BackgroundMulti-walled carbon nanotubes (MWCNTs) are new manufactured nanomaterials with a wide spectrum of commercial applications. To address the hypothesis that MWCNTs cause persistent pulmonary pathology, C57BL/6J mice were exposed by pharyngeal aspiration to 10, 20, 40 or 80 μg of MWCNTs (mean dimensions of 3.9 μm × 49 nm) or vehicle. Lungs were preserved at 1, 7, 28 and 56 days post- exposure to determine the potential regions and target cells for impact by MWCNT lung burden. Morphometric measurement of Sirius Red staining was used to assess the connective tissue response.ResultsAt 56 days post-exposure, 68.7 ± 3.9, 7.5 ± 1.9 and 22.0 ± 5.1 percent (mean ± SE, N = 8) of the MWCNT lung burden were in alveolar macrophages, alveolar tissue and granulomatous lesions, respectively. The subpleural tissues contained 1.6% of the MWCNT lung burden. No MWCNTs were found in the airways at 7, 28 or 56 days after aspiration The connective tissue in the alveolar interstitium demonstrated a progressive increase in thickness over time in the 80 μg exposure group (0.12 ± 0.01, 0.12 ± 0.01, 0.16 ± 0.01 and 0.19 ± 0.01 μm for 1, 7, 28 and 56 days post-exposure (mean ± SE, N = 8)). Dose-response determined at 56 days post-exposure for the average thickness of connective tissue in alveolar septa was 0.11 ± 0.01, 0.14 ± .02, 0.14 ± 0.01, 0.16 ± 0.01 and 0.19 ± 0.01 μm (mean ± SE, N = 8) for vehicle, 10, 20, 40 and 80 μg dose groups, respectively.ConclusionsThe distribution of lung burden was predominately within alveolar macrophages with approximately 8% delivery to the alveolar septa, and a smaller but potentially significant burden to the subpleural tissues. Despite the relatively low fraction of the lung burden being delivered to the alveolar tissue, the average thickness of connective tissue in the alveolar septa was increased over vehicle control by 45% in the 40 μg and 73% in the 80 μg exposure groups. The results demonstrate that MWCNTs have the potential to produce a progressive, fibrotic response in the alveolar tissues of the lungs. However, the increases in connective tissue per μg dose of MWCNTs to the interstitium are significantly less than those previously found for single-walled carbon nanotubes (SWCNTs).

BackgroundPrior studies have demonstrated a rapid and progressive acute phase response to bolus aspiration of multi-walled carbon nanotubes (MWCNTs). In this study we sought to test the hypothesis that inhalation exposure to MWCNT produces a fibrotic response and that the response is chronically persistent. To address the hypothesis that inhaled MWCNTs cause persistent morphologic changes, male C57BL/6 J mice were exposed in a whole-body inhalation system to a MWCNT aerosol and the fibrotic response in the alveolar region examined at up to 336 days after termination of exposure.MethodsInhalation exposure was to a 5 mg/m3 MWCNT aerosol for 5 hours/day for 12 days (4 times/week for 3 weeks). At the end of inhalation exposures, lungs were either lavaged for analysis of bronchoalveolar lavage (BAL) or preserved by vascular perfusion of fixative while inflated with air at 1, 14, 84, 168 and 336 days post inhalation exposure. Separate, clean-air control groups were also studied. Light microscopy, enhanced darkfield microscopy and field emission electron microscopy (FESEM) of tissue sections were used to analyze the distribution of lung burden following inhalation exposure. Morphometric measurements of Sirius Red staining for fibrillar collagen were used to assess the connective tissue response. Serial section analysis of enhanced darkfield microscope images was used to examine the redistribution of MWCNT fibers within the lungs during the post-exposure period.ResultsAt day 1 post-exposure 84 ± 3 and 16 ± 2 percent of the lung burden (Mean ± S.E., N = 5) were in the alveolar and airway regions, respectively. Initial distribution within the alveolar region was 56 ± 5, 7 ± 4 and 20 ± 3 percent of lung burden in alveolar macrophages, alveolar airspaces and alveolar tissue, respectively. Clearance reduced the alveolar macrophage burden of MWCNTs by 35 percent between 1 and 168 days post-exposure, while the content of MWCNTs in the alveolar tissue increased by 63 percent. Large MWCNT structures containing greater than 4 fibers were 53.6 percent of the initial lung burden and accounted for the majority of the decline with clearance, while lung burden of singlet MWCNT was essentially unchanged. The mean linear intercept of alveolar airspace, a measure of the expansion of the lungs, was not significantly different between groups. Pulmonary inflammation and damage, measured as the number of polymorphnuclear leukocytes (PMNs) or lactate dehydrogenase activity (LDH) and albumin in BAL, increased rapidly (1 day post) after inhalation of MWCNTs and declined slowly with time post-exposure. The fibrillar collagen in the alveolar region of MWCNT-exposed mice demonstrated a progressive increase in thickness over time (0.17 ± 0.02, 0.22 ± 0.02, 0.26 ± 0.03, 0.25 ± 0.02 and 0.29 ± 0.01 microns for 1, 14, 84, 168 and 336 days post-exposure) and was significantly different from clean-air controls (0.16 ± 0.02) at 84 and (0.15 ± 0.02) at 336 days post-exposure.ConclusionsDespite the relatively low fraction of the lung burden being delivered ...

Engineered carbon nanotubes are newly emerging manufactured particles with potential applications in electronics, computers, aerospace, and medicine. The low density and small size of these biologically persistent particles makes respiratory exposures to workers likely during the production or use of commercial products. The narrow diameter and great length of single-walled carbon nanotubes (SWCNT) suggest the potential to interact with critical biological structures. To examine the potential of nanotubes to induce genetic damage in normal lung cells, cultured primary and immortalized human airway epithelial cells were exposed to SWCNT or a positive control, vanadium pentoxide. After 24 hr of exposure to either SWCNT or vanadium pentoxide, fragmented centrosomes, multiple mitotic spindle poles, anaphase bridges, and aneuploid chromosome number were observed. Confocal microscopy demonstrated nanotubes within the nucleus that were in association with cellular and mitotic tubulin as well as the chromatin. Our results are the first to report disruption of the mitotic spindle by SWCNT. The nanotube bundles are similar to the size of microtubules that form the mitotic spindle and may be incorporated into the mitotic spindle apparatus.

The ability of a cell to move requires the asymmetrical organization of cellular activities. To investigate polarized cellular activity in moving endothelial cells, human endothelial cells were incubated in a Dunn chamber to allow migration toward vascular endothelial growth factor. Immunofluorescent staining with a specific antibody against caveolin-1 revealed that caveolin-1 was concentrated at the rear of moving cells. Similarly, monolayer scraping to induce random cell walk resulted in relocation of caveolin-1 to the cell rear. These results suggest that posterior polarization of caveolin-1 is a common feature both for chemotaxis and chemokinesis. Dual immunofluorescent labeling showed that, during cell spreading, caveolin-1 was compacted in the cell center and excluded from nascent focal contacts along the circular lamellipodium, as revealed by integrin ␤ 1 and FAK staining. When cells were migrating, integrin ␤ 1 and FAK appeared at polarized lamellipodia, whereas caveolin-1 was found at the posterior of moving cells. Notably, wherever caveolin-1 was polarized, there was a conspicuous absence of lamellipod protrusion. Transmission electron microscopy showed that caveolae, similar to their marker caveolin-1, were located at the cell center during cell spreading or at the cell rear during cell migration. In contrast to its unphosphorylated form, tyrosine-phosphorylated caveolin-1, upon fibronectin stimulation, was associated with the focal complex molecule phosphopaxillin along the lamellipodia of moving cells. Thus, unphosphorylated and phosphorylated caveolin-1 were located at opposite poles during cell migration. Importantly, loss of caveolin-1 polarity by targeted down-regulation of the protein prevented cell polarization and directional movement. Our present results suggest a potential role of caveolin polarity in lamellipod extension and cell migration.

Inhalation of butter flavoring vapors by food manufacturing workers causes an emerging lung disease clinically resembling bronchiolitis obliterans. Diacetyl, an α-diketone, is a major component of these vapors. In rats, we investigated the toxicity of inhaled diacetyl at concentrations of up to 365 ppm (time weighted average), either as six-hour continuous exposures or as four brief, intense exposures over six hours. A separate group inhaled a single pulse of ~1800 ppm diacetyl (92.9 ppm six-hour average). Rats were necropsied 18 to 20 hours after exposure. Diacetyl inhalation caused epithelial necrosis and suppurative to fibrinosuppurative inflammation in the nose, larynx, trachea, and bronchi. Bronchi were affected at diacetyl concentrations of 294.6 ppm or greater; the trachea and larynx were affected at diacetyl concentrations of 224 ppm or greater. Both pulsed and continuous exposure patterns caused epithelial injury. The nose had the greatest sensitivity to diacetyl. Ultrastructural changes in the tracheal epithelium included whorling and dilation of the rough endoplasmic reticulum, chromatin clumping beneath the nuclear membrane, vacuolation, increased intercellular space and foci of denuded basement membrane. Edema and hemorrhage extended into the lamina propria. These findings are consistent with the conclusion that inhaled diacetyl is a respiratory hazard.