Connecting glutathione with immune responses to occupational methylene diphenyl diisocyanate exposure

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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 quasi-stable 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 (10μM–10mM), 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.