When Dealing with Carbon Nanotubes: Does Diameter Matter?

SWCNTs and MWCNTs and Genotoxicity

Carbon nanotubes (CNTs) are an allotrope of carbon. Since their discovery in 1991 they have been used for a variety of applications including fiber optics, conductive plastics, and molecular electronics, as well as biological and biomedical applications. Their unique properties make them commercially important. They exhibit extraordinary strength and are used in both consumer and industrial products such as electronic devices, protective clothing, sports equipment and medical devices, as well as vehicles for drug delivery. However, because of their low density and small size, respiratory exposures are likely during their production or processing because they can be aerosolized under workplace conditions. In this study, “Genotoxicity of Multi-Walled Carbon Nanotubes at Occupationally Relevant Doses,” the authors examined whether multi-walled carbon nanotubes cause mitotic spindle damage in cultured cells at doses equivalent to 34 years of exposure at the NIOSH Recommended Exposure Limit (REL).

There are two main types of carbon nanotubes: single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs). The low density, fiber-like geometry and durability of carbon nanotubes are characteristics shared with asbestos. And although concerns have been raised that carbon nanotubes may have effects similar to asbestos (mitotic spindle disruption and aneuploidy) and both SWCNTs and MWCNTs have been shown to enter cells and induce DNA damage, the health effects have not been fully investigated.

The Study: Instrumentation, Technology and Methodology
The overall objective of the study was to examine the role of the carbon nanotube diameter in nanotube-induced genetic damage. Carbon nanotubes were prepared with the same acid washing procedure and the same 1µm length used in previous studies in which the potential genotoxicity of the narrower SWCNT was evaluated. Inhalation exposure is the route that most closely resembles occupational exposure and so the lung is the principal target organ for carbon nanotube exposure. The study exposed immortalized and primary human lung epithelial cells to occupationally relevant doses and then examined for the potential of MWCNTs to cause aneuploidy, mitotic spindle disruption, centrosome fragmentation, and cell cycle distribution.

    • Raman spectroscopy was used to characterize the chemical structure of pristine and acid-washed MWCNT and to determine the degree of MWCNT functionalization after acid treatment.
    • Energy dispersive X-ray spectroscopy (EDS) was used to examine the elemental composition of the pristine and acid-washed MWCNT and confirmed the increase in the oxygen content due to the acid treatment and thus the increase in the MWCNT degree of functionalization with free carboxylic acid groups.
    • Inductively coupled plasma mass spectrometry (ICP-MS) was performed to analyze the metal content of the MWCNTs.
    • Transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) were used to document the diameter and morphology of the MWCNTS as well as show evidence of monopolar mitosis.
    • 3D reconstructed images created from serial optical laser scanning confocal microscopy sections demonstrated the strong physical associations between the carbon nanotubes, the microtubules and DNA and the centrosomes.
    • Atomic force microscopy (AFM) was used to investigate the length of both pristine and acid-washed MWCNT and showed that acid treatment led to shortening of the fibers.

Study Results
Data reported here are the first to show induction of monopolar mitotic spindles, aneuploidy, and a G1/S block in the cell cycle as well as a dramatic increase in colony formation following exposure to 10–20nm diameter MWCNTs. Cells exposed to 0.024μg MWCNT/cm2 resulted in errors in chromosome number and mitotic spindle aberrations in greater than 40% of the cells examined. The proliferation of cells with a high degree of genetic damage could result in the expansion of a population of genetically-altered cells, which is important in pulmonary carcinogenesis and tumor promotion. MWCNTs were found in association with the DNA, the microtubules, the centrosomes as well as inside the centrosome structure. In this study, fragmented centrosomes clustered into a single pole were observed. These results are in sharp contrast to the multipolar mitotic spindles that were observed with narrower SWCNTs. The mitotic disruption following exposure to MWCNTs may be due to a number of factors including incorporation of the nanotubes into the centrosome and microtubules of the mitotic spindle. Although both SWCNTs and MWCNTs had a strong association with the microtubules that make up the mitotic spindle and induced aberrant mitotic spindles, the data suggests that the type of damage may be determined by the diameter of the carbon nanotubes. The stiffness of the nanotubes is determined by their diameter. Although carbon nanotubes have similar mechanical properties to the microtubules, the stiffness of the carbon nanotubes is a thousand-fold greater than that of the microtubules. The incorporation of the more rigid MWCNTs into the microtubules that make up the mitotic spindle fibers and the centrosome may reduce the elasticity of the mitotic spindle apparatus to a greater degree than the SWCNT. The elasticity of the mitotic apparatus is a critical factor in the separation of the centrosomes to organize two spindle poles as well as in the separation of the chromosomes during cell division.

Although lung cancer or mesothelioma have not been observed in humans exposed to MWCNTs, centrosome disruption, aneuploidy and mitotic spindle aberrations -as well as recent data indicating mesothelioma and lung tumor promotion and progression – are a concern. Indications are that caution should be used to prevent respiratory exposure to workers during the production or use of commercial products using carbon nanotubes.

For more information about this study:

Genotoxicity of Multi-Walled Carbon Nanotubes at Occupationally Relevant Doses
Katelyn J Siegrist, Steven H Reynolds, Michael L Kashon, David T Lowry, Chenbo Dong, Ann F Hubbs, Shih-Houng Young, Jeffrey L Salisbury, Dale W Porter, Stanley A Benkovic, Michael McCawley, Michael J Keane, John T Mastovich, Kristin L Bunker, Lorenzo G Cena, Mark C Sparrow, Jacqueline L Sturgeon, Cerasela Zoica Dinu and Linda M Sargent, Published: 30 January 2014 © 2014 Siegrist et al; licensee BioMed Central Ltd.

Kristin L. Bunker, Ph.D.

About Kristin L. Bunker, Ph.D.

Kristin L. Bunker, Ph.D. in Materials Science and Engineering, is currently a Senior Scientist at RJ Lee Group, Inc. where she specializes in the analysis and characterization of materials utilizing ultra high-resolution electron microscopy and X-ray spectroscopy techniques. She directs and conducts forensic examinations of particulate, nanomaterials, coatings, and thin films in advanced materials, environmental, manufacturing, and pharmaceutical projects. Dr. Bunker works closely with industry, national laboratories, and government agencies in the evaluation of nanoparticles from an industrial hygiene and environmental perspective. She is an expert in the characterization of various nanomaterials used in sensors, diagnostics, aerospace composites, architectural products and protective coatings among others.  
In 2012 Dr. Bunker became the President of the internationally recognized Microanalysis Society (MAS). She is the author or co-author of more than 40 publications covering topics in semiconductors, life sciences, mineralogy, and nanomaterials. Prior to receiving her Ph.D., Dr. Bunker served as an expert witness in California courts for the analysis of Gunshot Residue (GSR).

Contact Kristin Bunker