This report demonstrates the ability of the aliphatic diisocyanate, HDI, to react with glutathione across a vapor/liquid phase boundary, and describes unique reaction products formed in the presence of physiologic GSH concentrations. Notably, different reaction products formed under identical exposure conditions, depending upon the GSH concentration and the ionic composition of the fluid phase. Among the different vapor HDI-GSH reaction products, mono(GSH)-HDI* exhibited particularly potent carbamoylating activity, based on immunochemical analyses, and was a major product present in HDI-vapor exposed GSH solutions proven to transfer HDI to albumin by LC-MS/MS. Together, the data support a hypothetical mechanism by which GSH in the airway fluid may mediate HDI vapor uptake from the respiratory tract, as reversible reaction products. The data further suggest that individual differences in airway fluid composition could modify this process.
The present study extends recent investigations modeling mixed (vapor/liquid) phase reactivity between GSH and asthma–causing diisocyanate chemicals used for commercial polyurethane production. The formation of bis(GSH)-diisocyanate conjugates, as observed here upon HDI vapor exposure of 10 mM GSH, is analogous to that previously reported with the aromatic diisocyanate, TDI (Day et al. 1997
; Wisnewski et al. 2011
). However, the mono(GSH)-HDI reaction products observed at lower GSH concentrations are distinct and include partially hydrolyzed HDI dimers and/or unique cyclized structures. Importantly, mono rather than bis(GSH)-HDI conjugates were the major reaction products resulting from HDI vapor exposure of 100 μM GSH, the approximate concentration of the lower airway fluid in vivo
The influence of the fluid phase ionic composition and [GSH] on the formation of GSH-HDI reaction products highlights the potential complexity of HDI reactivity in vivo
upon inhalational exposure. Inorganic ions such as H2
, which affect pH levels, also alter reactivity of GSH's thiol toward NCO, and may participate in hydrolysis of NCO and solvolysis of GSH-NCO thiocarbamates (Ng et al. 2004
). Inter-individual differences in airway fluid GSH levels, due to genetic and/or other environmental exposures (e.g. smoking), might further result in different HDI reaction products (Day et al. 2004
; Duan et al. 1993
; Rahman and MacNee 1999
). Thus, variability in airway fluid GSH and ion composition might alter the clinical response to HDI vapor exposure, and could help explain individual differences in HDI asthma susceptibility.
The carbamoylating activity of the presently described (aliphatic) HDI-GSH conjugates differs from that of (aromatic) TDI-GSH conjugates (Day et al. 1997
; Wisnewski et al. 2011
). While bis(GSH)-TDI is more potent than mono(GSH)-TDI* at transferring TDI to albumin, the opposite appears to be true for the corresponding GSH-HDI conjugates, at least based on immunochemical (anti-HDI IgG) analysis. Perhaps more importantly, the kinetics of GSH-HDI mediated albumin carbamoylation, at neutral pH, are substantially delayed compared with those of GSH-TDI. Such differences may reflect inherent differences in stability of thiocarbamate linkages between aromatic vs. aliphatic isocyanate (e.g. TDI vs. HDI) as mentioned earlier, and may be especially relevant to “detoxification” and elimination in vivo
(Chipinda et al. 2006
). As HDI is excreted rapidly (within hours) from exposed workers, the relative stability of GSH-HDI conjugation (vs. GSH conjugates with aromatic isocyanates) may serve an overall protective, rather than pathogenic role, consistent with previous short-term in vitro HDI vapor studies (Liu et al. 2004
; Wisnewski et al. 2005
Differences between albumin carbamoylated by GSH-HDI, and albumin directly conjugated by HDI, are further suggested by the present data. Under the current experimental conditions, the amount of HDI per albumin molecule, resulting from transfer by GSH-HDI, was four times lower than that resulting from direct HDI vapor exposure. However, while over 25 of albumin's lysines could be modified by GSH-HDI at neutral pH, vapor exposure appears to target a limited number of sites (Wisnewski et al. 2004
). By native gel electrophoretic analysis, albumin conjugated by GSH-HDI was virtually unaffected, while the migration of albumin directly conjugated by HDI vapor (and liquid, not shown) was dose-dependently increased, suggesting differences in conformation and/or charge. Such data are consistent with the identification of partially hydrolyzed HDI, vs. cross-linked HDI, as the major modification resulting from carbamoylation via GSH-HDI. Further studies will be needed to better understand the difference(s) between albumin directly reacted with HDI vapor vs. GSH-HDI, including, structural/conformational changes and preferential loci of HDI conjugation in vivo
Particular strengths and weaknesses of the present study are important considerations in the data interpretation. The immunology-based assessment of HDI conjugation, using polyclonal HDI-specific rabbit serum, facilitated high-throughput analysis of albumin carbamoylation in a cost and time-effective manner. However, while the approach recognizes “antigenic” HDI, it is possible that HDI carbamoylation may alter albumin's structure in a way that affects antiserum binding in an HDI dose-independent manner. Although this possibility cannot be ruled out entirely, quantitation of hydrolyzable HDA and the LC/MS-MS data from studies performed at different pH levels are consistent with anti-HDI serology, and positively identify numerous sites of HDI conjugation. It should be noted that the workup (reduction/acetylation) of protein samples for LC/MS/MS, prevented potential identification of albumin carbamoylation on cys-34, the only free thiol in albumin, which could have occurred by a thiol-exchange mechanism. Similar S-linked HDI-albumin conjugates, if they occur in vivo, are likely to be relatively unstable, susceptible to further thiol-exchange as they are in vitro.
Inherent limitations of the in vitro mixed-phase exposure system, in modeling the airway microenvironment, should be also recognized. In particular, the exposure system lacks many components essential to airway fluid's functional activity (surfactant, protein, alveolar macrophages, etc.). Furthermore, while the system recapitulates a mixed (vapor/liquid) phase exposure, the HDI vapor concentration is difficult to compare with that in vivo, given differences in the volume:surface area, and lack of fluid phase mixing. As a starting point, we relied upon a fixed HDI vapor concentration established in previous studies, and evaluated the potential influence of variability in (airway) fluid composition. Future studies with titrated levels of HDI vapor, and other structural/functional components of the airway lining fluid mentioned above, should better reflect GSH-HDI interactions in vivo under “normal” working conditions. Ultimately however, in vivo studies will be necessary to confirm the biological relevance of the present in vitro findings.
In summary, the ability of HDI to react with glutathione across a vapor/liquid phase, as exists in the airway microenvironment in vivo, was documented using a mixed-phase exposure system. The concentration of GSH, across a physiologic range, and the ionic composition of the fluid phase markedly influenced the reaction, with five distinct reaction products observed depending upon the experimental conditions. Notably, HDI vapor exposure of liquid solutions with GSH concentrations similar to normal human airway fluid (100 μM) resulted in predominately mono(GSH)-HDI conjugates and the capacity to carbamoylate human albumin in a pH-dependent manner. Together, the data support the hypothesis that GSH serves as a primary reaction target for HDI in vivo, with a potentially important role in the clinical response to respiratory tract exposure.