NORA Manufacturing Sector Strategic Goals
927ZGFV - Genetic Fingerprint of Mouse Lung CancerStart Date: 10/1/2008
End Date: 9/30/2013
Principal Investigator (PI)Name: Steve Reynolds
Funded By: NIOSH
Primary Goal Addressed5.0
Secondary Goals Addressed6.0, 9.0
Attributed to Manufacturing
We are using in vitro and in vivo models to determine if there are different carcinogen-specific chromosomal (genetic) markers in spontaneously-occurring and chemically-induced mouse lung adenocarcinomas. The mice were exposed by inhalation to vanadium pentoxide, nickel oxide or cumene (a benzene derivative). Workers in the construction and manufacturing sectors are exposed to these compounds. Results from these experiments should directly benefit workers in the Manufacturing and Construction sectors. We are also planning to analyze mouse lung tumors induced by single-wall carbon nanotubes. If these experiments are successful we plan to extend these findings to tumors from occupationally-exposed human populations. Results from these studies will be used to establish biomarkers for early detection and therapeutic intervention of lung cancer in worker populations.
Our hypothesis is that there are distinct genetic differences between spontaneously-occurring and chemically-induced tumors. We propose to analyze chromosomal aberrations and gene expression in spontaneously-occurring mouse lung adenocarcinomas and compare those changes to changes observed in chemically-induced mouse lung adenocarcinomas which were induced by carcinogens that act through different mechanisms. If our hypothesis is correct this will allow identification of biomarkers for detection, diagnosis and intervention of occupationally-related lung cancers.
Long-term exposure to benzene, nickel compounds and vanadium pentoxide are a significant risk factor for development of lung cancer. Benzene is metabolized through reactive intermediates that form bulky adducts which bind to DNA. Vanadium pentoxide is thought to increase cancer risk by inhibiting the assembly and the breakdown of tubulin resulting in aneuploidy while nickel oxide is thought to increase cancer risk via reactive oxygen damage, stabilization of tubulin by acetylation and increased assembly of tubulin. Both vanadium pentoxide and nickel oxide exposure result in binucleate and polyploid cells, mitotic spindle aberrations and aneuploidy by different mechanisms. Nickel compounds induce a bundling of tubulin that is perinuclear. Our preliminary data shows that vanadium pentoxide exposure in vitro induces fragmentation of the centriole and perinuclear bundling in addition to the published spindle disruption. We have also shown that single walled carbon nanotubes (SWCNT) induce tubulin bundling, binucleate cells and polypoloid cells, mitotic spindle aberrations and fragmentation of the centriole similar to vanadium pentoxide. Centriole disruption can result from global DNA damage or inhibition of spindle motors. 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.
1. Analyze the genomic copy number of spontaneously-occurring and chemically-induced mouse lung tumors to determine if there is a distinct difference in genomic fingerprint between spontaneously-occurring and chemically-induced tumors.
2. Determine if the genomic fingerprint of tumors induced by different chemicals that act through different carcinogenic mechanisms produces a genomic fingerprint that is distinct for each carcinogen.
3. Examine the mechanism of mitotic spindle disruption by vanadium pentoxide, nickel oxide and SWCNT.
4. Determine whether the mitotic spindle disruption and centriole disruption by SWCNT is capable of inducing lung cancer in mice.
Research design and methods
Tumor DNA samples for Specific Aims 1 and 2 will be analyzed utilizing NimbleGen high resolution CGH arrays. Gene expression analysis will be performed by real time PCR and immunohistochemistry. Spontaneously-occurring and chemically-induced mouse lung adenocarcinoma samples from mice exposed to cumene (a benzene derivative), nickel oxide and vanadium pentoxide will be obtained from the National Toxicology Program through a collaboration with Dr. Sills.
In Specific Aim 3, primary human and mouse respiratory cells will be exposed in vitro to vanadium pentoxide, nickel oxide or SWCNT. The outcome of the experiments will be measured by analysis of inhibition of kinesin movement, kinesin activity, tubulin protein modification and DNA damage.
In Specific Aim 4, mice will be exposed to SWCNT by pharyngeal aspiration. The animals will be sacrificed for pathological and genetic analysis after 12 and 18 months to determine if nanotubes produce an excess of mouse lung cancer. These copy number and expression changes will be analyzed as described in Aims 1 and 2.
The objectives of these studies are to:
1) Analyze the changes in gene expression and gene copy number that occur in spontaneously-occuring and chemically-induced mouse lung adenocarcinoma. The expression of Tubulin alpha 4 and NUCKS were increased at greater levels in the chemically-induced mouse lung adenocarcinomas as compared to spontaneously-occurring mouse lung adenocarcinomas. The carcinogen exposures were mixed and the sample size was small. We intend to expand the sample size with defined chemical exposures to determine the significant changes in spontaneously-occurring tumors versus spontaneously-occurring tumors.
2) Analyze the specific genetic changes induced in mouse lung adenocarcinoma by exposure to carcinogens of different mechanisms to determine if there are carcinogen specific biomarkers.
3) Determine the mechanism of centriole and tubulin disruption of in vtiro and in vivo exposure to SWCNT, vanadium and nickel oxide.
4) The positive results in the in vitro system demonstrated potential for mitotic spindle events in the absence of inflammation. The combined genetic effects of nanotube exposure and inflammation are unknown. 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 genotoxicity exposure of mouse and human primary cells to in vivo carcinogenicity in the mouse model.
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. It is conservatively estimated that approximately 2,000 women and 10,000 men develop lung cancer each year from past occupational exposures. Studies in both human and mouse suggest there may be distinct genomic differences between spontaneously-occurring and carcinogen-induced lung tumors.
We propose to test the hypothesis that there are distinct genomic differences between spontaneously-occurring and chemically-induced tumors. We further propose to test the hypothesis that there may be distinct genomic differences between tumors induced by carcinogens which act through different mechanisms. The mouse lung adenocarcinoma model is the only experimentally manipulable model for human lung adenocarcinoma. We propose to analyze chromosomal aberrations and gene expression in spontaneously-occurring mouse lung adenocarcinomas and compare those changes to changes observed in chemically-induced mouse lung adenocarcinomas which were induced by carcinogens that act through different mechanisms.
Our approach to testing our hypothesis will be to analyze spontaneously-occurring mouse lung adenocarcinomas and mouse lung adenocarcinomas induced by nickel oxide, vanadium pentoxide and cumene, lung carcinogens in mice which are believed to act through different mechanisms. We also plan to induce mouse lung tumors with single-walled carbon nanotubes and analyze these tumors (preliminary evidence from NIOSH studies indicates that single-walled carbon nanotubes induced centrosomal fragmentation in in vitro experiments and polyploidy and binucleate cells in exposed animals, similar to what is seen with vanadium pentoxide treatment). Construction and manufacturing workers are exposed to nickel, vanadium, cumene and nanoparticles. Spontaneously-occurring mouse lung adenocarcinomas and mouse lung adenocarcinomas induced by nickel oxide, vanadium pentoxide and cumene have already been obtained from the National Toxicology Program (NTP). We will analyze these tumors for genomic changes using a combination of high density DNA array, gene expression array analysis, Fluorescence In Situ Hybridization (FISH), real time PCR and immunohistochemistry.
The 5-year relative survival rate for all stages of lung cancer combined is only 15%. If our hypothesis is correct this will eventually allow identification of biomarkers for detection, diagnosis and intervention of occupationally-related lung cancers in human worker populations.
Sector Goals Cross-sector and sub-sectors and goals of the proposed research:
1) Cancer, Reproductive and Cardiovascular Diseases Cross-Sector Program
Research results from this project will address: Interim Strategic Goal 1 of the Cancer, Reproductive and Cardiovascular Diseases Cross-Sector Program (50%) "Reduce the incidence of work related cancer"; Intermediate Goal 1.1 (09PPCRCIG1.1), Conduct research to reduce work-related cancer; Activity/output goal, 1.1.2 (09PPCRCAPOG.1.2). Assess worker exposures, pre-cancerous effects of exposure or susceptibility to high-priority carcinogens through industry-wide surveys, population-based studies, or analysis of biological specimens;
2) Activity/Output Goal 1.2.3 (09PPCRCAOG1,2,3) of the Cancer, Reproductive, Cardiovascular Diseases Cross-Sector program "Promote carcinogen-free workplaces in the US; "Enhance the relevance and utility of interventions and recommendations by transferring research findings, technologies, and information into practice by publishing in technical and trade journals, developing methods for inclusion into NMAM or similar collections, and developing workplace or trade documents"; Intermediate Goal 1.3 (O9PPCRCG 1.3), Enhance global workplace safety and health through international collaborations; Activity/Output Goal 1.3.2 (09PPCRCAOG2.2), "Address global workplace hazards through information sharing and research collaborations"; 2) Strategic Goal 2 (09PPCRCSG2), Reduce mortality from work-related cancer through early interventions; Intermediate Goal 2.1 (09PPCRCIG2.1), Develop improved methods for early detection of work-related cancer.
3) Strategic Goal 4 (09PPRDRSG4) of the Respiratory Disease Research Program Cross-Sector (50%) "Prevent and reduce work-related cancer through research"; Strategic Goal 4, Intermediate Goal 4.2 (09PPRDRIG4.2): reduce mortality from work-related cancer by developing, testing and implementing methods for early detection of work-related cancer. Strategic Goal 5 (09PPRDRSG5) of the Respiratory Disease Research Program Cross Sector (50%) "Prevent respiratory and other diseases potentially resulting from occupational exposures to nanomaterials." Activity/Output Goal 5.1.1 (09PPRDRAOG5.1.1) perform basic in vitro and in vivo toxicology studies to evaluate for respiratory toxicity of nanoparticles and, if present , to characterize nanoparticle characteristics and mechanisms of action underlying toxic effects.
4) Strategic Goal 5 (09PPMNFSG5) of the Manufacturing Sector program (75%) "Reduce the number of respiratory conditions and diseases due to exposures in the manufacturing sector. Strategic Goal 6 (09PPMNFSG6) of the Manufacturing Sector "Reduce the prevalence of cancer due to exposures in the manufacturing sector. Strategic Goal 9 (09PPMNFSG7) Enhance the state of knowledge related to emerging risks to occupational safety and health in manufacturing.
6) Exposure Assessment (25%). Strategic Goal 2 (09PPEXASG2) "Develop or improve specific methods and tools to asses worker exposures to critical occupational agents and stressors. Intermediate goal 2.82 Initiation of new toxicity studies to fill gaps in knowledge so that better-designed exposure assessment strategies can be developed.
7) Global Collaborations (25%): Strategic Goal 1 (09PPGLCSG1): Enhance global occupational safety and health through international collaborations. Strategic Goal 5 (09PPGLCSG5): Prevent worker illness globally from exposure to nanomaterials by sharing information.
8) Nanotechnology (25%) Strategic Goal 1 (09PPNANSG1). Determine if nanoparticles and nanomaterials pose risks for work-related injuries and illnesses. Performance measure 2.1 Key factors and mechanisms. Intermediate Goal 2.1 (09PPNANIG2.1) Systematically investigate the physical and chemical properties of particles that influence their toxicity (Size shape, surface area, solubility, chemical properties. Performance measure 2.1 Key factors and mechanisms. Determine the pulmonary response (dose dependence and time course) to single-walled carbon nanotubes within the next two years and multi-walled carbon nanotubes within the next three years. Strategic Goal 4 (09PPNANSG4). Enhance global workplace safety and health through national and international collaborations on nanotechnology research and guidance.
9) Construction (25%) Strategic Goal 14.0 (09PPCONSG14) Improve surveillance at the Federal, State, and private level to support the identification of hazards and associated illnesses and injuries; the evaluation of intervention and organizational program effectiveness; and the identification of emerging health and safety priorities in construction.
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