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
). In fact, MWCNT are man-made fiber-shaped materials. The concern of their potential health hazards has been raised due to their unique physical properties, such as high aspect ratio (length/diameter), nanoscale diameters, micrometer length fiber like-shape and durability, which are analogous to asbestos fibers (Muller et al., 2005
; Donaldson et al., 2006
; Kobayashi et al., 2010
). Particularly, the high aspect ratio and micrometer length are two important features that may contribute to the fiber toxicity (Stanton et al., 1981
). Whether MWCNT fit the classical fiber ‘paradigm’ has not yet been fully established. Poland et al. (Poland et al., 2008
) reported an asbestos-like acute inflammatory effect of long MWCNT when they were administered into abdominal cavity of mice. They found that the asbestos-like effects were fiber length-dependent since results showed that short fibers failed to induce abdominal inflammation or granulomas. Conversely, long fibers induced inflammation and granulomas at the peritoneal side of the diaphragm at 24 h and 7 days post-exposure. Another study by Takagi et al. (Takagi et al., 2008
) reported that intra-abdominal exposure to MWCNT induced mesothelioma, a form of cancer of the lining of the body cavities, in p53+
mice. In this study, the p53+
mouse was chosen because it is sensitive to induction of tumors. Histopathological analysis of the peritoneal cavity of MWCNT-treated p53+
mice found the presence of several asbestos-like pathological changes, such as fibrous scars, granulomas, and typical epithelial mesotheliomas. Particularly, the characteristics of MWCNT-induced mesotheliomas were hobnail appearance to large tumors along with high mitotic rate cells and central necrosis due to high grade of malignant mesothelioma. Furthermore, recent two studies show that MWCNT can reach the pleural tissue in mice, the site of mesothelioma, after pulmonary exposure to occupationally relevant burdens of MWCNT (Ryman-Rasmussen et al., 2009
; Mercer et al., 2010
) These studies conclude that the fibrous structure of MWCNT may pose a carcinogenic risk similar to that for asbestos fibers.
It has been well-established that the biopersistence and pulmonary penetration potential of asbestos are the critical factors associated with asbestos pathogenicity and carcinogenicity (Vallyathan et al., 1998
; Shukla et al., 2003
). Studies have shown that MWCNT exposure has a similar pattern of biopersistence and pulmonary penetration as asbestos (Elgrabli et al., 2008
; Kim et al., 2010
). Recent studies found that MWCNT were persistent in lungs up to 60 days post-exposure (Muller et al., 2005
; Porter et al., 2010
). In addition, histopathological analyses found that upon pulmonary exposure to MWCNT the particles penetrated through alveolar walls and were transported to the alveolar interstitium, subpleural tissue and subpleural lymphatics, finally reaching the intrapleural space (Porter et al., 2010
Several in vitro studies have shown that MWCNT exposure induces genotoxicity. Zhu et al. (Zhu et al., 2007
) found that MWCNT exposure induces DNA damage and increases mutation frequency in mouse embryonic stem cells. Karlsson et al.(Karlsson et al., 2008
) demonstrated that MWCNT exposure significantly damages DNA in A549 type II epithelial cells. Sargent et al. (Sargent et al., 2011
) found that MWCNT induce mitotic abnormality with one mitotic spindle pole, showing the evidence of the mechanism responsible for the disruption of cell division by MWCNT. Their results indicate that MWCNT are genotoxic, which may result in carcinogenic potential for MWCNT. These results are in agreement with our present results of MWCNT-induced lung cancer biomarker gene expression changes. Although whether MWCNT would induce tumors can’t be addressed in the present study, our data indicate an association between MWCNT exposure and lung cancer biomarker changes. Such an association has been used to justify a current project within NIOSH to evaluate lung tumor formation over the course of 1 year after a 2 week inhalation of MWCNT in a cancer susceptible mouse strain.
Taken together, the fibrous characteristics of MWCNT, their biopersistence, their ability to penetrate the lungs, and their genotoxicity have prompted the hypothesis that exposure to MWCNT may contribute to the initiation and progression of asbestos-like pathological responses, such as lung carcinogenesis (Donaldson et al., 2006
; Pacurari et al., 2010
In the present study, we sought to determine whether MWCNT exposure would affect lung cancer biomarker gene expression through profiling a set of lung cancer biomarker genes in mouse lungs following exposure to MWCNT. The design of this study was established based on our previous in vivo mouse model for pharyngeal aspiration of MWCNT (Porter et al., 2010
). The mouse lung tissue specimens at both 7 and 56 days post-MWCNT exposure were taken from this previous investigation and were analyzed for the expression changes of a set of 63 lung cancer biomarker genes using RT-PCR analysis (). Of these 63 genes, 47 were previously shown to be prognostic markers of human lung cancer recurrence and metastasis (Guo et al., 2006
; Guo et al., 2008
; Wan et al., 2010
), and the remaining 16 genes are well established hallmarks of cancer signaling pathways. The 47 lung cancer prognostic biomarkers were from a 35-gene (Guo et al., 2006
) and a 12-gene signature (Guo et al., 2008
), respectively. Both lung cancer gene signatures could identify more aggressive tumors from stage I non-small cell lung cancer tumors, which were at the early stage of cancer formation by the time of diagnosis. We have data showing that 45 out of 47 prognostic markers were also significantly differentially expressed in NSCLC tumors vs. normal lung tissues (currently unpublished results). Specifically, Msx2
were both over-expressed in lung cancer tumor tissues with a fold change of 2.3 and 1.8, respectively, which are concordant with the over-expression patterns observed in the MWCNT-treated mouse lungs in this study. The functions of these 63 genes and the associated signaling transduction pathways were extracted from the literatures and are listed in .
The results obtained from our study demonstrated that 7 genes out of these 63 lung cancer biomarker genes showed differential expression changes at 7 days post-exposure to MWCNT. These 7 genes are Arpgap19, Nos2, Shh, Wif1, Mt3, Ccdc99,
The functional analysis of 7 genes showed that: 1) Arpgap19, Nos2, Shh,
are involved in signaling transduction, 2) Mt3
is involved in metal detoxification, cell proliferation, and cell protection against damage, 3) Ccdc99
is involved in cell cycle regulation, and 4) Msx2
is a transcription factor and an oncogene (). Particularly, Shh,
a tissue developmental and angiogenic factor, is involved in tumor angiogenesis in a variety of cancers, and Mt3,
modulated by oxidative stress, plays a role in carcinogenesis (Pola et al., 2001
; Garrett et al., 2002
). The increase in expression of both genes upon MWCNT exposure in mouse lungs may indicate the potential molecular mechanisms related to MWCNT-induced potential carcinogenic effects. Such a relationship warrants further study.
At 56 days post-exposure to MWCNT, the gene profiling showed that 11 genes underwent different expression changes. These 11 genes are: Dhh, Nos2, Ptch1, Pik3r1, Zak, Wif1, Ccdc99, Cav1, Bcl2, Msx2,
. The functional analysis of these 11 genes showed that: 1) Dhh, Nos2, Ptch1, Pik3r1, Zak,
are involved in signaling transduction, 2) Ccdc99
is a cell cycle protein, 3) Cav1
is associated with plasma membrane, 4) Bcl2
is an anti-apoptotic gene, 5) Msx2
is a transcription factor and an oncogene, and 6) Gpx3
is involved in antioxidant activity (). Notably, Bcl2
is an anti-apoptotic gene and plays a major role in tumorigenesis (Coultas and Strasser, 2003
). The decrease in Bcl-2
gene expression in this study indicates that MWCNT exposure may damage anti-oncogenic suppressor genes in mouse lungs.
Furthermore, these two sets of significant genes at 7 and 56 days post-exposure share 4 common genes between them. These genes are: Ccdc99, Msx2, Nos2,
(). The data analysis showed that the expression changes of these 4 overlapping genes at 7 days post-exposure were attenuated at 56 days post-exposure. Interestingly, our previous in vivo studies have shown that MWCNT-induced pulmonary inflammation and damage peaked at 7 days post-exposure of MWCNT (Porter et al., 2010
); while interstitial fibrosis was significantly elevated at 28 days post-exposure and peaked at 56 days (Mercer et al., 2011
). Therefore, the time-dependent changes in gene expression profiling correlate with the previously published results of histopathological measurements in mouse lungs, indicating a transient inflammatory/damage response and persistent fibrosis. These results demonstrate that gene profiling may well reveal alterations in the molecular processes at the level of the gene transcription and signaling transduction pathways upon MWCNT exposure in mouse lungs.
The notion of predicting chemical toxicity by gene expression profiling has been proposed and applied in genotoxicity for several decades (Aubrecht and Caba, 2005
). Particularly, the recent development of high-throughput technologies has made it possible to identify key predictive biomarkers through global gene expression profiling. It has become increasingly important to use gene expression signatures identified with bioinformatics methods for toxicity predication (Shi et al., 2010
), risk assessment, and screening (Afshari et al., 2011
). A similar gene expression and copy number profiling study was conducted to identify important allelic imbalance in asbestos-related lung cancer (Wikman et al., 2007
). Blood gene expression signatures have been used to predict exposure levels of APAP (Bushel et al., 2007
). The unique advantage of these studies, as well our present study, is the ability to detect toxic injury at the molecular level and to identify the signaling pathways that lead to organ injury long before the clinical symptoms occur. In these biomarker and gene signature studies, the emphasis is on the prediction of potential risk or toxicity. In this study, the lung tissues were collected from the mice at 7 and 56 days post-exposure, which are not long enough for mice to develop lung cancer. In the case of asbestos exposure, the latency period between the first exposure and diagnosis of mesothelioma ranges from 20 to 40 years (Pacurari et al., 2010
). An in vivo animal study demonstrated that asbestos exposure induces malignant mesothelioma in mouse lungs at 60 weeks post-exposure (Vaslet et al., 2002
). Thus, neither asbestos nor MWCNT would be able to induce lung carcinogenesis in mice at 56 days post-exposure. In vivo animal model-generated gene profiling reveals information that approximates the complexity of the human body and its cellular, biochemical, and molecular systems that are involved in responses to chemical agents. Several studies have shown that animal model-based gene expression profiling can predict human target organ toxicities reliably for numerous human diseases, including cancer (Nuwaysir et al., 1999
; Newton et al., 2004
). The combined results from the gene expression profiling in this study and the histopathological changes observed in the previous study (Porter et al., 2010
) indicate that exposure to MWCNT may potentially enhance the risk of pulmonary diseases, including lung carcinogenesis, in humans. Thus, results from the present study have been used to justify a further investigation by NIOSH to address whether the changes observed in gene expression in lung target cells are involved in the pathogenesis of lung cancer. Specifically, NIOSH is currently conducting a two week inhalation exposure study with histological evaluation through 1 year post-exposure in a cancer susceptible mouse strain.
Ingenuity Pathway Analysis (IPA) software analyzes the gene information from more than 200,000 published full text articles in order to create global gene networks, an interactome. At the same time, IPA relates specific biological functions and potential diseases and functional disorders to individual genes (Calvano et al., 2005
; Long et al., 2008
). The software is used to identify significant networks, biological functions, potential diseases and canonical signaling pathways associated with the differentially expressed gene profiles. The results obtained from IPA show a correlation between our gene expression profiling and an empirical classification of biological functions and potential diseases. Among the top five most significant canonical pathways associated with the 7-gene biomarker set, 4 pathways (corticotrophin releasing hormone signaling, Sonic hedgehog signaling, MIF regulation of innate immunity, and MSP-RON signaling pathway) are involved in carcinogenesis. Furthermore, the results of IPA showed that cancer is one of top 5 diseases and functional disorders associated with the 7-gene biomarker set. Similarly, 4 out of the 5 most significant canonical pathways associated with 11-gene biomarker set are involved in carcinogenesis and cancer is ranked as the second most significant of the potential diseases and functional disorders listed. Moreover, the comparison of results from analyses of both gene sets showed that there are 12 overlapping significant canonical signaling pathways and that basal cell carcinoma signaling is ranked as the most significant pathway.
Our IPA results also showed that MWCNT induced potentially carcinogenic alterations in the genes of mice and that these changes were time dependent. The Signaling Pathway of Molecular Mechanisms of Cancer is ranked as the most significant among the 11-gene biomarker set-associated canonical pathways at 56 days post-exposure. Notably, it is ranked much lower among the 7-gene biomarker set-associated canonical pathways at 7 days post-exposure. Furthermore, cancer is ranked as thesecond most significant among the 11-gene set-associated diseases and functional disorders whereas in the 7-gene set analysis, its significance falls to the fifth. These results are consistent with those of our histopathological measurements (Porter et al., 2010
; Mercer et al., 2011
). They demonstrate a shift from rapid/transient inflammation/damage to a persistent fibrotic response involving remodeling signals and growth factor damages, and as well as epithelial hyperplasia and hypertrophy (Porter et al., 2010
). Such changes have been associated with initiation of carcinogenesis.
The IPA results were focused on the 7- and 11-gene sets identified in this study. As only cancer-related genes were initially analyzed in the experiments, the results would be biased toward cancer related pathways and functions, as opposed to those obtained from genome-wide expression studies. However, the identified biomarkers are involved in multiple diseases. Pathological disorders, such as organismal disorders and injuries (including fibrosis), were also retrieved from the IPA database as significant functions, indicating that the genes identified in this study were associated with the observed MWCNT-induced lung pathology.
Overall, the results obtained from this study have demonstrated that MWCNT exposure induces changes in lung cancer biomarker gene expression in mouse lungs, which may indicate a potential association between MWCNT exposure-induced lung inflammatory, damage and fibrotic responses and lung carcinogenesis. The results of the IPA also indicate that MWCNT exposure may induce alterations in several disease-related signaling transduction pathways. In light of this finding, long term inhalation studies of MWCNT to monitor possible induction of lung tumors and/or mesothelioma are justified. NIOSH is currently conducting a two week inhalation exposure to MWCNT in a cancer susceptible mouse strain with histological evaluation through 1 year post-exposure to address this issue. Additionally, the results obtained from this study indicate these gene signature alterations of MWCNT exposure and could conceivably be used for the medical surveillance of occupationally-exposed MWCNT workers.