NORA Manufacturing Sector Strategic Goals
927ZJKCa - Mechanism of carbon nanotube-induced aneuploidy (NTRC)
Principal Investigator (PI)
Primary Goal Addressed
Secondary Goals Addressed
Attributed to Manufacturing
We are also planning to use in vitro and in vivo models to examine the mechanism and time course of Single Walled Carbon Nanotube-induced aneuploidy, mitotic spindle disruption, centrosomal damage and the potential risk of lung cancer. If these experiments are successful we plan to extend these findings to nanomaterials with other chemical and physical properties. Results from these experiments should directly benefit workers in the Manufacturing, Construction and Mining sectors.
Preliminary data shows that single walled carbon nanotubes (SWCNT) induce tubulin bundling, binucleate cells and polypoloid cells, mitotic spindle aberrations and fragmentation of the centriole and aneuploidy at higher levels than the positive control vanadium pentoxide. The level of mitotic spindle disruption and anueploidy is equal to that published with the chemotherapeutic agent, Tamoxifen [66,67]. Centriole disruption can result from global DNA damage, inhibition of spindle motors or inhibition of protein degradation. The centriole determines the shape of the mitotic spindle and the cytoskeleton. Tubulin disruption, centriole fragmentation, aneuploidy and changes in cell shape are characteristics of tumor cells. The associated human health hazards of SWCNT have not been investigated, including their potential for carcinogenicity. We have therefore proposed to examine the mechanism, time course and potential carcinogenicity of carbon nanotube exposure.
Specific Aim 1. Determine the mechanism(s) of SWCNT-induced aneuploidy and mitotic spindle disruption. Specific Aim 2. Determine the dose-response, time-course, and progression of mitotic spindle, DNA and centrosome disruption. Specific Aim 3. Determine the potential aneuploidy and carcinogenicity of in vivo SWCNT exposure.
Research to Practice (r2p) sections:
Involvement of Stakeholders: We will present our findings at scientific meetings. The work will be published in scientific journals. In the future we will use the NANOTECH Report, 4th edition from the Investment Overview and Market Research for Nanotechnology document to locate companies who are using nanotechnology. The published results from this study will provide information on biomarkers for occupationally-induced lung cancer as well as further examine the mechanism of genetic damage induced by vanadium pentoxide and nanoparticles. Updated data available on the NIOSH webpage will provide information for exposed workers and their employers on the hazard identification and toxicity of nanotube exposure. The results of this research have the potential to establish exposure standards and recommended handling practices to avert significant human health risks in the future. The findings of these proposed studies will be of interest to the National Toxicology Program, the National Institute of Environmental Health Sciences and the Environmental Protection Agency.
The goals of the project are as follows: 1) Study the mechanism of SWCNT induced centrosome and mitotic spindle aberrations and associated abnormal chromosome number (aneuploidy) using in vitro systems. 2) Determine the dose-response, time-course, and progression of mitotic spindle, DNA and centrosome disruption. 3) Determine the potential of acute in vivo exposure of SWCNT and the more rigid MWCNT to induce mitotic spindle aberration and aneuploidy in exposed mice. 4) Examine the potential of single walled carbon nanotubes to induce cancer in exposed mice. A few potential confounders may arise and require consideration in the extrapolation of the in vitro tissue culture studies and in vivo mouse exposure studies to human disease. The in vitro studies do not involve the inflammatory response that occurs in vivo. The positive results in the in vitro system would show potential for genotoxicity events in the absence of inflammation. The combined effects of nanotube exposure and inflammation are unknown. The examination of the genotoxicity in vivo will be timed to the full inflammatory response. Rodent models are used in the scientific community to examine the toxicity, genotoxicity and carcinogenicity of environmental exposures. Our study will allow a comparison of the results from the in vitro exposure of human primary cells to the in vivo mouse exposure. The data will be published in scientific journals. The results from the published studies will be used in the NIOSH “Approaches to Safe Nanotechnology” document to influence recommended prevention measures and good work practices by industry.
Carbon nanotubes are currently used in many consumer and industrial products such as electronics, drug delivery devices, protective clothing, sports equipment, and in research of genetically modified crops and space exploration. The nanotechnology industry is a multi-billion dollar industry that is expected to reach a trillion dollars by 2015. The low density and small size of these particles makes respiratory exposures likely. Nanotubes are degraded slowly and may stay in the body for long periods of time following exposure. Carbon particles are organic structures which generally have low toxicity based upon chemical composition.
However, new technology allows the production of manufactured carbon nanotubes, which are narrow, hollow, fibrous tubes of graphene carbon. Manufactured carbon nanotubes with one layer are known as single-walled carbon nanotubes (SWCNT), while those composed of multiple layers are known as multi-walled carbon nanotubes (MWCNT). The durability and physical characteristics (high aspect ratio) of carbon nanotubes resemble that of asbestos and suggest similar toxicity. Although a number of occupational exposures to particles are often regulated based upon particle composition, the toxicity of durable inorganic mineral fibers is often determined by particle dimensions rather than chemical composition. Both SWCNT and MWCNT can be aerosolized under workplace conditions. Occupational exposures to nanoparticles are likely to increase as their use in manufacturing, electronics, and medicine increases; however, the human health hazards associated with exposure to carbon nanotubes have not been fully investigated, especially their potential for carcinogenicity.
The in vitro studies observations demonstrate that SWCNT induce genetic damage in primary and immortalized respiratory epithelial cells. The SWCNT-induced microtubule disruption, centriole fragmentation, aneuploidy and changes in cell shape will be examined in vitro and in vivo. This type of genetic damage is characteristic of cancer cells. The low density and small size of these particles makes respiratory exposures likely. Nanotubes are degraded slowly and may stay in the exposed body for long periods of time. The highest exposures are expected to occur occupationally, either in the production or use of various new products. The human health hazards associated with exposure have not been fully investigated, including their potential for carcinogenicity. Lung cancer is the leading cause of cancer death not only in the United States but worldwide, with the number of annual deaths in the U.S.A., 157,000, being greater than that from breast, ovarian and prostate cancer combined. Although the majority of lung cancers are linked to tobacco use, lung cancer is ranked second only to bladder cancer in proportion of cases thought to be due to occupational exposures. Due to the lethality of the disease, there is an urgent need to establish whether carbon nanotubes induce lung cancer.
1. The genetic damage in vivo will be examined by determination of the centrosome damage, the mitotic spindle disruption and the chromosome number following exposure of mice by pharyngeal aspiration.
2. Animals will be dosed with carbon nanotubes based on the acute experiments and followed for 1 and 2 years to determine the potential for carcinogenicity.
3. The genetic damage that is induced by nanotubes with other physical properties has begun with the exposure of primary and immortalized epithelial cells in vitro. Preliminary data indicates that MWCNT exposure results in monopolar mitotic spindles.
4. Nanohybrid molecules of nanotubes (SWCNT and MWCNT) and microtubules have been produced. An assay will be performed using SWCNT and MWCNT to determine if they inhibit the cellular motor kinesin and if the transport of the cellular motor across the hybrid molecules occurs and if the efficiency is the same as the transport across the microtubule.
5. In collaboration with Kevin Vaughan, sedimentation of SWCNT followed by a Western Blot indicates an interaction between dynein, alpha-tubulin, and gamma-tubulin. Interaction with dynein supports the hypothesis that SWCNT interrupt normal mitotic dynein function resulting in metaphase arrest. Since SWCNT gamma-tubulin is a critical centrosomal protein and alpha-tubulin is one of the major subunits of the microtubule, association with alpha-tubulin and gamma-tubulin may lead to spindle fragmentation.
6. Live cell imaging will be used to determine the dynamic interaction with subcellular structures, the chromosomes, the tubulin, the microtubules and the centrosome.