Multi-walled carbon nanotube-induced gene expression in the mouse lung: Association with lung pathology

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Abstract

Due to the fibrous shape and durability of multi-walled carbon nanotubes (MWCNT), concerns regarding their potential for producing environmental and human health risks, including carcinogenesis, have been raised. This study sought to investigate how previously identified lung cancer prognostic biomarkers and the related cancer signaling pathways are affected in the mouse lung following pharyngeal aspiration of well-dispersed MWCNT. A total of 63 identified lung cancer prognostic biomarker genes and major signaling biomarker genes were analyzed in mouse lungs (n = 80) exposed to 0, 10, 20, 40, or 80 μg of MWCNT by pharyngeal aspiration at 7 and 56 days post-exposure using quantitative PCR assays. At 7 and 56 days post-exposure, a set of 7 genes and a set of 11 genes, respectively, showed differential expression in the lungs of mice exposed to MWCNT vs. the control group. Additionally, these significant genes could separate the control group from the treated group over the time series in a hierarchical gene clustering analysis. Furthermore, 4 genes from these two sets of significant genes, coiled-coil domain containing-99 (Ccdc99), muscle segment homeobox gene-2 (Msx2), nitric oxide synthase-2 (Nos2), and wingless-type inhibitory factor-1 (Wif1), showed significant mRNA expression perturbations at both time points. It was also found that the expression changes of these 4 overlapping genes at 7 days post-exposure were attenuated at 56 days post-exposure. Ingenuity Pathway Analysis (IPA) found that several carcinogenic-related signaling pathways and carcinogenesis itself were associated with both the 7 and 11 gene signatures. Taken together, this study identifies that MWCNT exposure affects a subset of lung cancer biomarkers in mouse lungs.

Research highlights

► Multi-Walled Carbon Nanotubes affect lung cancer biomarkers in mouse lungs. ► The results suggest potentially harmful effects of MWCNT exposure on human lungs. ► The results could potentially be used for the medical surveillance of workers.

Introduction

Carbon nanotubes (CNT) represent an important class of engineered nanomaterials. They can be synthesized either as single-walled carbon nanotubes (SWCNT) or as multi-walled carbon nanotubes (MWCNT). SWCNT consist of a single sheet of graphite rolled-up in the form of a cylinder with a diameter of 1–2 nm and lengths ranging up to several micrometers. MWCNT consist of several stacked single wall carbon nanotubes with diameters up to 100 nm and lengths up to several micrometers (Pacurari et al., 2010). Due to their unique physicochemical properties, MWCNT have been widely used for various industrial applications, including, but not limited to, supercapacitors, batteries, automotive industry, aerospace industry, electronics, pharmaceutics, bio-engineering, medical devices, and biomedicine (Pacurari et al., 2010).

Concern over potential MWCNT-induced toxicity has emerged, particularly due to the structural similarity between asbestos and MWCNT. Asbestos is a group of naturally occurring hydrated silicate minerals with fibrous morphology (Pacurari et al., 2010). They consist of fibers of various lengths and diameters, with lengths ranging from 0.1 to greater than 200 μm. It has been well known that asbestos exposure leads to the development of pulmonary fibrosis (asbestosis), bronchogenic lung cancer, pleural plaques, and malignant mesothelioma (Pacurari et al., 2010). Numerous studies have demonstrated that fiber length, diameter, and durability are critical factors involved in the pathogenicity of asbestos (Pacurari et al., 2010). Although MWCNT have a tendency to bundle together, MWCNT aerosols may contain many long, thin, fiber-like nanotube structures similar to asbestos (Pacurari et al., 2010). In addition, the resistance of MWCNT to high temperature or acid treatment indicates that these engineered fibers are durable. It has been proposed that these unique asbestos-like properties of MWCNT may potentially induce pathogenic and carcinogenic effects (Service, 1999, Donaldson et al., 2006, Tian et al., 2006). Indeed, several studies reported that MWCNT induce inflammation, granuloma formation, and biopersistence in the rodent lung after 60 days post-exposure (Muller et al., 2005, Elgrabli et al., 2008, Porter et al., 2010). Other studies have shown that MWCNT exposure increases the genotoxic potential both in vivo and in vitro (Muller et al., 2008).

Recently, our group conducted an in vivo dose–response and time course study of MWCNT exposure in mice in order to investigate the ability of MWCNT to induce pulmonary inflammation, damage, and fibrosis (Porter et al., 2010). Mice were exposed to 0, 10, 20, 40, or 80 μg of MWCNT by pharyngeal aspiration. At 1, 7, 28, and 56 days post-exposure, MWCNT-induced pulmonary toxicity was evaluated. The results demonstrate that pulmonary inflammation and damage were dose-dependent, appeared 1 day post-exposure, and peaked 7 days post-exposure. In contrast, morphometric analysis of lung tissue from this study by Mercer et al. (submitted for publication) indicates that MWCNT-induced interstitial fibrosis increased at day 28 and progressed through 56 days post-exposure. These results indicate that MWCNT exposure rapidly produces significant pulmonary inflammation, damage, and fibrosis.

Several studies have indicated that fibrosis, a pulmonary fibrotic scarring, could be a precursor to lung cancer. A clinical study found that lung scarring was associated with elevated lung cancer risk, and furthermore, they found that pulmonary scarring and lung cancer occurred to the same lung and extended over time (Yu et al., 2008). In a review over a 21 year period of lung cancer patients, it was found that 45% of all peripheral lung cancers originated in a lung scar (Auerbach et al., 1979). A study with computed tomographic (CT) scans and pathologic specimen analysis found that 47 patients of 57 histologically proved lung cancers had pulmonary fibrosis, indicating a possible association between lung cancer and fibrosis (Sakai et al., 2003).

A casual relationship between lung fibrosis and lung cancer has been observed in crystalline silica exposed patients. Literature review indicated that an increased risk of lung cancer among patients with silicosis, a progressive lung fibrosis, might be an effect of the lung fibrosis rather than a direct effect of silica exposure (Peretz et al., 2006). Analysis of the relationship between tuberculosis-induced lung fibrosis and lung cancer indicated that there is an association between tuberculosis and increased lung cancer risk (Shiels et al., 2011), which implicates chronic pulmonary scarring in the etiology of lung cancer.

One study indicated that pulmonary fibrosis may not be a precursor for lung cancer, rather it may increase the susceptibility of fibrosis patients to develop a lung cancer over time. A comparative study of metaplastic epithelia in lungs of usual interstitial pneumonia (UIP), idiopathic pulmonary fibrosis, with or without lung cancer showed that 32 of 70 UIP autopsy cases had lung cancer, and quantitative assessment of the metaplastic epithelia in lungs demonstrated that squamous metaplasia occurred more frequently in UIP with lung cancer than in UIP without lung cancer (Hironaka and Fukayama, 1999).

Molecular biological analysis also supports that lung fibrosis and lung cancer could be associated. Immunohistochemical analysis found that TGF-beta expression is increased in asbestos-induced fibrosis (Jagirdar et al., 1997). TGF-beta is a ubiquitous and essential regulator of cellular proliferation, differentiation, migration, cell survival, and angiogenesis (Elliott and Blobe, 2005). Alterations in the TGF-beta have been associated with human cancers, including lung cancer. TGF-beta signaling promotes epithelial to mesenchymal transition, a process by which cells lose their epithelial characteristics and acquire more migratory and metastatic mesenchymal properties (Toonkel et al., 2010). An increase in TGF-beta expression in fibrosis may imply that fibrotic scarring may increase the risk of carcinogenesis. Several biological and pathological characteristics of lung fibrosis are similar to that of lung cancer, including genetic alterations, uncontrolled proliferation, and tissue invasion (MacKinnon et al., 2010, Vancheri et al., 2010).

Although more research is needed for the assessment of the clinical outcome of lung fibrosis, Vancheri et al.(2010) suggested that the abnormal fibroblast proliferation observed in idiopathic pulmonary fibrosis may be associated with the development of lung cancer. The current study was designed to follow up our previous investigation of MWCNT-induced pulmonary inflammation, damage, and fibrosis in the mouse. The aim of this study was to successfully profile MWCNT-induced gene expression changes within a set of previously identified lung cancer biomarkers (Guo et al., 2006, Guo et al., 2008) using MWCNT-exposed mouse lungs from a previous study (Porter et al., 2010). Ultimately, the association between MWCNT-induced pulmonary inflammation, damage and fibrosis and genetic markers for lung carcinogenesis was investigated.

In our previous lung cancer-related genome-wide DNA microarray analysis, a 35-gene signature was identified from 86 lung adenocarcinoma patients in order to predict tumor recurrence (Guo et al., 2006). This 35-gene prognostic signature was further validated in 348 non-small cell lung cancer (NSCLC) patients using their transcriptional profiles generated from microarray studies (Guo et al., 2008). The gene expression of the 35 biomarkers was confirmed with real-time RT-PCR analysis of independent snap-frozen human lung cancer tumors (Guo et al., 2008). In the Director's Challenge Study, a separate set of patient cohorts from multiple hospitals was analyzed, and a new 12-gene signature was identified from genome-wide DNA microarray analysis. This 12-gene signature provides accurate prognostic stratification of 442 lung adenocarcinoma patients including those with early stage tumors (Wan et al., 2010). We hypothesize that expression profiling of lung cancer biomarkers in MWCNT-exposed mouse lungs will identify a subset of biomarkers affected by the exposure, suggesting a possible association between MWCNT exposure and lung cancer risk. These biomarkers could be relevant for early detection or medical surveillance of lung cancer risk in occupationally-exposed workers. In the current study, in order to identify biomarkers relevant for risk prediction of lung carcinogenesis after MWCNT exposure, quantitative RT-PCR low density arrays (LDA) were designed to measure the gene expression profiles of the identified 47 lung cancer prognostic markers (Guo et al., 2006, Guo et al., 2008, Wan et al., 2010) as well as several major signaling transduction pathway genes in lung tissues from mice exposed to different doses of MWCNT at 7 and 56 days post-exposure (Table 1).

Section snippets

MWCNT

MWCNT used in this study were a gift from Mitsui-&-Company (MWCNT-7, lot # 05072001K28). The characterization of MWCNT has been published (Porter et al., 2010). Briefly, the bulk MWCNT exhibit a distinctive crystalline structure with the number of walls ranging from 20 to 50 walls. Overall, MWCNT trace metal contamination was 0.78%, including sodium (0.41%) and iron (0.32%) with no other metals present above 0.02%. Transmission electron microscopy (TEM) micrographs of MWCNT dispersed in

A 7-gene biomarker set identified at 7 days post-exposure

A total of 63 lung cancer prognostic and major signaling pathway biomarker genes were analyzed using qPCR assays in mouse lungs exposed by aspiration to 0, 10, 20, 40, or 80 μg of MWCNT at 7 days post-exposure. Overall, it was found that only 7 genes had different expression levels compared to the control group. These 7 genes were Rho GTPase activating protein-19 (Arhgap19), coiled-coil domain containing-99 (Ccdc99), muscle segment homeobox gene-2 (Msx2), methalothionein-3 (Mt3), nitric oxide

Discussion

Several studies have demonstrated that exposure to CNT substantially induces harmful effects on the lungs, including inflammatory granulomas and lung fibrosis in animal models (Lam et al., 2004, Muller et al., 2005, Shvedova et al., 2005, Porter et al., 2010). Furthermore, it has been proposed that CNT exposure can induce an asbestos-like pathogenicity and may pose a similar carcinogenic risk as exposure to asbestos fiber does (Takita et al., 1986, Donaldson et al., 2006, Poland et al., 2008).

Acknowledgments

We thank Rebecca Raese for her help in editing the manuscript. This study is supported by NIH/NLMR01LM009500 (PI: Guo) and NCRRP20RR16440 and Supplement (PD: Guo).

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