title: Formation, solvolysis, and transcarbamoylation reactions of bis(S-glutathionyl) adducts of 2,4- and 2,6-diisocyanatotoluene.
abstract: During our ongoing studies of the reactions of toluene diisocyanate (2,4- and 2,6-diisocyanatotoluene, TDI) in vivo, it became apparent that reactive form(s) of these diisocyanates reach(es) the circulatory system after passage through the respiratory system. Based on recent work by others regarding the transcarbamoylation reactions of monoisocyanates, we hypothesized that the reactive form could be masked as an S-thiocarbamoylglutathione adduct of one or more of the isocyanato moieties. In this study, the glutathione adducts of 2,4- and 2,6-diisocyanatotoluene were synthesized under physiological conditions. Bis adducts were the major products when near-equimolar amounts of glutathione and the individual diisocyanato compounds were mixed at physiological pH, and were formed in high yield. Little to no mono adducts formed under these reaction conditions. The masses of the bis adducts were confirmed by electrospray mass spectrometry (MS), and 1H NMR analysis strongly suggested that the thiol of the cysteine residue of glutathione was the nucleophile in each case. The rates of solvolysis of the two bis adducts in aqueous buffer under conditions of physiological temperature and pH were determined, and electrospray MS analysis showed that the corresponding mono(glutathionyl)-TDIs were formed in these reactions. Incubation in vitro of each of the bis(glutathionyl)-TDI adducts with a 12 amino acid peptide (Thr-Cys-Val-Glu-Trp-Leu-Arg-Arg-Tyr-Leu-Lys-Asn) at pH 7.5 resulted in transfer of one mono(glutathionyl)-toluylisocyanato moiety to the peptide as detected by HPLC and on-line electrospray MS analyses. In both the solvolysis and transfer experiments, the 2,4-TDI-derived bis(glutathionyl) adduct reacted most quickly, while both the bis(glutathionyl)-2,6-TDI adduct and its transfer product with the peptide were more stable than their 2,4-TDI-derived counterparts. The results indicate high stoichiometry in formation and ready transfer to nucleophilic sites of protein, and suggest that the isocyanato moiety of both 2,4- and 2,6-TDI may be regenerated in vivo from their bis(glutathionyl) adducts. As a consequence, the thiol status of particular tissues may be a contributing factor to individual TDI toxicity susceptibility, and a mechanism by which toxicity at sites distant to the initial point of contact may be proposed.
authors.affiliation: Department of Environmental & Occupational Health, University of Pittsburgh, Pennsylvania 15238, USA.
authors.forename: B W
authors.initials: BW
authors.lastname: Day
authors.forename: R
authors.initials: R
authors.lastname: Jin
authors.forename: D M
authors.initials: DM
authors.lastname: Basalyga
authors.forename: J A
authors.initials: JA
authors.lastname: Kramarik
authors.forename: M H
authors.initials: MH
authors.lastname: Karol
mesh_terms: D000595:Amino Acid Sequence; D002219:Carbamates / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D005978:Glutathione / Q000737:chemistry*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D012997:Solvents / Q000737:chemistry*; D014051:Toluene 2,4-Diisocyanate / Q000737:chemistry*
chemical_list: D002219:Carbamates; D012997:Solvents; D014051:Toluene 2,4-Diisocyanate; C026942:2,6-diisocyanatotoluene; D005978:Glutathione