The commercial interest in single walled carbon nanotubes (SWCNT) is rapidly increasing due to their superior mechanical, electric and thermal properties [1]. With the development of mass production and handling facilities for SWCNT, it is critical to fully understand the risk associated with exposure since it is likely that increasing human exposure will occur [2]. Moreover, it would be highly desirable to identify specific biomarkers of SWCNT exposure in order to identify individuals who are exposed occupationally or consumers who become exposed to products containing SWCNT. Individual SWCNT are less than 2 nm in diameter and form fibrous ropes that range up to several microns in length [3]. Ropes of SWCNT further aggregate by means of electrostatic charge to form larger, micron-sized agglomerates.

Several recent studies indicate fibrogenic effects of SWCNT instilled into the lungs of mice or rats. Lam et al reported that SWCNT instilled into the lungs of mice caused the formation of fibroproliferative lesions to the same extent as toxic quartz particles [4]. In a similar study, Warheit and colleagues reported that SWCNT exposure in rats by intratracheal instillation caused pulmonary granulomas, although there was a lack of lung toxicity as assessed by lavage parameters and cell proliferation parameters [5]. Shvedova and coworkers reported that pharyngeal aspiration of SWCNT in mice caused progressive intersitital fibrosis and granulmonas, along with increased levels of cytokines (IL-1β and TNF-α) within 1 day and elevated TGF-β1 after 1 week [6]. All of these studies indicate that SWCNT cause some lung toxicity when instilled or aspirated as a bolus of agglomerated nanoropes. Nevertheless, it remains unclear from these reports whether SWCNT have the potential to stimulate pulmonary fibrogenesis under conditions of occupational or environmental exposure.

The overall goal of this study was to investigate changes in the expression of genes encoding known profibrogenic mediators in vivo following SWCNT exposure and correlate gene expression patterns with the formation of fibrotic lesions in the lung. Surprisingly, no inflammation was observed in SWCNT-exposed rats, although focal interstitial fibrotic lesions were observed near clusters of macrophages containing micron-sized aggregates of SWCNT within the alveolar region of the lung. Moreover, small but significant increases in lung mRNAs encoding three PDGF ligands (PDGF-A, -B, -C) were observed at 1 day post-SWCNT exposure but were not elevated in the lungs of rats exposed to carbon black (CB) nanoparticles with a similar size and specific surface area. Interestingly, SWCNT formed unique intercellular structures that bridged alveolar macrophages in situ. These structures were not observed in the lungs of CB-exposed rats. While our results indicate relatively weak fibrogenic potential of SWCNT in the lungs of rats, carbon bridge structures between macrophages allowed easy identification of animals exposed to SWCNT as compared to those exposed to CB. To our knowledge this is the first report of a biomarker of carbon nanotube exposure.

SWCNT have been reported to cause the formation of granulomas, a type of pulmonary fibrotic reaction in the lung. The progression of granulomas in the lung interstitium is mediated in large part by alveolar macrophages, which accumulate at sites of particle deposition and become activated by particle phagocytosis [8, 9]. Once activated, macrophages produce polypeptide growth factors that stimulate the proliferation of interstitial fibroblasts, which are the principal collagen-producing cell that drives fibrogenesis [10]. In the present study, we observed small, interstitial fibrotic lesions in the lungs of rats 21 days after exposure to SWCNT. These fibroproliferative lesions were associated with clusters of alveolar macrophages containing carbonaceous material. CB nanoparticles of similar size and specific surface area also caused focal clustering of macrophages with carbon inclusions but did not induce fibrotic lesions. Our findings extend the work of others in that we report 1) the induction of lung mRNAs encoding PDGF ligands after SWCNT exposure, and 2) the formation of intercellular carbon bridge structures between alveolar macrophages in the lungs of SWCNT-exposed rats.

Key growth factors that stimulate fibroblasts to proliferate and/or migrate to sites of lung injury are PDGF [11], CTGF [12], and OPN [13]. Other growth factors such as TNF-α and TGF-β1 stimulate fibroblasts to deposit a collagen matrix, which defines a fibrotic lesion [14]. We found that SWCNT induced small but significant increases in the levels of mRNAs encoding three PDGF ligands (PDGF-A, -B, and -C) at day 1 post-exposure. SWCNT also increased OPN mRNA levels ~5-fold at day 1 post-exposure. At 21 days after SWCNT exposure we observed slight increases PDGF-B and OPN, whereas V₂O₅ induced robust and significant increases in mRNAs encoding PDGF-A, PDGF-C, PDGFRα, TGF-β1, OPN, and CTGF. In particular, the induction of PDGFRα by V₂O₅ confirmed our earlier observation that this growth factor receptor is up-regulated in vivo during fibrogenesis [7]. In general, the induction of mRNA levels for profibrogenic factors by V₂O₅ 21 days post-exposure correlated with the formation of diffuse interstitial and airway fibrotic lesions within the lung. Moreover, the induction of mRNAs encoding PDGFs and OPN by SWCNT at day 1 correlated with the formation of small, focal fibrotic lesions at day 21. In contrast, CB nanoparticles caused neither induction of genes nor fibrotic lesions. The increase in mRNA encoding PDGF family members observed at day 1 after SWCNT exposure suggest that PDGF could play a role in the formation of SWCNT-induced fibrotic lesions as PDGF and its receptors are known to mediate fibroblast chemotaxis and proliferation during fibrogenesis [11].

In agreement with other studies of SWCNT instillation or pharyngeal aspiration in mice or rats, we observed interstitial fibrotic lesions, albeit these lesions were relatively small (~100 μm) and sparsely distributed. Other investigators have reported more severe fibrotic or granulomatous lesions within the lungs of mice or rats after SWCNT exposure. Differences between the toxicity of SWCNT in this study as compared to some earlier studies could be due to differences in the delivered dose of aggregated SWCNT or differences in the size of SWCNT aggregates delivered to the distal lung. We aspirated 2 mg/kg SWCNT into the lungs rats in our experiments, whereas others have used concentrations that range from 4 to 20 mg/kg [4, 5]. Shvedova and colleagues used a dose of SWCNT comparable to the present study and reported significant progressive interstitial fibrosis at deposition sites of more dispersed nanostructures [6]. While we observed most fibrotic lesions closely adjacent to macrophage clusters and micron-sized SWCNT aggregates, we also observed interstitial thickening within alveolar walls characterized by increased trichrome staining at sites distant from SWCNT aggregates and macrophages. While our study did not show lesions as severe as those reported by Shvedova, there were several potentially important differences in our materials. First, our SWCNT were synthesized by a different method (chemical vapor deposition) and contained different metal catalysts (cobalt and molybdenum). The SWCNT used by Shvedova and coworkers contained trace levels of iron catalyst. Second, while we used similar suspension concentrations of SWCNT to those used by Shvedova et al (i.e., 2 mg/kg), based on histopathologic evaluation it is clear that we did not achieve the same efficiency of delivery. Finally, we dispersed our SWCNT with nonionic surfactant that could have decreased the bioactivity of nanoparticles. Finally, we exposed rats to SWCNT, while Shvedova and colleagues used mice.

The dose of SWCNT used in our rodent exposures is presumably higher than would be encountered in human exposure situations assuming that the delivered dose reached the lung. Estimates of airborne concentration of nanotubes material generated during handling are below 53 μg/m³ [3]. However, a limitation of oropharyngeal aspiration and intratracheal instillation techniques is that a bolus of particles is delivered in suspension and may not be evenly distributed within the lung or may inadvertently pass into the esophagus and swallowed. This could significantly reduce the predicted dose to the distal lungs. A far superior technique to deliver nanoparticles and nanotubes is inhalation exposure, especially for SWCNT that are known to have a high electrostatic potential and are therefore predisposed to agglomeration. However, even when aerosolizing SWCNT for inhalation exposure, micro-sized particles are formed that will likely deposit in the lung according to the principles of sedimentation rather than diffusion. Nevertheless, inhalation studies will no doubt be invaluable in addressing health effects of nanomaterials.

A potentially important factor in determining the toxicity of carbon nanotubes is the presence of contaminating metals that were used as catalysts in the manufacturing process. While much emphasis has been given to particle size, particle shape, and increased specific surface area as a determinant of particle toxicity, our study showed that 8 nm diameter carbon black (CB) nanoparticles (composed entirely of elemental carbon) caused no inflammation or fibrosis in the lungs of rats, although macrophages accumulated at sites of CB deposition in the lung and engulfed these nanoparticle aggregates. The SWCNT and CB used in this study possessed similar specific surface areas (~300 to 600 m²/g) and yet at equivalent doses of CB and SWCNT, only the SWCNT stimulated fibrotic responses at day 21 post-exposure. Therefore, the fibrogenic activity of SWCNT in comparison to CB is likely due to either differences in shape or elemental composition. The SWCNT used in this study were synthesized by chemical vapor deposition using cobalt and molybdenum as catalysts. While the raw nanotubes formed by this process are subsequently acid washed to remove contaminating metal catalysts, residual cobalt and molybdenum remained as 2.6% and 1.7% of the total elemental composition, respectively. Individuals exposed to occupationally to cobalt are at risk of developing "hard metal disease", which includes the formation of interstitial pulmonary fibrotic lesions [15]. Other reports of SWCNT-induced interstitial fibrosis have used materials synthesized by laser ablation or a high-pressure carbon monoxide disproportionation (HiPCO) process, both of which require metal catalysts [4–6]. Warheit and colleagues used SWCNT synthesized by laser ablation that contained 5% nickel and 5% molybdenum, while studies by Shvedova et al and Lam et al used SWCNT synthesized by HiPCO that contained residual iron concentrations of 0.23% and 2.14%, respectively. Iron has been proposed to mediate the toxic effects of air pollution particles and asbestos fibers by generating reactive oxygen species through the Fenton reaction [15, 16]. Moreover, loading nonfibrogenic titanium dioxide particles with iron has been shown to make these particles fibrogenic in a rat tracheal explant model [17]. Therefore, metals such as cobalt and iron likely contribute to the surface reactivity of SWCNT. This is supported by our observation that CB nanoparticles, which possessed the same surface area as SWCNT and yet did not contain contaminating metals, caused no fibrotic effects in the lungs of rats. Warheit and colleagues recently reported that various quartz particles produced differential degrees of pulmonary toxicity correlated with surface activity rather than particle size [18]. We further propose that low levels of contaminating metals coupled with high surface area determine the toxicity and fibrogenic potential of SWCNT.

We discovered unique carbon structures formed of SWCNT that bridged a small percent of alveolar macrophages within the lung. Approximately 5% of alveolar macrophages collected by BAL contained carbon inclusions after exposure to either CB or SWCNT. Approximately one fourth of the macrophages with carbon inclusions in the SWCNT-exposed rats were joined by carbon bridges at 21 days post-exposure. The unique hourglass structure of carbon bridges that joined macrophages were not readily apparent in BAL cytospins at day 1 post-SWCNT exposure, although some groups of macrophages were clustered around SWCNT aggregates thereby indicating the early stages of bridge formation. Carbon bridges were not observed in any CB-exposed rats despite similar numbers of lung macrophages that engulfed carbon aggregates composed of either CB and SWCNT in a comparable size range of 0.5 to 3 μm. The appearance of micron-sized carbon bridges between macrophages at 21 days was so reliable that we were able to correctly identify all SWCNT-exposed rats by evaluating the BAL cytospins using light microscopy in a blinded analysis. These structures may have important future implications for determining human exposure to SWCNT.

How carbon bridges form in the lung after SWCNT is not entirely clear. Preliminary observations with NR8383 macrophages indicate that some bridges form during cytokinesis with the resulting daughter cells joined by aggregated bundles of SWCNT (Mangum and Bonner, unpublished observation). We did not detect carbon bridges between macrophages in situ in the lungs of rats or in BAL cytospins 1 day after SWCNT exposure. This observation suggests that phagocytosis by two or more macrophages is an unlikely mechanism of bridge formation. In addition, bridge formation is not akin to "frustrated" or incomplete phagocytosis of long (>17 μm) asbestos or man-made fibers, where macrophages viewed in real time avidly move along the length of the fiber [19]. Furthermore, the biological or functional consequence of carbon bridge formation has yet to be determined. Carbon bridges could affect the phagocytic function of macrophages or inhibit their ability to release or respond to cytokines. While potentially important, these issues could be difficult to address as only about one percent of alveolar macrophages within the lung at 21 days post-exposure were linked by carbon bridges. Because of the low percentage of macrophages linked by carbon bridges, it is also not likely that these structures would have a deleterious effect on the overall lung macrophage population.

Despite similar size and specific surface area of carbon black nanoparticles and SWCNT used in this study, only SWCNT caused 1) interstitial fibrotic lesions in the lungs of rats, 2) significant increases in lung PDGF mRNA levels, and 3) formation of unique carbon bridge structures between alveolar macrophages in situ. These differences between carbon black nanoparticles and SWCNT suggest that the fibrogenic activity of SWCNT is due to factors that make them unique from carbon black nanoparticles, including their unique shape or the presence of contaminating metal catalysts used in the manufacturing process. Finally, carbon bridge structures that form between macrophages are likely due to unique structural characteristics of SWCNT. Intercellular carbon bridge structures that link macrophages serve as a useful biomarker of SWCNT exposure in macrophages retrieved by bronchoalveolar lavage.

We thank Carol Bobbitt, David Weil, and Victoria Wong for excellent technical expertise. We are also grateful to Dr. David Dorman for invaluable comments during the preparation of this manuscript. This work was funded by The American Chemistry Council's Long Range Research Initiative provided to CIIT Centers for Health Research.