System-based identification of toxicity pathways associated with multi-walled carbon nanotube-induced pathological responses

Abstract

The fibrous shape and biopersistence of multi-walled carbon nanotubes (MWCNT) have raised concern over their potential toxicity after pulmonary exposure. As in vivo exposure to MWCNT produced a transient inflammatory and progressive fibrotic response, this study sought to identify significant biological processes associated with lung inflammation and fibrosis pathology data, based upon whole genome mRNA expression, bronchoaveolar lavage scores, and morphometric analysis from C57BL/6J mice exposed by pharyngeal aspiration to 0, 10, 20, 40, or 80 µg MWCNT at 1, 7, 28, or 56 days post-exposure. Using a novel computational model employing non-negative matrix factorization and Monte Carlo Markov Chain simulation, significant biological processes with expression similar to MWCNT-induced lung inflammation and fibrosis pathology data in mice were identified. A subset of genes in these processes was determined to be functionally related to either fibrosis or inflammation by Ingenuity Pathway Analysis and were used to determine potential significant signaling cascades. Two genes determined to be functionally related to inflammation and fibrosis, vascular endothelial growth factor A (vegfa) and C-C motif chemokine 2 (ccl2), were confirmed by in vitro studies of mRNA and protein expression in small airway epithelial cells exposed to MWCNT as concordant with in vivo expression. This study identified that the novel computational model was sufficient to determine biological processes strongly associated with the pathology of lung inflammation and fibrosis and could identify potential toxicity signaling pathways and mechanisms of MWCNT exposure which could be used for future animal studies to support human risk assessment and intervention efforts.

MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs

“…Fibrosis is a well-established endpoint in MWCNT-induced toxicity (Aiso et al, 2010;Mercer et al, 2011;Muller et al, 2005;Porter et al, 2010;Snyder-Talkington et al, 2013). In the present study, fibrosis (hepatic fibrosis/hepatic stellate cell activation) was observed as the top pathway hit following exposure to both CNT Small and CNT Large .…”

“…Erdely et al reported workplace exposure levels up to 10.6 μg/ m 3 , resulting in a calculated deposited dose of approximately 4.07 μg/ day in a human, equivalent to 2 ng/day in the mouse (Erdely et al, 2013). Thus, although within dose ranges of other instillation/aspiration studies (Park et al, 2009;Porter et al, 2010;Shvedova et al, 2008;Snyder-Talkington et al, 2013), the doses used in present study are to be considered high in a workplace environment.…”

“…This indicates activation of fibrotic processes 28 days after exposure, but with an effect that was most prominent following CNT Large exposure. Also, by using a list of genes linked to fibrosis described by Snyder-Talkington et al (2013) and a by conducting a literature search, we identified 14 fibrosis-associated genes uniquely expressed on post-exposure day 28 following high dose CNT Large exposure; Arg1 (6.98-fold), Igf1 (5.02-fold), Lgals3 (3.13-fold), Mmp12 (6.69-fold), Mmp13 (2.39-fold), Pde3a (− 1.95-fold), Ptgir (3.33-fold), Smurf2 (− 1.45-fold), Tnfrsf1b (1.77-fold), Vegfa (− 1.66-fold), Eng (− 1.61-fold), Jun (1.87-fold), Smad6 (− 2.17-fold) and Spp1 (6.41-fold). This unique expression pattern could indicate a chronic response related to the physicochemical properties of CNT Large .…”

Inflammation in the pleural cavity following injection of multi-walled carbon nanotubes is dependent on their characteristics and the presence of IL-1 genes

“…Fibrosis as an endpoint of exposure to MWCNTs and changes in gene expression have been reported in the lungs (Snyder-Talkington et al, 2013;Dong et al, 2015;Poulsen et al, 2015). Several interleukins were upregulated in WT mice.…”

Stromelysin-2 (MMP-10) facilitates clearance and moderates inflammation and cell death following lung exposure to long multiwalled carbon nanotubes

“…This dose of MWCNT was based on exposures used on other studies. [28][29][30] Lungs were collected 24 h later and processed for mRNA isolation. In lungs from unchallenged wild-type mice (ie, no MWCNTs, no DM alone), we detected a Ct range for Mmp10 mRNA between 36 and 37 indicating essentially no or very low levels of expression.…”