Cheng-Chung Chou

Titanium dioxide nanoparticles induce emphysema-like lung injury in mice

Titanium dioxide nanoparticles (nanoTiO2) have been widely used as a photocatalyst in air and water cleaning. However, these nanoparticles inhalation can induce pulmonary toxicity and its mechanism is not fully understood. In this study we investigated the pulmonary toxicity of nanoTiO2 and its molecular pathogenesis. The adult male ICR mice were exposed to intratracheal single dose of 0.1 or 0.5 mg nanoTiO2 (19–21 nm) and lung tissues were collected at 3rd day, 1st wk, and 2nd wk for morphometric, microarray gene expression, and pathway analyses. NanoTiO2 can induce pulmonary emphysema, macrophages accumulation, extensive disruption of alveolar septa, type II pneumocyte hyperplasia, and epithelial cell apoptosis. NanoTiO2 induced differential expression of hundreds of genes include activation of pathways involved in cell cycle, apoptosis, chemokines, and complement cascades. In particular, nanoTiO2 up‐regulates placenta growth factor (PlGF) and other chemokines (CXCL1, CXCL5, and CCL3) expressions that may cause pulmonary emphysema and alveolar epithelial cell apoptosis. Cultured human THP‐1 cell‐derived macrophages treated with nanoTiO2 in vitro also resulted in up‐regulations of PlGF, CXCL1, CXCL5, and CCL3. These results indicated that nanoTiO2 can induce severe pulmonary emphysema, which may be caused by activation of PlGF and related inflammatory pathways.—Chen, H‐W., Su, S‐F., Chien, C‐T., Lin, W‐H., Yu, S‐L., Chou, C‐C., Chen, J. J. W., Yang, P. C. Titanium dioxide nanoparticles induce emphysema‐like lung injury in mice. FASEB J. 20, E1732–E1741 (2006)

Single-walled carbon nanotubes can induce pulmonary injury in mouse model.

Carbon nanotubes are a nanomaterial that is extensively used in industry. The potential health risk of chronic carbon nanotubes exposure has been raised as of great public concern. In the present study, we have demonstrated that intratracheal instillation of 0.5 mg of single-walled carbon nanotubes (SWCNT) into male ICR mice (8 weeks old) induced alveolar macrophage activation, various chronic inflammatory responses, and severe pulmonary granuloma formation. We then used Affymetrix microarrays to investigate the molecular effects on the macrophages when exposed to SWCNT. A biological pathway analysis, a literature survey, and experimental validation suggest that the uptake of SWCNT into the macrophages is able to activate various transcription factors such as nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1), and this leads to oxidative stress, the release of proinflammatory cytokines, the recruitment of leukocytes, the induction of protective and antiapoptotic gene expression, and the activation of T cells. The resulting innate and adaptive immune responses may explain the chronic pulmonary inflammation and granuloma formation in vivo caused by SWCNT.

Molecular characterization of toxicity mechanism of single-walled carbon nanotubes

Carbon nanotubes (CNTs) are one of widely used nanomaterials in industry and biomedicine. The potential impact of single-walled carbon nanotubes (SWCNTs) was evaluated using Caenorhabditis elegans (C. elegans) as a toxicological animal model. SWCNTs are extremely hydrophobic to form large agglomerates in aqueous solutions. Highly soluble amide-modified SWCNTs (a-SWCNTs) were therefore used in the present study so that the exact impact of SWCNTs could be studied. No significant toxicity was observed in C. elegans due to the amide modification. a-SWCNTs were efficiently taken up by worms and caused acute toxicity, including retarded growth, shortened lifespan and defective embryogenesis. The resulting toxicity was reversible since C. elegans could recover from a-SWCNT-induced toxicity once the exposure terminates. Chronic exposure to low doses of a-SWCNTs during all development stages could also cause a toxic accumulation in C. elegans. Genome-wide gene expression analysis was performed to investigate the toxic molecular mechanisms. Functional genomic analysis and molecular biology validation suggest that defective endocytosis, the decreased activity of the citrate cycle and the reduced nuclear translocation of DAF-16 transcription factor play key roles in inducing the observed a-SWCNT toxicity in worms. The present study presents an integrated approach to evaluating the toxicity of nanomaterials at the organism and molecular level for human and environmental health and demonstrates that traditional toxicological endpoints associated with functional genomic analysis can provide global and thorough insight into toxicity.

Human kallikrein 8 protease confers a favorable clinical outcome in non-small cell lung cancer by suppressing tumor cell invasiveness

The human kallikrein 8 (KLK8) gene, a member of the human tissue kallikrein gene family, encodes a serine protease. The KLK8 protein (hK8) is known to be a favorable prognostic marker in ovarian cancer, but the biological basis of this is not understood. We found that overexpressing the KLK8 gene in highly invasive lung cancer cell lines suppresses their invasiveness. This role in invasiveness was further confirmed by the fact that inhibition of endogenous KLK8 expression with a specific short hairpin RNA reduced cancer cell invasiveness. In situ degradation and cell adhesion assays showed that proteins produced from KLK8 splice variants modify the extracellular microenvironment by cleaving fibronectin. DNA microarray experiments and staining of cells for actin filaments revealed that the degradation of fibronectin by hK8 suppresses integrin signaling and retards cancer cell motility by inhibiting actin polymerization. In addition, studies in a mouse model coupled with the detection of circulating tumor cells by quantitative PCR for the human Alu sequence showed that KLK8 suppresses tumor growth and invasion in vivo. Finally, studies of clinical specimens from patients with non-small cell lung cancer showed that the time to postoperative recurrence was longer for early-stage patients (stages I and II) with high KLK8 expression (mean, 49.9 months) than for patients with low KLK8 expression (mean, 22.9 months). Collectively, these findings show that KLK8 expression confers a favorable clinical outcome in non-small cell lung cancer by suppressing tumor cell invasiveness.

Design of microarray probes for virus identification and detection of emerging viruses at the genus level.

BackgroundMost virus detection methods are geared towards the detection of specific single viruses or just a few known targets, and lack the capability to uncover the novel viruses that cause emerging viral infections. To address this issue, we developed a computational method that identifies the conserved viral sequences at the genus level for all viral genomes available in GenBank, and established a virus probe library. The virus probes are used not only to identify known viruses but also for discerning the genera of emerging or uncharacterized ones.ResultsUsing the microarray approach, the identity of the virus in a test sample is determined by the signals of both genus and species-specific probes. The genera of emerging and uncharacterized viruses are determined based on hybridization of the viral sequences to the conserved probes for the existing viral genera. A detection and classification procedure to determine the identity of a virus directly from detection signals results in the rapid identification of the virus.ConclusionWe have demonstrated the validity and feasibility of the above strategy with a small number of viral samples. The probe design algorithm can be applied to any publicly available viral sequence database. The strategy of using separate genus and species probe sets enables the use of a straightforward virus identity calculation directly based on the hybridization signals. Our virus identification strategy has great potential in the diagnosis of viral infections. The virus genus and specific probe database and the associated summary tables are available at http://genestamp.sinica.edu.tw/virus/index.htm

Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection

This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD)-induced fluorescence resonance energy transfer (FRET) reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment) of avian influenza viruses were used as the targets in this work. Two oligonucleotides (16 mers and 18 mers) that specifically recognize two separate but neighboring regions of the H5 sequences were served as the capturing and reporter probes, respectively. The capturing probe was conjugated to QD655 (donor) in a molar ratio of 10:1 (probe-to-QD), and the reporter probe was labeled with Alexa Fluor 660 dye (acceptor) during synthesis. The sandwich hybridization assay was done in a 20 μL transparent, adhesive frame-confined microchamber on a disposable, temperature-adjustable indium tin oxide (ITO) glass slide. The FRET signal in response to the sandwich hybridization was monitored by a homemade optical sensor comprising a single 400 nm UV light-emitting diode (LED), optical fibers, and a miniature 16-bit spectrophotometer. The target with a concentration ranging from 0.5 nM to 1 μM was successfully correlated with both QD emission decrease at 653 nm and dye emission increase at 690 nm. To sum up, this work is beneficial for developing a portable QD-based nucleic acid sensor for on-site pathogen detection.

Single-Walled Carbon Nanotubes Induce Airway Hyperreactivity and Parenchymal Injury in Mice

Inhalation of single-walled carbon nanotubes (SWCNTs) has raised serious concerns related to potential toxic effects in the respiratory system. This study examined possible SWCNT-induced toxic mechanisms in vivo in mice. The results indicated that a single intratracheal instillation of SWCNTs could induce airway hyperreactivity and airflow obstruction and confirmed previous findings of granulomatous changes in the lung parenchyma that persisted from 7 days to 6 months after exposure. The irreversible lung pathology and functional airway alterations in the mouse model mimicked obstructive airway disease in humans. Transcriptomic analysis showed that SWCNTs might up-regulate proteinases (cathepsin K and matrix metalloproteinase [MMP]12), chemokines C-C motif ligands (CCL2 and CCL3), and several macrophage receptors (Toll-like receptor 2, macrophage scavenger receptor 1). Pathway analyses showed that NF-κB-related inflammatory responses and downstream signals affecting tissue remodeling dominated the pathologic process. The NF-κB inhibitor pyrrolidine dithiocarbamate attenuated SWCNT-induced airway hyperreactivity, chronic airway inflammation, and MMP12 and cathepsin K expression when administered in vivo, whereas a cathepsin K inhibitor could partially reduce airway hyperreactivity and granulomatous changes in the SWCNT-treated group. The up-regulation of cathepsin K and MMP12 by SWCNTs was further confirmed via in vitro coculture of bronchoalveolar macrophages with lung epithelial/mesenchymal cells but not in macrophages without coculture, indicating that SWCNT-induced MMP12 and cathespin K were cell-type specific and cell-cell interaction dependent. In conclusion, exposure to SWCNTs may cause irreversible obstructive airway disease. Nanotoxicogenomics uncovered novel mechanisms underlying SWCNT-induced lung diseases, implicating MMP12 and cathepsin K in the pathologic injury as potential biomarkers or therapeutic targets.

Molecular Elucidation of Biological Response to Mesoporous Silica Nanoparticles in Vitro and in Vivo

Biomedical applications of mesoporous silica nanoparticles (MSNs) require efficient cellular uptake and low toxicity. The purpose of this study is to investigate the cellular uptake and toxicity of MSNs with different sizes and charges (50, 100, and 250 nm with a positive surface charge and 100 nm with a negative surface charge) exposed to human monocyte-derived macrophages, lung epithelium BEAS-2B cells, and mice using genome-wide gene expression analysis and cellular/animal-level end point tests. We found that MSNs can be taken up into cells through endocytosis in a charge- and size-dependent manner, with positively charged and larger MSNs being more easily taken up into the cells by recruiting more types of endocytotic pathways for more cellular uptake. Moreover, the cytotoxicity of MSNs could be correlated with the amount of MSNs taken up by cells, which positively correlates to the particle size and dosage. Therefore, only positively charged and larger MSNs (≥100 nm) during higher treatment doses (≥500 μg mL-1) resulted in a sufficient accumulation of internalized MSNs in cells to induce significant release of reactive oxygen species (ROS) and oxidative stress, inflammatory gene upregulation through NF-κB and AP-1, and eventually autophagy-mediated necrotic cell death. Furthermore, genome-wide gene expression analysis could reflect the above in vitro cellular damages and corresponding in vivo injuries in mice, indicating that specific gene expression footprints may be used for assessing the safety of nanoparticles. The present finding provides some insights into the rational design of effective MSN-based drug/gene delivery systems and biomedical applications.

Aquatic toxicity assessment of single-walled carbon nanotubes using zebrafish embryos

Zebrafish embryos selected at the 64-cell stage were exposed to various concentrations of amide functionalized single-walled carbon nanotubes (SWCNTs) ranging from 1 to 10 μg/ml dissolved in 1% Pluronic F-68 (a cell culture grade surfactant), and the development of embryos was examined from 24 to 120 hours post fertilization (hpf). Incubation of embryos in 1% F-68 did not induce overt abnormal phenotype as compared to the wild-type; neither did it cause significant mortality during the exposure period. Generally, there was a slight developmental delay in larvae treated with SWCNTs of 5 μg/ml or above. Only larvae exposed to ≥ 5 μg/ml SWCNTs showed significantly reduced survival rates. About 50% of the embryos exposed to 5 μg/ml showed abnormal phenotypes at 24 hpf as compared to the control group. As development proceeds to 120 hpf, more embryos displayed defective morphology. A slight hatching delay was observed in embryos exposed to concentrations above 5 μg/ml. There was a general reduction of body axes, including narrowed somite and shortened yolk stalk. In addition, pigmentation in the ventral trunk area was less than that observed in control group. The body lengths of the exposed embryos were decreased significantly at 48 hpf (3.11 mm in control vs. 3.00 mm in SWCNTs-exposed embryos). However, exposure to SWCNTs did not affect the number of somites. Other features that were noticed in the SWCNTs-exposed embryos included edema and shrinkage and blebbling of the epidermal lining. Most of these observed phenotypes persisted from 48 hpf through 120 hpf. Overall, the aforementioned results indicate that soluble amide-functionalized SWCNTs are toxic to zebrafish embryos at a minimum concentration of 5 μg/ml.

Design and Fabrication of Spotted Long Oligonucleotide Microarrays for Gene Expression Analysis

DNA microarray technology has advanced rapidly since the first use of cDNA microarrays almost a decade ago. For gene expression studies on organisms, for which the genomes have been sequenced, cDNA microarrays are being gradually replaced by gene-specific oligonucleotide microarrays. Although, cDNA microarrays give higher signal intensity than oligonucleotide microarrays, they cannot be used for the measurement of gene-specific expression, whereas, oligonucleotide microarrays can. To obtain both a high signal intensity and specificity in gene expression measurements, gene-specific oligonucleotide probes as long as 150-mers, designed using sequence databases and algorithms to identify unique sequences of genes, are used as microarray probes. In order to achieve a high signal intensity, specificity, and accurate measurement of expression, in addition to the length and sequence of the probes, it is necessary to optimize other parameters such as the surface chemistry of the microarray slides, the addition of spacers and linkers to the probes, and the composition of the hybridization solution.