Multiwalled carbon nanotubes intratracheally instilled into the rat lung induce pleural malignant mesothelioma and lung tumors
Hiroyuki Tsuda, M.D., Ph.D. – Professor, Nagoya City University
Carbon nanotubes (CNTs) are hollow cylindrical tubes composed of one-atom thick sheets of carbon. These sheets, called graphene, are composed of a hexagonal lattice of carbon atoms, one carbon atom at each vertex. The carbon-carbon bonds of graphene are exclusively sp2 bonds, the same type of C-C bond as in benzene. These bonds are shorter (0.142 nm) and stronger (4.93 eV) than the carbon-carbon bonds of diamonds (0.154 nm and 3.69 eV) and give structures derived from graphene sheets, such as single-walled carbon nanotubes (SWCNTs), double-walled carbon nanotubes (DWCNTs), and multi-walled carbon nanotubes (MWCNTs), their unique physical proterties. A SWCNT is basically a rolled up graphene sheet that forms a tube; a DWCNT has a second graphene sheet wrapped around the first sheet, forming two coaxial tubes; MWCNTs are composed of three or more coaxial CNTs. MWCNTs typically have diameters of 10 – 100 nm, and their hollow structure gives them low densities, ranging from approximately 0.6 to 3 g/cm3, depending on the diameter (decreasing the diameter increases the density) and number of CNT layers; for comparison, the density of aluminium is 2.7 g/cm3. Because of the sp2 character of the bonds that make up MWCNTs, MWCNTs have a higher thermal stability than diamonds and a higher tensile strength than steel: MWCNTs with tensile strengths of 20 to 63 GPa have been manufactured and a tensile strength of 150 GPa has been theoretically predicted; steels generally have tensile strengths of 0.3 to 1.7 GPa, and the strongest steels (maraging steels) have tensile strengths of up to 2.4 Gpa. In addition, MWCNTs have high flexibility, high electrical conductivity, and high resistance to many chemical agents. These properties make MWCNTs useful in a wide range of applications such as use in sport instruments, semiconductors, high capacity lithium batteries, fuel-cell batteries, screen displays, aerospace materials, fire protection, heat sinks, and electromagnetic interference shielding. Furthermore, in vivo applications as bio-sensors and drug delivery tools are curently being explored. The approximate production of MWCNT exceeds 100 tons in Japan and 300 tons globally.
MWCNTs have a fibrous structure and are biopersistent (i.e., they can be maintained in the body for extended periods of time), physical properties that MWCNTs share with asbestos. This raises concern that like asbestos, MWCNTs may be carcinogenic to the lung and pleura in humans. Studies have shown that injection of MWCNT-7 (M-H company) into the peritoneal cavity or the scrotum induces malignant mesotheliomas in rats and mice (Takagi et al., J Tox Sci, 2008, 33, 105-16; Sakamoto et al., J Tox Sci, 2009, 34, 65-76). In addition, inhalation of MWCNT-7 promotes lung carcinogenesis in mice (Sargent et al. Particle and Fibre Tox, 2014, 11, 3). Based on these findings, WHO/IARC (International Agency for Research on Cancer) evaluated MWCNT-7 as a Group 2B carcinogen, “sufficient evidence of carcinogenicity to animals” and thus “possibly carcinogenic to humans”.
Recently, using whole-body inhalation exposure chambers, rats were exposed to MWCNT-7 for 2 years. This study found that MWCNT-7 was carcinogenic to the rat lung (Report of a carcinogenicity test of MWCNT by systemic exposure to rats. Japan Bioassay Research Center 2015, Contract by the Ministry of Health, Labour and Welfare, Japan). While MWCNT-7 has been found to be carcinogenic, there are considerable differences in metal content, length, diameter, and layers among the MWCNTs produced by different companies and even in different lots of the same MWCNT produced by a single company. These differences can influence the physical properties and the biological effects, including carcinogenicity, of the different MWCNTs. However, it is a practical impossiblity to test all of the MWCNTs by whole-body inhalation exposure because facilities capable of conducting such tests are severely limited due to the enormous cost of construction and operation of such facilities.
We have developed the Trans-tracheal Intra-pulmonary Spraying (TIPS) method of administration of inhalable particles. Using TIPS, inhalable particles can be delivered into the lung alveoli without using an inhalation chamber (Xu et al., Carcinogenesis, 2010, 31, 927-35). In earlier studies using this method, we demonstrated that MWCNTs induced lung inflammation and mesothelial proliferation (Xu et al., Cancer Sci, 2012, 103, 2045-50; Xu et al., Cancer Sci, 2014, 105, 763-9). Based on these findings, we tested the carcinogenicity of MWCNT-N (N company) in a 2-weeks TIPS administration protocol: 125μg MWCNT-N in 0.5 ml solvent was administered to each rat by TIPS every other day for 2 weeks (8 doses for a total of 1 mg per rat), followed by observation for 107 weeks without further administration of MWCNT-N. The incidence of malignant mesothelioma in the mediastinal cavity including the pericardium was 6/38 (15.8%, p < 0.05), and the incidence of lung tumors, bronchiolo-alveolar adenoma and carcinoma combined, was 14/38 (36.8%, p < 0.01). No mesotheliomas or lung tumors were found in the control group, i.e., rats not administered MWCNT-N. The combined tumor incidence of the lung and pleura was 20/38 (52.6%, p < 0.01), clearly indicating that MWCNT-N induced tumors in more than half of the rats. The amount of MWCNT-N in the lung at week 109 was 38 – 80% of the initial dose (Cancer Sci, 2016, in Press).
We are currently setting up studies to validate the TIPS system using known fiber carcinogens including asbestos, MWCNT-7, MWCNT-N, and other carcinogenic particles/fibers at lower doses including the “No Observed Adverse Effect Level” (NOAEL) dose. Our simple and low cost testing system is suitable for examining the toxicity and carcinogenicity of inhalable particles, including the numerous types of MWCNTs being produced. Since the TIPS method does not require access to whole body inhalation facilities, it can be used by a large number of groups. Consequently, this method can greatly accelerate the safety assessment of MWCNTs.