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Summary Carbon nanotubes have fibre-like shape1 and stimulate inflammation at the surface of the peritoneum when injected into the abdominal cavity of mice2, raising concerns that inhaled nanotubes3 may cause pleural fibrosis and/or mesothelioma4. Here we show that multi-walled carbon nanotubes reach the sub-pleura in mice after a single inhalation exposure of 30 mg/m3 for 6 hours. Nanotubes were embedded in the sub-pleural wall and within sub-pleural macrophages. Mononuclear cell aggregates on the pleural surface increased in number and size after 1 day and nanotube-containing macrophages were observed within these foci. Sub-pleural fibrosis increased after 2 and 6 weeks following inhalation. None of these effects were seen in mice that inhaled carbon black nanoparticles or a lower dose of nanotubes (1 mg/m3). This work advances a growing literature on pulmonary toxicology of nanotubes5 and suggests that minimizing inhalation of nanotubes during handling is prudent until further long term assessments are conducted.

We developed a dispersal method for multi-walled carbon nanotubes (MWCNTs) that allows quantitative assessment of dispersion on pro-fibrogenic responses in tissue culture cells as well as in mouse lung. Here we demonstrate that the dispersal of as-prepared (AP), purified (PD), and carboxylated (COOH) MWCNTs by bovine serum albumin (BSA) and dipalmitoylphosphatidylcholine (DPPC) influences TGF-β1, PDGF-AA and IL-1β production in vitro and in vivo. These biomarkers were chosen based on their synergy in promoting fibrogenesis and cellular communication in the epithelial-mesenchymal cell trophic unit in the lung. The effect of dispersal was most noticeable in AP- and PD-MWCNTs, which are more hydrophobic and unstable in aqueous buffers than hydrophilic COOH-MWCNTs. Well-dispersed AP- and PD-MWCNTs were readily taken up by BEAS-2B, THP-1 cells and alveolar macrophages (AM), and induced more prominent TGF-β1 and IL-1β production in vitro as well as TGF-β1, IL-1β and PDGF-AA production in vivo than non-dispersed tubes. Moreover, there was good agreement between the pro-fibrogenic responses in vitro and in vivo as well as the ability of dispersed tubes to generate granulomatous inflammation and fibrosis in airways. Tube dispersal also elicited more robust IL-1β production in THP-1 cells. While COOH-MWCNTs were poorly taken up in BEAS-2B and induced little TGF-β1 production, they were bio-processed by AM and induced less prominent collagen deposition at sites of non-granulomatous inflammation in the alveolar region. Taken together, these results indicate that the dispersal state of MWCNTs affects pro-fibrogenic cellular responses that correlate with the extent of pulmonary fibrosis and are of potential use to predict pulmonary toxicity.

Carbon nanotubes are gaining increasing attention due to possible health risks from occupational or environmental exposures. This study tested the hypothesis that inhaled multiwalled carbon nanotubes (MWCNT) would increase airway fibrosis in mice with allergic asthma. Normal and ovalbumin-sensitized mice were exposed to a MWCNT aerosol (100 mg/m(3)) or saline aerosol for 6 hours. Lung injury, inflammation, and fibrosis were examined by histopathology, clinical chemistry, ELISA, or RT-PCR for cytokines/chemokines, growth factors, and collagen at 1 and 14 days after inhalation. Inhaled MWCNT were distributed throughout the lung and found in macrophages by light microscopy, but were also evident in epithelial cells by electron microscopy. Quantitative morphometry showed significant airway fibrosis at 14 days in mice that received a combination of ovalbumin and MWCNT, but not in mice that received ovalbumin or MWCNT only. Ovalbumin-sensitized mice that did not inhale MWCNT had elevated levels IL-13 and transforming growth factor (TGF)-beta1 in lung lavage fluid, but not platelet-derived growth factor (PDGF)-AA. In contrast, unsensitized mice that inhaled MWCNT had elevated PDGF-AA, but not increased levels of TGF-beta1 and IL-13. This suggested that airway fibrosis resulting from combined ovalbumin sensitization and MWCNT inhalation requires PDGF, a potent fibroblast mitogen, and TGF-beta1, which stimulates collagen production. Combined ovalbumin sensitization and MWCNT inhalation also synergistically increased IL-5 mRNA levels, which could further contribute to airway fibrosis. These data indicate that inhaled MWCNT require pre-existing inflammation to cause airway fibrosis. Our findings suggest that individuals with pre-existing allergic inflammation may be susceptible to airway fibrosis from inhaled MWCNT.

Background: Differences in interlaboratory research protocols contribute to the conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity.Objectives: Grantees of a National Institute of Health Sciences (NIEHS)-funded consortium program performed two phases of in vitro testing with selected ENMs in an effort to identify and minimize sources of variability.Methods: Consortium program participants (CPPs) conducted ENM bioactivity evaluations on zinc oxide (ZnO), three forms of titanium dioxide (TiO2), and three forms of multiwalled carbon nanotubes (MWCNTs). In addition, CPPs performed bioassays using three mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1) selected in order to cover two different species (rat and human), two different lung epithelial cells (alveolar type II and bronchial epithelial cells), and two different cell types (epithelial cells and macrophages). CPPs also measured cytotoxicity in all cell types while measuring inflammasome activation [interleukin-1β (IL-1β) release] using only THP-1 cells.Results: The overall in vitro toxicity profiles of ENM were as follows: ZnO was cytotoxic to all cell types at ≥ 50 μg/mL, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells. MWCNTs did not produce cytotoxicity, but stimulated lower levels of IL-1β production in THP-1 cells, with the original MWCNT producing the most IL-1β.Conclusions: The results provide justification for the inclusion of mechanism-linked bioactivity assays along with traditional cytotoxicity assays for in vitro screening. In addition, the results suggest that conducting studies with multiple relevant cell types to avoid false-negative outcomes is critical for accurate evaluation of ENM bioactivity.

Exposure to urban airborne particulate matter (PM) is associated with adverse health effects. We previously reported that the cytotoxic and proinflammatory effects of Mexico City PM10 (less than or equal to 10 micro m mean aerodynamic diameter) are determined by transition metals and endotoxins associated with these particles. However, PM2.5 (less than or equal to 2.5 micro m mean aerodynamic diameter) could be more important as a human health risk because this smaller PM has the potential to reach the distal lung after inhalation. In this study, we compared the cytotoxic and proinflammatory effects of Mexico City PM10 with those of PM2.5 using the murine monocytic J774A.1 cell line in vitro. PMs were collected from the northern zone or the southeastern zone of Mexico City. Elemental composition and bacterial endotoxin on PMs were measured. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production by J774A.1 cells was measured in the presence or absence of recombinant endotoxin-neutralizing protein (rENP). Both northern and southeastern PMs contained endotoxin and a variety of transition metals. Southeastern PM10 contained the highest endotoxin levels, 2-fold higher than that in northern PM10. Northern and southeastern PM2.5 contained the lowest endotoxin levels. Accordingly, southeastern PM10 was the most potent in causing secretion of the proinflammatory cytokines TNF-alpha and IL-6. All PM2.5 and PM10 samples caused cytotoxicity, but northern PMs were the most toxic. Cytokine secretion induced by southeastern PM10 was reduced 50-75% by rENP. These results indicate major differences in PM10 and PM2.5. PM2.5 induces cytotoxicity in vitro through an endotoxin-independent mechanism that is likely mediated by transition metals. In contrast, PM10 with relatively high levels of endotoxin induces proinflammatory cytokine release via an endotoxin-dependent mechanism.

Because of their unique physicochemical properties, engineered nanoparticles have the potential to significantly impact respiratory research and medicine by means of improving imaging capability and drug delivery, among other applications. These same properties, however, present potential safety concerns, and there is accumulating evidence to suggest that nanoparticles may exert adverse effects on pulmonary structure and function. The respiratory system is susceptible to injury resulting from inhalation of gases, aerosols, and particles, and also from systemic delivery of drugs, chemicals, and other compounds to the lungs via direct cardiac output to the pulmonary arteries. As such, it is a prime target for the possible toxic effects of engineered nanoparticles. The purpose of this article is to provide an overview of the potential usefulness of nanoparticles and nanotechnology in respiratory research and medicine and to highlight important issues and recent data pertaining to nanoparticle-related pulmonary toxicity.

Lung carcinomas and pulmonary fibrosis (asbestosis) occur in asbestos workers. Understanding the pathogenesis of these diseases is complicated because of potential confounding factors, such as smoking, which is not a risk factor in mesothelioma. The modes of action (MOA) of various types of asbestos in the development of lung cancers, asbestosis, and mesotheliomas appear to be different. Moreover, asbestos fibers may act differentially at various stages of these diseases, and have different potencies as compared to other naturally occurring and synthetic fibers. This literature review describes patterns of deposition and retention of various types of asbestos and other fibers after inhalation, methods of translocation within the lung, and dissolution of various fiber types in lung compartments and cells in vitro. Comprehensive dose-response studies at fiber concentrations inhaled by humans as well as bivariate size distributions (lengths and widths), types, and sources of fibers are rarely defined in published studies and are needed. Species-specific responses may occur. Mechanistic studies have some of these limitations, but have suggested that changes in gene expression (either fiber-catalyzed directly or by cell elaboration of oxidants), epigenetic changes, and receptor-mediated or other intracellular signaling cascades may play roles in various stages of the development of lung cancers or asbestosis.