Toluene Diisocyanate Reactivity with Glutathione Across a Vapor/Liquid Interface and Subsequent Transcarbamoylation of Human Albumin

Abstract

Glutathione has previously been identified as a reaction target for toluene diisocyanate (TDI) in vitro and in vivo, and has been suggested to contribute to toxic and allergic reactions to exposure. In this study, the reactivity of reduced glutathione (GSH) with TDI in vitro was further investigated using a mixed phase (vapor/liquid) exposure system to model the in vivo biophysics of exposure in the lower respiratory tract. HPLC/MS/MS was used to characterize the observed reaction products. Under the conditions tested, the major reaction products between TDI vapor and GSH were S-linked bis(GSH)-TDI and to a lesser extent mono(GSH)-TDI conjugates (with one N=C=O hydrolyzed). The vapor phase generated GSH-TDI conjugates were capable of transcarbamoylating human albumin in a pH-dependent manner, resulting in changes in the self-protein’s conformation/charge, based on electrophoretic mobility under native conditions. Specific sites of human albumin-TDI conjugation, mediated by GSH-TDI, were identified (Lys73, Lys159, Lys190, Lys199, Lys212, Lys351, Lys136/137, Lys413/414, Lys524/525) and overlap with those susceptible to direct conjugation by TDI. Together, the data extend proof-of-principle for GSH to act as a “shuttle” for a reactive form of TDI, which could contribute to clinical responses to exposure.

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

“…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.…”

Connecting glutathione with immune responses to occupational methylene diphenyl diisocyanate exposure

“…Evidence supporting a “shuttling” capacity of GSH has been derived largely from in vitro studies of toluene diisocyanate (TDI) and aryl mono-isocyanates, demonstrating that GSH conjugates can carbamoylate other peptides/proteins, including albumin [11, 38, 41, 42]. In contrast, GSH mediated transcarbamoylation of MDI, which possesses unique physical properties compared with other mono- and di-isocyanates, remains relatively under-studied.…”

Vapor conjugation of toluene diisocyanate to specific lysines of human albumin

“…The majority of in vitro studies on diisocyanate–albumin reactivity have been performed with liquid phase chemical; however, for volatile diisocyanates such as TDI, the airway microenvironment is exposed to vapor rather than liquid phase chemical [19]. TDI–albumin conjugates that form under such mixed (vapor/liquid) phase exposure conditions differ structurally and conformationally from those formed in liquid phase and have been hypothesized to more closely reflect those that form in vivo, based on immune recognition by IgE from diisocyanate asthma patients [16].…”