 proteins for control versus U-MWCNT, 13 proteins for control versus A-MWCNT, and 2 proteins for U-MWCNT versus A-MWCNT. Finally, an in-vitro binding experiment was performed yielding 4 common proteins statistically different (p<0.05) for both the in-vitro and in-vivo study. Several of the proteins found to be significantly different between exposed and control groups are known to play a key role in inflammatory and immune response. A comparison between the in-vitro and in-vivo CNT exposure emphasized a true biological response to CNT exposure.

Mechanisms of carbon nanotube‐induced pulmonary fibrosis: a physicochemical characteristic perspective

“…Peroxidases have also been implicated in the biodegradation of CNTs 128 . Bronchoalveolar lavage fluid from B6 mice following a 24 hour exposure to uncoated or aluminum oxide coated MWCNTs experienced a significant increase in MPO, indicating that it is involved in the CNT host immune response 129 . Changes to CNTs, by adding a metal oxide coating, may alter the biodegradation process and should be studied further.…”

“…128 Bronchoalveolar lavage fluid from B6 mice following a 24-h exposure to uncoated or aluminum oxide-coated MWCNTs experienced a significant increase in MPO, indicating that it is involved in the CNT host immune response. 129 Changes to CNTs, by adding a metal oxide coating, may alter the biodegradation process and should be studied further. Degradation of CNTs by neutrophilic MPO can decrease inflammation, as modeled from in vitro testing; a loss or reduction of MPO could then result in increased inflammation and resulting fibrosis from a CNT insult.…”

Characterization of the proteome and lipidome profiles of human lung cells after low dose and chronic exposure to multiwalled carbon nanotubes

“…For instance, human epidermal keratinocytes displayed differential expression of more than 30 and 100 proteins after 24 and 48 h of MWCNT exposure, respectively (Witzmann and Monteiro-Riviere 2006). Similarly, other proteomic analyses have identified alterations in the expression of several proteins upon MWCNT exposure (Haniu et al 2010;Ju et al 2014;Hilton et al 2015Hilton et al , 2017. These findings indicate that toxicoproteomic analyses may provide valuable information on the mechanisms underlying toxicity of MWCNTs.…”

“…The aim of this study was to investigate molecular mechanisms associated with long-term but lowdose exposure to a well characterized MWCNT. Toxicoproteomic analysis of pulmonary effects of MWCNT has shown changes in proteins in bronchoalveolar lavage fluid of the exposed mice (Hilton et al 2015). We thus hypothesized that monitoring proteomic changes associated with chronic exposure of the human lung cells might reveal mechanisms contributing to the adverse effects of MWCNT.…”

Impact of single-walled carbon nanotubes on the embryo: a brief review

“…50 However, their toxic effect on human health could have an important impact on their use worldwide; presently, it was demonstrated that SWCNTs have a toxic effect on cells, including human normal cells, and living organisms. 24,[51][52][53] The toxic effect of these NPs could be influenced by a number of factors including the surface chemistry, surface area, functional groups, shape, photochemistry, charge, and aggregation as well as preparation method. 6,24,54 Hence, we will review the recent publications related to the effect of SWCNTs on embryo development, which is unfortunately limited to a few number of studies including one from our group.…”