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NORA Manufacturing Sector Strategic Goals

927ZGFX - WC-Co nanoparticles in initiating angiogenesis by reactive oxygen species

Start Date: 10/1/2008
End Date: 9/30/2012

Principal Investigator (PI)
Name: Min Ding
Phone: 304-285-6229
Organization: NIOSH
Sub-Unit: HELD
Funded By: NIOSH

Primary Goal Addressed

Secondary Goal Addressed

Attributed to Manufacturing


Project Description

Short Summary

This project will investigate the potential pulmonary carcinogenesis in response to WC-Co particles exposure using cell culture and animal model. Exposure to WC-Co has been show to causes pulmonary disease and induces lung cancer. Our preliminary results indicated that nano–WC–Co generated more ROS than the fine particles when incubated with the cells. Both fine and nanoparticles of WC-Co stimulate angiogenesis using (CEM) assay. The underline mechanisms of WC-Co-induced carcinogenesis effects have not been investigated. The mechanistic investigations (gene mutation, activation of transcription factors, ROS generation) will be conducted to explain the events of WC-Co-induced tumor initiation, promotion, and progression. Determining the mechanisms involved in WC-Co-induced carcinogenesis in parallel with the manipulation of target signaling could provide insights for the development of biomarkers and possible prevention strategies for WC-Co-induced diseases, thus, targeting cancer, respiratory disease, and nanotechnology issues important in the construction and manufacturing sectors.


Workers in producing and using hard metal products are exposed to WC-Co. The proposed project is a cancer research methods project that will examine the mechanisms of carcinogenesis and angiogenesis of WC-Co in pulmonary tissue.

Angiogenesis is important in cancer development and is required for tumor growth. Tumors cannot grow larger than 1-2 millimeters in diameter without an efficient blood supply provided by angiogenesis. Tumor angiogenesis can be triggered by extracellular signals, such as environmental carcinogens or growth factors, and by genetic alterations, such as activation of oncogenes or deletion of tumor suppressor genes. Angiogenesis is the process by which new blood capillaries are generated from the pre-existing vasculature. The process involves multiple steps including dissolution of the basement membrane of the vessel, endothelial cell proliferation and migration, formation of a new vessel lumen and vessel branches, and maturation of the new vessel by the recruitment of pericytes and the formation of basement membrane

The hypothesis is that WC-Co may react with cellular systems and generate reactive oxygen species (ROS). The ROS trigger cellular signal cascades leading to the oncogene expression, gene mutation, and alternation in the function of tumor suppressor genes, and unregulated cell proliferation which may eventually result in cancer development or progression. The genes or gene products we are planning to investigated are; AP-1, Akt, NF-?B, PI3 kinase, and MAPKs. Those gene products are known to play pivotal role in regulation of angiogenesis, cell proliferation, and transformation. Both cell cultures and animal models will be used in the present study.

The specific aims of the project are: (1) Identify ROS production in lung epithelial cells exposed to WC-Co nanoparticles and the mechanism of ROS generation in the cells. (2) Determine the effects of WC-Co nanoparticles in lung epithelial cells for inducing angiogenesis, and roles of ROS and other signalling molecules in the nanoparticles-inducing angiogenesis. (3) Determine which signalling pathways and molecules are regulated by WC-Co nanoparticles through ROS production. (4) Use mouse model to study the roles of WC-Co nanoparticles in inducing signalling molecules, effectors, and angiogenesis.

The cultured cell lines will be used as an in vitro model to study gene alteration induced by WC-Co. ROS generation will be measured by ESR. Angiogenesis assay in chicken embryo (CAM) will be used to study the effects of WC-Co on cultured cells angiogenesis. The AP-1 luciferase reporter transgenic mice will be used as an in vivo model for this study. The role of transcription factors, AP-1, AKT, PI3 kinase, and NF-kB, as well as the ROS, in angiogenesis will also be evaluated by using the specific inhibitors or antioxidant reagents.

The project will be carried out by staffs at PPRB with a combination of expertise in toxicology, physiology, chemistry, free radical biology, and molecular biology. Extramural collaboration will involve researches at West Virginia University, New York University, and Minnesota University.


1. Identify ROS production in lung epithelial cells exposed to WC-Co nano- or fine-particles and the mechanism of ROS generation in the cells. We will determine which species of ROS are generated by the exposure of cells to WC-Co particles, and what signaling molecules are required for ROS induction by the particles. We will specifically test the involvement of membrane-bound NADPH for the ROS production in the cells.

2. Determine the effects of WC-Co particles in lung epithelial cells for inducing angiogenesis, and roles of ROS and other signalling molecules in the particle-inducing angiogenesis. We will analyze whether WC-Co particles induce angiogenesis through ROS production, PI3K, AKT, and ERK1/2 signalling pathways in lung epithelial cells. We will further test whether AP-1, VEGF, or NF-?B are downstream effectors for mediating particle-inducing angiogenesis in the cells.

3. Determine which signalling pathways and molecules are regulated by WC-Co nanoparticles through ROS production. We will study whether the particles regulate EGF receptor activation, PI3K, AKT, and ERK1/2 signaling through ROS production, and whether the particles regulate AP-1, MAPKs, and NF-?B expression through ROS. We will then study the connection of these signaling molecules induced by the nanoparticles in the cells.

4. Use mouse model to study the roles of WC-Co nanoparticles in inducing signalling molecules, effectors, and angiogenesis. To further understand the mechanism of WC-Co nanoparticles in inducing angiogenesis, we will determine whether the nanoparticles induced ROS and the activation of AKT and ERK1/2, and their downstream effectors, AP-1, VEGF and NF-?B activities in the lung using the transgenic mice. We will then use mouse model and transgenic mice to test whether the nanoparticles regulate ROS and other related signaling molecules for inducing angiogenesis in the lung. This study will provide further insight into the molecular mechanism underlying the particle-inducing angiogenesis.

Mission Relevance

Hard metals or cemented carbide consists of a powder mixture of tungsten carbide (WC, 85%) and metallic cobalt (Co, 5–15%). WC–Co was evaluated as a probable carcinogen in humans (Group 2A) by IARC in 2003. Currently hard metals are widely used in a wide range of products from aerospace, automobile, to home appliances. For these applications, it is important to have the hard metals with the following mechanical properties: hardness, toughness, compressive strength, transverse rupture strength, and wear resistance. There is a special interest to use finer and finer cemented carbide particles because it is found that hardness and wear resistance increase with decreasing the size of hard metals. It is indicated that the WC-Co nanoparticles have many advantages over the fine particles including the greatly improved toughness and hardness, wear resistance. Currently WC-Co nanoparticles can be produced in bulk quantities, and is in the process of commercializing in USA (Inframat Corporation, Willington, CT). It is estimated that application of WC-Co nanoparticles is emerging and growing to replace the WC-Co fine particles in various applications and products for improving the mechanical properties of cemented carbide hard metals in the future.

There are increasing public concerns over the adverse health effects of exposure to nanoparticles and environmental impact of emerging nanotechnology. Occupational exposure to WC-Co dust, such as WC-Co manufacturing industry, outdoor construction, mining, and road construction, may place these workers at increased risk of lung cancer. The molecular mechanisms involved in WC-Co-induced toxicity and carcinogenesis are not fully understood. Thus, this study is to investigate the carcinogenic effects and mechanisms of fine or ultra-fine particles of WC-Co in the lung using cultured cell lines and experimental animals.

The result from the present study will provide solid scientific information concerning health risks posed by occupational exposure to WC-Co and improve our understanding by employing in vitro and in vivo models of pulmonary carcinogenesis induced by WC-Co. Mechanistic information may identify biomarkers for early cancer detection which could be employed in surveillance studies.

The project supports the following goals:

Construction Sector (50%), Goal 5: Reduce silica exposure and future silicosis risks among construction workers by increasing the availability and use of silica dust controls and practices for tasks associated with important exposures. Intermediate Goal 5.5 (09PPCONIG5.5): Evaluate hazard and exposure assessment research gaps associated with silica in construction. Research Goal 5.5.1(09PPCONAOG5.5.1): Reactive species hazard component. Support research to improve understanding of health effects . . . . with mixed exposures to silica particles co-generated with metal exposures.

Manufacturing Sector (50%), Goal 6: Reduce the prevalence of cancer due to exposures in the manufacturing sector; and Goal 5: Reduce the number of respiratory conditions and diseases due to exposures in the manufacturing sector.

Cancer Cross Sector (50%), Goal 1: Reduce the incidence of work-related cancer. Intermediate Goal 1.1 (09PPCRCIG1.1): Conduct research to reduce work-related cancer.

Respiratory Disease Cross Sector (50%), Goal 4: Prevent and reduce work-related respiratory malignancies. Intermediate Goal 4.2 (09PPRDRAOG4.2): Reduce mortality from work-related cancer by developing testing, and implementing methods for early detection of work-related cancer. Activity/Output Goal 4.2.2: Develop and validate biomarkers of exposure to occupational carcinogens or biomarkers for early detection that address needs of specific occupational groups at high lung cancer risk. (09PPRDRSG5) "Prevent respiratory and other diseases potentially resulting from occupational exposures to nanomaterials". Intermediate Goal 5.1 (09PPRDRIG5.1) "Determine the potential respiratory toxicities of nanomaterials" Activity/Output Goal 5.1.1 (09PPRDRAOG5.1.1) "Perform basic in vitro and in vivo toxicology studies"

Nanotechnology (100%), Goal 1: Determine if nanoparticles and nanomaterials pose risks for work-related injuries and illnesses. Intermediate Goal 2.2 (09PPNANIG2.1): Key factors and mechanisms…Evaluate acute and chronic effects in the lung and in other organs systems and tissues. Performance Measure 2.1: Determine the pulmonary and systemic effects of other nanoparticles within the next five years.

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