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.

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.

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 cm3 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.

Research Significance-Toxicological evidence suggests the potential for a wide range of health effects, which could result from exposure to carbon nanotubes (CNTs) and carbon nanofibers (CNFs). The National Institute for Occupational Safety and Health (NIOSH) has proposed a recommended exposure limit (REL) for CNTs/CNFs at the respirable size fraction. The current literature is lacking exposure information, with few studies reporting results for personal breathing zone (PBZ) samples in occupational settings. To address this gap, exposure assessments were conducted at six representative sites identified as CNT/CNF primary or secondary manufacturers.Methods-Personal and area filter-based samples were collected for both the inhalable mass concentration and the respirable mass concentration of elemental carbon (EC) as well as CNT structure count analysis by transmission electron microscopy to assess exposures. When possible, full-shift PBZ samples were collected; area samples were collected on a task-based approach.Results-The vast majority of samples collected in this study were below the proposed REL (7 μg m −3 ). Two of the three secondary manufacturers' surveyed found concentrations above the proposed REL. None of the samples collected at primary manufacturers were found to be above the REL. Visual and microscopy-based evidence of CNTs/CNFs were found at all sites, with the highest CNT/CNF structure counts being found in samples collected at secondary manufacturing sites. The statistical correlations between the filter-based samples for the mass concentration of EC and CNT structure counts were examined. A general trend was found with a P-value of 0.01 and a corresponding Pearson correlation coefficient of 0.44.Conclusions-CNT/CNF concentrations were above the proposed NIOSH REL for PBZ samples in two secondary manufacturing facilities that use these materials for commercial * Author to whom correspondence should be addressed. Tel: +1-513-458-7136; fax: +1-513-841-4486; mdahm@cdc.gov. Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the NIOSH. Mention of any company name or product does not constitute endorsement by NIOSH. applications. These samples were collected during dry powder handling processes, such as mixing and weighing, using fairly large quantities of CNTs/CNFs. HHS Public Access

Inhalable rather than respirable CNT/F was more consistently associated with fibrosis, inflammation, oxidative stress, and cardiovascular biomarkers.

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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.Electronic supplementary materialThe online version of this article (10.1186/s12989-018-0258-0) contains supplementary material, which is available to authorized users.

A classifier for separating conductive fibers according to length using dielectrophoresis has been developed and tested. The classifier consisted of two concentric steel cylinders with an annular space having a width of 3.18 mm. A high-voltage alternating field applied between the cylinders caused fibers to drift to the central cylinder. In these experiments, chrysotile fibers were generated from a fluidized bed generator and collected on the central cylinder in the classifier. Fibers collected on the central cylinder were washed off from ten equal length segments and sized by transmission electron microscopy. The median lengths of classified groups of fibers ranged from 8 to 31 p.m. The length distributions of these fibers had coefficients of variation ranging from 0.17 to 0.33. The quantity of classified fibers was limited by the aerosol flow rate, which in the current system was 2.13 L/min (total flow rate through the classifier was 5.35 L/min). Limitations in the classification process prevent increasing the fiber throughput significantly except by increasing the concentration of the challenge aerosol. Fiber conductivity is important for accurate classification, but can be readily achieved for fibers of low bulk conductivity by using high humidity air in the classifier.