FORMATION, SOLVOLYSIS, AND TRANSCARBAMOYLATION REACTIONS OF BIS(S-GLUTATHIONYL) ADDUCTS OF 2,4-DIISOCYANATOTOLUENE AND 2,6-DIISOCYANATOTOLUENE

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 sys tem after passage through the respiratory system. Based on recent work by others regarding the transcarbamoylation reactions of monoisocyana tes, we hypothesized that the reactive form could be masked as an S-th iocarbamoylglutathione adduct of one or more of the isocyanato moietie s. In this study, the glutathione adducts of 2,4- and 2,6-diisocyanato toluene were synthesized under physiological conditions. Bis adducts w ere 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 confirme d by electrospray mass spectrometry (MS), and H-1 NMR analysis strongl y suggested that the thiol of the cysteine residue of glutathione was the nucleophile in each case. The rates of solvolysis of the two bis a dducts 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)-toluylis ocyanato moiety to the peptide as detected by HPLC and on-line electro spray MS analyses. In both the solvolysis and transfer experiments, th e 2,4-TDI-derived bis(glutathionyl) adduct reacted most quickly, while both the bis(glutathionyl)-2,6-TDI adduct and its transfer product wi th the peptide were more stable than their 2,4-TDI-derived counterpart s. The results indicate high stoichiometry in formation and ready tran sfer 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 part icular 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.