Joseph E. Fernback

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.

Carbon nanotube dosimetry: from workplace exposure assessment to inhalation toxicology

BackgroundDosimetry for toxicology studies involving carbon nanotubes (CNT) is challenging because of a lack of detailed occupational exposure assessments. Therefore, exposure assessment findings, measuring the mass concentration of elemental carbon from personal breathing zone (PBZ) samples, from 8 U.S.-based multi-walled CNT (MWCNT) manufacturers and users were extrapolated to results of an inhalation study in mice.ResultsUpon analysis, an inhalable elemental carbon mass concentration arithmetic mean of 10.6 μg/m3 (geometric mean 4.21 μg/m3) was found among workers exposed to MWCNT. The concentration equates to a deposited dose of approximately 4.07 μg/d in a human, equivalent to 2 ng/d in the mouse. For MWCNT inhalation, mice were exposed for 19 d with daily depositions of 1970 ng (equivalent to 1000 d of a human exposure; cumulative 76 yr), 197 ng (100 d; 7.6 yr), and 19.7 ng (10 d; 0.76 yr) and harvested at 0, 3, 28, and 84 d post-exposure to assess pulmonary toxicity. The high dose showed cytotoxicity and inflammation that persisted through 84 d after exposure. The middle dose had no polymorphonuclear cell influx with transient cytotoxicity. The low dose was associated with a low grade inflammatory response measured by changes in mRNA expression. Increased inflammatory proteins were present in the lavage fluid at the high and middle dose through 28 d post-exposure. Pathology, including epithelial hyperplasia and peribronchiolar inflammation, was only noted at the high dose.ConclusionThese findings showed a limited pulmonary inflammatory potential of MWCNT at levels corresponding to the average inhalable elemental carbon concentrations observed in U.S.-based CNT facilities and estimates suggest considerable years of exposure are necessary for significant pathology to occur at that level.

Carbon Nanotube and Nanofiber Exposure Assessments: An Analysis of 14 Site Visits

Recent evidence has suggested the potential for wide-ranging health effects that could result from exposure to carbon nanotubes (CNT) and carbon nanofibers (CNF). In response, the National Institute for Occupational Safety and Health (NIOSH) set a recommended exposure limit (REL) for CNT and CNF: 1 µg m(-3) as an 8-h time weighted average (TWA) of elemental carbon (EC) for the respirable size fraction. The purpose of this study was to conduct an industrywide exposure assessment among US CNT and CNF manufacturers and users. Fourteen total sites were visited to assess exposures to CNT (13 sites) and CNF (1 site). Personal breathing zone (PBZ) and area samples were collected for both the inhalable and respirable mass concentration of EC, using NIOSH Method 5040. Inhalable PBZ samples were collected at nine sites while at the remaining five sites both respirable and inhalable PBZ samples were collected side-by-side. Transmission electron microscopy (TEM) PBZ and area samples were also collected at the inhalable size fraction and analyzed to quantify and size CNT and CNF agglomerate and fibrous exposures. Respirable EC PBZ concentrations ranged from 0.02 to 2.94 µg m(-3) with a geometric mean (GM) of 0.34 µg m(-3) and an 8-h TWA of 0.16 µg m(-3). PBZ samples at the inhalable size fraction for EC ranged from 0.01 to 79.57 µg m(-3) with a GM of 1.21 µg m(-3). PBZ samples analyzed by TEM showed concentrations ranging from 0.0001 to 1.613 CNT or CNF-structures per cm(3) with a GM of 0.008 and an 8-h TWA concentration of 0.003. The most common CNT structure sizes were found to be larger agglomerates in the 2-5 µm range as well as agglomerates >5 µm. A statistically significant correlation was observed between the inhalable samples for the mass of EC and structure counts by TEM (Spearman ρ = 0.39, P < 0.0001). Overall, EC PBZ and area TWA samples were below the NIOSH REL (96% were <1 μg m(-3) at the respirable size fraction), while 30% of the inhalable PBZ EC samples were found to be >1 μg m(-3). Until more information is known about health effects associated with larger agglomerates, it seems prudent to assess worker exposure to airborne CNT and CNF materials by monitoring EC at both the respirable and inhalable size fractions. Concurrent TEM samples should be collected to confirm the presence of CNT and CNF.

Single-Walled Carbon Nanotubes Induce Fibrogenic Effect by Disturbing Mitochondrial Oxidative Stress and Activating NF-κB Signaling

Single-walled carbon nanotubes (SWCNTs) are newly discovered material of crystalline carbon that forms single-carbon layer cylinders with nanometer diameters and varying lengths. Although SWCNTs are potentially suitable for a range of novel applications, their extremely small size, fiber-like shape, large surface area, and unique surface chemistry raise potential hazard to humans, including lung toxicity and fibrosis. The molecular mechanisms by which SWCNTs cause lung damage remain elusive. Here we show that SWCNTs dose and time-dependently caused toxicity in cultured human bronchial epithelial (BEAS-2B), alveolar epithelial (A549), and lung fibroblast (WI38) cells. At molecular levels, SWCNTs induced significant mitochondrial depolarization and ROS production at subtoxic doses. SWCNTs stimulated the secretion of proinflammatory cytokines and chemokines TNFα, IL-1β, IL-6, IL-10 and MCP1 from macrophages (Raw 264.7), which was attributed to the activation of the canonical signaling pathway of NF-κB by SWCNT. Finally, SWCNTs stimulated profibrogenic growth factors TGFβ1 production and fibroblast-to-myofibroblast-transformation. These results indicate that SWCNTs has a potential to induce human lung damage and fibrosis by damaging mitochondria, generating ROS, and stimulating production of proinflammatory and profibrogenic cytokines and growth factors.

Airborne fiber size characterization in exposure estimation: Evaluation of a modified transmission electron microcopy protocol for asbestos and potential use for carbon nanotubes and nanofibers.

BACKGROUND Airborne fiber size has been shown to be an important factor relative to adverse lung effects of asbestos and suggested in animal studies of carbon nanotubes and nanofibers (CNT/CNF). MATERIALS AND METHODS The International Standards Organization (ISO) transmission electron microscopy (TEM) method for asbestos was modified to increase the statistical precision of fiber size determinations, improve efficiency, and reduce analysis costs. Comparisons of the fiber size distributions and exposure indices by laboratory and counting method were performed. RESULTS No significant differences in size distributions by the ISO and modified ISO methods were observed. Small but statistically-significant inter-lab differences in the proportion of fibers in some size bins were found, but these differences had little impact on the summary exposure indices. The modified ISO method produced slightly more precise estimates of the long fiber fraction (>15 μm). CONCLUSIONS The modified ISO method may be useful for estimating size-specific structure exposures, including CNT/CNF, for risk assessment research.

Presence of Airborne Fibers in Tungsten Refining and Manufacturing Processes: Preliminary Characterization

In tungsten refining and manufacturing processes, a series of tungsten oxides (WO X ) are typically formed as intermediates in the production of tungsten powder. Studies in the Swedish tungsten refining and manufacturing industry have shown that intermediate tungsten refining processes can create WOX fibers. The purpose of the present study was to identify and provide a preliminary characterization of airborne tungsten-containing fiber dimensions, elemental composition, and concentrations in the U.S. tungsten refining and manufacturing industry. To provide the preliminary characterization, 10 static air samples were collected during the course of normal employee work activities and analyzed using standard fiber sampling and counting methods. Results from transmission electron microscopy analyses conducted indicate that airborne fibers with length > 0.5 μ m, diameter > 0.01 μ m, and aspect ratio ≥ 3:1, with a geometric mean (GM) length of ∼ 2.0 μ m and GM diameter of ∼ 0.25 μ m, were present on 9 of the 10 air samples collected. Energy dispersive X-ray spectrometry results indicate that airborne fibers prior to the carburization process consisted primarily of tungsten and oxygen, with other elements being detected in trace quantities. Results from an air sample collected at the carburization process indicated the presence of fibers composed primarily of tungsten with oxygen and carbon, and traces of other elements. Based on National Institute for Occupational Safety and Health standard fiber counting rules, airborne fiber concentrations ranged from below the limit of detection to 0.14 f/cm 3 . The calcining process was associated with the highest airborne fiber concentrations. More than 99% (574/578) of the airborne fibers identified had an aerodynamic diameter ≤10 μ m, indicating that they were capable of reaching the thoracic regions. Until more is known about the durability and potential health effects associated with airborne tungsten-containing fibers, it would be prudent to take steps to limit or eliminate occupational exposures.

Workplace Monitoring of Airborne Carbon Nanomaterials by HRTEM

Growing production and use of carbon nanomaterials may pose health risks for exposed workers. The National Institute for Occupational Safety and Health (NIOSH) set a recommended exposure limit (REL) for carbon nanotubes and nanofibers (CNT and CNF): an 8-hr time weighted average (TWA) of 1 μg/m as respirable elemental carbon (EC) [1]. As other EC sources may interfere, complementary techniques (e.g., metals, organics, and microscopy analyses) have been used to better characterize exposure [1-4]. High resolution transmission electron microscopy (HRTEM) is especially useful because it provides visualization of particle size, shape, structure, and agglomeration state.

Association of pulmonary, cardiovascular, and hematologic metrics with carbon nanotube and nanofiber exposure among U.S. workers: a cross-sectional study

BackgroundCommercial use of carbon nanotubes and nanofibers (CNT/F) in composites and electronics is increasing; however, little is known about health effects among workers. We conducted a cross-sectional study among 108 workers at 12 U.S. CNT/F facilities. We evaluated chest symptoms or respiratory allergies since starting work with CNT/F, lung function, resting blood pressure (BP), resting heart rate (RHR), and complete blood count (CBC) components.MethodsWe conducted multi-day, full-shift sampling to measure background-corrected elemental carbon (EC) and CNT/F structure count concentrations, and collected induced sputum to measure CNT/F in the respiratory tract. We measured (nonspecific) fine and ultrafine particulate matter mass and count concentrations. Concurrently, we conducted physical examinations, BP measurement, and spirometry, and collected whole blood. We evaluated associations between exposures and health measures, adjusting for confounders related to lifestyle and other occupational exposures.ResultsCNT/F air concentrations were generally low, while 18% of participants had evidence of CNT/F in sputum. Respiratory allergy development was positively associated with inhalable EC (p=0.040) and number of years worked with CNT/F (p=0.008). No exposures were associated with spirometry-based metrics or pulmonary symptoms, nor were CNT/F-specific metrics related to BP or most CBC components. Systolic BP was positively associated with fine particulate matter (p-values: 0.015-0.054). RHR was positively associated with EC, at both the respirable (p=0.0074) and inhalable (p=0.0026) size fractions. Hematocrit was positively associated with the log of CNT/F structure counts (p=0.043).ConclusionsMost health measures were not associated with CNT/F. The positive associations between CNT/F exposure and respiratory allergies, RHR, and hematocrit counts may not be causal and require examination in other studies.

Exposure assessments for a cross-sectional epidemiologic study of US carbon nanotube and nanofiber workers

BACKGROUND Recent animal studies have suggested the potential for wide-ranging health effects resulting from exposure to carbon nanotubes and nanofibers (CNT/F). To date, no studies in the US have directly examined the relationship between occupational exposure and potential human health effects. OBJECTIVES Our goal was to measure CNT/F exposures among US workers with representative job types, from non-exposed to highly exposed, for an epidemiologic study relating exposure to early biologic effects. METHODS 108 participants were enrolled from 12 facilities across the US. Personal, full-shift exposures were assessed based on the mass of elemental carbon (EC) at the respirable and inhalable aerosol particle size fractions, along with quantitatively characterizing CNT/F and estimating particle size via transmission electron microscopy (TEM). Additionally, sputum and dermal samples were collected and analyzed to determine internal exposures and exposures to the hands/wrists. RESULTS The mean exposure to EC was 1.00 μg/m3 at the respirable size fraction and 6.22 μg/m3 at the inhalable fraction. Analysis by TEM found a mean exposure of 0.1275 CNT/F structures/cm3, generally to agglomerated materials between 2 and 10 μm. Internal exposures to CNT/F via sputum analysis were confirmed in 18% of participants while ∼70% had positive dermal exposures. CONCLUSIONS We demonstrated the occurrence of a broad range of exposures to CNT/F within 12 facilities across the US. Analysis of collected sputum indicated internal exposures are currently occurring within the workplace. This is an important first step in determining if exposures in the workforce have any acute or lasting health effects.

In vivo formation of Ce-phosphate Nanoparticles following Intratracheal Instillation of CeCl3: Subcellular sites, Nanostructures, Precipitation Mechanisms and Nanoparticle 3D-Alignment

We demonstrate the in vivo formation of nano-particulate Ce-containing structures in lungs instilled with 5 mg/kg CeCl3 using high resolution electron microscopy (HRTEM), energy loss spectroscopy (EELS), and elemental mapping (EDS). The observed high lung retention of Ce after instillation of CeCl3 (75-92% retained at 28 days) is unexpected since metal ions are usually readily transported across the air-blood barrier. The binding of cerium ions to lung constituents has been suggested [1], and the formation of cerium phosphate has been shown previously, but without discussions on the mechanisms involved in nanoparticle nucleation and growth [2]. Determining the form of Ce after lung exposure to CeCl3 has been challenging due to the difficulties in distinguishing ions from particulate forms when using radioactivity or ICP-MS. We have identified cerium nanoparticles at the sub-cellular level in lung macrophages after CeCl3 instillation. This observation provides insights on the cell structures and components that will help distinguish which cellular areas are the sites of in vivo nanoparticle formation.