In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 108, No. 49 ( 2011-12-06)
Abstract:
In conclusion, our findings show that MWCNTs differ from asbestos fibers in how they enter mesothelial cells (i.e., membrane piercing versus active internalization) and suggest that MWCNT toxicity should be evaluated differently from asbestos toxicity in the context of mesothelial injury. Specifically, MWCNTs showed diameter- and rigidity-dependent piercing of mesothelial cells, which is important in mesothelial injury, inflammation, and mesotheliomagenesis ( Fig. P1 ). These data along with the conventional paradigm indicate that short, flexible, thick, and chemically functionalized NTs are less biologically harmful, and that such fibers should be favored for safe practices. There are several limitations in our study. As in other studies, we used an injection model to test the in vivo effects of MWCNTs. However, the health risks of carbon NTs are posed by respiratory exposure, and not injection. Moreover, the dosing in the current study was relatively high, corresponding to chronic inhalation. Therefore, careful considerations, including translocation of fibers and their interaction with mesothelial cells in vivo, are necessary before reaching conclusions about the potent health risks of NTs. Finally, we performed long-term carcinogenicity experiments, which again showed the greater harm caused by thin, rigid MWCNT fibers. Moreover, we identified a common genetic aberration underlying both MWCNT- and asbestos-induced mesothelioma in humans and rodents. To further study the toxicological effects of each MWCNT type, we injected 1 mg or 5 mg of MWCNTs into rats intraperitoneally. In agreement with the in vitro results, thin MWCNTs induced stronger fibrous inflammation. We also found that thin and thick MWCNTs similarly affected macrophages in terms of inflammatory responses and cellular viability, which suggests that different degrees of direct mesothelial injury by thin and thick MWCNTs are responsible for the extent of inflammation caused, at least when MWCNTs are injected. We propose that injected MWCNTs immediately injure mesothelial cells, which causes the body to respond with inflammation. To corroborate the hypothesis that mesothelial membrane penetration by thin MWCNTs was a key event in mesothelial injury, we investigated other characteristics such as length, structural defects, and free radical generation activity. However, none of these features could explain the difference in cytotoxicity between thin and thick MWCNTs, confirming that diameter is the key factor in determining whether MWCNTs penetrate mesothelial cell membranes and injure the cells. Collectively, these data show that NT toxicity and asbestos toxicity should be evaluated in a different manner, at least in the context of mesothelial injury. Because of the inverse correlation between cytotoxicity and MWCNT diameter, we hypothesized that thin MWCNTs were toxic because they penetrated mesothelial cell membranes. To test this hypothesis, we observed mesothelial cells after incubation with fibers by using EM. Confirming our hypothesis, we found that thin MWCNTs were more likely to pierce mesothelial plasma and nuclear membranes than thick or tangled MWCNTs. We also found that there was a distinct difference between MWCNTs and asbestos fibers in how they entered mesothelial cells. Asbestos was surrounded by vesicular membranous structures, which means that the internalization of the fiber was mediated by active signaling pathways, such as phagocytosis. In contrast, thin MWCNTs with high rigidity directly penetrated cell plasma membranes without a surrounding membranous structure, indicating that the MWCNTs pierced the plasma membrane via physical force and/or physical interaction. We investigated how MWCNTs cause mesothelial injury in comparison with asbestos fibers. We used five different types of MWCNTs of different diameters and three types of asbestos. First, by using confocal microscopy and flow cytometry (a method for counting microscopic particles), we evaluated how cells internalize each fiber type. Consistent with previous literature, two types of mesothelial cells and macrophages internalized asbestos fibers. However, we found that mesothelial cells did not internalize MWCNTs, whereas macrophages did. In other words, MWCNTs behaved differently from asbestos fibers. Based on this observation, we expected limited mesothelial cell toxicity by MWCNTs because cellular internalization of fibers is an important step in asbestos-induced cytotoxicity. Surprisingly, we did find mesothelial cell toxicity, with each MWCNT type showing varying levels of toxicity. Specifically, we found that the toxicity was inversely associated with MWCNT diameter. Thin MWCNTs [e.g., nanotube (NT) 50a] showed strong cytotoxicity, whereas thick MWCNTs (e.g., NT145) showed weak cytotoxicity. (The number following “NT” refers to the mean diameter in nanometers.) Notably, not only the thinness but also the rigidity of MWCNTs was an important factor in cytotoxicity because tangled MWCNTs (e.g., NTtngl) did not injure mesothelial cells despite having the thinnest diameter among the NTs tested. During mesothelial carcinogenesis, several events occur: direct and indirect effects caused by the fibers, mesothelial injury, and macrophage activation ( 1 ). MWCNTs, like asbestos fibers, induce inflammation in a length-dependent manner ( 2 ) because long fibers are not fully engulfed by macrophages, cells that defend the body against large invaders. This results in persistent macrophage activation and chronic inflammation as the body's immune response attempts to respond to the invaders. This failure to completely engulf, i.e., cause phagocytosis, is referred to as “frustrated phagocytosis” and is a key event in the length-dependent theory of how mesothelioma develops ( 3 ). This theory describes how MWCNTs behave like asbestos to activate macrophages, but the mechanism of mesothelial injury remains elusive. The discovery and development of new materials opens the way to fascinating applications, but these materials may also pose potential health risks. For example, asbestos has been shown to increase cancer risks as a result of the unique properties of asbestos fibers, including their needle-like shape and high durability. Recently, another new material with similar features, the multiwalled carbon nanotube (MWCNT), has been discovered, developed, and even commercially distributed. To prevent similar environmental health problems, there is a pressing need to determine the toxicological and carcinogenic (i.e., cancer-causing) features of MWCNTs. Genetic damage in mesothelial cells, which cover body cavities in a single cellular layer, is a necessary step in carcinogenesis; however, the mechanism by which MWCNTs induce mesothelial cell injury is unsolved. Here, we demonstrate that the diameter and rigidity of MWCNT fibers plays a role in their toxicity and carcinogenicity levels.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1110013108
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2011
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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