Connecting glutathione with immune responses to occupational methylene diphenyl diisocyanate exposure

Abstract

Methylene diphenyl diisocyanate (MDI) is among the leading chemical causes of occupational asthma world-wide, however, the mechanisms of disease pathogenesis remain unclear. This study tests the hypothesis that glutathione (GSH) reacts with MDI to form quasistable conjugates, capable of mediating the formation of MDI-conjugated “self” protein antigens, which may participate in pathogenic inflammatory responses. To test this hypothesis, an occupationally relevant dose of MDI (0.1% w/v) was reacted with varying concentrations of GSH (10 μM-10 mM), and the reaction products were characterized with regard to mass/structure, and ability to carbamoylate human albumin, a major carrier protein for MDI in vivo. LC-MS/MS analysis of GSH-MDI reaction products identified products possessing the exact mass of previously described S-linked bis(GSH)-MDI and its partial hydrolysis product, as well as novel cyclized GSH-MDI structures. Upon co-incubation of GSH-MDI reaction products with human albumin, MDI was rapidly transferred to specific lysines of albumin, and the protein's native conformation/charge was altered, based on electrophoretic mobility. Three types of modification were observed, intra-molecular MDI cross-linking, addition of partially hydrolyzed MDI, and addition of “MDI-GSH”, where MDI's 2nd NCO had reacted with GSH's “N-terminus”. Importantly, human albumin carbamoylated by GSH-MDI was specifically recognized by serum IgG from MDI exposed workers, with binding dependent upon the starting GSH concentration, pH, and NaCl levels. Together, the data define a non-enzymatic, thiol-mediated transcarbamoylating mechanism by which GSH may promote immune responses to MDI exposure, and identify specific factors that might further modulate this process.

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

“…GSH was reacted with MDI as previously described (Wisnewski, Liu et al 2013). Briefly, 50 µl of 10% (w/v) MDI in acetone from JT Baker (Phillipsburg, NJ) was added dropwise with stirring to 25 ml of 10 mM GSH in 200 mM sodium phosphate, pH 7.4 (final acetone concentration 0.2% v/v).…”

“…Experiments with GSH-MDI were initially performed for 60 minutes at a temperature lower than optimal (e.g. 22°C) as previously described (Sener and Yardimci 2005), since MDI thiocarbamates are more susceptible than aliphatic thiocarbamates to hydrolysis and/or transcarbamoylation reactions at 37°C (Chipinda, Stetson et al 2006, Wisnewski, Liu et al 2013, Wisnewski, Mhike et al 2013). However, subsequent studies at 37°C (data not shown) yielded identical results.…”

“…MS/MS analysis was performed on an LTQ Orbitrap XL (Thermo Scientific; Waltham, MA) by the Yale Keck Center as previously described (Stone, DeAngelis et al 1998, Wisnewski, Liu et al 2013). The fragmentation patterns of monoisotopic ions corresponding to specific GSH-diisocyanate metabolites were analyzed following collision-induced dissociation (CID).…”

“…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.…”