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NIOSH Program Portfolio

 

Manufacturing

NORA Manufacturing Sector Strategic Goals

927ZJFU - Characterization of toluene diisocyanate exposure biomarkers

Start Date: 10/1/2009
End Date: 9/30/2012

Principal Investigator (PI)
Name: Justin Hettick
Organization: NIOSH
Sub-Unit: HELD
Funded By: NIOSH

Primary Goals Addressed
5.0 , 9.0

Secondary Goal Addressed
None


Attributed to Manufacturing
75%

Project Description

Short Summary

The overall goal of the proposed project is to isolate and characterize specific proteins that react with isocyanates when exposed to occupationally-relevant levels of TDI. Diisocyanates are highly reactive chemicals used in production of polyurethane products that represent a significant occupational exposure hazard to workers. This project seeks to reduce isocyanate-induced respiratory disease by developing improved detection methods for isocyanate exposure and characterizing the chemical changes to proteins that cause isocyanate-induced disease. Experiments are proposed that will result in the identification of specific biomarkers of TDI exposure and better reagents for identification of dNCO sensitized workers (e.g. appropriate hapten-protein conjugate to screen workers for TDI-specific antibodies). This project should result in peer reviewed journal articles which will provide scientific data regarding the mechanism of haptenation by isocyanates. The knowledge gained from this study will address specific NIOSH research goals for reducing respiratory disease and occupational skin disease in the manufacturing and service sectors.



Description

Diisocyanates (dNCOs) are widely used in manufacturing for the production of polyurethane products, including wood binders, elastomers, coatings and paints. They are utilized in the automotive service industry in undercoatings, spray-in truck bed liners and paints. They are also found in commercially available glues, paints and lacquers used in construction and in the home. Because of the highly reactive chemical nature of dNCOs, they represent a significant occupational exposure hazard to workers. Documented health effects of dNCO exposure include immune mediated hypersensitivity pneumonitis and asthma, as well as irritation of the skin, respiratory and gastrointestinal systems. According to SENSOR data, isocyanate exposure is responsible for 8.3% of all reported work-related asthma cases. This project seeks to reduce isocyanate-induced respiratory disease by developing improved detection methods for isocyanate exposure and characterizing the chemical changes to proteins may contribute to isocyanate-induced diseases.

Of the diisocyanates, toluene diisocyanate (TDI) is the second-most common dNCO used in industry. In spite of the documented health hazards of dNCO exposure, the ultimate allergenic form of TDI (hapten-protein conjugate) is not known. This has made diagnosis and exposure assessment problematic. Diisocyanates have been reported to show selectivity for target proteins in vivo; however the specific protein targets or nature of protein modification have not been identified. Our laboratory has considerable experience in the fields of TDI bio-organic chemistry, animal modeling, and proteomics/mass spectrometry. Recently, we succeeded in developing a unique set of monoclonal antibodies (mAbs) that recognize TDI-bound bio-molecules. While previous efforts to identify TDI target proteins have been unsuccessful, these mAbs provide a new tool to specifically isolate and identify TDI target proteins.

The overall goal of the proposed project is to isolate and characterize specific proteins of the respiratory tract and skin that react when exposed to occupationally-relevant levels of TDI. In Specific Aim 1 we will utilize quadrupole time-of-flight tandem mass spectrometry (qTOF-MS/MS) to characterize the binding characteristics (binding site, number of molecules bound) of TDI to model peptides and proteins under different mole ratios in vitro. In Specific Aim 2, we will use samples from TDI-exposed mice to identify specific proteins conjugated to TDI using immuno-blotting and immuno-precipitation with the mAbs followed by high performance liquid chromatography /tandem mass spectrometry (HPLC-MS/MS). In Specific Aim 3 these targets will be further characterized to identify the number of TDI molecules bound per protein molecule using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In Specific Aim 4 we will identify specific binding sites (e.g. identify amino acid residue bound to TDI and its position within the protein) on TDI-reactive proteins by tandem mass spectrometry (MS/MS)-based peptide sequencing. Furthermore, we will investigate whether changes are observed in the conjugated proteins (e.g. more and/or different proteins bound to TDI, changes in the number of TDI molecules bound per protein molecule) with changes in exposure time and/or dose. Similarly, we will investigate whether protein or TDI polymerization is observed. Finally, the TDI mAbs will be used to develop an immunoassay for TDI exposure with the identified target proteins.

This project will result in the identification of specific biomarkers of TDI exposure and better reagents for identification of dNCO sensitized workers (e.g. appropriate hapten-protein conjugate to screen workers for TDI-specific antibodies). Furthermore, it will lead to a better understanding of the relationship between exposure and formation of allergenic TDI-protein conjugates within the workers following occupational exposures.



Objectives

The proposed project will be evaluated on the bases of publication of scientific manuscripts, presentations at scientific conferences, and attaining the specific aims as described above. The outcomes will be determined by monitoring citations using internet and journal resources.



Mission Relevance

Diisocyanates are the most commonly reported causes of occupational asthma, the fundamental chemical causes of which are not completely understood. Diisocyanates are widely used in manufacturing for the production of polyurethane products, including wood binders, elastomers, coatings and paints. They are utilized in the automotive service industry in undercoatings, spray-in truck bed liners and paints. They are also found in commercially available glues, paints and lacquers used in construction and in the home. Because of the highly reactive chemical nature of dNCOs, they represent a significant occupational exposure hazard to workers. Documented health effects of dNCO exposure include immune mediated hypersensitivity pneumonitis and asthma, as well as irritation of the skin, respiratory and gastrointestinal systems. Of the diisocyanates, toluene diisocyanate (TDI) is the second-most common dNCO used in industry. In spite of the documented health hazards of dNCO exposure, the ultimate allergenic form of TDI (hapten-protein conjugate) is not known. This has made diagnosis and exposure assessment problematic. Diisocyanates have been reported to show selectivity for target proteins in vivo; however the specific protein targets or nature of protein modification have not been identified.

According to SENSOR data, isocyanate exposure is responsible for 8.3% of all reported work-related asthma cases. This project seeks to reduce isocyanate-induced respiratory disease by developing improved detection methods for Isocyanate exposure and characterizing the chemical changes to proteins may contribute to isocyanate-induced diseases. This project will contribute to the overall understanding of isocyanate-induced occupational disease by (A) delineating the chemical species produced when diisocyanates covalently modify biologically relevant proteins, (B) studying the ultimate disposition of Isocyanates in vivo and identifying the protein targets to which diisocyanates are covalently bound, (C) providing information that can lead to the development of better laboratory diagnostic tools for diisocyanate exposure and asthma. Our laboratory has considerable experience in the fields of TDI bio-organic chemistry, animal modeling, and proteomics/mass spectrometry. Recently, we succeeded in developing a unique set of monoclonal antibodies (mAbs) that recognize TDI-bound bio-molecules. While previous efforts to identify TDI target proteins have been unsuccessful, these mAbs provide a new tool to specifically isolate and identify TDI target proteins. The knowledge gained from this study will address specific NIOSH research goals for reducing respiratory disease particularly in the manufacturing and service sectors.



Page last updated: June 3, 2011
Page last reviewed: May 23, 2011
Content Source: National Institute for Occupational Safety and Health (NIOSH) Office of the Director

 

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Manufacturing