Formation, Solvolysis, and Transcarbamoylation Reactions of Bis(S

During our ongoing studies of the reactions of toluene diisocyanate (2,4- and 2,6-diisocyanatotoluene, TDI) in vivo, it became apparent that reactive ...
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Chem. Res. Toxicol. 1997, 10, 424-431

Formation, Solvolysis, and Transcarbamoylation Reactions of Bis(S-glutathionyl) Adducts of 2,4- and 2,6-Diisocyanatotoluene Billy W. Day,†,‡,§ Ruhzi Jin,† Dina M. Basalyga,† Jean A. Kramarik,† and Meryl H. Karol*,†,§ Department of Environmental & Occupational Health, Department of Pharmaceutical Sciences, and University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15238 Received December 11, 1996X

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-GluTrp-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.

Introduction Diisocyanatotoluene (TDI)1 is the most prevalent cause of occupational asthma in the Western world (1). Individuals are most often exposed to vapors of the 4:1 molar mixture of the 2,4-isomer (1; see Scheme 1) and the 2,6isomer (2), and 5-10% develop respiratory hypersensitivity to the chemical (2). In order to understand the mechanism(s) by which TDI exerts its asthmogenic effects, we have been performing studies to determine the tissues and soluble proteins of the respiratory tract with which these isocyanates react. * To whom correspondence should be addressed at the Department of Environmental & Occupational Health, Center for Environmental & Occupational Health & Toxicology, University of Pittsburgh, 260 Kappa Dr., Pittsburgh, PA 15238. Telephone: (412) 967-6530. FAX: (412) 967-6611. E-mail: [email protected]. † Department of Environmental & Occupational Health. ‡ Department of Pharmaceutical Sciences. § University of Pittsburgh Cancer Institute. X Abstract published in Advance ACS Abstracts, March 15, 1997. 1 Abbreviations: BAL, bronchoalveolar lavage; BCNU, N,N′-bis(2chloroethyl)-N-nitrosourea; CEIC, 2-chloroethyl isocyanate; DAD, diode array detector; GSH, reduced glutathione; HBSS, Hank’s balanced salts solution; MIC, methyl isocyanate; RADS, reactive airways disorder syndrome; tR, retention time; TDI, toluene diisocyanate.

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Studies of guinea pigs exposed to TDI vapors have shown formation of adducts localized predominantly to the apical surface of the respiratory tract epithelium (3), as well as adduction of soluble proteins in bronchoalveolar lavage (BAL) fluid (4). We also identified TDI-adducted hemoglobin, both in BAL and in intact erythrocytes in peripheral blood (5). The mechanism by which these highly reactive diisocyanates are transported across the epithelial layer of the respiratory tract, into the blood, and through the erythrocyte membrane to react with hemoglobin is unknown, but the presence of carbamoylated hemoglobin implies that a reactive form of TDI survives the transport process. Studies by others on the biochemistry of monoisocyanato compounds have shown that, after carbamoylation of the cysteine sulfur of glutathione (GSH), subsequent transfer of the isocyanato moiety to protein thiols can occur (6,7). Thus, GSH adducts of isocyanato compounds may serve as masked, reactive forms of isocyanates, and may contribute to toxicities at sites other than that of original absorption into the body (8). In the current study, we investigated the reactions of TDI with GSH to determine if reasonably stable, monomeric or dimeric thiocarbamoyl adducts would form with © 1997 American Chemical Society

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Chem. Res. Toxicol., Vol. 10, No. 4, 1997 425 Scheme 1

the tripeptide, and if these would transfer isocyanato equivalents to other molecules. We found that bis(Sglutathionyl) adducts of 2,4- and 2,6-TDI readily form without enzymatic assistance under physiological conditions (3 and 8; see Scheme 1), and that their rates and mechanism of solvolysis allow transfer of one mono(glutathionyl)toluylisocyanato moiety to a different cysteinecontaining peptide.

Experimental Procedures Caution: TDI is a skin and mucus membrane irritant and should be handled in a chemical vapor hood with appropriate protective equipment. Its hydrolysis products are aromatic amines, potential human carcinogens. Materials. GSH and Thr-Cys-Val-Glu-Trp-Leu-Arg-Arg-TyrLeu-Lys-Asn (MHC antigen H-2Kb) were purchased from Sigma Chemical Co. (St. Louis, MO). 2,4- and 2,6-TDI were gifts from Bayer Chemical Corp. (Pittsburgh, PA). Trifluoroacetic acid (sequencing grade), C2H3O2H, and 2H2O were purchased from Aldrich Chemical Co. (Milwaukee, WI). Solvents of the highest available purity were obtained from Fisher Scientific (Pittsburgh, PA). Synthesis of BisGS-2,4-TDI and BisGS-2,6-TDI Adducts. Two flasks were each charged with GSH (307 mg, 1

mmol) dissolved in 10 mL of 0.1 M NH4HCO3 (final pH 7.73). Neat 1 or 2 (175 µL, 1.2 mmol each) was added to individual flasks containing the GSH solutions and shaken at ambient temperature for 10 min, and then centrifuged at 1000 rpm for 10 min. Residual unreacted TDIs were removed (droplets on the upper surfaces of the solutions) by pipet; the remaining solutions were again centrifuged to pellet the small amount of white precipitate that formed in each reaction. The supernatants were decanted to borosilicate test tubes and lyophilized in a Speedvac, in each case yielding a white powder. The yield of 3 was 355 mg (68%), while that of 8 was 345 mg (66%). Each of the bis adducts yielded both 1H NMR (Varian XL-200 and Bruker AM500 spectrometers) and pneumatically-assisted electrospray MS (vide infra) spectral data consistent with the proposed structures (data shown under Results). Reactions performed at 37 °C gave identical results. HPLC Analyses. Analytical HPLC was performed on a Hewlett Packard 1090 Series II LC equipped with a Hewlett Packard 1040 UV-vis diode array detector (DAD), both controlled by a Hewlett Packard Series 300 Chemstation computer. All separations were performed on a 10 µm particle size, 250 × 4 mm, Hibar LiChrosorb C18 column (E. Merck Darmstadt, Germany) using a 2 mL/min flow rate of the following mobile phase and gradient: solvent A, H2O containing 0.1% (v/v) CF3CO2H; solvent B, CH3CN containing 0.1% (v/v) CF3CO2H; 0-7 min, 16% B; 7-30 min, linear change to 100% B; 30-35

426 Chem. Res. Toxicol., Vol. 10, No. 4, 1997 min, linear return to 16% B. Column effluent was monitored at 250 nm, and spectra over the range of 200-400 nm were collected every 1.28 s. Rates of BisGS-2,4-TDI and BisGS-2,6-TDI Solvolysis. The kinetics of the solvolyses of 3 and 8 in aqueous medium (see Scheme 1) were determined as follows. Compounds 3 and 8 (4.55 mg each) were separately dissolved in 1 mL of Hank’s balanced salts solution (HBSS), and the resulting solutions were maintained at constant temperature (25 or 37 °C) in a jacketed water bath. At selected time points, 20 µL of each sample solution was removed and analyzed by HPLC and then by electrospray MS when necessary (vide infra). The integral under each chromatographic peak was converted to its percentage of all peaks in the chromatogram, and the molar concentration that this percentage represented was calculated from the initial concentration of the bis adduct. The compounds were thus determined to convert first to the intermediate 2-GSthiocarbamoyl-4(or 6)-isocyanato adducts (4 and 9, respectively), followed by hydrolysis to the 2-GS-thiocarbamoyl-4(or 6)-amino adducts (5 and 10, respectively). Mass Spectrometry. A Perkin Elmer/Sciex API I mass spectrometer with an atmospheric pressure ionization source and an articulated IonSpray interface linked in tandem with glass capillary tubing to the Hewlett-Packard HPLC/DAD system was employed to determine the molecular masses of 3 and 8 and decomposition products (4-7 and 9-12, respectively). The collected and lyophilized LC fractions were redissolved in 25-50 µL of 1:1 H2O-CH3CN containing 0.1% CF3CO2H, 5-20 µL of which was injected directly through the LC without a column and introduced into the ion source of the mass spectrometer in 1:1 H2O-CH3CN containing 0.1% CF3CO2H flowing at 40 µL/min. High-purity air was used for nebulization at an operating pressure of 40 psi. High-purity N2 heated to 55 °C was used as the curtain gas, flowing at 0.6 L/min. The ionspray interface was maintained at 5 kV and the orifice voltage at 70 V. The quadrupole was scanned over the required range (i.e., gm/z 70 to em/z 2400) in 9-11 s per scan at a resolution of m/z 0.1. Twenty microliters of the crude time ) ∼0 and time ) 3 day solutions of 3 and 8 prepared at 4.55 mg/mL in HBSS was also separately injected into the flowing 1:1 H2O-CH3CN/ CF3CO2H stream described above and analyzed by MS. Transcarbamoylation Reactions. The MHC antigen H-2Kb peptide (0.5 mg, 316 nmol) was incubated in two separate reactions with 10-fold molar excesses of 3 and 8 (2.5 mg, 3.16 µmol). Each reaction was carried out in 0.5 mL of 0.1 M NH4HCO3 (final pH 7.73) at 25 °C for 24 h. Aliquots (15 µL) of the reaction mixtures were withdrawn at given time points and immediately stored at -70 °C until analyzed. For “time zero” determinations, 1 mM solutions of 3, 8, GSH, and the peptide were individually prepared in 0.1% aqueous CF3CO2H. The appropriate mixtures of three components were prepared, or the reaction aliquots were thawed and immediately injected for HPLC-DAD/UV-MS analysis. GSH was detected with a monitoring wavelength of 214 nm, toluyl-containing materials at 250 nm, and the 12-mer peptide and any products containing it at 280 nm. A 5 µL portion of each of the reaction mixture aliquots was analyzed in each HPLC-DAD/UV-MS run, and the vial containing the remainder of each aliquot was immediately stored at -70 °C. The column, flow rate, and MS parameters used in the analysis of the solvolysis reactions were employed with the following changes: for the LC gradient, time ) 0-15 min isocratic, 86% solvent A; linear change to 30 min, 10% solvent A; linear change to 35 min, 86% solvent A; for the mass spectrometer, N2 curtain gas flow was set at 0.65 L/min and the quadrupole was scanned from m/z 250 to m/z 2300 in 10.29 s/scan at a resolution of m/z 0.1.

Results Synthesis of BisGS-TDI Adducts. We initially attempted the syntheses of GS-TDI adducts with a method described useful for preparation of glutathionemonoisocyanate adducts (6). Reactions were performed

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in CH3CN-H2O in both acidic (apparent pH 2) and nearneutral (apparent pH 6.8) conditions. HPLC analysis indicated formation of over 30 products in each case. The early eluting products were characterized by ionspray MS (vide infra). Yields of these products were low (1500 amu) in MS experiments (data not shown), we concluded they were various polymers of the reactants. We then performed the syntheses in aqueous bicarbonate, pH 7.7, to simulate reactions that might occur in the lung. The diisocyanates 1 and 2 were individually added, neat, to bicarbonate solutions containing 0.8 molar equiv of GSH. Rapid reactions ensued, each yielding essentially one product. Reactions at 25 °C and at 37 °C gave product profiles and yields that were indistinguishable. No attempts were made to optimize yields. The identity of each of the major products was determined by electrospray mass spectrometry and proton NMR spectroscopy (Figure 1). Each of the two major products yielded ion signals of m/z 789, consistent with [M+H]+ quasi-molecular ions of bis(glutathionyl) addition products of the diisocyanato compounds (compounds 3 and 8 in Scheme 1). The proton NMR spectra of these adducts exhibited identical signals for the two attached glutathionyl moieties, and the aromatic signals gave no indication of unsymmetrical substitution. Further, the signals for the cysteinyl protons were shifted downfield relative to unsubstituted GSH, while the signals for the remaining glutathione-derived protons were essentially unchanged. Because of the masses detected, the symmetries evident in the NMR spectra, and the lowered energies of the cysteinyl proton signals, it was clear that the products were the bis(thiocarbamoyl) structures assigned to 3 and 8 as shown in Scheme 1. Solvolysis of BisGS-2,4-TDI and BisGS-2,6-TDI Adducts. The solvolyses of 3 and 8 in a medium mimicking physiological pH and ion strength (Hank’s balanced salts solution, no pH indicator) were followed over time by HPLC-DAD/UV-electrospray MS analyses. The reactions of the bis adducts followed the paths shown in Scheme 1 when incubated at 25 or 37 °C. Example HPLC chromatograms obtained during the course of these incubations are shown in Figure 2, and the kinetics of the solvolysis reactions are depicted in Figure 3. Solvolysis of the 2,4-isomer 3 was more rapid than that of the 2,6-positional isomer 8. Each reaction followed pseudo-first-order kinetics in the initial phases of the reactions. Over the entire course of the reactions, disappearance of 3 and 8 followed apparent pseudo-secondorder kinetics. For the 2,4-isomer 3, the instantaneous first-order rate constant kapp,unimolecular at 25 °C was 4.7 × 10-5 s-1. Overall, solvolysis of 3 at 25 °C followed secondorder kinetics with a kapp,bimolecular of 0.013 M-1 s-1. For 8, kapp,unimolecular at 25 °C was 4.7 × 10-6 s-1, while kapp,bimolecular was 1.2 × 10-3 M-1 s-1. The rate of conversion of 3 was accelerated by the studied increase in temperature to 37 °C, yielding a kapp,unimolecular of 7.4 × 10-5 s-1 and a kapp,bimolecular of 0.022 M-1 s-1. Mass Spectrometric Analysis of Solvolysis Products. HPLC fractions from solvolysis reactions were collected and analyzed individually by direct injection ionspray mass spectrometry. Aliquots from the later time points of the reaction mixtures were also analyzed

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Chem. Res. Toxicol., Vol. 10, No. 4, 1997 427

Figure 1. Proton NMR spectra of 3 (A) and 8 (B). Spectra were recorded at ambient temperature at 500.121 MHz in 9:1:0.009 (v/v/v) H2O-2H2O-CF3CO2H. Similar shifts and coupling were observed when spectra were recorded at 200.057 MHz in 199:1 (v/v) 2H O-CF CO H, and at 200.058 MHz in C2H O2H using standard decoupling (solvent suppression and assignment of coupling 2 3 2 3 constants) routines. Shifts and multiplicities for the Cys R signals were determined to be δ 4.58 (dd, 2H, J ) 5.3 and 2.9 Hz) for 3 and δ 4.54 (dd, 2H, J ) 5.4 and 3.1 Hz) for 8 from the 200 MHz spectra obtained in C2H3O2H.

by on-line LC-MS to verify results from the collected fractions. As stated, the products from the synthetic reactions performed in aqueous-only medium consisted almost entirely of the bis adducts 3 and 8. Our earlier but aborted attempts at synthesis in mixed organicaqueous media had given us indications of other possible structures, including GSH, that could be formed during solvolysis (i.e., 4-7 and 9-12). Each of these products

was detected over the course of the solvolysis reactions as their [M+H]+ (4, 5, 9, 10, GSH), [M+Na]+ (4, 6, 9, 11), [M+NH4]+ (5, 10), and/or [M+2H]2+ (7, 12) quasimolecular ions. We found no firm indications of higher order products (i.e., arylamino group addition to a freed isocyanato group) in these reactions, although there were a few minor but unexplained hydrophobic products noted during the solvolysis of 8 (see Figure 2B).

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Figure 3. Time dependencies of the solvolyses of the bis(glutathionyl) adducts 3 and 8 (closed symbols) and formation of their corresponding mono(glutathionyl) mono(isocyanato) products 4 and 9 (open symbols) in a physiological bicarbonate buffer (Hank’s balanced salts solution): circles, 8 f 9 at 25 °C; triangles, 3 f 4 at 25 °C; diamonds, 3 f 4 at 37 °C.

Figure 2. Example HPLC chromatograms during the course of solvolyses of 3 and 8 at 25 °C in a physiological bicarbonate buffer (Hank’s balanced salts solution). Peaks are labeled according to Scheme 1, and the principal cationized molecular ions noted for each analyte in pneumatically-assisted electrospray experiments are listed.

Transcarbamoylation by BisGS-TDI Adducts. The appearance of 4, 6, 9, and 11 during incubation of 3 and 8 in physiological medium suggested that glutathione-TDI adducts could act as masked but reactive intermediates, and might serve as protoxins capable of transferring isocyanato equivalents to other biological molecules. A commercially available major histocompatibility complex-derived 12 amino acid peptide (Thr-CysVal-Glu-Trp-Leu-Arg-Arg-Tyr-Leu-Lys-Asn, MHC antigen H-2Kb) containing one free cysteine sulfhydryl was incubated separately with 10-fold molar excesses of 3 and

8 under physiological conditions (Scheme 2). Reactions of the 2,4-isomer 3 will be discussed first. The MHC H-2Kb peptide was detected by LC-electrospray-MS as its doubly charged quasi-molecular ion [retention time (tR) ca. 27 min, m/z 791.5, [M+2H]2+], while GSH (tR 4 min, m/z 308), 3 (tR ) 14 min, m/z 789), and the intermediate mono(glutathionyl) mono isocyanate 4 (tR 8 min, m/z 482) were detected as their [M+H]+ ions. At time zero, the bis adduct and the MHC peptide were evident. After 15 min, GSH, 3, and the unmodified MHC peptide were detected. The transcarbamoylation product of 3 [GS-2,4-TDI-peptide (13), tR 25 min] appeared at the 30 min time point as its doubly charged quasi-molecular ion [m/z 1032, [M+2H]2+], as did 4. GSH, 3, and the unreacted peptide were also evident at this time. All five of these analytes were detected up to and including the 2 h time point of the reaction. After 6 h of reaction, neither 3 nor 4 was detected, but GSH, 13, and the unmodified MHC peptide were still evident. The latter three analytes were all detected at the 12 and 24 h time points, but the signal of 13 showed a timedependent decay in intensity. An example chromatogram after 30 min of reaction is shown in Figure 4A. The reaction between 8 and the MHC peptide under the same conditions was analyzed as above. Under these conditions, compound 8 (tR 7 min) eluted more quickly than did 3. This was also the case with its mono(glutathionyl) mono(isocyanato) intermediate solvolysis product 9 (tR 5 min). From the 15 min time point up to and including the 2 h time point of this reaction, GSH, 8, 9, and the unmodified 12-mer peptide were detected. After 6 h of reaction, 9 was not detected, but GSH, 8, the 12mer peptide, and the transcarbamoylation product GS2,6-TDI-peptide (14) (tR 25 min; again, m/z 1032, [M+2H]2+) were detected. At the 12 and 24 h points, these four analytes were all detected, with the signal for 8 showing a time-dependent decay in intensity, but the signal for the transcarbamoylation product 14 remained relatively constant. An example chromatogram after 12 h of reaction is shown in Figure 4B. Note that the structure of 4 in Scheme 1 was originally assumed based on the more rapid solvolysis of 3 vs that of 8. The more rapid transfer reaction of 3 vs that of 8 further supports this assumption, as well as the assumed structure 13 given in Scheme 2.

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Chem. Res. Toxicol., Vol. 10, No. 4, 1997 429 Scheme 2

Figure 4. Example HPLC chromatograms obtained during the transcarbamoylation reactions of the MHC antigen H-2Kb peptide Thr-Cys-Val-Glu-Trp-Leu-Arg-Arg-Tyr-Leu-Lys-Asn with 3 (A, 30 min of reaction) and 8 (B, 12 h of reaction) in NH4CO3buffered medium at 25 °C. Peaks are labeled with the analyte contained therein and with the base ion in the pseudo-molecular ion clusters detected in pneumatically-assisted electrospray experiments.

Discussion TDI has been associated with several pulmonary disorders, including sensory and pulmonary irritation (9), reactive airways disorder syndrome (RADS) (10), hypersensitivity pneumonitis (11), and asthma (1). Although there is evidence that each disorder is dependent on the TDI exposure concentration and, for some individuals, on the frequency of exposure, the molecular mechanisms involved in each of these syndromes remain largely unknown. The initial step in chemical sensitization is believed to be reaction of the chemical with an endogenous protein (1). Animal models have been employed to identify the macromolecules with which TDI reacts following inhalation exposure. There is evidence that laminin is adducted

in guinea pigs exposed to vapors of 14C-TDI (12). In a guinea pig model of TDI asthma, TDI adducts have been found localized to the apical surface of the upper respiratory tract epithelium by immunohistochemical methods (3). Additionally, TDI-adducted proteins can be isolated from the BAL (4) and TDI-adducted hemoglobin from circulating erythrocytes of such guinea pigs (5). The latter finding suggests that a reactive form of TDI reaches the circulation and penetrates the erythrocyte membrane to carbamoylate hemoglobin. The present study sought to investigate a potential mechanism by which such a reaction could occur by addressing the hypothesis that, under physiologic conditions, TDI may react reversibly with sulfhydryl moieties, and, in the presence of appropriate nucleophiles, the product(s) of such reactions could react further to yield transcarbamoylation products. Glutathione is the major intracellular non-protein thiol that is protective against reactive electrophiles. Its concentrations in epithelial lining fluid can reach 10 mM (13). Due to its free sulfhydryl group, GSH may serve as a nucleophile, an activity facilitated by enzymatic catalysis of glutathione transferases. Conjugation of electrophilic moieties with GSH is generally considered a detoxification process. Such S-linked adducts are excreted, or are transformed into the corresponding cysteine or N-acetylcysteine conjugates before elimination (14). If the conjugation process is reversible, however, such adducts may be transformed back to the original electrophilic species, and thus remain toxic (15). From the current study, we propose that reaction of glutathione-TDI adducts also involves subsequent liberation of the original electrophile; this is a consequence of the relative lability of the thiocarbamoyl bond linking the alkyl sulfur to the isocyanato carbon (6, 7, 14-16). Reactions of monoisocyanates with GSH, N-acetylcysteine, and cysteine have been studied. Under physiologic conditions, methyl isocyanate (MIC) reacts with GSH to form S-(N-methylcarbamoyl)glutathione (7). Rats injected ip with MIC excrete large amounts of the carbamoylated GSH adduct in the bile (7) and its N-acetylcysteinyl transformation product in the urine (17), with the latter accounting for 25% of the administered MIC. The current study demonstrated rapid reaction of GSH with TDI under physiological conditions, possibly favored by the carbonate-buffered medium in which reactions were performed (18). The reactions did not require enzymatic assistance from glutathione transferase. A

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similar situation has been found with MIC; the resulting MIC carbamate thioester adducts, however, decompose rapidly (7). We observed extremely rapid formation but comparatively slower solvolysis of and transcarbamoylation by the TDI-GSH adducts as compared to the rates observed by others for MIC-GSH adducts. Additionally, the 2,4 diisocyanato adduct 3 solvolyzed more rapidly (about 10-fold) than the 2,6 adduct 8. The higher reactivity of 3 is no doubt due to the steric availability of the 4-position of the toluyl moiety, and adds credence to our hypothesis shown in Schemes 1 and 2 that the thiocarbamoyl system at this position solvolyzes before its counterpart at the 2-position. To our knowledge, reaction products of diisocyanates with GSH have not been reported. We observed high yields of bis adducts 3 and 8 and their slow but efficient transfer of isocyanato equivalents to the 12-mer peptide in carbonate-buffered aqueous media. Explanation of the preferred reaction of isocyanates with GSH and their subsequent transfer of isocyanato moieties has been offered as being due to preferential isocyanate reaction with the soft nucleophile GSH versus reaction with the hard nucleophile water (hydrolysis) (15, 16). For example, preferred reaction of the GSH-MIC adduct S-(Nmethylcarbamoyl)glutathione with cysteine in aqueous media has been shown (7). In the present case, rapid and essentially complete reaction of 1 and 2 with GSH to form only the bis adducts 3 and 8 can be attributed to the TDIs’ insolubilities in purely aqueous systems. Their dissolution into aqueous phases requires cosolvent or physical mixing assistance, and their hydrolysis to diamines requires catalytic assistance (e.g., carbonate or bicarbonate ions) (18). We thus envision that reaction of 1 and 2 with GSH occurs at the organic-aqueous interface to form 4 (or, more likely, its 2-isocyanato-4thiocarbamoyl isomer) and 9, which then react rapidly with GSH (as per the hard/soft argument) to form 3 and 8. Our view of this reaction mechanism is strengthened by the fact that we consistently observed 1 and 2 remaining as water-insoluble droplets at the end of the synthetic reactions. Although reactions in vivo and in vitro of monoisocyanates with sulfhydryl-containing peptides and proteins have been reported (7, 16), there is little information on the protein specificity of such reactions. For example, incubation of [1-14C]MIC with rat erythrocyte membrane proteins for 15 min at 37 °C results in a 59% reduction in the free sulfhydryl content of the membrane proteins, and MIC administered ip to rats results in a 27% decrease in the free thiol content of erythrocyte membranes 24 h after treatment (19). Carbamoylation of proteins in vitro by the S-linked glutathione conjugate of MIC has been shown by Pearson et al. (7), who found that Cys-6 of reduced oxytocin is the preferred site of carbamoylation, with secondary reaction sites at Cys-1 and Tyr-2. Bovine serum albumin, which contains one free sulfhydryl, has also been shown to be carbamoylated after incubation with GSH-MIC (7). These findings indicate that S-conjugates of monoisocyanates can donate their masked isocyanato moiety to nucleophilic sites on peptides and proteins and that the preferred target residue is cysteine and, since such thiocarbamate esters are formed reversibly under physiological conditions, suggest that the toxicity of isocyanates may in part be a result of such carbamoylation processes. To evaluate the transcarbamoylation potential of the bisGS-TDI adducts, we selected a 12 amino acid, single

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cysteine-containing, MHC antigen-derived peptide as a model target for such transfers (20). The proteins involved in the initial stages of sensitization to TDI are not known, but are believed to engage in antigen presentation to appropriate T lymphocytes (21). We noted that the bis adducts transcarbamoylated this peptide under physiological conditions, with the bis 2,4-TDIGSH adduct 3 demonstrating a greater rate of transfer relative to the bis 2,6-TDI-GSH adduct 8. These results suggest a potential relevance of the thiocarbamoylation reaction in human sensitization to TDI. Glutathione conjugation may offer protection against isocyanate toxicity, or may enhance the toxicity of such xenobiotics. Detection of transcarbamoylation reactions of TDI suggests that regeneration of the reactive electrophilic isocyanato functionality may occur in the presence of an appropriate second nucleophile. Precedence for such activity has been shown with N,N′-bis(2-chloroethyl)-N-nitrosourea (BCNU), an antitumor agent that also possesses toxic properties. BCNU degrades in aqueous solution to yield reactive alkylating intermediates that are at least partly responsible for its antitumor activity, and to carbamoylating moieties, notably 2-chloroethyl isocyanate (CEIC), which may be the mediator of some of BCNU’s toxicity. Davis et al. (6) identified the GSH conjugate of CEIC in rats following ip injection of BCNU, suggesting that these thiocarbamoyl adducts contribute to the toxicity of the parent drug. With MIC, survivors of the catastrophic accident in Bhopal, India, in 1984 have displayed numerous toxicities even though the chemical exposure was likely restricted to an inhalation route. The reports of cardiovascular, gastrointestinal, and reproductive effects in the Bhopal victims may well be ascribable to transcarbamoylation of distant proteins. Our detection of the transcarbamoylation reactions of 3 and 8 with the MHC antigen H-2Kb peptide suggests that the toxicity of TDI can be regenerated in the presence of appropriate nucleophiles, and may occur at sites distant to the original location of TDI exposure. It must also be considered that enhanced toxicity may result simply from the rapid reaction of GSH with TDI; intracellularly, such reaction would immediately result in a lowering of cellular thiol levels. GSH protects cells not only from electrophiles but also from damaging free radicals by its intimate involvement in the maintenance of cellular redox balance. A consequence of TDI exposure may thus be toxicity not only due to the isocyanate functionality but also as a result of reduced levels of GSH and the consequent oxidative stress. Recent work has also suggested a more insidious action that is less dependent on thiol isocyanate stoichiometry: the isocyanato and diisocyanato GSH adducts may, via the sitedirecting glutathionyl moiety, irreversibly inactivate enzymes in the GSH-regenerating pathway (16, 22). The reaction kinetics determined and shown in Figure 3 indicate that the thiocarbamoyl adducts 3 and especially the 2,6-adduct 8 are stable enough to diffuse from their site of formation. Several implications can be drawn from these data. First, the possibility exists that TDI can be transported as a bis carbamoylated GSH adduct to sites distant from the initial site of isocyanate contact. As such, the data offer an explanation for our previous finding of TDI-adducted hemoglobin inside red blood cells following inhalation exposure of guinea pigs to TDI vapor (5). Second, the isocyanato functionality can be regenerated from the bisGSH adduct, resulting in carbamoylation of a second nucleophile, and perhaps

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causing toxicity and/or the formation of immunogens. Such transfers would be favored at sites of altered micropH, as the thiocarbamates are most stable at ca. pH 5. As a consequence, the recognized toxicity of TDI to the lung not only may result from direct inhalation of TDI but also may reflect the acidic pH of the respiratory tract and thus the stability of thiocarbamoyl-TDI adducts in the lung. Third, the rapid and direct reaction of TDI with GSH and the transfer of the isocyanate moiety to protein relate to the possible sites of isocyanate toxicity following an inhalation exposure, as well as the possibility of oxidant/antioxidant status contributing to individual susceptibility to these important industrial chemicals.

Acknowledgment. This research was supported in part by PHS Grant ES 05651 from the National Institute of Environmental Health Sciences. We thank Dr. FuTyan Lin at the Department of Chemistry for acquiring the 500 MHz NMR spectra.

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