Shih-Houng Young

Single-walled carbon nanotube-induced mitotic disruption

Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 μg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 μg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.

Cardiovascular Effects of Pulmonary Exposure to Single-Wall Carbon Nanotubes

Background Engineered nanosized materials, such as single-wall carbon nanotubes (SWCNT), are emerging as technologically important in different industries. Objective The unique physical characteristics and the pulmonary toxicity of SWCNTs raised concerns that respiratory exposure to these materials may be associated with cardiovascular adverse effects. Methods In these studies we evaluated aortic mitochondrial alterations by oxidative stress assays, including quantitative polymerase chain reaction of mitochondrial (mt) DNA and plaque formation by morphometric analysis in mice exposed to SWCNTs. Results A single intrapharyngeal instillation of SWCNTs induced activation of heme oxygenase-1 (HO-1), a marker of oxidative insults, in lung, aorta, and heart tissue in HO-1 reporter transgenic mice. Furthermore, we found that C57BL/6 mice, exposed to SWCNT (10 and 40 μg/mouse), developed aortic mtDNA damage at 7, 28, and 60 days after exposure. mtDNA damage was accompanied by changes in aortic mitochondrial glutathione and protein carbonyl levels. Because these modifications have been related to cardiovascular diseases, we evaluated whether repeated exposure to SWCNTs (20 μg/mouse once every other week for 8 weeks) stimulates the progression of atherosclerosis in ApoE−/− transgenic mice. Although SWCNT exposure did not modify the lipid profiles of these mice, it resulted in accelerated plaque formation in ApoE−/− mice fed an atherogenic diet. Plaque areas in the aortas, measured by the en face method, and in the brachiocephalic arteries, measured histopathologically, were significantly increased in the SWCNT-treated mice. This response was accompanied by increased mtDNA damage but not inflammation. Conclusions Taken together, the findings are of sufficient significance to warrant further studies to evaluate the systemic effects of SWCNT under workplace or environmental exposure paradigms.

Oxidative stress and inflammatory response in dermal toxicity of single-walled carbon nanotubes

Single-walled carbon nanotubes (SWCNT) represent a novel material with unique electronic and mechanical properties. The extremely small size ( approximately 1 nm diameter) renders their chemical and physical properties unique. A variety of different techniques are available for the production of SWCNT; however, the most common is via the disproportionation of gaseous carbon molecules supported on catalytic iron particles (high-pressure CO conversion, HiPCO). The physical nature of SWCNT may lead to dermal penetration following deposition on exposed skin. This dermal deposition provides a route of exposure which is important to consider when evaluating SWCNT toxicity. The dermal effects of SWCNT are largely unknown. We hypothesize that SWCNT may be toxic to the skin. We further hypothesize that SWCNT toxicity may be dependent upon the metal (particularly iron) content of SWCNT via the metals ability to interact with the skin, initiate oxidative stress, and induce redox-sensitive transcription factors thereby affecting/leading to inflammation. To test this hypothesis, the effects of SWCNT were assessed both in vitro and in vivo using EpiDerm FT engineered skin, murine epidermal cells (JB6 P+), and immune-competent hairless SKH-1 mice. Engineered skin exposed to SWCNT showed increased epidermal thickness and accumulation and activation of dermal fibroblasts which resulted in increased collagen as well as release of pro-inflammatory cytokines. Exposure of JB6 P+ cells to unpurified SWCNT (30% iron) resulted in the production of ESR detectable hydroxyl radicals and caused a significant dose-dependent activation of AP-1. No significant changes in AP-1 activation were detected when partially purified SWCNT (0.23% iron) were introduced to the cells. However, NFkappaB was activated in a dose-dependent fashion by exposure to both unpurified and partially purified SWCNT. Topical exposure of SKH-1 mice (5 days, with daily doses of 40 microg/mouse, 80 microg/mouse, or 160 microug/mouse) to unpurified SWCNT caused oxidative stress, depletion of glutathione, oxidation of protein thiols and carbonyls, elevated myeloperoxidase activity, an increase of dermal cell numbers, and skin thickening resulting from the accumulation of polymorphonuclear leukocytes (PMNs) and mast cells. Altogether, these data indicated that topical exposure to unpurified SWCNT, induced free radical generation, oxidative stress, and inflammation, thus causing dermal toxicity.

Molecular mechanism of tumor necrosis factor-alpha production in 1-->3-beta-glucan (zymosan)-activated macrophages.

The molecular details of 1→3-β-glucans, a fungal cell wall component, induced inflammatory responses are not well understood. In the present study, we conducted a systematic analysis of the molecular events leading to tumor necrosis factor (TNF)-α production after glucan stimulation of macrophages. We demonstrated that activation of nuclear factor κB (NF-κB) is essential in zymosan A (a source of 1→3-β-glucans)-induced TNF-α production in macrophages (RAW264.7 cells). Zymosan A-induced TNF-α protein production was associated with an increase in the TNF-α gene promoter activity. Activation of the TNF-α gene promoter was dependent on activation of NF-κB. Time course studies indicated that DNA binding activity of NF-κB preceded TNF-α promoter activity. Inhibition of NF-κB activation led to a dramatic reduction in both TNF-α promoter activity and TNF-α protein production in the response to zymosan A. Mutation of a major NF-κB binding site (κ3) in the gene promoter resulted in a significant decrease in the induction of the gene promoter by zymosan A, while mutation of Egr or CRE sites failed to inhibit the response to zymosan. Together, these results strongly suggest that NF-κB is involved in signal transduction of 1→3-β-glucans-induced TNF-α expression.

Multiwalled Carbon Nanotubes Induce a Fibrogenic Response by Stimulating Reactive Oxygen Species Production, Activating NF-κB Signaling, and Promoting Fibroblast-to-Myofibroblast Transformation

Carbon nanotubes (CNTs) are novel materials with unique electronic and mechanical properties. The extremely small size, fiberlike shape, large surface area, and unique surface chemistry render their distinctive chemical and physical characteristics and raise potential hazards to humans. Several reports have shown that pulmonary exposure to CNTs caused inflammation and lung fibrosis in rodents. The molecular mechanisms that govern CNT lung toxicity remain largely unaddressed. Here, we report that multiwalled carbon nanotubes (MWCNTs) have potent, dose-dependent toxicity on cultured human lung cells (BEAS-2B, A549, and WI38-VA13). Mechanistic analyses were carried out at subtoxic doses (≤20 μg/mL, ≤ 24 h). MWCNTs induced substantial ROS production and mitochondrial damage, implicating oxidative stress in cellular damage by MWCNT. MWCNTs activated the NF-κB signaling pathway in macrophages (RAW264.7) to increase the secretion of a panel of cytokines and chemokines (TNFα, IL-1β, IL-6, IL-10, and MCP1) that promote inflammation. Activation of NF-κB involved rapid degradation of IκBα, nuclear accumulation of NF-κBp65, binding of NF-κB to specific DNA-binding sequences, and transactivation of target gene promoters. Finally, MWCNTs induced the production of profibrogenic growth factors TGFβ1 and PDGF from macrophages that function as paracrine signals to promote the transformation of lung fibroblasts (WI38-VA13) into myofibroblasts, a key step in the development of fibrosis. Our results revealed that MWCNTs elicit multiple and intertwining signaling events involving oxidative damage, inflammatory cytokine production, and myofibroblast transformation, which potentially underlie the toxicity and fibrosis in human lungs by MWCNTs.

Titanium Dioxide (TiO2) Nanoparticles Induce JB6 Cell Apoptosis Through Activation of the Caspase-8/Bid and Mitochondrial Pathways

Titanium dioxide (TiO2), a commercially important material, is used in a wide variety of products. Although TiO2 is generally regarded as nontoxic, the cytotoxicity, pathogenicity, and carcinogenicity of TiO2 nanoparticles have been recently recognized. The present study investigated TiO2 nanoparticle-induced cell apoptosis and molecular mechanisms involved in this process in a mouse epidermal (JB6) cell line. Using the 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, TiO2 nanoparticles were found to exhibit higher cytotoxicity than fine particles. YO-PRO-1 iodide (YP) staining demonstrated that both TiO2 nanoparticles and fine particles induced cell death through apoptosis. The signaling pathways involved in TiO2 particle-induced apoptosis were investigated. Western-blot analysis showed an activation of caspase-8, Bid, BAX, and caspase-3 and a decrease of Bcl-2 in JB6 cells treated with TiO2 particles. Time-dependent poly(ADP)ribose polymerase (PARP) cleavage induced by TiO2 nanoparticles was observed. TiO2 particles also induced cytochrome c release from mitochondria to cytosol. Further studies demonstrated that TiO2 nanoparticles induced significant changes in mitochondrial membrane permeability, suggesting the involvement of mitochondria in the apoptotic process. In conclusion, evidence indicated that TiO2 nanoparticles exhibit higher cytotoxicity and apoptotic induction compared to fine particles in JB6 cells. Caspase-8/Bid and mitochondrial signaling may play a major role in TiO2 nanoparticle-induced apoptosis involving the intrinsic mitochondrial pathway. Unraveling the complex mechanisms associated with these events may provide further insights into TiO2 nanoparticle-induced pathogenicity and potential to induce carcinogenicity.

Factoring-in agglomeration of carbon nanotubes and nanofibers for better prediction of their toxicity versus asbestos

BackgroundCarbon nanotubes (CNT) and carbon nanofibers (CNF) are allotropes of carbon featuring fibrous morphology. The dimensions and high aspect ratio of CNT and CNF have prompted the comparison with naturally occurring asbestos fibers which are known to be extremely pathogenic. While the toxicity and hazardous outcomes elicited by airborne exposure to single-walled CNT or asbestos have been widely reported, very limited data are currently available describing adverse effects of respirable CNF.ResultsHere, we assessed pulmonary inflammation, fibrosis, oxidative stress markers and systemic immune responses to respirable CNF in comparison to single-walled CNT (SWCNT) and asbestos. Pulmonary inflammatory and fibrogenic responses to CNF, SWCNT and asbestos varied depending upon the agglomeration state of the particles/fibers. Foci of granulomatous lesions and collagen deposition were associated with dense particle-like SWCNT agglomerates, while no granuloma formation was found following exposure to fiber-like CNF or asbestos. The average thickness of the alveolar connective tissue - a marker of interstitial fibrosis - was increased 28 days post SWCNT, CNF or asbestos exposure. Exposure to SWCNT, CNF or asbestos resulted in oxidative stress evidenced by accumulations of 4-HNE and carbonylated proteins in the lung tissues. Additionally, local inflammatory and fibrogenic responses were accompanied by modified systemic immunity, as documented by decreased proliferation of splenic T cells ex vivo on day 28 post exposure. The accuracies of assessments of effective surface area for asbestos, SWCNT and CNF (based on geometrical analysis of their agglomeration) versus estimates of mass dose and number of particles were compared as predictors of toxicological outcomes.ConclusionsWe provide evidence that effective surface area along with mass dose rather than specific surface area or particle number are significantly correlated with toxicological responses to carbonaceous fibrous nanoparticles. Therefore, they could be useful dose metrics for risk assessment and management.

Direct effects of carbon nanotubes on dendritic cells induce immune suppression upon pulmonary exposure.

Pharyngeal aspiration of single-walled carbon nanotubes (SWCNTs) caused inflammation, pulmonary damage, and an altered cytokine network in the lung. Local inflammatory response in vivo was accompanied by modified systemic immunity as documented by decreased proliferation of splenic T cells. Preincubation of naïve T cells in vitro with SWCNT-treated dendritic cells reduced proliferation of T cells. Our data suggest that in vivo exposure to SWCNT modifies systemic immunity by modulating dendritic cell function.

Effect of stainless steel manual metal arc welding fume on free radical production, DNA damage, and apoptosis induction

Questions exist concerning the potential carcinogenic effects after welding fume exposure. Welding processes that use stainless steel (SS) materials can produce fumes that may contain metals (e.g., Cr, Ni) known to be carcinogenic to humans. The objective was to determine the effect of in vitro and in vivo welding fume treatment on free radical generation, DNA damage, cytotoxicity and apoptosis induction, all factors possibly involved with the pathogenesis of lung cancer. SS welding fume was collected during manual metal arc welding (MMA). Elemental analysis indicated that the MMA-SS sample was highly soluble in water, and a majority (87%) of the soluble metal was Cr. Using electron spin resonance (ESR), the SS welding fume had the ability to produce the biologically reactive hydroxyl radical (•OH), likely as a result of the reduction of Cr(VI) to Cr(V). In vitro treatment with the MMA-SS sample caused a concentration-dependent increase in DNA damage and lung macrophage death. In addition, a time-dependent increase in the number of apoptotic cells in lung tissue was observed after in vivo treatment with the welding fume. In summary, a soluble MMA-SS welding fume was found to generate reactive oxygen species and cause DNA damage, lung macrophage cytotoxicity and in vivo lung cell apoptosis. These responses have been shown to be involved in various toxicological and carcinogenic processes. The effects observed appear to be related to the soluble component of the MMA-SS sample that is predominately Cr. A more comprehensive in vivo animal study is ongoing in the laboratory that is continuing these experiments to try to elucidate the potential mechanisms that may be involved with welding fume-induced lung disease.

Identification of systemic markers from a pulmonary carbon nanotube exposure.

Objective: Interest exists for early monitoring of worker exposure to engineered nanomaterials. Here, we highlight quantitative systemic markers of early effects after carbon nanotube (CNT) exposure. Methods: Mice were exposed by pharyngeal aspiration to 40-&mgr;g CNT and harvested 24 hours, 7 days, and 28 days postexposure for measurements of whole blood, lung and extrapulmonary tissue gene expression, blood and bronchoalveolar lavage (BAL) differentials, and serum protein profiling. Results: Early effects included increased inflammatory blood gene expression and serum cytokines followed by an acute phase response (eg, CRP, SAA-1, SAP). Beyond 24 hours, there was a consistent increase in blood and BAL eosinophils. At 28 day, serum acute phase proteins with immune function including complement C3, apolipoproteins A-I and A-II, and &agr;2-macroglobulin were increased. Conclusions: Carbon nanotube exposure resulted in measurable systemic markers but lacked specificity to distinguish from other pulmonary exposures.