Characterization of a Valine− Lysine Thiourea Cross-Link on Rat

Previous in vivo studies have supported protein cross-linking by CS2 as both a mechanism of neurotoxicity and a potential biomarker of effect through ...
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Chem. Res. Toxicol. 1998, 11, 1128-1136

Articles Characterization of a Valine-Lysine Thiourea Cross-Link on Rat Globin Produced by Carbon Disulfide or N,N-Diethyldithiocarbamate in Vivo John C. L. Erve,*,† Venkataraman Amarnath,† Robert C. Sills,‡ D. L. Morgan,‡ and William M. Valentine† Department of Pathology and Center in Molecular Toxicology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2561, and Environmental Toxicology Program, National Institute of Environmental Health Science, Research Triangle Park, North Carolina 27709 Received April 15, 1998

Previous in vivo studies have supported protein cross-linking by CS2 as both a mechanism of neurotoxicity and a potential biomarker of effect through the detection of a structure responsible for CS2-mediated protein cross-linking, namely, lysine-lysine thiourea. In this study, the structure of a previously uncharacterized stable protein cross-link produced by CS2 in vivo involving lysine and the N-terminal valine of globin has been determined. Rats were exposed to 50, 500, and 800 ppm CS2 for 2, 4, 8, and 13 weeks by inhalation or to 3 mmol/kg N,N-diethyldithiocarbamate administered orally on alternating days for 8 and 16 weeks. Acid hydrolysis, using 6 N HCl, of globin from control and exposed rats caused cyclization of the valine-lysine thiourea cross-link in treated rats to isopropyl norleucyl thiohydantoin. The hydrolysate was separated by size-exclusion chromatography, and the fraction that coeluted with the synthetic deuterated isopropyl norleucyl thiohydantoin internal standard was derivatized with 3-[4′-(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate and analyzed by liquid chromatography/tandem mass spectrometry using selected reaction monitoring detection. Derivatized isopropyl norleucyl thiohydantoin obtained from CS2-treated rats displayed a cumulative dose response and was detectable at the lowest exposure (50 ppm, 2 weeks) at levels of approximately 50 pmol/g of globin. N,N-Diethyldithiocarbamate-treated rats, but not controls, also contained a CS2-generated valine-lysine thiourea cross-link on globin. In vitro incubation of human hemoglobin with either CS2 or N,N-diethyldithiocarbamate also resulted in the formation of CS2-generated valine-lysine thiourea. These observations demonstrate the potential of thiourea cross-linking involving a free amino terminus and -amino groups of lysine to accumulate in a long-lived globular protein and suggest that cross-linking of globin may provide a specific dosimeter of internal exposure for CS2 capable of assessing exposure over subchronic periods.

Introduction Carbon disulfide is a chemical used in the production of rayon and cellophane and has been known to be neurotoxic for more than a century. N,N-Diethyldithiocarbamate (DEDC1), which is a metabolic product of the drug disulfiram, and the active component of certain dithiocarbamate-containing pesticides, may also be a source of CS2 exposure to humans. Currently, several * To whom correspondence should be addressed. † Vanderbilt University Medical Center. ‡ National Institute of Environmental Health Science. 1 Abbreviations: ACN, acetonitrile; CID, collision-induced dissociation; DEDC, N,N-diethyldithiocarbamate; ESI, electrospray ionization; HCCA, R-cyano-4-hydroxy-trans-cinnamic acid; iP-NLeuTH, isopropyl norleucyl thiohydantoin; Lys-Lys thiourea, di--lysylthiourea; MALDI, matrix-assisted laser desorption/ionization; PETAPITC, 3-[4′-(ethyleneN,N,N-trimethylamino)phenyl]-2-isothiocyanate; PFPITC, pentafluorophenyl isothiocyanate; SRM, selected reaction monitoring; TTCA, 2-thiothiazolidine-4-carboxylic acid; TFA, trifluoroacetic acid; Val-Lys thiourea, valyl--lysylthiourea.

methods are available for estimating recent CS2 exposure, such as levels of blood CS2, CS2-derivatized blood proteins (1, 2), and, the most widely used method, urinary CS2 metabolites (3). All of these methods have recognized limitations. Although urinary 2-thiothiazolidine-4-carboxylic acid (TTCA) can be detected after low levels of exposure, the metabolic pathway leading to TTCA appears to be saturable (4). Furthermore, dietary components (5) and exposure to some types of fungicides, such as Captan [N-[(trichloromethyl)thio]-4-cyclohexene-1,2dicarboximide], can also contribute to TTCA levels, thereby reducing the specificity of this marker (6). In addition, all of these methods appear to be limited to the assessment of subacute exposure and have no demonstrated correlation to pathogenetic mechanisms of toxicity. More recently, CS2-mediated covalent modification of erythrocyte spectrin and globin has been investigated as

10.1021/tx980077p CCC: $15.00 © 1998 American Chemical Society Published on Web 08/27/1998

CS2 Thiourea Cross-Linking of Globin in Vivo

a potential biomarker of effect (7). The advantage of measuring cross-linked protein over current techniques is that it can be applied to chronic exposure scenarios with the potential to integrate exposure over the life of the erythrocyte (120 days in humans and 60 days in rats) and may reflect relevant biochemical events occurring in the axon. Previous studies on CS2 (7-9) and DEDC (10) have demonstrated a cumulative dose response for CS2mediated cross-linking of spectrin (9), and for a covalent modification of globin (7), that were correlated with the amount of neurofilament protein cross-linking occurring in the spinal cord. Moreover, detection of cross-linked spectrin and the modified globin was possible before manifestations of neurotoxicity, and thus, these compounds appear to be suitable for use as preneurotoxic markers of exposure. The chemical identity of one crosslinking structure produced by CS2 and DEDC on spectrin has been determined to be di--lysylthiourea (Lys-Lys thiourea) (11). One drawback of utilizing spectrin as a surrogate for cross-linked axonal proteins is that it takes a significant amount of time and blood to obtain spectrin from erythrocytes. Hemoglobin, however, can be isolated more easily in significantly greater quantities than spectrin and can be chromatographed by reverse-phase HPLC. For these reasons, hemoglobin is expected to be the preferred biomarker and was chosen for further investigation in this study. The purpose of this work was to elucidate the cross-linking structures present in globin obtained from CS2- (inhalation) and DEDC-exposed (po) rats. To this end, we used a combination of liquid chromatography and electrospray ionization (ESI) tandem mass spectrometry to detect the presence of an intramolecular CS2-generated Val-Lys thiourea crosslink involving N-terminal Val on both the R- and β-components of rat hemoglobin.

Materials and Methods Chemicals. Constantly boiling hydrochloric acid solution (6 N), trifluoroacetic acid (TFA), amino acid standard solutions, Drabkin’s reagent, human sickle cell β-chain N-terminal peptide (Val-His-Leu-Thr-Pro-Val-Glu-Lys), and R-cyano-4-hydroxytrans-cinnamic acid (HCCA) were from Sigma Chemical Co. (St. Louis, MO). HPLC grade acetonitrile (ACN) was from EM Science (Gibbstown, NJ). Triethylamine was distilled before use. 3-[4′-(Ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate (PETAPITC) was a gift from R. Aebersold (Department of Molecular Biotechnology, University of Washington, Seattle, WA). Human N-terminal β-chain peptide (Val-His-Leu-Thr-ProGlu2-Lys) was synthesized by Research Genetics, Inc. (Huntsville, AL). DL-Valine-d8 (98% pure) was purchased from Cambridge Isotope Labs (Andover, MA). Outdated human packed red blood cells were obtained from the Vanderbilt Blood Bank. 3-[5-(Acetylamino)-5-carboxypentyl]-5-(2-isopropyl)-2thioxoimidazolidin-4-one (10). NR-Acetyllysine (190 mg, 1 mmol) was dissolved in water (10 mL), and CS2 (90 µL, 1.5 mmol) in 1 mL of ethanol was added. The solution was stirred, and 1 N NaOH (2 mL) was added in 10 portions over a period of 2 h. Stirring was continued for 2 h, and excess CS2 was removed by rotoevaporation. Val (235 mg, 2 mmol) was added, and the mixture was heated to reflux in an oil bath (110 °C) for 10 h. The reaction mixture was acidified to pH 3 and concentrated to 1 mL, and the residue was purified on a column of silica (10% water in ACN). The product having an Rf of 0.3 was isolated: 230 mg (40%); 13C NMR δ 16.2 and 18.9 [(CH3)2CH], 23.0 (COCH3), 31.6 [(CH3)2CH], 23.6, 27.7, 32.0, 41.7 and 55.8 (lysyl chain), 66.2 (C-5), 174.6 (COCH3), 178.3 (C-4), 179.8

Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1129 (CO2H), 185.0 (C-2); MS m/z 330.0. Similarly, starting with NRacetyllysine (95 mg, 0.5 mmol) and Val-d8 (90 mg, 0.75 mmol), we obtained 10-d8.

In Vitro Cross-Linking of Model Peptides. Peptide (ValHis-Leu-Thr-Pro-Val-Glu-Lys or Val-His-Leu-Thr-Pro-Glu2-Lys) was dissolved in 40 µL of 0.4 M NH4HCO3 buffer to give a concentration of 5 mM. The pH was raised to approximately 11 by the addition of 0.5 µL of TEA, and CS2 (1 µL, 16.6 µmol) was added. The reaction mixture was incubated at room temperature for approximately 18 h to generate dithiocarbamate, after which CS2 was removed by lyophilization. Following lyophilization, the reaction mixture was reconstituted with NH4HCO3 buffer to give a more dilute peptide concentration of approximately 2.5 mM before heating (85 °C for 4 h). UV scanning revealed the disappearance of peaks at 286 and 252 nm characteristic of dithiocarbamate and the appearance of a new peak at 240 nm signifying the presence of thiourea. The reaction mixture was lyophilized and reconstituted with the mobile phase prior to HPLC analysis as described below. The peptides Val-His-Leu-Thr-Pro-Val-Glu-Lys and cross-linked ValHis-Leu-Thr-Pro-Val-Glu-Lys were acetylated by adding 1 µL of peptide (∼50 pmol/µL) to 20 µL of 50 mM ammonium bicarbonate buffer (pH 7.8) and 50 µL of acetylating reagent (100 µL of acetic anhydride and 300 µL of methanol). The reaction mixture was incubated at 37 °C for 10 min before lyophilizing to dryness. The peptides were reconstituted with 10 µL of water prior to matrix-assisted laser desorption/ ionization (MALDI) MS analysis. In Vivo and in Vitro Exposures. Animal studies were conducted in accordance with the NIH Guide For Care and Use of Animals and approved by the institutional animal use and care committee. For the CS2 exposures, male Fisher 344 rats (Charles River Breeding Laboratories, Raleigh, NC) were used. The exposure levels were control and 50, 500, and 800 ppm for a duration of 2, 4, 8, or 13 weeks. CS2 (>99%) was mixed with conditioned air and the mixture introduced into the inhalation chambers 5 days per week; the daily exposure time was 6 h. Actual chamber CS2 concentrations were monitored continuously by infrared spectroscopy so that mean daily exposures were within 3% of the desired concentrations. Further details can be found elsewhere (9). For the DEDC exposures, male Sprague-Dawley rats (Harlan, Sprague-Dawley, Indianapolis, IN) were used. The rats were administered 3 mmol/kg every other day orally for either 8 or 16 weeks. DEDC concentrations were checked spectrophotometrically; the purity was >99%. Dosing was accomplished by dissolving DEDC in 0.1 M phosphate buffer (pH 7.5) and introducing the solution into the rat by intragastric gavage once a day on alternate days throughout the exposure period. Further details can be found elsewhere (3). Within 24 h of the last exposure, rats were exsanguinated under deep anesthesia, and globin was purified from isolated erythrocytes as described previously (12). After washing four times at 4 °C with isotonic Tris (pH 7.6), the erythrocytes were lysed with a 6 volume excess of 20 mOsm Tris (pH 7.6). Globin was precipitated from acetone containing 2.5% oxalic acid, washed with the same solvent, dried under a stream of argon, and stored at - 78 °C. Human Hemolysate. Human hemolysate was prepared from packed cells according to the method of Antonini and Brunori (13). The hemolysate was dialyzed against three changes of 0.05 M phosphate buffer (pH 7.4) and stored at 4

1130 Chem. Res. Toxicol., Vol. 11, No. 10, 1998 °C. The final concentration was determined using Drabkin’s reagent (A540,  ) 44 M-1 cm-1) to be 27 mg/mL. For in vitro incubations, 5 µL of CS2 was added to 1 mL of hemolysate (28 mM), or DEDC was added to give a concentration of 0.9 mM, and the mixture allowed to react at room temperature for 24 h. Acid Hydrolysis of Globin. Approximately 15 mg of globin (or 3-4 mg of purified R- or β-components) was placed in a hydrolysis tube (6 mm × 50 mm) and hydrolyzed for approximately 18 h at 110 °C under vacuum on a Picotag workstation (Waters, Milford, MA). Following removal of residual HCl, samples were reconstituted with 85 µL of 20% ACN and 0.1% TFA and filtered through a disposable Centrex MF 0.4 microcentrifuge filter with 0.2 µm pores (Schleicher and Schuell, Keene, NH). Analysis of Lys-Lys thiourea was performed as previously described (11). Derivatization of iP-NLeuTH with PETAPITC. Fractions containing iP-NLeuTH were derivatized in 1.5 mL Eppendorf tubes. Briefly, samples were reconstituted with 25 µL of solvent (50:49:1 v/v/v methanol/water/ethyl acetate), and the pH was adjusted to approximately 10 by the addition of 3 µL of triethylamine. Next, 3 µL of a 90 mM solution of PETAPITC was added, followed by heating at 60-65 °C for 15 min. Samples were dried by rotoevaporation with a Speed-vac (Savant Instruments, Holbrook, NY) before the addition of 5 µL of 50:50 TFA/water and further heating at 45 °C for 30 min. The samples were dried, filtered (Centrex MF 0.4), and then reconstituted with 55 µL of 5% ACN, 0.1% acetic acid, and 0.02% TFA. Chromatography. The peptide reaction mixtures were injected onto a RP column (Zorbax SB C8, 2.1 mm × 150 mm, 3.5 µm, 80 Å, MacMod Analytical, Chadds Ford, PA) and eluted at 0.2 mL/min with a linear gradient from 5 to 37% B [solvent A is ACN/H2O/TFA (5:95:0.1 v/v/v); solvent B is ACN/H2O/TFA (80:20:0.1 v/v/v)] over 37 min. The eluant was monitored at 220 nm, and modified peptides were collected and lyophilized before analysis by ESI/MS as described below. Separation of rat globin into R- or β-components was accomplished by dissolving globin in mobile phase A to give a concentration of approximately 1 mg/mL, and 100 µL was injected onto a semipreparative polymeric reverse-phase column (7.1 mm × 305 mm, 7 µm, 100 Å, Hamilton, Reno, NV). The elution gradient was linear from 44 to 56% B over 45 min with a flow of 1 mL/min [solvent A is ACN/H2O/TFA (20:80:0.1 v/v/ v); solvent B is ACN/H2O/TFA (60:40:0.1 v/v/v)]. The eluant was monitored at 220 nm, allowing protein to be collected, lyophilized, and weighed. Enrichment of iP-NLeuTH was accomplished by size-exclusion HPLC with a G2500PWXL column (7.8 mm × 300 mm i.d., 6 µm, TosoHaas, Montgomeryville, PA). The elution conditions were isocratic with 20% ACN and 0.1% TFA at a flow rate of 1 mL/min. The iP-NLeuTH could be easily detected by its dual absorbance at 235 (thiourea) and 270 nm (thiohydantoin). For each analysis, approximately 3 µL of the iP-NLeuTH-d8 (20 mM) deuterated standard was spiked into 85 µL of hydrolysate (1200 nmol/µL), which was then injected onto the column. The deuterated standard, which had the same retention time as the authentic iP-NLeuTH, was detected easily and collected, along with any coeluting iP-NLeuTH present in the treated globin. The liquid chromatography system included a Waters 996 photodiode array detector and model 2690 liquid chromatograph (Waters) equipped with an autosampler coupled to a fraction collector (Foxy Jr., Isco, Lincoln, NE). Mass Spectrometry. Liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis of derivatized iP-NLeuTH with selected reaction monitoring (SRM) detection was performed with a Finnigan TSQ 7000 triple-quadrupole ESI/MS system (Finnigan, San Jose, CA). Forty microliters of sample was chromatographed on a reverse-phase C18 column (1 mm × 50 mm i.d., 5 µm, Monitor, Ontario, CA) with a linear elution gradient from 35 to 70% B over 4 min followed by 70% B for 2 min with a flow rate of 50 µL/min [solvent A is ACN/H2O/acetic acid/TFA (5:95:0.1:0.02 v/v/v/v); solvent B is ACN/H2O/acetic

Erve et al. acid/TFA (80:20:0.1:0.02 v/v/v/v)] and the eluant directed into the mass spectrometer. The electrospray voltage of the atmospheric pressure ionization positive ion source was maintained at 4.2 kV and the capillary temperature fixed at 200 °C; the nebulizer pressure was 70 psi. Collision-induced dissociation (CID) occurred in Q2, with argon as a collision gas (2.8 mT) at a collision energy of 32 eV (laboratory frame of reference). SRM experiments to detect iP-NLeuTH were conducted by monitoring the m/z 490.3 f 431.3 transition arising from the loss of the quaternary amine from the molecular ion (m/z 498.3 f 439.3 for the deuterated standard). The total scan time was 1.4 s. To increase sensitivity, resolution was decreased by raising the voltages on Q1 (parent resolution, 10.5 V) and Q3 (daughter resolution, 6.1 V). Cross-linked peptides were dissolved in methanol/H2O/acetic acid (50:49:1) to give a concentration of approximately 5-10 pmol/µL and analyzed by flow infusion at a rate of 20-30 µL/ min. Tandem MS/MS spectra were obtained by CID in the second quadrupole (Q2, RF-only) with a collision energy in the range of 20-25 eV and a collision gas pressure of 2.5 mT. Mass spectra were collected between m/z 200 and 1000 (or m/z 501000 for MS/MS) and averaged over 1 min at one scan per second. MALDI/MS analysis with delayed extraction and an ion mirror was performed on a Voyager Elite (PerSeptive Biosystems Inc., Framingham, MA) time-of-flight mass spectrometer. A 1 µL drop of saturated HCCA matrix was applied to the sample well, followed by the addition of 1 µL of peptide solution with an approximate concentration of 2-5 pmol/µL. The ion acceleration voltage was 22 kV, and the instrument was operated with delayed extraction (100 ns delay time) in the reflectron mode. The pressure in the time-of-flight analyzer was approximately 2.3 × 10-8 Torr. Mass spectra were acquired as the sum of ions generated by irradiation of the target with 20100 laser pulses (337 nm N2 laser) and processed using GRAMS/ 386 software (Galactica Industries Corp., Salem, NH). Statistics. One-way ANOVA, Bonferroni’s multiple comparison test (post hoc), and linear regression were performed using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). The level of significance was taken to be p < 0.01, unless otherwise noted. For CS2 exposures, four rats were analyzed at each time point and exposure level for a total of 64 rats. For DEDC exposures, five rats were analyzed at 8 weeks and three rats analyzed at 16 weeks. The sixteen control rats were pooled since there were no significant differences between any of the control groups. Normalized values were obtained by dividing the measured mass spectrometric response (peak height) for iP-NLeuTH by the internal standard response and the amount of protein hydrolyzed and then multiplying by 10 000.

Results Model Peptide Cross-Linking by CS2. Upon chromatography on a C8 RP column, Val-His-Leu-Thr-ProGlu-Val-Lys eluted at approximately 23 min. In the reaction mixture produced with Val-His-Leu-Thr-ProGlu-Val-Lys and CS2, in addition to the starting peptide, there were several late eluting peptides. One of these peptides had a retention time of 37 min and was mass analyzed by ESI/MS to reveal a doubly charged molecular ion (m/z 482.9) consistent with modification of the peptide by the carbon and sulfur atoms of CS2 (Figure 1A). Following purification, this peptide was submitted for N-terminal analysis by Edman analysis, but it could not be sequenced, indicating that the N-terminal Val had become blocked. The CS2-modified peptide, and the peptide from which it was derived (Val-His-Leu-Thr-ProGlu-Val-Lys), were incubated with an acetylating mix-

CS2 Thiourea Cross-Linking of Globin in Vivo

Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1131

Figure 2. Size-exclusion HPLC analysis of amino acid hydrolysate. Globin obtained from a CS2-treated rat without an internal standard (A) or with iP-NLeuTH-d8 as an internal standard that elutes at 17 min (B). The chromatogram represents elution of 270 nm-absorbing material from the sizeexclusion column as described in Materials and Methods.

Figure 1. ESI/MS of the intramolecularly cross-linked model peptide. Panel A shows the ESI mass spectrum of the peptide Val-His-Leu-Thr-Pro-Glu-Val-Lys containing a thiourea crosslink between Val and Lys. Panel B shows the mass spectrum following CID as described in Materials and Methods. Fragment ions at m/z 279, 392, and 493 contain isothiocyanate on Val generated following cleavage of the thiourea bond.

ture, and products were measured separately by MALDI/ MS. The unmodified peptide displayed an 84 Da mass increment indicative of the addition of two acetyl groups, while the CS2-modified peptide was unchanged. The CS2modified peptide was subjected to CID which resulted in generation of the ions shown in Figure 1B. The large ion at m/z 279 corresponds to Val-His with retention of the isothiocyante group derived from cleavage of the thiourea bond. Likewise, peaks at m/z 392 and 493 correspond to Val-His-Leu and Val-His-Leu-Thr, with retention of isothiocyanate on the N-terminal Val. Fragment ions with charge retained on the C terminus (y4y6) were also identified. Incubation of the N-terminal peptide of the human hemoglobin β-chain, Val-His-LeuThr-Pro-Glu2-Lys, also led to the identification of a CS2modified peptide as revealed by the doubly charged molecular ion (m/z 497.8) and produced similar diagnostic fragment ions following CID (data not shown). Size-Exclusion Analysis of Amino Acid Hydrolysates and iP-NLeuTH. Previously, size-exclusion chromatography was used successfully to purify Lys-Lys thiourea from spectrin (11), and therefore, the same approach was applied to the purification of iP-NLeuTH from globin hydrolysates in this study. Unlike Lys-Lys thiourea which eluted between the two major amino acid peaks using 10% ACN, iP-NLeuTH eluted last and required 20% ACN for elution to occur in a timely fashion. Since iP-NLeuTH is expected to be considerably more hydrophobic than Lys-Lys thiourea because of the isopropyl side chain, its longer retention time might be

explained by column-analyte interactions that are involved in addition to separation by size. As with spectrin (11), globin from either control or exposed rats displayed several additional unidentified peaks compared to an amino acid standard mixture. Nevertheless, the iPNLeuTH-d8 internal standard could be baseline resolved in the globin hydrolysates, which allowed its collection along with coeluting iP-NLeuTH thiourea originating from the globins (Figure 2). Liquid Chromatography/Mass Spectrometry Analysis of Derivatized iP-NLeuTH. iP-NLeuTH enriched on the size-exclusion column was derivatized with PETAPITC. PETAPITC is a first-generation novel Edman reagent that contains a quaternary amine group that allows for efficient ionization and sensitive detection by ESI/MS (14) and was used successfully for the mass spectrometric analysis of Lys-Lys thiourea in spectrin (11). Authentic derivatized iP-NLeuTH (calculated mass of 490 Da) produced a singly charged molecular ion at m/z 490.3, which when fragmented by collision with argon produced numerous ions, including a major ion at m/z 431.3 (Figure 3). In our SRM analysis, the fragment ion at m/z 431.3, arising from the loss of the quaternary amine, was measured. The internal standard had a molecular mass 8 Da higher, resulting in a singly charged molecular ion and the corresponding major fragment ion at m/z 498.3 and 439.3, respectively. The retention time of PETAPITC-derivatized iP-NLeuTH was approximately 3 min when it was chromatographed on a C18 reversedphase column. The lower limit of detection was determined to be approximately 500 pg, and the response for derivatized iP-NLeuTH was linear over the range of 0-500 ng (based on 0, 1, 5, 50, 250, and 500 ng of iPNleuTH standards). Coefficients of variation for the exposure groups ranged from 1 to 25% and averaged about 10%. The standards could be run repeatedly without any apparent deterioration. Reconstructed ion chromatograms obtained from representative control rats produced a negligible signal for iP-NLeuTH (Figure 4A). In contrast, a reconstructed ion chromatogram from a representative CS2-treated rat (Figure 4B) or DEDC-

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Figure 3. Tandem mass spectrum of the [M+] parent (m/z 490.3) of iP-NLeuTH derivatized with PETAPITC. The fragment ion at m/z 431.3 arises from the loss of the quaternary amine moiety. The spectrum was obtained on a Finnigan TSQ7000 triple-quadrupole mass spectrometer with conditions as described in Materials and Methods.

Figure 4. LC/MS/MS with SRM of globin hydrolysate. The internal standard, iP-NLeuTH-d8 (m/z 498.3), is shown in the upper trace of each panel. The lower traces show the absence of iP-NLeuTH in the control sample (A) and the presence of derivatized iP-NLeuTH in the sample obtained from the CS2exposed (B) and DEDC-exposed rats (C).

treated rat (Figure 4C) revealed peaks with signal-tonoise ratios of approximately 20:1 having the same retention time as the internal standard. To ascertain whether the CS2-generated Val-Lys thiourea cross-link was specific to either the R- or β-components of globin, the R- or β-components were separated and analyzed independently for iP-NLeuTH content by LC/MS/MS. iPNLeuTH was detected in both samples, indicating that at least one subtype of the R- and β-globin chains of rat

Erve et al.

Figure 5. CS2-mediated globin cross-linking as a function of exposure level and duration. Dose-response curves relate the quantity of iP-NLeuTH detected for control and 50, 500, and 800 ppm exposure levels vs the duration of exposure (0, 2, 4, 8, and 13 weeks, 6 h/day for 5 days/week). The iP-NLeuTH intensity was normalized to the amount of both the protein hydrolyzed and the internal standard. Error bars represent standard errors (n ) 4).

globin had been cross-linked by CS2 through the formation of Val-Lys thiourea (data not shown). Human hemoglobin, also possessing N-terminal Val on both Rand β-chains, treated with either CS2 or DEDC in vitro also produced iP-NLeuTH in quantities significantly greater than control quantities.2 Dose Response of Val-Lys Thiourea Cross-Linking of Globin. Quantities of iP-NLeuTH isolated from rats administered CS2 by inhalation are shown in Figure 5 as a function of exposure level and duration. Relative to controls, significantly greater amounts of iP-NLeuTH were measured at all exposure levels and durations except for 50 ppm at 4 weeks. Within an exposure period, the quantity of iP-NLeuTH detected demonstrated a linear dose response (R2 of 0.977, 0.981, 0.963, and 0.994 for 2, 4, 8, and 13 weeks, respectively). Using regression analysis, the level of CS2 exposure corresponding to the amount of protein cross-linking resulting from oral DEDC administration for exposure for 8 and 16 weeks was calculated to be 292 ( 66 and 233 ( 27 ppm, respectively (Figure 6). Estimates of the amounts of iP-NLeuTH produced from globin were obtained on the basis of comparison with calibration standards of known quantities of iP-NLeuTH. The range of iP-NLeuTH amounts produced in globin in this study was estimated to be 53 ( 20 to 4880 ( 530 pmol/g of globin, corresponding to CS2 exposure at 50 ppm for 2 weeks and 800 ppm for 13 weeks, respectively. Liquid Chromatography/Mass Spectrometry Analysis of Derivatized Lys-Lys Thiourea. [13C]Lys-Lys thiourea-d8 standard (98% pure) synthesized in our laboratory was spiked into globin hydrolysates and collected off the size-exclusion column, derivatized with PETAPITC, and analyzed as described previously (11). Lys-Lys thiourea was not detected in CS2- or DEDCexposed rats. 2

Unpublished data.

CS2 Thiourea Cross-Linking of Globin in Vivo

Figure 6. Comparison of the quantities of iP-NLeuTH produced by oral DEDC administration and CS2 inhalation. Doseresponse curves for Val-Lys cross-links produced on globin were generated from values obtained for CS2 inhalation exposure for 8 and 13 weeks (6 h/day for 5 day/week). The levels of inhalation exposure calculated by regression analysis corresponding to the amount of iP-NLeuTH produced by DEDC administered orally for 8 and 16 weeks (3 mmol kg-1 day-1 on alternating days) were 292 ( 66 ppm at 8 weeks and 233 ( 27 ppm at 13 weeks, respectively.

Discussion The chemistry responsible for protein thiourea crosslinking structures produced by CS2 has been previously described in detail (15, 16). The first step in the reaction between globin (1) and CS2 begins with the reversible derivatization of an -amino group on Lys which forms -dithiocarbamate (2) (Scheme 1), followed by the generation of isothiocyanate (3), through the loss of hydrogen sulfide ion. From previous work, it is not expected that dithiocarbamate present on R-amino groups will generate isothiocyanate under physiological conditions (16). Nucleophilic addition by Lys can produce a stable Lys-Lys thiourea cross-link on protein (5), while an unblocked N-terminal amino group, such as that found on hemoglobin, is also capable of reacting with isothiocyanate to give a Val-Lys thiourea cross-link (4). Due to the lower pKa, R-amino groups may be more reactive toward isothiocyanate at physiologic pH than the -amino group of Lys. CS2 and DEDC have been shown to produce highmolecular mass species of spectrin (8, 9) and neurofilament proteins in vivo. These high-molecular mass proteins were interpreted to be the result of intermolecular cross-links, and the structure of one cross-link on spectrin has now been determined to be Lys-Lys thiourea (11). We initially analyzed globin for the presence of Lys-Lys thiourea (7) but were unable to detect it. Subsequently, in vitro experiments with the model peptides Val-His-Leu-Thr-Pro-Glu2-Lys and Val-His-LeuThr-Pro-Glu-Val-Lys showed, by ESI/MS, that CS2 can modify these two peptides as evidenced by the observation of a molecular ion 42 Da greater than the respective parent peptide. The 42 Da mass increment is consistent with three possible structures, including an isothiocyanate functional group on either Val or Lys, or an intramo-

Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1133

lecular thiourea cross-link. Structures containing free isothiocyanate are unlikely due to the reactivity of this electrophilic group, and the inability of acetic anhydride to acetylate the CS2-modified peptide. Unlike the unmodified peptide, Val-His-Leu-Thr-Pro-Glu-Val-Lys, which displayed a mass increment of 84 Da upon acetylation consistent with two free amino groups, the CS2-modified peptide showed no mass increment after incubation with the acetylating reagent, indicating that both amino groups were no longer free. Such an observation is consistent only with the formation of an intramolecular thiourea cross-link between Val and Lys. Tandem MS with CID of peptides produces a series of b and y fragment ions that provide amino acid sequence information about the peptide (17). Structural information on modified peptides can also be obtained by this technique if the adduct is not completely removed from the peptide during fragmentation (18, 19). Under the low-energy CID conditions used here, fragmentation of the thiourea bond generated an isothiocyanate group that was retained on one of the amino groups, thus marking a site of modification. In our experiments, the presence of isothiocyanate on Val indicates that Val is involved in the cross-link. Presently, it is unclear why isothiocyanate is not also retained on the -amino group of Lys. Nevertheless, in both model peptides, Lys is the only other possible nucleophile capable of thiourea bond formation, indicating that an intramolecular cross-link can form between residues separated by up to six amino acids. The sequence of the model peptide Val-His-Leu-ThrPro-Glu2-Lys is identical to the first eight residues from the N terminus of the β-chain of human hemoglobin (20), and also has a similar distance between the N terminus and the first internal Lys as all the R- and β-components of rat globin possess sequences of either Val-Leu-Ser-AlaAsp2-Lys or Val-His-Leu-Thr-Asp-Ala-Glu-Lys, respectively (21). Quantitative analysis of hydrolysates from rats exposed to CS2 for the presence of Val-Lys thiourea required an internal standard. To obtain an authentic sample for comparison, NR-acetyllysine dithiocarbamate was heated with excess Val. From the reaction mixture, instead of the expected thiourea, the thiohydatoin (10) was detected, which on hydrolysis yielded iP-NLeuTH (8). During acid hydrolysis of globin in 6 N HCl, ValLys thiourea was liberated from protein (6) but quickly underwent cyclization to form the more stable compound, iP-NLeuTH, which in this study, was derivatized with PETAPITC to form (9). Formation of Val-Lys thiourea, like that of Lys-Lys thiourea, is expected to be irreversible under physiological conditions, and therefore, the compound is also expected to accumulate with continued exposure. The globular tertiary structure of globin, as opposed to the fibrous nature of neurofilament and erythrocyte spectrin subunits, may favor intramolecular cross-linking versus the intermolecular cross-linking suspected to occur between neurofilament subunits and between the R- and β-subunits of erythrocyte spectrin. Although intermolecular cross-linking of human hemoglobin subunits has been accomplished using reagents with greater spacing between electrophilic groups, such as bis(3,5-dibromosalicyl) succinate (22), the cross-linking bridge formed by CS2 is relatively small, on the order of 3 Å. Hence, CS2 appears to be less likely to link Lys residues on adjacent globin subunits to form Lys-Lys thiourea, but does

1134 Chem. Res. Toxicol., Vol. 11, No. 10, 1998

Erve et al. Scheme 1

appear to be capable of linking N-terminal Val with a closely positioned Lys several residues away on the same chain to form an intramolecular Val-Lys thiourea crosslink as seen here for our work with the model peptides. Interatomic distances, calculated from the X-ray crystallographic structure of human hemoglobin, between the N terminus and the nearest -nitrogen on Lys are approximately 7.2 and 9.4 Å for the β- and R-chains, respectively. Although our work does not address the specific Lys residue in hemoglobin involved in the crosslink, it suggests that the Lys nearest the N terminus, i.e., Lys-7 and Lys-8 on the rat R- and β-chains (21), or the R- and β-chains of human hemoglobin, can participate in cross-link formation. The reactivity of the N-terminal valine of hemoglobin with several known carcinogens, such as ethylene oxide (23) and acrylonitrile (24), forms the basis of biomonitoring human exposure to these and other electrophilic chemicals through an approach known as the “N-alkyl Edman method” (25). With this method, the alkylated N-terminal Val is selectively cleaved after derivatization with pentafluorophenyl isothiocyanate (PFPITC) and the

resulting thiohydantoin level is measured by GC/MS. For Val-Lys thiourea, however, the reactivity of the Nterminal nitrogen is greatly reduced due to the involvement in the thiourea bond and is not expected to react appreciably with PFPITC. Hence, our method utilized total protein hydrolysis to liberate a free thiohydantoin possessing a Lys R-amino group that was subsequently derivatized by PETAPITC prior to LC/MS/MS analysis. The current sensitivity of our method allowed detection of approximately 50 pmol/g of globin for iP-NLeuTH in rats exposed to 50 ppm CS2 for 2 weeks. This level of sensitivity may be sufficient for monitoring human exposure to levels of CS2 encountered in the workplace. Although occupational exposures are not expected to exceed the current Occupational Health and Safety Administration’s time-weighted average of 20 ppm, the exposure duration could extend over the lifetime of the erythrocyte in humans, resulting in the accumulation of Val-Lys thiourea. If a higher sensitivity is required, however, derivatization with PFPITC and negative ion chemical ionization tandem MS may prove efficacious as N-terminal Val adducts of 1,2-epoxy-3-butene at levels

CS2 Thiourea Cross-Linking of Globin in Vivo

of 0.16 pmol/g have been detected (26). Size-exclusion purification of iP-NLeuTH from the hydrolysate may cause a substantial loss of iP-NLeuTH and limit the sensitivity compared to methods where this purification step is unnecessary. This limitation may be offset by analyzing a larger amount of protein and/or improving the efficiency of the derivatization step which may not be optimized and might only reach 35-40%, as was found for the derivatization step of the modified alkyl Edman method (25). Oral administration of DEDC produced Val-Lys crosslinks identical to those resulting from exposure to CS2 via inhalation, consistent with previous studies that have demonstrated the in vivo release of CS2 from DEDC (10). The degree of Val-Lys cross-linking observed in globin resulting from administration of DEDC was equivalent to that expected from an inhalation level of approximately 230-290 ppm CS2 for a comparable duration of exposure. Similarly, the quantity of erythrocyte spectrin and neurofilament cross-linking, the amount of modified R-globin produced, and the severity of the morphological lesions in the nervous system reported for DEDC exposure at this level also correspond to CS2 exposure via inhalation within a similar range. These results demonstrate a good correlation between the CS2-mediated cross-linking of globin and other proteins, including the putative target proteins within the axon, and suggest that globin cross-linking can be used as a specific dosimeter for internal exposure to CS2 regardless of the route or source of exposure. The detection of iP-NLeuTH derived from CS2-generated Val-Lys thiourea on human hemoglobin in vitro warrants further evaluation of hemoglobin obtained from humans exposed to CS2, or compounds that can release CS2 such as disulfiram. Such investigations, along with the analytical methods developed in this work, may serve to advance human biomarker studies of CS2 exposure, thereby protecting humans from this toxic chemical.

Conclusion A major finding of this investigation is the identification of iP-NLeuTH derived from a CS2-generated ValLys thiourea cross-linking structure produced in rats exposed to CS2 or DEDC in vivo. The observation of a dose-response relationship for CS2-exposed rats supports the potential relevance of protein cross-linking chemistry as a marker of exposure to CS2. This work also demonstrates the ability of CS2 and DEDC to produce a thiourea protein cross-link in vivo involving the N terminus of globular proteins, suggesting that other unblocked proteins may also be potential targets of CS2-mediated intramolecular cross-linking.

Acknowledgment. We thank Dr. Ruedi Aebersold (Department of Molecular Biotechnology, University of Washington) for the generous gift of PETAPITC used in these experiments. We also thank Deadre Johnson and Holly Valentine for providing the globin from the DEDCtreated rats, the Vanderbilt Mass Spectrometry Resource for use of the mass spectrometer, and the Vanderbilt University NMR Center for the use of the NMR instrument. We acknowledge the Protein Chemistry Laboratory (P30 ES00267) and Eric Howard for amino acid sequencing. These studies were supported by NIH Grant ES06387

Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1135

and Center in Molecular Toxicology Grant P30 ES00267. J.C.L.E. was supported, in part, by NRSA Grant ES05764.

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