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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Chem Res Toxicol. Author manuscript; available in PMC 2012 April 7.
Published in final edited form as:
PMCID: PMC3321232

Toluene Diisocyanate Reactivity with Glutathione Across a Vapor/Liquid Interface and Subsequent Transcarbamoylation of Human Albumin


Glutathione has previously been identified as a reaction target for toluene diisocyanate (TDI) in vitro and in vivo, and has been suggested to contribute to toxic and allergic reactions to exposure. In this study, the reactivity of reduced glutathione (GSH) with TDI in vitro was further investigated using a mixed phase (vapor/liquid) exposure system to model the in vivo biophysics of exposure in the lower respiratory tract. HPLC/MS/MS was used to characterize the observed reaction products. Under the conditions tested, the major reaction products between TDI vapor and GSH were S-linked bis(GSH)-TDI and to a lesser extent mono(GSH)-TDI conjugates (with one N=C=O hydrolyzed). The vapor phase generated GSH-TDI conjugates were capable of transcarbamoylating human albumin in a pH-dependent manner, resulting in changes in the self-protein’s conformation/charge, based on electrophoretic mobility under native conditions. Specific sites of human albumin-TDI conjugation, mediated by GSH-TDI, were identified (Lys73, Lys159, Lys190, Lys199, Lys212, Lys351, Lys136/137, Lys413/414, Lys524/525) and overlap with those susceptible to direct conjugation by TDI. Together, the data extend proof-of-principle for GSH to act as a “shuttle” for a reactive form of TDI, which could contribute to clinical responses to exposure.


The health hazards of TDI exposure are well recognized (13). Airborne concentrations >2.5 ppm are considered immediately dangerous to life or health based on animal studies, while concentrations <20 parts/per billion (ppb), below the short-term permissible exposure limits set by regulatory agencies, have been shown to trigger asthma attacks in sensitized individuals (46). Globally, TDI is the 2nd most abundantly produced diisocyanate, and is commonly used for making polyurethane foam (7).

The process by which TDI exposure causes disease remains unclear, in part due to TDI’s potent chemical reactivity, which has hampered mechanistic studies (8, 9). A possible role for GSH in TDI asthma pathogenesis is suggested by several lines of evidence. In vitro studies with primary human bronchial cells, and in vivo mouse studies, have shown that TDI vapors can cause marked decreases in cellular thiol levels, and induce oxidative stress (1014). Genetic studies in humans have shown an association of TDI asthma, and TDI metabolites in urine and serum, with specific GSH S-transferase polymorphisms (1517). Furthermore, the overall importance of GSH in human asthma caused by other occupational and environmental allergens is increasingly being recognized (18).

Generally, GSH plays a protective role against exposure to “xenobiotics” (19, 20). However, for TDI it is hypothesized that GSH plays a pathogenic role by acting as a “shuttle” that stabilizes a reactive form (of TDI) for delivery to another location, potentially extending the target range for protein modification and immune presentation (21, 22). This hypothesis is supported by in vitro studies demonstrating that GSH can form conjugates with TDI, capable of transcarbamoylating a model peptide (10). Similar findings have been demonstrated in studies with other mono-isocyanates, most notably, 2-chloroethyl-isocyanate, released by the cancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea, methylisocyanate, the cause of the catastrophic industrial disaster in Bhopal India in 1984, and phenyl isocyanate, a sensitizer in animal studies (2326). Thus, substantial evidence for reversible formation of thiocarbamate linkages between isocyanates and GSH, suggest this process might contribute to TDI’s toxicity and/or allergenicity.

To date, liquid phase reactions have been used almost exclusively for in vitro studies of TDI, and other isocyanates’, reactivity with GSH (10, 2327). However, vapor, rather than liquid TDI, is the major phase to which GSH in the lower airway fluid is exposed in vivo (28). Vapor phase TDI may react with compounds in solution differently than liquid phase TDI, as evidenced by recent studies with human albumin (9). It remains unclear if differences between TDI vapor vs. liquid phase are due to biophysics, maximum/minimum achievable TDI concentration, or technical issues, especially (a) TDI’s insolubility and tendency to “self-aggregate” in aqueous solution, creating localized areas of high concentrations and a “self-encapsulating” physical barrier, as well as (b) TDI’s reactivity with water and conversion of its N=C=O groups to NH2, which may further co-polymerize with unhydrolyzed TDI.

In the present study, we investigated the interaction of GSH (in solution) with TDI in its vapor phase, to better model exposure biophysics in vivo in the lower respiratory tract. The mixed phase exposure approach alleviated many of the technical challenges previously noted in studying TDI / GSH reactivity in solution; namely problems with mixing, precipitation formation, and large numbers of reaction products (10). GSH-TDI conjugates formed via mixed phase exposure, and subsequent transcarbamoylation of human albumin (a major carrier protein mediating TDI allergenicity), were further studied through tandem mass-spectrometry approaches. The data support the hypothetical pathogenic role of GSH in response to TDI exposure.


TDI Vapor Phase Exposure of GSH

Ten mM solution of reduced or oxidized glutathione (GSH or GSSG respectively) from Sigma (St. Louis, MO) in 20 mM phosphate buffered saline (PBS, pH 7.4) from Gibco (Grand Island, NY), were exposed to room air, or TDI vapor (≤200 ppb) for 18 hrs, in 35 × 10 mm Petri dishes obtained through VWR International (Bridgeport, NJ) as depicted in Fig. 1A. All solutions and reaction products were 0.2 μm filtered (Millipore; Billerica, MA) to ensure sterility. The TDI solution used was an 80/20 mixture of 2,4/2,6-isomers, obtained from Sigma-Aldrich.

Figure 1
Depiction of methodology used in the study. Panel A (top) depicts vapors of reactive TDI interacting with GSH in solution. Panel B (bottom) depicts the co-incubation of GSH-TDI reaction products with human albumin in solution.

Reverse phase HPLC analysis of GSH-TDI reaction products

Samples were initially fractionated by the Yale Keck Center as previously described, on a Hewlett Packard 1090 HPLC system equipped with an Isco Model 2150 Peak Separator and a 1 mm × 25 cm Vydac C-18 (5 micron particle size, 300 Å pore size) reverse phase column (29). Following equilibration with 98% buffer A (0.06% TFA) and 2% buffer B (0.052% TFA, 80% acetonitrile), TDI-GSH reaction products were eluted over the course of 1 hour, by increasing buffer B from 2 to 37%.

Mass spectrometry analysis of GSH-TDI reaction products

GSH-TDI reaction products were initially analyzed by the Yale Keck Center on a Micro Q-Tof MS instrument (Agilent Technologies; Santa Clara, CA). Twenty five microliters of each HPLC purified sample was diluted with 25 μL 0.1% formic acid, and injected onto a column (RP C4 micro column: PROTO 300 C4 5 μm 50 × 0.3 mm) for desalting. The bound analyte was eluted over a 30 minute gradient (mobile phase is acetonitrile with 0.1% TFA) directly into the Micro Q-Tof MS instrument where accurate mass was recorded. In other studies a separate aliquot (2 μL) of the HPLC purified reaction products were diluted in 60 μL of 60% acetonitrile/ 0.1% formic acid and directly infused via Triversa NanoMate into a Bruker 9.4T FT-ICR MS (Bruker Daltonics; Billerica, MA) (30).

GSH-TDI mediated transcarbamoylation of human albumin

HPLC purified TDI-GSH reaction products, or total (10 mM) GSH solutions exposed to TDI vapors, were incubated 1:2 (v/v) with a 5 mg/mL solution of human albumin at 37°C, as depicted in Fig. 1B. Initial reactions, including those analyzed by native gel (Fig. 7) and MS/MS (Table 1), were performed in isotonic saline (140 mM NaCl) buffered to pH 8.8 with 0.1 M carbonate. Subsequent experiments, exploring pH-dependence, were performed using 0.1 M buffers made with citric acid, phosphate or carbonate (Sigma) to achieve pH levels of 3, 7, and 9 respectively, or varying ratios of mono-/di-basic phosphate to achieve pH values ranging from 5.8 to 8.2.

Figure 7
Native gel analysis of human albumin transcarbamoylated by purified GSH-TDI reaction products. Human albumin co-incubated with HPLC purified GSH-TDI reaction products, or controls (labeled above the lane) was subjected to electrophoresis under native ...
Table 1
Sites of TDI conjugation to human albumin mediated via GSH-TDI.

Native gel and anti-TDI Western blot

TDI conjugation to human albumin was detected in native gels based on characteristic changes in electrophoretic mobility as previously described (31, 32). For native protein analysis, samples were prepared in a glycerol buffer, run on 10% polyacrylamide gels, and stained with Imperial protein stain from Pierce (Rockford, IL). For Western blot analysis, samples were electrophoresed under reducing conditions on precast 4–15% gradient gels and a nitrocellulose trans-blot system from BioRad (Hercules, CA). Nitrocellulose membranes were blocked with 3% dry milk in PBS, probed with 1 μg/mL of the anti-TDI mAb 79G7 (33), followed by anti-mouse IgG1 from Pharmingen (San Diego, CA), and developed with electrochemical luminescence reagent from Thermo Fisher Scientific (Rochester, NY).

Reduction, alkylation, and trypsin digestion of albumin samples

In preparation for MS/MS analysis of GSH-mediated TDI conjugation sites of human albumin, 50 μL aliquots of GSH-TDI / albumin co-culture reactions were treated with tributylphosphine for 30 min at room temperature, followed by alkylation with iodoacetamide for 1 hour at room temperature. Alkylation was quenched by further addition of tributylphosphine for 15 min at room temperature and samples were twice dialyzed against 3 L of 25 mM NH4HCO3 using 3500 molecular weight cutoff mini dialysis units (Slide-A-Lyzer, Thermo Scientific, Waltham, MA). Porcine trypsin in 25 mM NH4HCO3 was then added at a 40:1 (protein:trypsin) ratio. Samples were incubated overnight at 37°C with shaking (400 rpm), and finally centrifuged at 14,000 × g in a microcentrifuge (MiniSpin, Eppendorf, Hamburg, Germany) to pellet any insoluble material.

Ultra-performance liquid chromatography

Tryptic peptides of albumin were separated on a Waters (Milford, MA) nanoACQUITY ultra-performance liquid chromatography system. Aliquots (1 μL) of the digest mixture were injected and trapped/desalted on a 5 μm SymmetryC18 (180 μm × 20 mm) trapping column with 99.5/0.5 A/B (A:0.1% formic acid; B:0.1% formic acid in acetonitrile) at a flow rate of 15 μL/min for 1 minute. Separation was performed on a 1.7 μm BEH130 C18 (100 μm × 100 mm) analytical column utilizing gradient elution at a flow rate of 400 nL/min and a gradient of 99/1 to 60/40 A/B over 60 min.

Tandem mass spectrometry of transcarbamoylated albumin peptides

The eluent from the ultra-performance liquid chromatography system was directed to the nanoelectrospray source of a Waters SYNAPT MS quadrupole time-of-flight (qTOF) mass spectrometer. Positive ion nanoelectrospray was performed utilizing 10 μm PicoTip (Waters) emitters held at a potential of +3.5 kV. The cone voltage was held constant at +40 V for all experiments. Dry N2 desolvation gas was supplied to the instrument via a nitrogen generator (NitroFlowLab, Parker Hannifin Corp., Haverhill, MA). [Glu]1-Fibrinopeptide B (100 fmol/μL in 75/25 A/B) was supplied to an orthogonal reference probe and the [M+2H]2+ ion (m/z = 785.84265 u) measured as an external calibrant at 30 sec intervals. Ultra-high purity argon was used as collision gas. Spectra were acquired in an “MSe” fashion (34). Alternating one-second mass spectra were acquired. The collision energy was set to 6 eV (1 sec low energy scan) and a 15–30 eV ramp (1 sec high energy scan).

Data analysis

Data were analyzed with BioPharmaLynx v. 1.2 (Waters), a software program for analysis of peptide mass maps and identification of sites of modification on known protein sequences. Default peptide mass map analysis criteria of 30 ppm mass error in both low and high collision energy mode were specified. Trypsin was specified as the digestion enzyme, and 2 missed cleavages were allowed. The submitted protein sequence was taken from P02768, “serum albumin precursor, Homo sapiens” ( excluding the signal and propeptides (residues 1–24). Two custom modifiers were created for TDI. The first (TDI: C9H6N2O2, m/z = 174.0429 u) represents TDI with both isocyanate moieties bound to a peptide via urea bonds. The second (TDI*: C8H8N2O, m/z = 148.0637 u) represents one isocyanate moiety bound to a peptide via a urea bond, while the second isocyanate moiety is hydrolyzed to the primary amine. Identification of an isocyanate binding site proceeded via a rigorous procedure that involved the following steps: 1) Observation of a potential peptide-isocyanate conjugation product with less than 30 ppm m/Δm mass error in the analyte peptide mass map. 2) Comparison of analyte and control peptide mass map from unmodified human serum albumin shows that observed m/z and chromatographic retention time are unique to analyte. 3) MS/MS data contains bn- and yn-type ions consistent with the assigned sequence and modifier.


TDI vapor phase exposure of GSH and purification of reaction products

Solutions of GSH that had been exposed to TDI vapors, as depicted in Fig. 1A, were subjected to reverse phase HPLC, and the UV light absorbance of each fraction of column eluent was measured. Comparison of the HPLC chromatograms of TDI vapor vs. room air exposed GSH revealed fractions that likely contained GSH-TDI reaction products, based on increases in A210 and A250 (Fig. 2). As highlighted, the bulk of the reaction products were contained in the fractions labeled peaks A through D, which eluted after unreacted GSH (peak [empty]), indicating increased hydrophobicity.

Figure 2
HPLC separation of GSH-TDI reaction products. The absorbance at 210 nm (top) and 245 nm (bottom) was measured (Y-axis) for each 1 min eluent sample collected over time (X-axis). Samples of TDI vapor (<200 ppb) exposed GSH (10mM), shown as blue ...

Identification and characterization of vapor GSH-TDI reaction products by mass spectrometry

The specific HPLC fractions with elevated A210 and A250 were further characterized by MS. As shown in Fig. 3, Peaks A and B contained primarily a singly charged 456.2187 or 456.2645 m/z species, while Peaks C and D contained predominantly the doubly charged 395.2007 m/z, and its singly charged species (m/z = 789.4146). Fragmentation of these species yielded the tandem mass spectra shown in Figs. 4 and and5,5, which are consistent with the chemical structures shown in Fig. 6. Together, the data indicate HPLC fractions A and B contain mono(GSH)-TDI conjugate, with one N=C=O group hydrolyzed to a free amine (indicated as mono(GSH)-TDI* here forward), while fractions C and D contain bis(GSH)-TDI. As shown in Fig 6, the fragmentation data (along with functional data, see below) are consistent with TDI conjugation via the thiol group of cysteine, rather than the α-amine of the glutamate residue. The reason that GSH-TDI conjugates with identical masses elute from the HPLC in 2 distinct peaks remains unclear, however, the 2 distinct peaks may represent GSH conjugates with different isomers of TDI (2,4 vs. 2,6) present in the starting vapors.

Figure 3
Mass spectrometry analysis of HPLC fractions containing GSH-TDI reaction products. Each panel A through B corresponds to samples from peaks A through D in Figure 2. Tandem MS/MS data (X-axis = m/z) and deduced structures for the singly charged m/z 456.2, ...
Figure 4
Tandem mass spectrometry analysis of peak A from HPLC purified GSH-TDI reaction products. A sample of peak A from the HPLC fractionation of GSH exposed to TDI vapor was analyzed by MS/MS (X-axis = m/z), focusing on the major m/z 456.2 product. The MS/MS ...
Figure 5
Tandem mass spectrometry analysis of peak C from HPLC purified GSH-TDI reaction products. A sample of peak C from the HPLC fractionation of GSH exposed to TDI vapor was analyzed by MS/MS (X-axis = m/z), focusing on the major m/z 789.4 product. The MS/MS ...
Figure 6
Chemical structures of the major reaction products between TDI vapors and GSH in solution. The structure of the major GSH-TDI reaction products and their predicted ionization fragments, including the m/z of the singly charged species, consistent with ...

Transcarbamoylation of human albumin by vapor GSH-TDI conjugates

To test the functional ability of GSH-TDI conjugates generated by TDI vapor exposure to transcarbamoylate another protein, human albumin was co-incubated with GSH-TDI conjugates (Fig 1B), and subsequently analyzed by native gel electrophoresis, for conformational/charge changes indicative of TDI conjugation. HPLC purified bis(GSH)-TDI, total (unpurified) GSH-TDI, and to a lesser extent, mono(GSH)-TDI, all caused electrophoretic changes in albumin consistent with TDI transcarbamoylation (Fig. 7). As further shown in Fig. 8, through native gel analysis as well as anti-TDI Western blot, GSH-TDI mediated transcarbamoylation of human albumin was strongly pH dependent regardless of buffer ion (phosphate, citrate, carbonate), and across a wide range (pH 3–9), with lower activity at acidic pH and higher activity at basic pH levels. Further support of the thiocarbamate-dependent mechanism of TDI transcarbamoylation via GSH is provided by the fact that oxidized GSH (GSSG) did not mediate TDI transcarbamoylation, when vapor exposed and co-cultured with albumin under identical conditions.

Figure 8
Dependence of human albumin transcarbamoylation by GSH-TDI on pH and free thiol of cysteine. Results of transcarbamoylation reactions of human albumin with total unfractionated GSH-TDI or GSSG-TDI reaction products. Co-incubations were performed at different ...

Identification of human albumin’s specific sites of TDI conjugation via GSH-TDI

It was hypothesized that specific amino acid side chains of human albumin would be most susceptible to TDI conjugation via GSH-TDI conjugates, and the identify of these putative targets was evaluated through tandem mass spectrometry analysis. Under alkaline conditions, which yielded maximal transcarbamoylation, 8 different lysine residues were consistently identified as targets of TDI conjugation by mono(GSH)-TDI* or bis(GSH)-TDI in replicate experiments (Table 1). In each case, the 2nd (unbound) N=C=O group of the transferred TDI molecule had been hydrolyzed to the free amine. Overall, the pattern of albumin conjugation via GSH-TDI is qualitatively distinct from that recently reported via direct exposure to low dose TDI in liquid phase titration studies (35). However, two “GSH-mediated” TDI conjugation sites (Lys199, Lys525) are also targeted by direct “low dose” TDI exposure, while the other six identified “GSH-mediated” TDI conjugation sites (Lys73, Lys159, Lys190, Lys212, Lys351, Lys137) can become directly conjugated with high enough TDI exposure concentrations (35).


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, 37, 38). 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, 32, 35). 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, 39, 40). 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.



The studies were supported by grants R42-ES016728 and R41-ES018021 from the National Institute of Environmental Health Safety. The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention or the National Institute for Occupational Safety and Health.

The authors would like to acknowledge Drs. Stone, Lam, Ted Voss, Tom Abbott, and Mary LoPresti, from the Yale Keck Center for their work on the HPLC, MS, and MS/MS, as well as Jian Liu for her work with the vapor exposures.


reduced glutathione
oxidized glutathione
hexamethylene diisocyanate
methylene diphenyl diisocyanate
parts per billion
toluene diisocyanate


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