DOI: 10.6084/m9.figshare.1626638.v1
title: <i>In vitro</i> cleavage of diisocyanate-glutathione conjugates by human gamma-glutamyl transpeptidase-1

description: Isocyanates differ from many other xenobiotics in their ability to form <i>S</i>-linked conjugates with glutathione (GSH) through direct nucleophilic addition reactions (e.g. without enzymatic “preactivation” and/or transferase activity), potentially predisposing them to metabolism via the mercapturic acid pathway. <i>In vivo</i>, mono-isocyanates are metabolized via the mercapturic acid pathway and excreted as N-acetylated cysteine conjugates, however, the metabolism of di-isocyanates remains unclear.We assessed the ability of purified human gamma-glutamyl transpeptidase-1 (GGT-1), a primary enzyme of the mercapturic acid pathway, to cleave <i>S</i>-linked GSH conjugates of 4,4′-methylene diphenyl diisocyanate (MDI) and 1,6-hexamethylene diisocyanate (HDI), two widely used industrial chemicals.A combination of liquid chromatography (LC), tandem mass spectrometry (MS/MS) and hydrogen-deuterium exchange studies confirmed GGT-1 mediated formation of the 607.2 and 525.2 <i>m</i>/<i>z</i> (M + H)<sup>+</sup> ions corresponding to bis(cys-gly)-MDI and bis(cys-gly)-HDI, respectively, the cleavage products expected from the corresponding bis(GSH)-diisocyanate conjugates. Additional intermediate metabolites and mono(cys-gly)-conjugates with partially hydrolyzed diisocyanate were also observed.Consistent with GGT enzyme kinetics, metabolism proceeded more rapidly under conditions that favored transpeptidation versus hydrolytic mechanisms of cleavage. Together the data demonstrate the capacity of human GGT-1 to cleave GSH conjugates of both aromatic and aliphatic diisocyanates, suggesting a potential role in their metabolism. Isocyanates differ from many other xenobiotics in their ability to form <i>S</i>-linked conjugates with glutathione (GSH) through direct nucleophilic addition reactions (e.g. without enzymatic “preactivation” and/or transferase activity), potentially predisposing them to metabolism via the mercapturic acid pathway. <i>In vivo</i>, mono-isocyanates are metabolized via the mercapturic acid pathway and excreted as N-acetylated cysteine conjugates, however, the metabolism of di-isocyanates remains unclear. We assessed the ability of purified human gamma-glutamyl transpeptidase-1 (GGT-1), a primary enzyme of the mercapturic acid pathway, to cleave <i>S</i>-linked GSH conjugates of 4,4′-methylene diphenyl diisocyanate (MDI) and 1,6-hexamethylene diisocyanate (HDI), two widely used industrial chemicals. A combination of liquid chromatography (LC), tandem mass spectrometry (MS/MS) and hydrogen-deuterium exchange studies confirmed GGT-1 mediated formation of the 607.2 and 525.2 <i>m</i>/<i>z</i> (M + H)<sup>+</sup> ions corresponding to bis(cys-gly)-MDI and bis(cys-gly)-HDI, respectively, the cleavage products expected from the corresponding bis(GSH)-diisocyanate conjugates. Additional intermediate metabolites and mono(cys-gly)-conjugates with partially hydrolyzed diisocyanate were also observed. Consistent with GGT enzyme kinetics, metabolism proceeded more rapidly under conditions that favored transpeptidation versus hydrolytic mechanisms of cleavage. Together the data demonstrate the capacity of human GGT-1 to cleave GSH conjugates of both aromatic and aliphatic diisocyanates, suggesting a potential role in their metabolism.