The lungs are the most likely route of exposure to carbon nanotubes (CNTs)5
. CNTs have a high aspect (length to width) ratio, a property shared with asbestos fibers, which has led to concern that inhalation of CNTs may cause similar lung pathology1
. These include pulmonary fibrosis (scarring of the lung tissue), and mesothelioma, a cancer of the pleura and peritoneum4
. The administration of multi-walled CNTs (MWCNT)6
and single-walled CNTs (SWCNT)7,8
by intratracheal instillation or aspiration9,10
in rodents causes lung injury. However, it is not known whether CNTs delivered to the lung reach the pleura, the outer mesothelial lining of the lung, to cause injury and disease.
Inhalation is the most relevant method for determining toxicity of CNTs for several reasons. First, airborne particles are subject to the physics of impaction, sedimentation, and diffusion, whereas instilled particles in aqueous suspension are not11
. Second, inhaled nano-sized particles reach the distal regions of the lung5
. Third, inhaled CNTs are more dispersed and less agglomerated as compared to an instilled bolus dose in aqueous liquid3,12,13
. In addition, inhalation3,12
is much more relevant than injection of materials into the body cavity2
, which bypass the lung and its defense and clearance mechanisms entirely. Therefore, inhalation studies more accurately model deposition and pathologic responses to ‘real world’ exposure scenarios.
We exposed male C57BL6 mice to an aerosol of MWCNT (1 mg/m3
or 30 mg/m3
) or carbon black nanoparticles (CB, 30 mg/m3
) for 6 h and then collected lung tissues at 1 dy, 2 wk, 6 wk, and 14 wk post-inhalation exposure. The physicochemical parameters of these MWCNT have been reported by our laboratory3
and are summarized in Table S1
). A schematic illustration and photograph of our inhalation setup is provided (Fig. S1 and S2
) and has been described3
. Transmission electron microscopy (TEM) of the bulk MWCNT showed a width range of 10–50 nm () confirmed by independent analysis (Table S1
). Aerosolized MWCNT were a mixture of agglomerated and individual nanotubes with lengths of <100 nm to >10 µm (). High dose MWCNT (30 mg/m3
), low dose MWCNT (1 mg/m3
), and CB (30 mg/m3
) had mass median aerodynamic diameters of 183 nm, 164 nm, and 209 nm, respectively, with a geometric standard deviation<2 (Fig. S3
). The calculated high and low MWCNT doses were 4 mg/kg and 0.2 mg/kg, respectively. Dose was based on a 10% estimated deposition fraction11
Aerosolization of carbon nanotubes
Inhaled MWCNT were engulfed by macrophages which migrated to the subpleural region (). MWCNT were observed in macrophages () and within subpleural mesenchymal cells and the collagen matrix of the subpleura 1 day after inhalation (). Serial sectioning of lung tissue revealed mononuclear cell aggregates, comprised mainly of lymphocytes with some monocytes/macrophages, on the pleural surface of mice exposed to MWCNT (). Macrophages containing MWCNT were present in these foci (). Occasional minor mononuclear cell aggregates were present in all exposure groups (data not shown). High dose MWCNT significantly increased the incidence (Table S2
) and number () of aggregates (1 to 2 per 10 sections) at 1 day and 2 wks post-exposure. The size of mononuclear cell aggregates was significantly increased by high dose MWCNT () as determined by image analysis2
and a point counting method14
(see a comparison of methods in Supplementary Information and Figs. S4, S5
Inhaled carbon nanotubes reach the sub-pleura in mice
Pleural immune response after carbon nanotube inhalation in mice
Sub-pleural fibrosis was also observed in mice that inhaled MWCNT (). There was a high incidence (9 out of 10 mice) of fibrotic lesions in this group (Table S3
). A point counting method14
was modified to derive a fibrosis score (Fig. S6
). High dose MWCNT exposure caused a significant increase in fibrosis at 2 wk and 6 wk post-inhalation (). MWCNT-laden macrophages were present beneath these sub-pleural lesions (). A caveat is that the fibrosis score relied on trichrome staining which, although commonly used, could stain other cell matrix components and contribute to the observed pleural wall thickness. It is also important to note that most of the sub-pleura in mice exposed to MWCNT was normal as fibrotic lesions were focal and regional. Nevertheless, sub-pleural fibrotic lesions were only observed in the high dose MWCNT group and maximal thickness of these lesions ranged from 20 to 70 µm compared to normal sub-pleural thickness which ranged from 3 to 15 µm.
Sub-pleural fibrosis in mice after carbon nanotube inhalation
Our study was motivated by reports of peritoneal inflammation in the abdominal cavities of mice injected with MWCNT that led investigators to propose “asbestos-like pathogenicity”2,15
. These investigators reported granulomas on the surface of the diaphragm, while we observed mononuclear cell aggregates at the pleura. Since minor aggregates were occasionally observed in control animals, these likely represent lymphoid tissue that is increased in number and size by MWCNT. We hypothesize that activated macrophages containing MWCNT traffic through the pleural lymphatic drainage and stimulate recruitment of mononuclear cells to enlarge these focal aggregates. Indeed, we observed macrophages containing MWCNT within mononuclear cell aggregates (). Increased migration of mononuclear cells to the pleura is consistent with our previous finding that the monocyte chemokine CCL2 (MCP-1) is increased in the lungs of mice after MWCNT inhalation3
. Bromodeoxyuridine labeling was not observed in these foci (data not shown), indicating that the mononuclear cells were not proliferating.
The development of sub-pleural fibrosis in mice suggest MWCNT could behave like asbestos in that they (1) are deposited at the alveolar level17
(), (2) reach the subpleura after inhalation18
(), and (3) remain in the sub-pleura intact for weeks to stimulate fibrosis19
(). However, the pathology we observed with MWCNT appears different from pleural pathology induced by asbestos fibers as MWCNT caused focal sub-pleural fibrosis and mononuclear cell aggregates, whereas asbestos causes pleural inflammation (granulomas) and diffuse pleural fibrosis18,20
. It is therefore important that further work compare CNTs and asbestos to carefully address these important pathologic issues and elucidate whether there are similar mechanisms of action for asbestos and CNTs. For example, both asbestos and MWCNT increase platelet-derived growth factor (PDGF) in the lungs of mice3,16
and PDGF is an important mediator of fibrosis21
. Investigation of other fibrotic mediators should provide insight into the molecular mechanisms underlying the pathologic responses to CNTs.
The issue of whether MWCNT cause mesothelioma cannot be answered from the results of this study. We showed no indication of neoplasia nor did we use asbestos as a positive control. However, mesothelioma does not readily develop in mice after asbestos inhalation, except in mice that are deficient in the p53 tumor suppressor gene20
. Asbestos and MWCNT both induce p5323,24
. Therefore, further inhalation studies should be performed with p53-deficient mice using relevant exposure concentrations of MWCNT.
It is unknown whether other types of MWCNT would cause the same response that we report here. Metal catalysts used in the manufacturing process of CNTs could be important determinants of pleural toxicity. The MWCNT used in our study contained nickel which is known to elicit immune responses and pleural fibrosis14
. In addition, agglomeration could affect lung deposition and toxicity of inhaled MWCNT. We used pluronic detergent to disperse the MWCNT, but it appears that the MWCNT were widely deposited in all aspects of the lung as would be expected when humans inhale such nanoparticles.
In our study some of the inhaled nanotubes remained in the sub-pleural wall for at least 14 wks, although most appeared to be cleared by this time and this could explain why fibrosis and mononuclear cell aggregates diminished. However, we did not carry out quantitative measures of clearance dynamics. Elgrabli et al
recently showed that the majority of MWCNT (~65%) instilled into the lungs of rats were eliminated after 3 months, and ~15% of instilled MWCNT remained in the lungs after 6 months25
. High aspect (length to width) ratio can influence biopersistence19
. Poland et al2
showed that fiber length was important for inducing granulomas on the peritoneal surface of the diaphragm after intraperitoneal injection of MWCNT. Our study used MWCNT that possessed a length range from 0.5 to 50 µm (Table S1
). Therefore, further inhalation studies should compare purified long and short MWCNT.
Whether CNTs cause pulmonary disease in humans is not known. There is currently no epidemiologic data to indicate that CNTs cause pulmonary fibrosis, mesothelioma, or other adverse effects. The individuals at most risk for inhalation exposure are those working with nanomaterials in occupational settings26
. We previously found that inhaled MWCNT caused airway fibrosis in mice with pre-existing allergic inflammation3
, suggesting that individuals with asthma are at greater risk.
In summary, inhaled MWCNT increased pleural mononuclear cell accumulation and sub-pleural fibrosis in mice. To our knowledge, this is the first report demonstrating that inhaled carbon nanotubes migrate to the sub-pleura to cause pathologic effects. These pathological changes could be unique to this type of MWCNT and may or may not persist under conditions of repeated, low-dose occupational exposures. Given this uncertainty, precautions minimizing inhalation during handling and use of MWCNT would be prudent until further studies can assess longer term responses of the lung and pleura after repeated exposures to MWCNT.