Transcriptomic Analysis Reveals Novel Mechanistic Insight into Murine Biological Responses to Multi-Walled Carbon Nanotubes in Lungs and Cultured Lung Epithelial Cells

There is great interest in substituting animal work with in vitro experimentation in human health risk assessment; however, there are only few comparisons of in vitro and in vivo biological responses to engineered nanomaterials. We used high-content genomics tools to compare in vivo pulmonary responses of multiwalled carbon nanotubes (MWCNT) to those in vitro in cultured lung epithelial cells (FE1) at the global transcriptomic level. Primary size, surface area and other properties of MWCNT- XNRI -7 (Mitsui7) were characterized using DLS, SEM and TEM. Mice were exposed via a single intratracheal instillation to 18, 54, or 162 μg of Mitsui7/mouse. FE1 cells were incubated with 12.5, 25 and 100 μg/ml of Mitsui7. Tissue and cell samples were collected at 24 hours post-exposure. DNA microarrays were employed to establish mechanistic differences and similarities between the two models. Microarray results were confirmed using gene-specific RT-qPCR. Bronchoalveolar lavage (BAL) fluid was assessed for indications of inflammation in vivo. A strong dose-dependent activation of acute phase and inflammation response was observed in mouse lungs reflective mainly of an inflammatory response as observed in BAL. In vitro, a wide variety of core cellular functions were affected including transcription, cell cycle, and cellular growth and proliferation. Oxidative stress, fibrosis and inflammation processes were altered in both models. Although there were similarities observed between the two models at the pathway-level, the specific genes altered under these pathways were different, suggesting that the underlying mechanisms of responses are different in cells in culture and the lung tissue. Our results suggest that careful consideration should be given in selecting relevant endpoints when substituting animal with in vitro testing

Gene network.

<p>Combined analysis of all significantly differentially expressed genes associated with angiogenesis, lipid metabolism, and cellular proliferation pathways in lungs in the high dose group. Analysis was conducted and the network was built using GeneGo Metacore map creator. The figure shows that the majority of the genes are commonly regulated by <i>E2f1</i>, <i>Il6</i> and <i>C-myc</i>. Hexagons represent physical and functional interactions: TR – transcription regulation, IE - influence on expression, B – binding, and GR – group. The green arrows represent positive regulation/activation, the brown arrows represent negative regulation/inhibition and the grey arrows represent unspecified regulation.</p

SEM images of dry, pristine Mitsui7.

<p>(A) Mitsui7 at high density. (B) A close-up of MWCNT at high density. (C) Mitsui7 behavior at low density.</p

Light microscopy images of Mitsui7 dispersed in the <i>in</i><i>vitro</i> exposure medium after sonication.

<p>(A) 100 μg/milliliter (31.19 µg/cm²) concentration (B) 12.5 μg/ml concentration (3.9 µg/cm²). Bars represent 50μm.</p

The total number of genes perturbed in the categories of gene expression and cellular signalling functions.

<p>Green: low dose (12.5 µg <i>in </i><i>vitro</i>, 18 µg <i>in </i><i>vivo</i>), blue: medium dose (25 µg <i>in </i><i>vitro</i>, 54 µg <i>in </i><i>vivo</i>), and red: high dose (100 µg <i>in </i><i>vitro</i>, 162 µg <i>in </i><i>vivo</i>). </p

Differentially expressed genes associated with five top canonical pathways derived from IPA analysis.

<p><b>Genes </b><b>are </b><b>categorized </b><b>into </b><b>four </b><b>major </b><b>functions</b> (cell cycle regulation-orange, xenobiotic metabolism-green, inflammation-violet, and tissue fibrosis-blue). All genes in both models are regulated by a common transcription factor, AHR. Genes highlighted with green indicate down-regulation and genes highlighted with red indicate up-regulation. The transcription factor AHR is down-regulated in cells, suggesting negative regulation of the downstream functions in cells. </p

TEM images.

<p>(A) Pristine Mitsui7 (B) Mitsui7 MWCNTs interacting with cells in the lung lining fluid (Black arrows). The MWCNTs have been partially dislodged during ultramicrotomy (C) Macrophage containing engulfed Mitsui7 in vesicle (red arrow).</p

Gene heatmap comparing differentially expressed genes in the categories of fibrosis, inflammation and oxidative stress.

<p>Red represents up-regulation, green represents down-regulation and grey represents no change in the expression. </p

Top three biological networks <i>in</i><i>vitro</i>.

<p>The networks were generated by merging the top three networks in each of the dose and were based on the connectivity between each gene and the molecular interaction knowledge base in IPA. Nodes are highlighted in large bold font. Green indicates down-regulation and red indicates up-regulation.</p

Overview of response in FE1 cells.

<p>(A) Venn diagram showing the overlapping differentially expressed genes (FDR P ≤ 0.05 and fold change ≥ 1.5) in response to low (12.5 µg, green), medium (25 µg, blue), and high (100 µg, red) doses of Mitsui7 <i>in </i><i>vitro</i>. (B-C) Gene ontology (GO) analysis of differentially expressed genes. (B) Commonly regulated GO biological processes (C) GO biological processes unique to the high dose group. Numbers in parentheses represent number of genes altered in that process.</p