NORA Manufacturing Sector Strategic Goals
927ZJFX - Vanadium Pentoxide – Motor Protein Interactions by 51V NMR SpectroscopyStart Date: 10/1/2009
End Date: 9/30/2011
Principal Investigator (PI)Name: David Murray
Funded By: NIOSH
Primary Goals Addressed5.06.0
Secondary Goal Addressed
Attributed to Manufacturing
51V NMR spectroscopy and cellular bioassays will be used to examine vanadium-protein interactions underlying the toxicity and carcinogenicity of airborne vanadium particulate dust. Vanadium in airborne particulate matter is associated with increased incidence of occupational respiratory disease and is a suspected carcinogen, though chemical mechanisms describing these hazards have not been determined. This project will contribute to efforts in the Manufacturing sector to reduce the number of work-related respiratory diseases and malignancies and both the respiratory disease and cancer cross sectors. Workers exposed to respirable vanadium in the manufacture of steel, aluminum or titanium alloys or in ceramics or glass suffer from short term respirable disease and may be at risk for cancer. Proteins studied will be those associated with vanadium transport (transferrin) or essential functions in mitotic spindle activities, including tubulin and several families of motor proteins (kinesin and dyneins). Their activities are disrupted by undetermined vanadate species which results in aneuploidy, a pathway to carcinogenesis. This project will provide four publications reporting on the state of vanadium in these interactions and the conditions in which transport or disruption processes occur. These results may suggest improved or more selective means of determining disease risk through the development of more effective exposure assessment measurement tools specific to vanadium toxic species or biomarkers of disease.
Chemical and bioassay techniques will be used to describe the chemical mechanisms involved in inhaled vanadium particulate toxicity. Vanadium-protein interactions will be examined to determine the active vanadium species involved and conditions that control their in vivo distribution from the lungs and their role in disrupting mitotic spindle processes. Vanadium pentoxide, a suspected carcinogen, may induce aneuploidy through motor protein disruption, tubulin breakage or other interactions. Identifying the active species and favorable chemical conditions is important for the development of improved and selective methods to assess exposure to species displaying greatest risk in disease processes based on known chemistry.
The project objective is to provide direct evidence using 51V NMR spectroscopy of the interaction of vanadium pentoxide, vanadate oligomers, or of vanadic or vanadyl anions with transport and motor proteins. Four types of protein will be examined: transferrin, tubulin, kinesins, and dynein. These interactions are suspected to be the basis for vanadium pentoxide in vivo transport and vanadium-induced carcinogenesis.
Specific Aim 1: 51V NMR studies will be incorporated into parallel assays to monitor vanadate-protein interactions in human small airway epithelial cells. Vanadium may interfere with mitotic spindle protein (kinesins and dynein) activity or motility, or may play a role in causing microtubule or chromosomal damage. Assays will be conducted at three levels of vanadium pentoxide exposure. Assays include kinesin and dynein activity; kinesin and dynein motility, acetylation of tubulin by immunofluorescent detection, Western blot (detecting acetylated tubulin by mouse monoclonal antibody), and a chromosomal breakage assay using fluorescent in situ hybridization (FISH) analysis (Aneuploidy, chromosome breakage and chromosomal translocations will be determined using inverted DAPI banding.).
Specific Aim 2: 51V NMR studies will be conducted for direct vanadium-protein interactions in 0.02 mM buffered solutions. 51V NMR spectroscopy will be used to determine vanadium species' oxidation states and concentrations, which vary according to pH, phosphate concentration, buffers, and temperature. The vanadium oxide species type will be varied over a range of pH conditions to favor specific vanadate oligomer types, and lower oxidation states (vanadyl or vanadic anions) will be included. Active species can then become targets for more selective monitoring methods or schemes to mitigate their toxic chemical behavior.
The inhalation of vanadium-containing dust is associated with occupational respiratory disease for workers in metal manufacturing and synthetic material manufacturing industries. Inhaled vanadium particulate transported from lungs produces extrapulmonary toxic effects, and the nature of this transport and the role in carcinogenicity is suspected to be a function of interaction with proteins. Vanadium pentoxide exhibited carcinogenic behavior in in vivo animal studies conducted as part of the National Toxicology Program. In separate in vivo studies, vanadium in the highest oxidation state V(V) was found to produce lung inflammation, cell apoptosis, and tissue injury, effects attributed to the generation of reactive oxygen species similar to other reducible transition metals such as chromium or nickel. Vanadium in vivo is present in a range of hydration, oxidation and oligomerization states depending on pH, and concentration, as soluble anionic orthovanadate (VO43-) and metavanadate (VO31-) and dimeric to decameric oligomers, and from cationic VO21+ species in the (V) state, to vanadyl (IV) cations (VO2+) and vanadic (III) V3+cations in lower states. These lower oxidation states exhibit lesser degrees of toxicity. Transferrin is a bloodborne metal transport protein known to translocate vanadium in living systems. Vanadate-transferrin interactions may account for the extrapulmonary distribution of vanadium. Tubulin, kinesins, and dynein are proteins with critical roles in the separation of chromosomes during mitosis. Vanadium has been shown to disrupt mitotic spindle assembly processes, but specific interactions with these proteins have not been established. Competition between orthovanadate and orthophosphate anions may disrupt kinesin or dynein motor protein activities which are powered by ATPase enzymatic activity. Knowledge of vanadium's interaction with these proteins may improve the development of disease prevention and treatment strategies for vanadium-induced cancers.
51V nuclear magnetic resonance (NMR) spectroscopy will be employed to provide direct detection of vanadium-protein interactions. This technique will be employed for the in vitro study of the nature and extent of protein interactions with vanadium under simulated in vivo conditions, including variations in vanadium concentration, oxidation state, pH, buffers, and phosphate concentration. Single pulse high resolution spectra will first be employed (first quarter of FY10) to yield chemical shift data that differentiates and quantifies the species generated by vanadium pentoxide hydration at sub-millimolar concentrations. 51V NMR spectroscopy will then be coordinated with bioassay techniques which yield observational evidence of motor protein motility, and/or microtubule or chromosomal breakage upon vanadium pentoxide exposure throughout the remainder of FY10. Vanadium-protein interactions in solution will be examined by 51V NMR starting in the second quarter of FY10 with transferrin. Vanadium-tubulin interactions will be conducted 3rd quarter FY10, followed by kinesins in the 4th quarter and throughout FY11. Interactions with dynein are planned for mid FY11.
The overall objective is to provide direct evidence using 51V NMR spectroscopy of mechanisms for inhaled vanadium to translocate in vivo and to disrupt motor protein activities involved in mitotic processes. These interactions are suspected to be the basis for vanadium pentoxide carcinogenesis and in its extrapulmonary toxicity. 51V NMR spectroscopic studies will be coordinated with bioassays to confirm vanadium pentoxide activity in mitotic spindle disruption. In vitro vanadium-protein interactions will be studied to observe the effects of vanadium species' transport, concentration, oxidation state, and oligomer formation, and to identify changes that may occur under different pH or temperature conditions, or upon exposure to phosphates or buffers. Factors affecting vanadium carcinogenesis and toxicity mechanisms will be reported in peer-reviewed publications, and these may contribute to improved measurement, prevention or treatment strategies.
Outputs from this project will be evaluated by:
(1) peer-review of the project proposal
(2) peer-review of reports prepared for journal publication or presentation
(3) requests from stakeholders for training and other assistance in the use of 51V NMR spectroscopic or in coordinating 51V NMR results with other bioassay monitoring of vanadium-protein interactions or monitoring of other vanadium-protein interactions.
Vanadium in airborne particulate matter is associated with increased incidence of occupational respiratory disease and is a suspected carcinogen, though the chemical bases for these effects are still undetermined. Knowledge of the chemical states, reaction products and factors affecting vanadium distribution may contribute to in vivo measurement, and improved disease prevention or treatment strategies. Approximately 500,000 workers were potentially subject to vanadium exposure in metal manufacturing or material synthetic processes in 2007. The major use of vanadium in the United States is in the production of steel or titanium alloys. It is used to make hardened or high speed steel, especially for use in automobile parts, springs, and ball bearings. Vanadium is alloyed with aluminum and titanium in airframes and aircraft engines. Vanadium oxide is a catalyst for sulfuric acid production. Vanadium is also incorporated into glasses, ceramics, or lithium ion batteries. Recent production of nanoparticulate vanadium oxide materials may potentiate increased or different toxic effects than those reported in vanadium toxicity studies of corresponding bulk materials. Workers in energy-producing industries may also be exposed to vanadium oxides in the airborne residual ash produced during combustion or refining of coal or petroleum. The Occupational Safety and Health Administration has set an exposure limit of 0.05 mg/m3 for vanadium pentoxide dust and 0.1 mg/m3 for vanadium pentoxide fumes in workplace air for an 8-hour workday, 40-hour work week.
Vanadium in airborne particulate matter is associated with increased incidence of occupational respiratory disease and is a suspected carcinogen, though chemical processes underlying vanadium biochemistry in lungs and its subsequent systemic distribution are poorly understood. Initial competitive vanadium interactions and complexes formed with lung fluid components influence complex stability and bioavailability. These interactions will be characterized using 51V NMR and ESR spectroscopies.
Coupling these monitoring techniques with a digestion method may extend utility of spectroscopic detection to permit the study of intracellular vanadium interactions. This may advance assessment of vanadium exposures in vivo and development of improved prevention and treatment strategies important to the reduction of respiratory disease and cancer among manufacturing workers, and to better assessment of toxic exposure to vanadium dust.
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