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. 2006 Nov 29:3:15.
doi: 10.1186/1743-8977-3-15.

Single-walled carbon nanotube (SWCNT)-induced interstitial fibrosis in the lungs of rats is associated with increased levels of PDGF mRNA and the formation of unique intercellular carbon structures that bridge alveolar macrophages in situ

James B Mangum  1 , Elizabeth A Turpin, Aurita Antao-Menezes, Mark F Cesta, Edilberto Bermudez, James C Bonner
Affiliations

Single-walled carbon nanotube (SWCNT)-induced interstitial fibrosis in the lungs of rats is associated with increased levels of PDGF mRNA and the formation of unique intercellular carbon structures that bridge alveolar macrophages in situ

James B Mangum et al. Part Fibre Toxicol. .

Abstract

Background: Nanotechnology is a rapidly advancing industry with many new products already available to the public. Therefore, it is essential to gain an understanding of the possible health risks associated with exposure to nanomaterials and to identify biomarkers of exposure. In this study, we investigated the fibrogenic potential of SWCNT synthesized by chemical vapor deposition using cobalt (Co) and molybdenum (Mo) as catalysts. Following a single oropharyngeal aspiration of SWCNT in rats, we evaluated lung histopathology, cell proliferation, and growth factor mRNAs at 1 and 21 days post-exposure. Comparisons were made to vehicle alone (saline containing a biocompatible nonionic surfactant), inert carbon black (CB) nanoparticles, or vanadium pentoxide (V2O5) as a known inducer of fibrosis.

Results: SWCNT or CB caused no overt inflammatory response at 1 or 21 days post-exposure as determined by histopathology and evaluation of cells (>95% macrophages) in bronchoalveolar lavage (BAL) fluid. However, SWCNT induced the formation of small, focal interstitial fibrotic lesions within the alveolar region of the lung at 21 days. A small fraction of alveolar macrophages harvested by BAL from the lungs of SWCNT-exposed rats at 21 days were bridged by unique intercellular carbon structures that extended into the cytoplasm of each macrophage. These "carbon bridge" structures between macrophages were also observed in situ in the lungs of SWCNT-exposed rats. No carbon bridges were observed in CB-exposed rats. SWCNT caused cell proliferation only at sites of fibrotic lesion formation as measured by bromodeoxyuridine uptake into alveolar cells. SWCNT increased platelet-derived growth factor (PDGF)-A, PDGF-B, and PDGF-C mRNA levels significantly at 1 day as measured by Taqman quantitative real-time RT-PCR. At 21 days, SWCNT did not increase any mRNAs evaluated, while V2O5 significantly increased mRNAs encoding PDGF-A, -B, and -C chains, PDGF-R alpha, osteopontin (OPN), connective tissue growth factor (CTGF), and transforming growth factor (TGF)-beta1.

Conclusion: Our findings indicate that SWCNT do not cause lung inflammation and yet induce the formation of small, focal interstitial fibrotic lesions in the alveolar region of the lungs of rats. Of greatest interest was the discovery of unique intercellular carbon structures composed of SWCNT that bridged lung macrophages. These "carbon bridges" offer a novel and easily identifiable biomarker of exposure.

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Figures

Figure 1
Figure 1
Lung histopathology from rats 21 days after a single oropharyngeal aspiration exposure to saline vehicle containing 0.1% Pluronic F-68 (A, B), carbon black ultrafine particles (C, D), aggregates of single walled carbon nanotubes, SWCNT (E, F), or vanadium pentoxide (V2O5) (G, H). Carbon inclusions are indicated by closed arrows in panels C, D, and F. Regions of alveolar wall thickening in the lungs of rats exposed to SWCNT are indicated by open arrows adjacent to carbon-filled macrophages in panesl E and F. Original magnification 20× (panels A, C, E, G) or 40× (panels B, D, F, H).
Figure 2
Figure 2
Cytospins of bronchoalveolar lavage (BAL) cells from rats exposed to for 21 days to carbon black ultrafine particles (A–C) or single wall carbon nanotubes, SWCNT (D–F). Carbon inclusions are indicated by arrows. Carbon bridges between macrophages formed only in SWCNT-exposed rats. Original magnification (40×), inset (80×).
Figure 3
Figure 3
Quantitation of BAL cells in cytospins for assessment of carbon bridge formation between macrophages from 21 day SWCNT-exposed or carbon black (CB)-exposed rats. Each bar represents the mean ± SD of five animals. Ten fields were counted per animal and at least 500 cells per animal were counted. (A) Total cells/field, (B) Numbers of cells per field containing carbon inclusions, (C) Carbon bridges per field (only found in SWCNT-exposed rats), and (D) Mitotic figures per field.
Figure 4
Figure 4
Bromodeoxyuridine (BrdU) immunohistochemistry showing a cell proliferative responses 21 days post-exposure in the lung alveolar region (panels A, C, E, G) and in the bronchus-associated lymphoid tissue (panels B, D, F, H). Saline vehicle containing 0.1% Pluronic F-68. (A, B). Carbon black treatment (C, D). SWCNT treatment (E, F). Vanadium pentoxide (V2O5) treatment (G, H). Solid arrows in panels C and E indicate aggregates of carbon black and SWCNT, respectively. Inset shows two macrophages joined by a carbon bridge within a nearby alveolar space. Open arrows indicate representative BrdU-positive nuclei. Original magnification for all panels, 40×.
Figure 5
Figure 5
Cell proliferation at (A) day 1 and (B) day 21 post-exposure in the lungs of mice exposed by oropharyngeal aspiration to saline vehicle containing 0.1% Pluronic F-68, carbon black (CB), single-walled carbon nanotubes (SWCNT) or vanadium pentoxide (V2O5). Open bars represent the labeling index in the alveolar region of the lung (open bars) and solid bars represent the labeling index in the bronchus-associated lymphatic tissue. Labelling indices were measured as described in Methods and are reported as the percentage of BrdU-labeled cells of the cells counted (minimum of 400 cells counted). *P < 0.05 compared to saline control.
Figure 6
Figure 6
Levels of growth factor and procollagen mRNAs in the lungs of rats exposed to SWCNT, CB, or V2O5. Whole lung RNA was extracted as described in Methods and mRNA expression measured by quantitative real-time RT-PCR. V2O5 was used as a positive control for inducing mRNA levels of profibrogenic mediators. (A) 1 day post-exposure. (B) 21 days post-exposure. Control animals were treated with vehicle alone (PBS with 0.1% Pluronic F-68). Data are presented as mean values ± SEM from three to five animals per group and expressed as the fold-change in mRNA relative to an 18S rRNA housekeeping gene. *P < 0.05 significantly increased or †P < 0.05 significantly decreased compared to control.
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