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Multi-walled carbon nanotubes have a fibrous structure similar to asbestos and induce mesothelioma when injected into the peritoneal cavity. In the present study, we investigated whether carbon nanotubes administered into the lung through the trachea induce mesothelial lesions. Male F344 rats were treated with 0.5 mL of 500 μg/mL suspensions of multi-walled carbon nanotubes or crocidolite five times over a 9-day period by intrapulmonary spraying. Pleural cavity lavage fluid, lung and chest wall were then collected. Multi-walled carbon nanotubes and crocidolite were found mainly in alveolar macrophages and mediastinal lymph nodes. Importantly, the fibers were also found in the cell pellets of the pleural cavity lavage, mostly in macrophages. Both multi-walled carbon nanotube and crocidolite treatment induced hyperplastic proliferative lesions of the visceral mesothelium, with their proliferating cell nuclear antigen indices approximately 10-fold that of the vehicle control. The hyperplastic lesions were associated with inflammatory cell infiltration and inflammation-induced fibrotic lesions of the pleural tissues. The fibers were not found in the mesothelial proliferative lesions themselves. In the pleural cavity, abundant inflammatory cell infiltration, mainly composed of macrophages, was observed. Conditioned cell culture media of macrophages treated with multi-walled carbon nanotubes and crocidolite and the supernatants of pleural cavity lavage fluid from the dosed rats increased mesothelial cell proliferation in vitro, suggesting that mesothelial proliferative lesions were induced by inflammatory events in the lung and pleural cavity and likely mediated by macrophages. In conclusion, intrapulmonary administration of multi-walled carbon nanotubes, like asbestos, induced mesothelial proliferation potentially associated with mesothelioma development.
Multi-walled carbon nanotubes (MWCNT) are structurally composed of cylinders rolled up from several layers of graphite sheets. They are several to tens of nanometers in diameter and several to tens of micrometers in length. This high length to diameter aspect ratio, a characteristic shared with asbestos fibers, has led to concern that exposure to MWCNT might cause asbestos-like lung diseases, such as lung fibrosis, lung cancer, pleural plaque and malignant mesothelioma.[1-6]
Pleural plaque and malignant mesothelioma are characteristic lesions in asbestos-exposed humans. Although fiber dimensions, biopersistence, oxidative stress and inflammation have all been implicated,[7-12] the exact mechanisms of pleural pathogenesis are unclear. According to a pathogenesis paradigm suggested by Donaldson et al., asbestos fibers penetrate into the pleural cavity from the alveoli and deposit in the pleural tissue. Unlike spherical particles, fibrous materials such as asbestos are not cleared effectively from the pleural cavity, resulting in deposition of the fibers in the parietal pleura. This deposition, in turn, causes frustrated phagocytosis-induced pro-inflammatory, genotoxic and mitogenic responses in the deposition sites.
Administration of MWCNT into the peritoneal cavity or scrotum in animals has been reported to induce mesothelial lesions, similar to those observed in asbestos cases.[13-15] The induction of mesothelioma in the peritonum is dose dependent, and is observed with as low as 3 μg/mouse in p53 heterozygous mice. These studies suggest a potential risk that inhaled MWCNT might lead to pleural mesothelioma. However, actual experimental evidence demonstrating induction of pleural mesothelioma by inhaled MWCNT fibers has not yet been shown. It has been shown that inhaled MWCNT induced subpleural fibrosis with macrophage aggregates on the surface of the visceral pleura. Notably, some of these macrophages contained MWCNT fibers. In addition, penetration of MWCNT administrated by pharyngeal aspiration into the pleural cavity was observed, and intrapleural injection of 5 μg/mouse of MWCNT has been shown to lead to sustained inflammation and length-dependent retention of MWCNT in the pleural cavity. Accordingly, direct interaction of MWCNT with the mesothelial tissue is postulated as an early pathogenic event.
In the present study, to examine whether MWCNT translocate into the pleural cavity and cause inflammation leading to proliferative change of the mesothelial tissue, we administered relatively high doses (five doses at 250 μg/rat) of two MWCNT samples (MWCNT-N and MWCNT-M) to the rat lung by intrapulmonary spraying (IPS)/intratracheal instillation; crocidolite (CRO; one kind of asbestos fiber) was used as a positive control. Intrapulmonary spraying has been shown to be an efficient method to deliver particle materials deep into the lung.[20-24] Our results demonstrated that MWCNT, like asbestos, translocated from the lung into the pleural cavity and induced inflammatory responses in the pleural cavity and, importantly, hyperplastic visceral mesothelial proliferation. These findings are important in understanding whether MWCNT have the potential to cause asbestos-like pleural lesions.
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In the present study, we compared the pleural translocation of MWCNT and CRO and examined the mesothelial lesions they induced. Our data demonstrate that after deposition in the lung, MWCNT, like CRO, translocated into the pleural cavity, mainly in pleural macrophages. Both MWCNT and CRO treatment also caused hyperplastic visceral mesothelial proliferation and marked pleural inflammation.
This is the first report to show that MWCNT administered into the rat lung causes mesothelial proliferative lesions. Adamson et al. reported that intratracheal instillation of asbestos in mice induced pleural mesothelial cell proliferation within several days; the degree of pleural mesothelial cell proliferation did not appear to correlate with the localization of asbestos fibers in the pleura. Similarly, we did not find fibers within the HVMP lesions. Thus, our findings suggest that HVMP lesions do not appear to be directly induced by the deposited MWCNT or CRO fibers. Also, in vitro exposure to MWCNT and CRO fibers did not lead to proliferation of TCC-MESO1 cells, but rather to cell death (Fig. S4). It has been reported that macrophages play a significant role in mesothelial cell proliferation caused by asbestos exposure and surgical injury,[28-31] and that the conditioned medium of macrophages exposed to MWCNT promotes mesothelial cell proliferation in vitro. Activated macrophages secrete a panel of growth factors and cytokines to regulate cell proliferation, which can augment transformation of mesothelial cells.[28, 30, 32, 33] Our observations that mesothelial cell proliferation is enhanced by conditioned macrophage culture media and by the supernatants of pleural cavity lavage are consistent with these results, although the factors that are involved need to be identified.
Translocation of asbestos[34, 35] and MWCNT fibers from the lung to the pleural cavity has been found in rodents. This translocation also probably occurs in humans since asbestos fibers have been detected in human pleural lesions. However, the mechanism and route of translocation are unclear. It has been suggested that penetration through the visceral pleura, possibly driven by increased pulmonary interstitial pressure and assisted by enhanced permeability of the visceral pleura due to asbestos-induced inflammation might be a major route. In the present study, only a few MWCNT and CRO fibers were observed penetrating through the visceral pleura, and a large number of the fibers in the pleural cavity was observed in macrophages. We also observed frequent deposition of MWCNT and CRO in the mediastinal lymph nodes, mostly phagocytosed by macrophages. These results suggest that a probable route of translocation of the fibers is lymphatic flow. Inflammation in the pleural cavity is probably a defense response against translocated fibers. Murphy et al. reported that intrapleural injection of 5 μg/mouse of long MWCNT or asbestos initiated sustained inflammation, including increased granulocyte number and protein level, in the pleural cavity. Thus, the observed proliferation of visceral mesothelial cells in the present study is probably caused by inflammatory reactions both in the lung and in the pleural cavity. In the present study, no MWCNT or crosidolite fibers or lesions were observed in the parietal pleura. This is possibly due to the short experimental period and limited amount of fibers in the pleural cavity, which would result in little inflammation in the parietal pleura.
Currently, the exposure level to MWCNT in the workplace is unknown and there are no administrational regulations for the occupational exposure limit for MWCNT. In November 2010, the National Institute of Occupational Safety and Health (NIOSH) released a non-official carbon nanotube exposure limit for peer review. The recommended exposure limit in the air was set at 7 μg/m3. Previously, we used a total dose of 1.25 mg/rat of titanium dioxide over a 9-day period and identified factors involved in titanium dioxide-induced lung lesions. In the present study, we used the same protocol for the purpose of induction of observable pleural lesions and inflammation in the pleural cavity as well to ensure the presence of a detectable number of fibers in the pleural cavity after short-term administration; this dose was higher than the NIOSH exposure limit. Time- and dose-dependent experiments are needed in future studies, and further investigation is also required to elucidate cytokines and other factors that cause parietal mesothelial proliferation in animal models that are more relevant to humans.
The IPS/intratracheal instillation is a widely used method to evaluate the respiratory toxicity of particles. It should be noted that IPS/intratracheal instillation is a non-physiological method and possibly affects the migration and distribution of particles in the lung due to the pressure from spraying. However, IPS/intratracheal instillation is relevant for identifying factors to be examined using long-term, more physiologically relevant methods of CNT administration.
In summary, MWCNT and CRO fibers were found to translocate from the lung to the pleural cavity after intrapulmonary administration. Importantly, MWCNT and CRO treatment caused visceral mesothelial cell proliferation and inflammation in the pleural cavity. This mesothelial proliferation was plausibly induced by inflammatory events in the lung and pleural cavity and mediated primarily by macrophages. The similarity between MWCNT-N, MWCNT-M and CRO in translocation to the pleural cavity, induction of pleural cavity inflammation and induction of visceral pleural mesothelial proliferation suggests that MWCNT might cause asbestos-like pleural lesions.