Hexamethylene diisocyanate (HDI) vapor reactivity with glutathione and subsequent transfer to human albumin

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 10 mM GSH solutions resulted in primarily S-linked, bis(GSH)-HDI reaction products. In contrast, lower GSH concentrations (100 μM) 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-/HPO42- 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, twenty-five 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 3 hours) 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, H2PO4-4/HPO42-, 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.

In vitrocleavage of diisocyanate-glutathione conjugates by human gamma-glutamyl transpeptidase-1

“…Glutathione, which is present in high concentrations in the lower respiratory tract is postulated to be a major reaction target for inhaled isocyanates, and readily forms S-linked thiocarbamate products (GSH-isocyanate “conjugates”) under physiologic conditions (Cantin, North et al 1987, Slatter, Rashed et al 1991, Wisnewski, Liu et al 2013, Wisnewski, Mhike et al 2013). The ability of N=C=O to directly conjugate with GSH, without the need for enzymatic “preactivation” or glutathione-S-transferases (required for many other xenobiotics) may predispose isocyanate chemicals to metabolism via the mercapturic acid pathway.…”

“…A single study of MDI exposed rats by Gledhill et al identified an (M+H) + ion at 388 m/z , consistent with that predicted for the N-acetylated-cysteine conjugate of partially hydrolyzed MDI, but did not characterize this metabolite (Gledhill, Wake et al 2005). In vitro studies provide clear evidence for rapid formation of S-linked GSH conjugates, however the ability of GSH-diisocyanate conjugates to be metabolized via the mercapturic acid pathway has yet to be assessed (Reisser, Schmidt et al 2002, Wisnewski, Hettick et al 2011, Wisnewski, Liu et al 2013, Wisnewski, Mhike et al 2013). In this report, we directly evaluate the ability of human GGT-1, the primary enzyme of the mercapturic acid pathway, to cleave GSH-diisocyanate conjugates under well controlled in vitro conditions.…”

Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin

“…This agrees with findings from Wisnewski et al . who found that the rate of HSA carbamoylation from TDI-GSH derived TDI was higher than carbamoylation from HDI-GSH derived HDI 20,28 . Table 2 also demonstrates that HSA was more reactive to TDI than Hb.…”

“…Apart from reacting with proteins at the site of exposure, protein conjugation by dNCOs may also occur via glutathione (GSH) thiocarbamate intermediates. GSH is abundant in the airways and Wisnewski and colleagues demonstrated that albumin can be conjugated to TDI and HDI by GSH-TDI and GSH-HDI, respectively 20 . HSA is the most common carrier protein used for dNCO antibody immunoassays 21 due to its prevalence in plasma 22 to form dNCO adducts.…”

“…Albumin was conjugated to TDI and HDI by GSH-TDI 28 and GSH-HDI 20 , respectively. From these two reports, the kinetics of GSH-HDI mediated albumin carbamoylation was substantially slower compared with those of GSH-TDI.…”