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,||,1,,1,2
* Department and Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan;
NTU Center for Genomic Medicine, National Taiwan University, Taipei, Taiwan;
Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan;
Institute of Life Sciences, College of Life Sciences, National Chung-Hsing University, Taichung, Taiwan;
|| Institutes of Biomedical Sciences and Molecular Biology, College of Life Sciences, National Chung-Hsing University, Taichung, Taiwan; and
Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
2Correspondence: Department of Internal Medicine National Taiwan University Hospital and National Taiwan University of Medicine College No. 7, Chung-Shan South Rd., Taipei, 100, Taiwan. E-mail: pcyang{at}ha.mc.ntu.edu.tw
SPECIFIC AIMS
Titanium dioxide nanoparticles (nanoTiO2) have been widely used as a photocatalyst in air and water cleaning. However, inhalation of these nanoparticles may cause pulmonary toxicity that is often ignored, and its mechanism is not fully understood. The specific aims of this study were to 1) investigate the pulmonary toxicity of nanoTiO2 in mice model and 2) study the pathological, cellular, and molecular mechanisms of nanoTiO2-induced pulmonary diseases in vivo and in vitro.
PRINCIPAL FINDINGS
1. NanoTiO2 can induce emphysema-like lung injury in mice (Fig. 1
)
We found that 1 wk after single intratracheal instillation with 0.1 mg nanoTiO2 in mice, the lungs showed significant changes in morphology and histology, including disruption of the alveolar septa and alveolar enlargement (emphysematous change), type II pneumocyte proliferation, increased alveolar epithelial thickness, and accumulation of particle-laden macrophages (Fig. 1A, B
). The mean linear intercept (MLI) of interalveolar wall distance, the air space area, and the septal chord length (a parameter that measures alveolar septal thickness) were significantly increased the first week (acute-phase) after instillation of nanoTiO2, and the pathological changes persisted until the second week (chronic phase) (Fig. 1C
). Immunostaining of the nanoTiO2-induced lung injury revealed a significant increase in macrophage accumulation, alveolar type II cell proliferation, and alveolar epithelial cell apoptosis 1 wk after nanoTiO2 treatment.
2. Microarray gene expression profiles of the nanoTiO2-induced injured lung and signaling pathway analysis
The nanoTiO2-induced injured mice lungs were examined by cDNA microarray. The differentially expressed genes (1.5-fold difference) of nanoTiO2-induced genes were categorized and integrated to fit the transduction signaling map using the KEGG and BIOCARTA pathway database. According to cDNA microarray analysis, 318 was up-regulated and 188 down-regulated at wk 1 in nanoTiO2-treated mice compared with normal saline-treated mice, including cell growth regulators (cdc2a, cyclin B, D, and E), vascular endothelial growth factor (VEGF) -related factors (PlGF), G-protein-coupled receptors (GPCR), chemokines (CXCL1, CXCL5, and CCL3), matrix metalloproteinases (MMP2 and 15), and other immune response factors. PlGF and these chemokines have been reported to be involved in the pathogenesis of pulmonary emphysema. According to the pathway analysis, four major pathways were up-regulated by nanoTiO2: the cell cycle regulatory pathway, apoptosis pathway, the chemokines pathway, and complement cascade (classical pathway).
3. The PlGF/chemokine pathway may involve in nanoTiO2-induced pulmonary injury (Fig. 2 )
Real-time quantitative RT-polymerase chain reaction (RT-PCR) analysis showed that plgf, chemokines (cxcl1, cxcl5, and ccl3), TRAIL, and prostaglandin E receptor 4 (ptger4, EP4) were significantly up-regulated in lung tissues of mice treated with nanoTiO2 for 1 wk (Fig. 2A
). Western blotting also showed that nanoTiO2 caused significant induction of PlGF expression in a dose-dependent manner (Fig. 2B
), while ELISA analysis showed that nanoTiO2-treated mice had higher serum levels of PlGF protein (Fig. 2C
), which might be produced mainly by infiltrating macrophages and some pulmonary epithelial cells, as shown by immunostaining with PlGF-specific antibodies (Fig. 2D
).
4. In vitro exposures of nanoTiO2 in macrophage cell line THP-1 dose-dependently induce significant increase of PlGF, Cxcl5, and Ccl2 (MCP1) expression
CONCLUSIONS AND SIGNIFICANCE
The results of this study indicate that single intratracheal instillation of 0.1 mg nanoTiO2 can induce severe pulmonary inflammation and emphysema in the mouse lung. Our results indicate that pulmonary emphysema is triggered by nanoTiO2 activation of macrophages, up-regulations of PlGF, and other inflammatory cytokines that resulted in disruption of alveolar septa, alveolar epithelial injury, alveolar epithelial cell proliferation, and apoptosis. This information may have important clinical implications regarding safety, as nanoTiO2 are widely used as a photocatalyst in air and water cleaning, and TiO2 is used as a pigment in the paint industry. Extra caution therefore should be taken in handling higher doses of nanoTiO2.
In this study, the microarray gene expressions and pathway analysis indicated that the cell cycle, apoptosis, chemokine, and complement pathways may be involved in nanoTiO2-induced pulmonary toxicity (Fig. 3
). Activation of the cell cycle pathway suggests that nanoTiO2 can regulate key factors for G2/M progression by increasing the expression of cdc2a and cyclins, which may explain the increase in the number of proliferating (PCNA-positive cells) type II pneumocytes and the increase in septal thickness seen in this study. Activation of the apoptosis pathway indicates that nanoTiO2 can increase TRAIL expression, which may account for the increased number of TUNEL-positive cells in nanoTiO2-treated samples, explaining the alveolar type II cell apoptosis, abnormal air space enlargement, and pulmonary emphysema.
The pathway analysis also shows that nanoTiO2 can stimulate the expression of several cytokines and chemokines, including PlGF, a prochemokine that can regulate the expression of MCP-1, IL-1, and TNF-
. These chemokines may also affect the expression of other C-C and C-X-C chemokines (Ccl3, Cxcl1, and Cxcl5) that modulate chemotaxis, neutrophil infiltration, macrophage accumulation, epithelial cell proliferation, and apoptosis to generate the inflammatory cascade, which may lead to the pathogenesis of pulmonary emphysema.
The results of this study add to our understanding of nanoTiO2-induced pulmonary toxicity and pulmonary emphysema. We suggest that PlGF, chemokines, and the complement cascade may cause inflammatory cell chemotaxis, cell proliferation, and apoptosis, resulting in serious lung injury. Further investigations are needed to elucidate the potential pulmonary toxicity of different nanoparticles and their pathogenesis.
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FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6485fje
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