Covalent Binding of Acrylonitrile to Specific Rat ... - ACS Publications

Chem. Res. Toxicol. 2001, 14, 799-806

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Covalent Binding of Acrylonitrile to Specific Rat Liver Glutathione S-Transferases in Vivo Donald E. Nerland,* Jian Cai, William M. Pierce, Jr., and Frederick W. Benz Department of Pharmacology & Toxicology, University of Louisville Medical School, Louisville, Kentucky 40292 Received January 5, 2001

Acrylonitrile (AN) is an industrial vinyl monomer that is acutely toxic. When administered to rats, AN covalently binds to tissue proteins in a dose-dependent but nonlinear manner [Benz, F. W., Nerland, D. E., Li, J., and Corbett, D. (1997) Fundam. Appl. Toxicol. 36, 149-156]. The nonlinearity in covalent binding stems from the fact that AN rapidly depletes liver glutathione after which the covalent binding to tissue proteins increases disproportionately. The identity of the tissue proteins to which AN covalently binds is unknown. The experiments described here were conducted to begin to answer this question. Male Sprague-Dawley rats were injected subcutaneously with 115 mg/kg (2.2 mmol/kg) [2,3-14C]AN. Two hours later, the livers were removed, homogenized, and fractionated into subcellular components, and the radioactively labeled proteins were separated on SDS-PAGE. One set of labeled proteins was found to be glutathione S-transferase (GST). Specific labeling of the µ over the R class was observed. Separation of the GST subunits by HPLC followed by scintillation counting showed that AN was selective for subunit rGSTM1. Mass spectral analysis of tryptic digests of the GST subunits indicated that the site of labeling was cysteine 86. The reason for the high reactivity of cysteine 86 in rGSTM1 was hypothesized to be due to its potential interaction with histidine 84, which is unique in this subunit.

Introduction The vinyl monomers are an important group of organic chemicals used commercially for the production of polymers, copolymers, and as synthetic intermediates. An intriguing toxicological facet of the vinyl monomers is the diversity of symptoms caused by these chemicals. Although they are structurally related, each of the monomers appears to possess a distinct toxicological profile. Vinyl chloride is a liver toxin (1) and carcinogen (2), acrylamide is a neurotoxin (3), the acrylates are dermal sensitizing agents (4-6) and acrylonitrile is an acute toxin (7,8) and animal carcinogen (9, 10). It appears that small changes in the functional group attached to the vinyl group can significantly alter the toxicological profile of the vinyl monomers. Acrylonitrile (AN)1 is used in the production of plastics, fibers and synthetic rubber. Industrial exposure of humans to AN can occur during the various phases of its production, polymerization, and transportation. The general public is also exposed to AN through air pollution and cigarette smoke (11, 12). Three separate mechanisms may contribute to the acute toxicity of AN: (a) cyanide formed by the oxidative metabolism of AN, (b) GSH * To whom correspondence should be addressed. Phone: (502) 8525560. Fax: (502) 852-7868. E-mail: [email protected]. 1 Abbreviations: AN, acrylonitrile; CEO, 2-cyanoethylene oxide; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; ESI-MS, electrospray ionization-mass spectrometry; GSTCBQ, 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone; GST, glutathione S-transferase; IAM, iodoacetamide; MALDI-TOF MS, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry; PAGE, polyacrylamide gel electrophoresis; PDB, Protein Data Bank; PVDF, poly(vinylidene difluoride); S-BDB-G, S-(4-bromo-2,3-dioxybutyl) glutathione; rGSTM, rat glutathione S-transferase µ family; rGSTA, rat glutathione Stransferase R family; SDS, sodium dodecyl sulfate.

depletion, and (c) covalent binding of AN to tissue proteins. Oxidation of AN to the labile metabolite CEO (2-cyanoethylene oxide) is catalyzed by the microsomal mixed function oxidase system (13, 14). In vitro studies using human and rat hepatic microsomes and in vivo studies using P450 2E1-null mice indicate that P450 2E1 is the major catalyst of AN epoxidation and that other P450 isoenzymes play only a minor role in the metabolism of AN (14-16). Hydration or nucleophilic addition to the methylene carbon of the epoxide of CEO converts it to a cyanohydrin (17). The cyanohydrin can spontaneously decompose liberating cyanide ion. Cyanide antidotes, however, cannot prevent AN-induced toxicity in rats even though they can drastically reduce or abolish the blood level of cyanide (18). This suggests that a mechanism(s) independent of cyanide formation plays a significant role in AN-induced toxicity. AN also reacts enzymatically and nonenzymatically with GSH to form S-(cyanoethyl)glutathione (17, 19). This reaction is believed to be responsible for the AN-induced depletion of GSH observed in several tissues (20, 21), and this depletion may predispose the animal to oxidative tissue damage (22). However, GSH depletion alone is unlikely to explain the acute toxicity of AN, but severe depletion of liver GSH appears to facilitate the covalent binding of AN to tissue proteins (23, 24). AN is a direct alkylating agent that can undergo Michael-like addition reactions with nucleophiles (19). The highly reactive nature of AN with proteins is illustrated by the observation that subcutaneously administered AN has the highest rat hemoglobin binding index (µmol of AN adduct‚g of globin-1/mmol of AN‚kg-1) of any compound reported (23). However, with the

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exception of hemoglobin, the identity of the proteins to which AN binds remains unknown (25, 26). In the present investigation we have initiated studies to identify the major protein adducts formed following subcutaneous administration of AN. Our results indicate that even though AN is highly reactive, it demonstrates selectivity in its reaction with liver proteins. The identification and characterization of the specific site of labeling of one class of these proteins is the subject of this report.

Experimental Procedures Chemicals. Acrylonitrile (>99% pure), containing 35-45 ppm hydroquinone monomethyl ether as a polymerization inhibitor, and R-cyano-4-hydroxy-trans-cinnamic acid were purchased from Aldrich Chemical Co. (Milwaukee, WI). [2,3-14C]AN (>99% radiochemical purity) with a specific activity of 5.5 Ci/mol was obtained from Sigma Chemical Co. (St. Louis, MO). Radiochemical purity was checked by HPLC prior to its use. Buffer reagents, GSH, glutathione agarose, dithiothreitol (DTT), iodoacetamide (IAM), and S-hexylglutathione were purchased from Sigma Chemical Co. SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) reagents and protein molecular weight markers were obtained from Bio-Rad Laboratories (Hercules, CA). All other chemicals were of the highest quality available from commercial sources. Animals and Tissue Treatments. Male Sprague-Dawley rats, weighing between 225 and 249 g were obtained from Harlan (Indianapolis, IN). All animals were maintained on a 12-h light-dark cycle at 22 ( 2 °C. The rats were acclimated for at least 1 week prior to use and were allowed Purina Rat Chow (Ralston Purina Co., St. Louis, MO) and water ad libitum. Rats were injected subcutaneously with 115 mg/kg (2.2 mmol/kg) of [2,3-14C]AN (500 µCi/kg). Two hours following the injection, the rats were sacrificed and the livers were perfused in situ and excised. A portion of the liver was solubilized using SDS lysis buffer. A second portion of the liver was homogenized in isotonic KCl containing 10 mM Tris-HCl buffer (pH 7.4), and the subcellular fractions were isolated by centrifugation. Electrophoresis, Blotting, and Detection. SDS-PAGE employing 12% gels was performed according to the method of Laemmli (27). The proteins were detected by staining with 0.5% Coomassie Brilliant Blue R250 in 30% methanol containing 10% acetic acid. Alternatively, the proteins were blotted onto PVDF (poly(vinylidene difluoride), Immobilon-P, Millipore) membranes using the method of Szewczyk and Kozloff (28) and the radiolabeled proteins were detected by autoradiography using Kodak BioMax MS (Rochester, NY) film with a Kodak BioMax LE intensifying screen. Immunodetection was performed using a rabbit primary polyclonal antibody to hGSTM1 (Calbiochem, LaJolla, CA) and visualized using Amersham-Pharmacia (Piscataway, NJ) chemiluminescence detection reagents. Purification of Rat Liver Glutathione S-Transferases. Glutathione S-transferases (GST) were isolated from rat liver cytosol by a modification of the method of Simons and Vander Jagt (29). A portion of the rat liver 100000g supernatant was applied to a glutathione-agarose affinity column equilibrated with 10 mM Tris (pH 7.8) containing 1 mM EDTA and 0.2 mM DTT. The column was washed with equilibration buffer containing 200 mM NaCl to remove proteins that were nonspecifically bound. The GSTs were eluted from the affinity column using the wash buffer containing 5 mM S-hexylglutathione. Further elution of the column with 5 mM GSH in 10 mM Tris (pH 9.6) did not elute any additional protein. The enzymatic activity in each fraction was assayed using 1 mM 2,4-dinitrochlorobenzene and 1 mM GSH in 100 mM sodium phosphate buffer, pH 6.5, according to the method of Habig et al. (30). HPLC Separation and Characterization of Glutathione S-Transferase Subunits. The GST subunits were separated by HPLC employing a Vydac 201 TP54 C18 reversed-phased column (4.6 × 250 mm) that was eluted with acetonitrile/water

Nerland et al. containing 0.1% trifluoroacetic acid at a flow rate of 1 mL/min (31). The elution conditions consisted of four successive linear gradients of acetonitrile; 40 to 45% in 5 min, 45 to 50% in 9 min, 50 to 60% in 16 min, and 60 to 75% in 5 min. Each of the five subunits was manually collected as it eluted from the HPLC column. An aliquot from each of the five fractions was concentrated in a Speed-Vac and the first 10 amino acids of the N-terminal sequence of each of the GST subunits were determined by automated peptide sequencing at the University of Louisville Protein Core Facility. The acetyl group was removed from the amino terminal serine of subunits rGSTA1 (rat glutathione S-transferase R 1) and rGSTA2 (rat glutathione S-transferase R 2) prior to sequencing by incubating the protein in anhydrous trifluoroacetic acetic acid for 1 h and then concentrating to dryness. The protein was then dissolved in a known volume of the starting HPLC buffer. The molar ratio of covalently bound radiolabel to each of the GST subunits was calculated from the protein concentration, determined using the calculated molar extinction coefficients at 214 nm for each of the transferases (32) and by liquid scintillation spectrometry. For those subunits containing radiolabel, an aliquot from the corresponding HPLC fraction was taken to dryness in a Speed Vac and redissolved in electrophoresis buffer and electrophoresed as described earlier. Protein spots were excised from the Coomassie-stained gels and in-gel digestion was performed as described below. HPLC/Mass Spectrometry of Intact Subunits. Mass spectral data were obtained using electrospray ionization-mass spectrometry (ESI-MS). For ESI-MS experiments, samples were introduced as the eluate from an HPLC separation using mixtures of acetonitrile:water:0.1% trifluoroacetic acid as described above. Source and quadrupole potentials and gas flows were optimized for ion current throughput and resolution using standard solutions of horse heart myoglobin. The mass spectrometer used was a Quattro LC (Micromass, Manchester, U.K.) with an orthogonal array ion source held at 80 °C. Data were collected by scanning only Q1 for m/z 600-1500 at 4.1 s/scan. Data obtained were deconvoluted using the maximum entropy algorithm in the Masslynx software package. In-Gel Digestion. SDS-PAGE separated protein bands were excised from the gel and the proteins were digested with the protocol reported by Jensen et al. (33). Briefly, the bands were cut into small pieces, destained with 50 mM NH4HCO3/ acrylonitrile (1:1, v/v), reduced with DTT, alkylated with IAM, and digested with sequencing grade modified trypsin (Promega, Madison, WI). MALDI-TOF Mass Spectrometry. Digests (1 µL) were mixed with 1 µL of R-cyano-4-hydroxy-trans-cinnamic acid (10 mg/mL in 0.1% TFA:acrylonitrile, 1:1, v/v). The mixture (1 µL) was deposited onto a fast evaporation nitrocellulose matrix surface (33), washed twice with 1.5 µL of 5% formic acid, and analyzed with a TofSpec 2E (Micromass, U.K.) MALDI-TOF mass spectrometer in reflectron mode. The mass axis was calibrated with peaks from trypsin autolysis and adjusted with trypsin peak (m/z 2211.10) as the lock mass. Database searches were performed by comparing the monoisotope peaks of the peptides with the theoretical molecular weights of peptides that were produced by digestion of the proteins in the Swiss-PROT database supplied with the instrument. The maximum error allowed was set to 0.15 Da. Amino Acid Sequencing. Amino acid sequencing was performed by the protein chemistry core facility in the Department of Biochemistry at the University of Louisville using an ABI 470A sequencer that is equipped with a 120A PTH-amino acid analyzer (Applied Biosystems) and Waters “Pico-Tag” amino acid analyzer.

Results An example of a SDS-PAGE gel of a rat liver homogenate and its subcellular fractions is illustrated in

Acrylonitrile-GST Adducts

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Figure 1. (A) Coomassie-stained SDS-gel prepared from the liver of a male Sprague-Dawley rat that was treated with a dose of 115 mg/kg of [2,3-14C]AN. Lane A, whole liver homogenate; lane B, 9000g supernatant; lane C, 100000g supernatant; lane D, proteins retained by glutathione-affinity column. (B) Autoradiogram of a SDS-gel prepared from the liver of a male Sprague-Dawley rat that was treated with a dose of 115 mg/kg of [2,3-14C]AN. Lane A, whole liver homogenate; lane B, 9000g supernatant; lane C, 100000g supernatant; lane D, proteins retained by the glutathione-affinity column.

Figure 1A. The samples were prepared from the liver of a rat injected sc with a dose of 115 mg/kg (2.2 mmol/kg) [2,3-14C]-acrylonitrile. An autoradiogram of an identical SDS-PAGE gel is illustrated in Figure 1B. The three major bands in lanes A-C of the autoradiogram (panel B) correspond to 12, 27, and 28 kDa cytosolic proteins (panel A). We noticed that the location of the two higher molecular weight proteins had a striking similarity to the migration pattern of the rat liver GSTs following SDSPAGE (34). Formation of a covalent adduct with the rGSTs would not be surprising since rat liver cytosol catalyzes the conjugation of AN with GSH (17) and other chemicals have been reported to form covalent adducts with GSTs (35-37). Western blotting combined with immunochemical detection was used to verify that the 27 kDa radiolabeled protein was coincident with the µ transferase subunits (data not shown). The rGSTs were isolated from liver cytosol using a glutathione-agarose affinity column. SDS-PAGE produced the expected 26, 27, and 28 kDa protein bands (Figure 1A, lane D) but only the 27 kDa protein was radiolabeled (Figure 1B, lane D). Both rat liver GST µ subunits migrate as a single band during SDS-PAGE, therefore it was not possible, using one-dimensional PAGE, to establish if one or both of the µ subunits was labeled with AN. Reversed-phase HPLC has been used previously by other groups to separate the rGST subunits (31, 32). The affinity-purified proteins were resolved into five major peaks using a C18 reversed-phase HPLC column (Figure 2). The peaks were manually collected and the first 10 amino acids of the N-terminal portion of each subunit were sequenced, using the Edman method, to verify the identity of the proteins (Table 1). Liquid scintillation spectrometry confirmed that both rGSTM1 (rat glutathione S-transferase µ 1) and rGSTM2 (rat glutathione S-transferase µ 2) were radiolabeled. However, the calculated molar ratio of 14C to protein indicated that rGSTM1 (0.69) was labeled to a much greater extent than rGSTM2 (0.10) (Table 1). We estimate that the rGSTM1

Figure 2. HPLC chromatogram of GSTs purified by affinity chromatography. The GSTs were purified from rat liver cytosol using GSH-agarose affinity chromatography. The proteins were concentrated by ultrafiltration and 30 µg of protein was applied to a Vydac 201 TP54 C18 reversed-phased column that was eluted with an increasing gradient of acetonitrile containing 0.1% trifluoroacetic and monitored at 214 nm. Gradient conditions are detailed in Experimental Procedures. The labeled peaks correspond to 1, rGSTM1; 2, rGSTM2; 3, rGSTA3; 4, rGSTA1; 5, rGSTA2.

adduct accounted for approximately 7% of the total radioactively labeled protein in liver at this dose. The molecular mass of each of the rGST subunits was also determined using ESI MS (Table 1). Except for rGSTM1, whose molecular mass was 53 Da greater than previously reported, the molecular mass of each subunit was within 1-3 Da of the average molecular masses

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Table 1. N-Terminal Sequences, ESI-MS Determined Masses, and AN Covalent Binding Molar Ratio for GST Subunits GST subunit

HPLC peak

N-terminal sequencea

mass (lit)b (Da)

mass (obs) (Da)

14C-AN/GSTc

rGSTM1 rGSTM2 rGSTA3 rGSTA1 rGSTA2

1 2 3 4 5

PMILGYWNVR PMTLGYWDIR PGKPVLHYGD SGKPVLHYFNd SGKPVLHYFNd

25 785 25 572 25 191 25 521 25 476

25 838 25 573 25 190 25 519 25 473

0.69 0.10 0.02 nce nc

molar ratio

a The N-terminal sequences determined by automated Edman degradation. The sequences were identical to the sequences translated from reported cDNA sequences; rGSTM1 (39-41), rGSTM2 (42), rGSTA1 (58), rGSTA2 (59), rGSTA3 (60). b Average of two previously reported values for rGST subunits determined by ESI-MS (31, 38); rGSTM1 (25 782 and 25 787 Da), rGSTM2 (25 571 and 25 573 Da), rGSTA1 (25 520 and 25 522 Da), rGSTA2 (25 473 and 25 478 Da), rGSTA3 (25 188 and 25 194 Da). c Ratio of moles of AN-derived 14C to moles of GST based on the calculated molar extinction coefficients of the GST monomer (32). d These two transferases cannot be distinguished on the basis of their N-terminal sequence. The acetyl group was removed from the N-terminal serines prior to sequencing. e The ratio was not calculated (nc) because radioactivity was not detected.

Table 2. Masses of GSTM1 Tryptic Peptides Isolated from Control and AN-Treated Rats peptidea T2 T18-T19 T30 T17 T6 T20 T21 T29 T9-T10 T23 T1 T5 T13*e T13 T15 T4-T5 T12-T13* T12-T13 T3 T3-T4 T8 T16 T22

residues

sequenceb

calcdc mass (Da)

11-17 129-135 211-217 122-128 43-49 136-143 144-151 202-210 68-77 173-181 1-10 32-42 83-93 83-93 96-107 31-42 82-93 82-93 18-30 18-31 52-67 108-121 152-172

GLTHPIR TIPEKMK LAQWSNK QKPEFLK SQWLNEK LYSEFLGK RPWFAGDK YLSTPIFSK KITQSNAIMR BLDAFPNLK PMILGYWNVR YAMGDAPDYDR HHLJGETEEER HHLBGETEEER ADIVENQVMDNR RYAMGDAPDYDR KHHLJGETEEER KHHLBGETEEER LLLEYTDSSYEEK LLLEYTDSSYEEKR LGLDFPNLPYLIDGSR MQLIMLBYNPDFEK VTYVDFLAYDILDQYHIFEPK

792.46 845.47 845.44 888.51 903.45 955.50 975.49 1054.57 1160.63 1076.53 1247.65 1272.51 1391.59 1395.58 1402.65 1428.61 1519.68 1523.68 1588.75 1744.85 1788.94 1800.82 2588.28

errord (Da) control AN treated 0.03 0.06 0.03 0.02 0.01 0.00 0.01 0.01 -0.02 -0.02 -0.01 -0.02 ndf -0.03 -0.03 -0.03 nd -0.03 -0.04 nd -0.04 0.05 0.02

0.01 0.02 -0.01 -0.00 -0.02 -0.03 -0.01 -0.02 -0.05 -0.02 -0.05 -0.07 -0.06 nd -0.06 -0.05 -0.06 nd -0.06 -0.11 -0.07 -0.04 -0.01

a Tryptic digestion fragments from GSTM1. b Letters B and J in the sequence are the cysteine residues alkylated by iodoacetamide (B) or AN (J). c Masses are the calculated monoisotopic masses for the sequences in the table. d Errors are the difference between calculated monoisotope masses and the masses detected in the samples. e (*) The asterisk indicates the peptide contains AN-adduct. f Not detected (nd).

previously determined using ESI MS (31, 38). The 53 Da discrepancy in the molecular mass of rGSTM1 we observed is identical to the calculated increase in the molecular mass expected for the cyanoethylation of the protein by AN. Although the radiolabeling experiments indicated that approximately 70% of rGSTM1 was adducted, we could not detect any ions that could be attributed to the native protein nor could we detect any ions for the approximately 10% adducted form of rGSTM2. To identify the tryptic fragments and site of AN incorporation we used matrix-assisted laser desorption ionization-time-of-flight mass specrometry (MALDI-TOF MS) to analyze tryptic digests of control and AN-labeled proteins. Samples of rGSTM1 and rGSTM2 purified from the livers of control and [2,3-14C]AN-treated rats were reduced with DTT and the cysteine residues were alkylated with IAM. Following trypsin digestion and mass analysis using MALDI-TOF MS, 23 peptide fragments were detected and could be assigned to peptides derived from rGSTM1 based on the amino acid sequence available from the Swiss Prot data bank (P04905). These fragments had mass errors ranging from -0.11 to 0.06 Da and covered 84% of the amino acid sequence (Table

2). The MALDI-TOF spectra of the rGSTM1 tryptic fragments from control and AN-treated rats were identical except that the control peptide signals at m/z 1396.58 (T13) and 1524.68 (T12-T13) were absent from the spectrum of the rGSTM1 digest from AN-treated rats and two new signals at m/z 1392.59 (T13*) and 1520.68 (T12-T13*) were present (Table 2 and Figure 3, only m/z region 1385-1415 is illustrated). Both new peptides had masses 4 Da less than the corresponding control peptides T13 and T12-T13. Each of these fragments contains a single cysteine residue (C86). Cyanoethylation of C86 by AN would yield an ion 4 Da less than predicted for carbaminomethylation of Cys86 by IAM. However, the fragments also contain a lysine and two histidines as potential nucleophilic sites. Cyanoethylation of one of these amino acid residues should yield ions with m/z of 1449.7 and 1577.7 due to the addition of 53 Da to the masses of T13 and T12-T13, respectively. However, careful examination of the mass spectra of samples from AN-treated rats did not indicate the presence of any ions at these m/z values. We conclude that rGSTM1 is alkylated at C86 exclusively. A similar analysis of rGSTM2 resulted in the assignment of 23 peptide fragments based on the amino acid



Table 3. Masses of GSTM2 Tryptic Peptides Isolated from Control and AN-Treated Rats peptidea

residues

sequenceb

calcdc mass (Da)

control

T2 T6 T30 T20 T19 T9 T29 T23 T1 T5 T12* e T12 T14 T4-T5 T16-T17 T11-T12* T11-T12 T3 T13-T14 T15 T3-T4 T8 T21

11-17 43-49 211-217 144-151 136-143 69-77 202-210 173-181 1-10 32-42 83-93 83-93 96-107 31-42 122-133 82-93 82-93 18-30 94-107 108-121 18-31 52-68 152-167

GLAHAIR SQWLSEK MAFWNPK QPWFAGNK LYSEFLGK ITQSNAILR FLSKPIFAK BLDAFPNLK PMTLGYWDIR YSMGDAPDYDR HNLJGETEEER HNLBGETEEER VDVLENQAMDTR KYSMGDAPDYDR KKPEYLEGLPEK KHNLJGETEEER KHNLBGETEEER LFLEYTDTSYEDK IRVDVLENQAMDTR LQLAMVBYSPDFER LFLEYTDTSYEDKK LGLDFPNLPYLIDGSHK ITYVDFLVYDVLDQHR

736.43 876.43 892.43 946.47 955.50 1014.58 1049.63 1076.53 1250.61 1288.50 1368.57 1372.57 1389.66 1416.60 1429.78 1496.67 1500.66 1622.74 1658.84 1727.80 1750.83 1897.99 1995.01

0.03 0.00 -0.00 0.01 -0.01 -0.04 -0.01 -0.04 -0.01 -0.05 ndf -0.05 -0.07 -0.04 -0.03 nd -0.04 -0.06 -0.04 -0.04 -0.05 -0.04 -0.04

errord (Da) AN treated 0.04 0.01 0.02 0.03 -0.00 -0.00 -0.00 -0.00 -0.00 -0.03 -0.02 -0.02 -0.01 0.00 -0.01 -0.02 -0.02 -0.02 -0.02 -0.01 -0.02 -0.01 -0.01

a Tryptic digestion fragments from GSTM2. b Letters B and J in the sequence are the cysteine residues alkylated by iodoacetamide (B) or AN (J). c Masses are the calculated monoisotopic masses for the sequences in the table. d Errors are the difference between calculated monoisotope masses and the masses detected in the samples. e (*) The asterisk indicates the peptide contains AN-adduct. f Not detected (nd).

Figure 4. A segment of the MALDI/TOF mass spectrum of a tryptic digest of rGSTM2 prepared from control (A) and ANtreated male rats (B). Figure 3. A segment of the MALDI/TOF mass spectrum of a tryptic digest of rGSTM1 prepared from control (A) and ANtreated male rats (B).

sequence from Swiss Prot (P08010). These fragments also covered 84% of the amino acid sequence and had mass errors ranging from -0.07 to 0.04 Da (Table 3). Tryptic digestion of rGSTM2 yields fragments T11-T12 and T12 that have primary sequences identical to T12-T13 and T13 in rGSTM1 except for a H84N substitution (39-42). The carbaminomethyl derivatives of these fragments correspond to m/z 1501.66 (T11-T12) and 1373.57 (T12)

and are present in the tryptic digests prepared from rGSTM2 isolated from both the control and AN-treated rats (Table 3 and Figure 4, only m/z region 1490-1510 is illustrated). Peptide signals at m/z 1497.67 (T11-T12*) and 1369.57 (T12*), i.e., 4 Da less than the corresponding carbaminomethylated fragments, are only present in ANtreated rats indicating that AN is also bound to C86 in rGSTM2. The presence of peptide signals from both the cyanoethylated and carbaminomethylated C86 reflects the lower extent of alkylation with [2,3-14C]AN in rGSTM2. This is consistent with the lower molar ratio of radioactivity to protein for rGSTM2 vs rGSTM1 (Table

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2). The apparent absence of ions for T13 and T12-T13 in the MALDI-TOF spectrum of rGSTM1 from AN-treated rats (Table 2 and Figure 3B), suggests that C86 is completely cyanoethylated and thus the molar ratio determined using [2,3-14C]AN, (Table 1), may underestimate the extent of covalent binding of AN to rGSTM1 in vivo. This underestimate is undoubtedly due to the fact that the molar extinction coefficients at 214 nm used to estimate the subunit concentrations are calculated values based solely on the individual subunit amino acid composition (32).

Discussion The results described in this report indicate that although AN is considered to be a highly reactive vinyl monomer, it demonstrates selectivity in its reaction with cytosolic liver proteins when administered sc to rats. Figure 1 (panel B, lanes A-C) shows that three proteins with approximate molecular masses of 12, 27, and 28 kDa bear the brunt of the covalent binding. Although the characterization of the 12 and 28 kDa proteins is currently in progress, the 27 kDa protein has been identified as GST. The liver of male rats contains GST isoenzymes of the R, µ, π, and θ classes, but the R and µ classes predominate (43). Within these two classes, our data indicate that AN displays not only specificity for the µ over the R class but also selectivity for rGSTM1 over rGSTM2. AN is known to be relatively specific for sulfhydryl groups (19), so it was not surprising that our data indicated that the site of labeling was a cysteine residue present in both µ subunits, specifically C86. Early studies using chemical modification reagents suggested that there might be a cysteine at the active site of GSTs (44, 45). Rat µ GSTs contain three cysteine residues at sequence positions 86, 114, and 173. Hsieh et al. reacted rGSTM1-1 with IAM, in vitro, and found that, despite the incorporation of 0.8 mol of reagent/mol of subunit on C86, the enzyme was fully active (46). The nonessentiality of C86 was confirmed by site-directed mutagenesis where the C86S mutant was enzymatically active (46). Later studies, also using site-directed mutagenesis, showed that none of the three cysteines in rGSTM1-1 were required for activity (47). However, although the reaction of rGSTM1-1 with IAM at 25 °C was specific for C86 with full activity retained, reaction at 50 °C led to labeling of both C86 and C114 with the concomitant loss of enzymatic activity. This suggested that, although C114 is much less reactive than C86, C114 might be near the active site. This notion was supported by the fact that S-dinitrophenyl GSH was able to protect C114 from labeling by IAM at elevated temperatures (47). The inherent high reactivity of C86 over C114 and C173 was confirmed by the work of Katusz and Colman, who synthesized S-(4-bromo-2,3-dioxybutyl) glutathione (S-BDB-G) as an affinity reagent for rGSTM2-2 (48). The X-ray crystal structures of GSTs indicate that the molecule is composed of two domains (49). Domain I contains the binding site of GSH (G site), while domain II binds the xenobiotic (usually hydrophobic) substrate (H site). S-BDB-G was designed so that the GSH portion would bind to the G site, placing the reactive portion of the molecule in the H site where it reacted with Y115 causing a loss of enzymatic activity. However, even though S-BDB-G bound to the active site of GST, these authors

Nerland et al.

reported that it reacted faster with C86, some 25 Å away from the active site (50), than with Y115 in the H site. When the same reagent was used against rGSTM1-1, it was found that C86 in this subunit reacted even faster than C86 in rGSTM2-2 and much faster than Y115 (51). Interestingly, no labeling was found on C114, also in the H site, just adjacent to Y115. All of these results point to a unique reactivity of C86, especially in rGSTM1. This inherent high reactivity of C86 over C114 (and Y115) can be overcome, however, with a better affinity reagent for GST, namely 2-(S-glutathionyl)-3,5,6-trichloro1,4-benzoquinone (GSTCBQ) (50, 52, 53). This inhibitor, which has a very high affinity for the active site of GST, primarily targets Y6, the active site tyrosine known to be involved in the lowering of the pKa of bound GSH (49, 54) and Y115 in the H site. However, this reagent, containing a quinone group with high reactivity toward sulfhydryl groups, also labels C114 adjacent to Y115, but does not label C86. Thus, the high reactivity of C86 can be overcome if the reagent has high enough affinity for the active site of GST some 25 Å away from C86. This principle is also illustrated by monobromobimane, a sulfhydryl reagent, in that it binds to the H site and is a substrate for GST but can, in the absence of GSH, inactivate the enzyme by labeling primarily Y115 and secondarily, C114. Like GSTCBQ, monobromobimane does not label the reactive C86 to any significant extent. In contrast to the studies described above, our data indicates that AN reacts exclusively on C86 in both rGSTM1 and rGSTM2. This suggests that AN has little if any affinity for the H site but rather reacts with the most reactive sulfhydryl group in these proteins. Two important questions then are, why is C86 the most reactive cysteine and why is it more reactive in rGSTM1 than in rGSTM2? The X-ray structure of rGSTM1-1 indicates that C86 is in a solvent exposed loop between domains I and II (49). However, we believe that accessibility is a necessary but not sufficient condition to explain the high reactivity of C86. The reaction of AN with hemoglobin provides a clue. AN reacts with rat hemoglobin, essentially on βC125 (55). βC125 is found in rat but not in human or mouse hemoglobin. Its higher reactivity over another solvent accessible and normally most reactive cysteine (βC93) found in the hemoglobin of humans, mice and rats, has recently been explained. Rossi et al. (56) have shown that βC125 in rat hemoglobin reacts with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) 100 times faster than GSH and ∼4000 times faster than βC93. This high reactivity is due to the abnormally low pKa ≈ 6.9 of βC125 due to an H-bond with βS123. This suggests that a similar structural relationship might explain the selectivity of AN for C86 in rGSTM1. Inspection of a space filling model of the crystal structure of rGSTM1, suggests that such an interaction may indeed exist. The static coordinates of the PDB entry 6GST indicates that H84 is in close proximity (∼7 Å) to C86 but not within hydrogen bonding distance (Figure 5). However, simple rotation about the β-R bond would point the sulfur atom of C86 toward the side chain Nδ of H84. The numerous water molecules surrounding C86 and H84 seen in the crystal structure would indicate that this region is solvent exposed, and therefore rotation about the β-R bond in C86 should not be impeded. The potential H-bond or ion-pair interaction between H84 and C86 would be expected to lower the pKa of C86, thereby increasing its reactivity. In fact, a more complex set of



Physical Facilities Trust Fund, the University of Louisville School of Medicine and the University of Louisville Research Foundation.

References

Figure 5. Space filling model of the region within 9.0 Å of cysteine 86 on chain A of rGSTM3-3 (PDB 6GST) illustrating the proximity of histidine 84. Cysteine 86 is labeled on its β carbon, while histidine 84 is labeled on its epsilon carbon. The red spheres surrounding cysteine 86 represent the oxygen atoms of water molecules. The model was drawn with RasMol.

interactions is used by GST itself to lower the pKa of the sulfhydryl group of bound GSH at the active site (49, 54). An interaction between H84 and C86 would account for the high reactivity of C86 in rGSTM1. As for the lower reactivity of C86 in rGSTM2, it is noted that the 84th residue in rGSTM2 is not a histidine but rather an asparagine, which would not be expected to favorably interact with C86 and affect its pKa as would a histidine. This hypothesis would allow the prediction of other likely protein targets for AN. For example, GST π, which is low in liver but high in other organs, such as kidney, might be predicted to be a target. This GST contains a C47 with an abnormally low pKa due to an interaction with K54. Adducts on this cysteine residue, unlike those on C86 of the µ transferase, have been shown to affect enzyme activity. Specifically, carboxymethylation of C47 increases the Km for GSH by 3 orders of magnitude (57). In summary, despite the high reactivity of AN, our data indicate that it demonstrates selectivity in its reaction with liver cytosolic proteins. In this study, we identified a highly labeled protein as a GST of the µ class with further selectivity for the M1 subunit. The selective reaction with the M1 subunit could be rationalized by a favorable environment around C86, potentially lowering its pKa and increasing its reactivity. A similar explanation has been used to account for the selectivity of AN for βC125 in rat hemoglobin. The data suggest that tissue proteins containing cysteine with an abnormally low pKa value would be a likely targets for AN. A systematic search for proteins labeled in this way by AN is in progress. It is anticipated that identification of one or more of these targets might help explain the acute toxicity of AN.

Acknowledgment. This research was supported by National Institute of Environmental Health Science Grants ES06141 (to F.W.B.) and P20 ES06832. The Biomolecular Mass Spectrometry Laboratory of the University of Louisville is supported in part by NIH grant 1 S10 RR11368-01 A1 (to W.M.P.), the State of Kentucky

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