Vapor conjugation of toluene diisocyanate to specific lysines of human albumin

Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Health Effects Laboratory Division, Morgantown, WV 26505, USA.
Analytical Biochemistry (Impact Factor: 2.22). 12/2011; 421(2):706-11. DOI: 10.1016/j.ab.2011.12.013
Source: PubMed


Exposure to toluene diisocyanate (TDI), an industrially important crosslinking agent used in the production of polyurethane products, can cause asthma in sensitive workers. Albumin has been identified as a major reaction target for TDI in vivo, and TDI-albumin reaction products have been proposed to serve as exposure biomarkers and to act as asthmagens, yet they remain incompletely characterized. In the current study, we used a multiplexed tandem mass spectrometry (MS/MS) approach to identify the sites of albumin conjugation by TDI vapors, modeling the air/liquid interface of the lung. Vapor phase TDI was found to react with human albumin in a dose-dependent manner, with up to 18 potential sites of conjugation, the most susceptible being Lys351 and the dilysine site Lys413-414. Sites of vapor TDI conjugation to albumin were quantitatively limited compared with those recently described for liquid phase TDI, especially in domains IIA and IIIB of albumin. We hypothesize that the orientation of albumin at the air/liquid interface plays an important role in vapor TDI conjugation and, thus, could influence biological responses to exposure and the development of in vitro assays for exposure and immune sensitivity.

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    • "as 4,4 0 -methylene diphenyl diisocyanate (MDI), are high-volume production chemicals used in the production of polyurethane foams, elastomers, paints, and other related products [1] [2]. Exposure to dNCOs has most commonly been reported in the occupational setting [3] [4]; however, concern about potential exposure through leaching of dNCOs from cured, semicured , or non-cured products has recently been raised in domestic settings [5]. "
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    ABSTRACT: Protein haptenation by polyurethane industrial intermediate methylene diphenyl diisocyanate (MDI) is thought to be an important step in the development of diisocyanate (dNCO)-specific allergic sensitization; however, MDI haptenated albumins used to screen specific antibody are often poorly characterized. Recently, the need to develop standardized immunoassays using a consistent, well characterized dNCO-haptenated protein to screen for the presence of MDI-specific IgE and IgG from workers' sera has been emphasized and recognized. This has been challenging to achieve due to the bivalent, electrophilic nature of dNCO leading to the capability to produce multiple cross-linked protein species and polymeric additions to proteins. In the present study, MDI was reacted with human serum albumin (HSA) and hemoglobin (Hb) at molar ratios ranging from 1:1 to 40:1 MDI: protein. Adducts were characterized by (1) loss of available trinitrobenzene sulfonic acid (TNBS) binding to primary amines, (2) electrophoretic migration in polyacrylamide gels, (3) quantification of methylene diphenyl diamine following acid hydrolysis and (4) immunoassay. Concentration dependent changes in all the above noted parameters were observed demonstrating increase in both number and complexity of conjugates formed with increasing MDI concentration. In conclusion, a series of bio-analytical assays should be performed to standardize MDI-antigen preparations across lots and laboratories for measurement of specific antibody in exposed workers which in total indicate degree of intra- and inter-molecular cross-linking, number of dNCO bound, number of different specific binding sites on the protein and degree of immuno-reactivity.
    Analytical Biochemistry 06/2013; 440(2). DOI:10.1016/j.ab.2013.05.022 · 2.22 Impact Factor
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    • "The potential sites of albumin modification by GSH–HDI were numerous compared with those directly conjugated by HDI vapors in previous reports, and overlapped with, but were qualitatively distinct from those conjugated by GSH–TDI, or direct exposure to TDI or MDI (Hettick and Siegel, 2012; Hettick et al., 2011; Wisnewski et al., 2004, 2010, 2011). Among the sites carbamoylated by GSH–HDI were all (four) of albumin's di-lysine site motifs, and Lys 351 , recently suggested to be most susceptible to direct TDI vapor conjugation (Hettick et al., 2011). It should be noted that although the number of potential HDI conjugation sites (via GSH–HDI) was numerous, the absolute amount of conjugation/albumin molecule was relatively low, based on HDA analysis as described above, suggesting conjugation to different lysine groups in different albumin molecules. "
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    ABSTRACT: Introduction: Airway fluid glutathione (GSH) reactivity with inhaled vapors of diisocyanate, a common occupational allergen, is postulated to be a key step in exposure-induced asthma pathogenesis. Methods: A mixed (vapor/liquid) phase exposure system was used to model the in vivo reactivity of inhaled HDI vapor with GSH in the airway fluid. HDI-GSH reaction products, and their capacity to transfer HDI to human albumin, were characterized through mass spectrometry and serologic assays, using HDI-specific polyclonal rabbit serum. Results: HDI vapor exposure of 10mM GSH solutions resulted in primarily S-linked, bis(GSH)-HDI reaction products. In contrast, lower GSH concentrations (100μM) resulted in mainly mono(GSH)-HDI conjugates, with varying degrees of HDI hydrolysis, dimerization and/or intra-molecular cyclization, depending upon the presence/absence of H2PO4(-)/HPO4(2-) and Na(+)/Cl(-) ions. The ion composition and GSH concentration of the fluid phase, during HDI vapor exposure, strongly influenced the transfer of HDI from GSH to albumin, as did the pH and duration of the carbamoylating reaction. When carbamoylation was performed overnight at pH 7, 25 of albumin's lysines were identified as potential sites of conjugation with partially hydrolyzed HDI. When carbamoylation was performed at pH 9, more rapid (within 3h) and extensive modification was observed, including additional lysine sites, intra-molecular cross-linkage with HDI, and novel HDI-GSH conjugation. Conclusions: The data define potential mechanisms by which the levels of GSH, H2PO4(-)/HPO4(2-), and/or other ions (e.g. H(+)/OH(-), Na(+), Cl(-)) affect the reactivity of HDI vapor with self-molecules in solution (e.g. airway fluid), and thus, might influence the clinical response to HDI respiratory tract exposure.
    Toxicology in Vitro 11/2012; 27(2). DOI:10.1016/j.tiv.2012.11.013 · 2.90 Impact Factor
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    ABSTRACT: Electrochemical oxidation of drug molecules is a useful tool to generate several different types of metabolites. In the present study we developed a model system involving electrochemical oxidation followed by characterization of the oxidation products and their propensity to modify peptides. The CB1 antagonist rimonabant was chosen as the model drug. Rimonabant has previously been shown to give high covalent binding to proteins in human liver microsomes and hepatocytes and the iminium ion and/or the corresponding aminoaldehyde formed via P450 mediated α-carbon oxidation of rimonabant were proposed to be likely contributors. This proposal was based on the observation that levels of covalent binding were significantly reduced when iminium species were trapped as cyanide adducts but also following addition of methoxylamine expected to trap aldehydes. Incubation of electrochemically oxidized rimonabant with peptides resulted in peptide adducts to the N-terminal amine with a mass increment of 64 Da. The adduct was shown to contain an addition of C5H4 originating from the aminopiperidine moiety of rimonabant. Formation of the peptide adducts required further oxidation of the iminium ion to short-lived intermediates, such as dihydropyridinium species. In addition, the metabolites and peptide adducts generated in human liver microsomes were compared with those generated by electrochemistry. Interestingly, the same peptide modification was found when rimonabant was co-incubated with one of the model peptides in microsomes. This clearly indicated that reactive metabolite(s) of rimonabant identical to electrochemically generated species are also present in the microsomal incubations. In summary, electrochemical oxidation combined with peptide trapping of reactive metabolites identified a previously unobserved bioactivation pathway of rimonabant that was not captured by traditional trapping agents and that may contribute to the in vitro covalent binding.
    Chemical Research in Toxicology 09/2014; 27(10). DOI:10.1021/tx500255r · 3.53 Impact Factor
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