The reactivity of TDI with GSH was studied under mixed (vapor/liquid) phase conditions, as exists in the human airways, an important site of occupational exposure. GSH-TDI reaction products formed readily, and were further capable of transferring TDI to albumin, altering the self-protein’s native conformation/charge. Specific lysine residues of human albumin were identified as the predominant targets for TDI conjugation, via GSH-TDI conjugates, and overlap with those susceptible to direct conjugation with TDI, a process previously shown to induce antigenic changes (9
). Together, these findings advance our understanding of the complex biochemistry that may connect TDI vapor inhalation with toxicity and/or allergy/asthma, and further suggest an important role for GSH in response to occupational TDI exposure.
The identification of bis(GSH)-TDI conjugates as a predominant reaction product via mixed phase exposure implies that reactivity of TDI with the thiol group of GSH, rather than reactivity with water, is a primary step though which the chemical vapor crosses the human airway’s fluid phase boundary. The reversible thiocarbamates that TDI forms, via S-linked conjugation with GSH, might shelter TDI from hydrolysis thereby allowing further penetration into the body in a reactive form, increasing toxicity or allergenicity, and creating the potential for systemic effects. Thus, GSH and/or other thiols at major sites of occupational TDI exposure (respiratory tract, as well as mucous membranes and skin) could play an important role in toxic and/or allergic responses. Airway fluid pH, which normally ranges from 6.5–7.5, and is acidified in some human conditions (e.g. asthma, pneumonia), may further modulate TDI exposure outcomes via its influence of transcarbamoylation reactions involving TDI-GSH conjugates, as demonstrated here (36
The present findings agree with the previously published studies of liquid phase TDI reactivity with GSH by Day et al, which also described predominately bis(GSH)-TDI reaction products, when using 10-fold higher GSH concentrations (100 mM), ~2% v/v TDI and alkaline pH-7.7 (10
). The present study extends these observations, by demonstrating the formation of GSH-TDI under more physiologic reaction conditions, as well as the ability of GSH-TDI to transfer TDI to human albumin, an important carrier protein for allergic (IgE) responses to TDI, and potential biomarker of exposure (9
). GSH-mediated transfer of TDI was shown to occur preferentially on specific lysine residues, some of which are also susceptible to direct conjugation by TDI, and have been associated with antigenic changes induced by other diisocyanates (HDI, MDI) (31
). Thus, the present findings raise new questions, which may be highly relevant to TDI asthma pathogenesis; e.g. the potential competition for isocyanate reactivity in vivo between thiols, such as GSH, and amines, such as those on albumin, as well as potential similarities and/or differences between TDI-albumin resulting from direct reaction with TDI vs. transcarbamoylation via GSH.
The present data differ from previous findings that GSH protects human albumin from vapors of another diisocyanate, HDI (39
). Such dual effects could be due to differences in the reaction and hydrolysis/disassociation rates of GSH with (aromatic) TDI vs. (aliphatic) HDI, as might be expected based on studies of their corresponding thiocarbamates prepared with cysteine methyl esters (kd
for TDI roughly 100X that of HDI). Differences in the effect of GSH might also relate to exposure conditions, which differed in the study on HDI, particularly the pH/temperature of the transcarbamoylation reaction, and the timing of albumin exposure relative to GSH (e.g. competitive vs. sequential). Studies reevaluating the interactions of GSH, albumin, and HDI vapors, under physiologic conditions (pH 7, temp. 37°C) more favorable to thiol-amine “isocyanate exchange”, such as those used in the present investigation, are underway in our laboratory.
The major strength of the present study is the application of a mixed (vapor/liquid) phase system to investigate the interaction of GSH with TDI vapors. The study design targeted a fixed GSH concentration and pH as starting points for investigation; however, the system should readily permit evaluation of a more dynamic range of physiologic factors (e.g. glutathione/ion concentration, pH, temperature) and other volatile occupational/environmental co-exposures (amine catalysts, solvents) that may influence TDI reactivity. The vapor/liquid exposure system should be similarly useful for studying other volatile diisocyanates (HDI, MDI), and differences that have been reported in their reactivity/stability with GSH and related thiols (21
). The basic system could be further developed with underlying human epithelial cell layers, and overlying surfactant, to more completely model the airway microenvironment. However, animal models or clinical investigations will likely be needed to ultimately determine potential systemic effects of GSH-TDI interactions in vivo.
In summary, it was demonstrated that GSH can mediate TDI vapor uptake across a fluid phase boundary, and subsequently transfer TDI onto human albumin, a protein recognized as a major carrier for TDI in vivo. GSH-mediated transfer of TDI onto human albumin occurred on specific lysine residues and altered the self-protein’s native conformation/charge, an effect previously associated with TDI’s antigenicity. Together, the data extend proof-of-principle for the hypothesis that GSH, and potentially other thiols in exposed tissue, act as a primary reaction route for entry of TDI into the body and could play an important role in pathogenic responses to exposure.