Proteomic Characterization of Metabolites, Protein Adducts, and

dicloroethylene of 99% plus purity was purchased from Aldrich. Chemical Co. ..... plus low abun- dance would account for our not finding proteins, suc...
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Chem. Res. Toxicol. 2003, 16, 1306-1317

Proteomic Characterization of Metabolites, Protein Adducts, and Biliary Proteins in Rats Exposed to 1,1-Dichloroethylene or Diclofenac Juliet A. Jones,† Lata Kaphalia,‡ Mary Treinen-Moslen,‡ and Daniel C. Liebler*,† Southwest Environmental Health Sciences Center, College of Pharmacy, University of Arizona, Tucson, Arizona, 85721-0207, and Departments of Pathology and Internal Medicine, University of Texas Medical Branch, Galveston, Texas Received April 22, 2003

A proteome profiling approach was used to compare effects of two toxicants, 1,1-dicloroethylene (DCE) and diclofenac, which covalently adduct hepatic proteins. Bile was examined as a potential source of protein alterations since both toxicants target the hepatic biliary canaliculus. Bile was collected before and after toxicant treatment. Biliary proteins were separated by one-dimensional SDS-PAGE and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) with data-dependent scanning. Comprehensive analysis of biliary proteins was performed by using SEQUEST and BLAST database searching, in combination with de novo interpretation. Bile not subjected to tryptic digestion was analyzed for DCE metabolites. DCE treatment resulted in a marked increase in the overall number of biliary proteins, whereas few changes in the proteomic profile were apparent in bile after diclofenac treatment. This is consonant with prior observations of more profound effects of DCE on canalicular membrane integrity. LC-MS-MS analyses for DCE metabolites revealed the presence of S-carboxymethyl glutathione, S-(cysteinylacetyl)glutathione, and a product of the intramolecular rearrangement of the DCE metabolite, ClCH2COSG, not previously described in vivo. In addition, several S-carboxymethylated proteins were identified in bile from DCEtreated animals. This investigation has produced the first comprehensive baseline characterization of the content of the rat biliary proteome and the first documentation of alterations in the proteome of bile by toxicant treatment. In addition, the results provide direct in vivo evidence for DCE metabolic routes proposed in the formation of covalent adducts.

Introduction Diverse xenobiotics can cause hepatobiliary toxicity by damaging the canalicular region of hepatocyte plasma membranes, which form the origin of the biliary tract (1). Canalicular membranes are rich in transporters that export conjugated metabolites of many xenobiotics into the bile (2-5). Some toxicants that target the bile canaliculus are biotransformed to chemically reactive glutathione and glucuronide conjugates, which are substrates for these transporters (1, 2). Selective damage to canalicular membrane-associated structures is thought to occur because these reactive conjugates must concentrate near the canalicular membrane enroute to their export into bile and after their export into the canalicular lumen. Thus, nucleophilic sites on canalicular transporters and other proteins would be exposed to high concentrations of the reactive electrophilic-conjugated metabolites. Two of the best-studied examples of xenobiotics that appear to act in this way are DCE,1 an industrial chemical and groundwater contaminant, and diclofenac, a widely prescribed nonsteroidal antiinflammatory drug. Rats exposed to 50 mg kg-1 DCE show selective injury * To whom correspondence should be addressed. Tel: (615)322-3063. Fax: (615)322-4349. E-mail: [email protected]. † University of Arizona. ‡ University of Texas Medical Branch.

to the biliary canalicular membrane, which is manifest by dilation of canalicular membranes, blunting of microvilli, and the presence of debris within the canalicular lumen. Associated biochemical changes include a decrease in total biliary protein content, diminished activity of multiple canalicular-localized enzymes, and functional changes consistent with damage to the canalicular membrane transport proteins (6, 7). The hypothesized mechanism for this injury is alkylation of canalicular membrane proteins by reactive, electrophilic metabolites of DCE (6, 7). Previous studies in vivo and in vitro have characterized the metabolism of DCE (Figure 1) (8-15). Cytochrome P450 bioactivation results in the formation of the reactive intermediates DCE oxide and ClCH2COCl. Reaction of the latter with glutathione forms ClCH2COSG, which is more stable than ClCH2COCl, yet is capable of alkylating GSH to form the bis-glutathionyl conjugate GSCOCH2SG. Alternatively, ClCH2COSG can alkylate cysteinecontaining peptides to form GSCOCH2-S-cys-peptide adducts (16, 17). GSCH2COSG can further hydrolyze to 1 Abbreviations: CID, collision-induced dissociation; ClCH COCl, 2 2-chloroacetyl chloride; ClCH2COSG, S-(2-chloroacetyl)glutathione; DCE, 1,1-dichloroethylene; ESI, electrospray ionization; GSCH2CO2H, S-carboxymethylglutathione; GSCOCH2-S-cys-peptide, S-((2-S-cysteinyl)acetyl) glutathione peptide adduct; GSH, γ-glutamylcysteinylglycine or reduced glutathione; GSCOCH2GS, S-((2-S-glutathionyl)acetyl) glutathione; GSSG, γ-glutamylcysteinylglycine disulfide; LC-MS-MS, liquid chromatography tandem mass spectrometry.

10.1021/tx0340807 CCC: $25.00 © 2003 American Chemical Society Published on Web 10/03/2003

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Figure 1. Metabolism of 1,1-DCE as determined in vitro showing reactions with cysteine-containing proteins and the resulting adducts. Pr-SH indicates a cysteine-containing protein; GS indicates a glutathionyl moiety. Dashed lines show pathways that occur in vivo based on observations from this investigation.

Figure 2. Metabolism of diclofenac via CYP oxidation and quinone intermediates leading to glutathione conjugates (left side) or via acyl glucuronide formation leading to acylated proteins (right upper) or glycation (right lower).

GSCH2CO2H, which presumably yields the observed in vivo urinary metabolites S-carboxymethylcysteine, Nacetyl-S-carboxymethyl cysteine, and thiodiglycolic acid. Another pathway to GSCH2CO2H is the direct reaction of DCE oxide with GSH (15). Although ClCH2COSG and GSCH2COSG are known to be formed in vitro and would appear to be metabolically and chemically competent precursors to the known in vivo metabolites, neither has been demonstrated in vivo. This question is particularly important because of their proposed roles in the selective canalicular injury caused by DCE (6, 7, 16). Diclofenac also targets hepatic canaliculi as documented by immunohistochemical detection of intense adduction of canalicular membranes (18-21). In contrast to DCE, diclofenac produces a more subtle perturbation of canalicular membrane integrity (18) and little evidence of acute hepatic injury or alterations in biliary formation (22, 23). Biotransformation of diclofenac involves both phase I and phase II reactions and apparently involves several different adduct-forming chemistries (Figure 2).

CYP-mediated biotransformation of diclofenac yields reactive benzoquinoneimine intermediates, which may react with GSH and protein nucleophiles (24-26). Of greater relevance to the canalicular membrance protein adducts is the conversion of diclofenac to a reactive acyl glucuronide (19), which may directly acylate nucleophiles (27, 28) or may rearrange with acyl migration followed by formation of Schiff base adducts with lysine residues and N-terminal amines (29, 30). The diclofenac acyl glucuronide is transported into the canalicular lumen by the rat canalicular conjugate export pump Mrp2 (19). This pathway is involved not only in damage to canalicular components (19) but also in intestinal toxicity of the drug (31). An identified membrane protein target of in vivo diclofenac treatment is dipeptidyl peptidase (DPP) IV (32), which has an exclusive localization to the canalicular membrane in liver. This adduction of DPP IV could account for the observed decrease in its activity after diclofenac treatment (32).

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Figure 3. One-dimensional gel of bile samples collected prior to (left three lanes) and 3 h after DCE treatment (right three lanes) to 1,1-DCE. The dotted lines indicate places at which the individual bands were cut. The corresponding bands from individual basal or posttreatment bile samples were combined for in-gel digestion.

DCE and diclofenac thus present an interesting contrast in severity of effect on canalicular integrity, despite the similarity in their conversion to reactive phase II metabolites that are transported across the canalicular membrane. We have applied LC-MS-MS analysis of the biliary proteomes of DCE- and diclofenac-treated rats to compare effects on biliary proteins and the formation of protein adducts and to further investigate the in vivo metabolism of DCE to reactive species. To our knowledge, no comprehensive characterization of the biliary proteome has been undertaken. The development of new proteomic technologies now allows high-throughput protein characterization. Here, we have applied wellestablished methods of protein identification involving one-dimensional SDS-PAGE and LC-MS-MS into a systematic approach to biliary proteome characterization.

Experimental Procedures Chemicals and Reagents. For animal treatments, 1,1dicloroethylene of 99% plus purity was purchased from Aldrich Chemical Co. (Milwaukee, WI) and the sodium salt of diclofenac was obtained from Sigma Chemical Co. (St. Louis, MO). For LCMS analyses, ultrahigh purity HPLC grade acetonitrile (Burdick & Jackson, Muskegon, MI), ultrahigh purity water, and formic acid (both from J. T. Baker, Phillipsburg, NJ) were used. For in-gel digestion and extraction, sequencing grade modified trypsin (Promega, Madison, WI) was used, as were HPLC grade acetonitrile (E. M. Science, Darmstadt, Germany), acetone (Fisher Scientific, Fairlawn, NJ), and dicloromethane (Burdick & Jackson). Ammonium bicarbonate (NH4HCO3, 99%) was purchased from Sigma. Trifluoroacetic acid (99+%) was purchased from Aldrich. Animal Treatments and Bile Collection. All animal experiments were performed with protocols approved by the Animal Care and Use Committee of the University of Texas Medical Branch at Galveston. Young male Sprague-Dawley rats were obtained from Harlan Teklad (Indianapolis, IN). Animals were acclimated in wire bottom cages on a 12 h day/ night cycle and were given rat chow and water ad libitum for about 1 week before experiments. Animals weighed 300-350 g at time of treatment. Animals were prepared for bile collection by an established procedure, which includes infusion of taurocholate into the duodenum to maintain bile flow (33). Bile samples were collected at 10-15 min intervals and then frozen at -80 °C.

After 45 min of basal bile collection, animals were treated by oral gavage with either 50 mg kg-1 DCE in a mineral oil vehicle at a volume of 2 mL kg-1 or 50 mg kg-1 diclofenac in 1% Tween vehicle at a volume of 2 mL kg-1. In the experiments described here, a single animal was dosed with DCE and another with diclofenac. Bile samples were collected for 3 h after DCE treatment and 6 h after diclofenac treatment. These doses of toxicant and times for subsequent bile collection were selected based on prior characterization of a DCE treatment with multiple effects on hepatic canalicular integrity (6, 7) in contrast to a diclofenac treatment with minor effects on bile formation (23). Analysis of 1,1-DCE Metabolites. For analysis of low molecular mass metabolites and glutathione conjugates, bile samples (approximately 200 µL) collected prior to exposure and 1, 2, and 3 h after exposure were dialyzed using a Slide-a-lyzer (Pierce) dialysis filter with a 3500 MW cutoff for 24 h against 0.1 M NH4HCO3. The buffer containing the low mass metabolites was lyophilized, resuspended in 200 µL of H2O, and analyzed by LC-MS-MS with data-dependent scanning. Protein Separation. The biliary proteins from both DCEand diclofenac-treated rats were separated by one-dimensional SDS-PAGE on a 12% Tris-Glycine gel (Bio-Rad Ready Gels). Each sample was loaded onto three separate lanes of the gel, with each lane containing 12.5 µL of bile and 12.5 µL of Laemmli sample buffer. The gels were then stained with Coomassie blue (Biosafe Coomassie, Bio-Rad) and were sliced into 10 bands as shown in Figure 3. Sets of three band slices corresponding to each sample were combined and subjected to in-gel tryptic digestion. Alkylation with iodoacetamide was omitted during digestion of the DCE samples to avoid confusion between the +57 amu modification resulting from iodoacemamide and the +58 modification resulting from possible S-carboxymethylation (see Results). The digested proteins were extracted by sonication for 30 min in each of the following solvents: (i) 0.1% trifluoroacetic acid in water, (ii) acetonitrile/water/trifluoroacetic acid (50:50:0.1, v/v/v), (iii) 0.001 M NH4HCO3, (iv) dichloromethane, and (v) acetone. The extracts were combined, lyophilized, and then resuspended in 200 µL of 0.1% formic acid in water and stored at -20 °C until further analysis. LC-MS Analysis. LC-MS-MS analyses were performed on a ThermoFinnigan LCQ Deca ion trap mass spectrometer equipped with a ThermoFinnigan Surveyor LC pump, microelectrospray source, and Xcalibur 1.2 instrument control and data analysis software. HPLC separation of the tryptic digests was achieved with fused silica capillary tips (Polymichro Technologies, 100 µm i.d., 5 µm tip) packed with Monitor C18 (Column Engineering) at approximately 300 psi. The flow from the pump was split

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Figure 4. MS-MS spectrum of S-(cysteinylacetyl)glutathione, [M + H]+ ) 469, found in bile samples collected after treatment with 1,1-DCE. to achieve flow rates between 500 nL and 1 µL min-1 in the packed tip. Solvent A was acetonitrile containing 0.1% formic acid, and solvent B was H2O with 0.1% formic acid. The gradient program began at 2% A for 5 min, and then increased to 60% solvent A by 45 min, followed by an increase to 80% solvent A by 47 min, and then continued at that composition until the end of the run at 65 min. For each gel band, the control sample was analyzed prior to the postexposure sample, and a blank (0.1% formic acid) was run in between. Each sample was subjected to five datadependent LC-MS-MS analyses. A full mass range of 300-2000 amu was used for the acquisition of precursors for MS-MS. In the next three runs, limited mass ranges of 300-700, 700-1200, and 1200-1800 amu were used for selection of precursors. The final run was a repeat of the full mass range (300-2000 amu) analysis. Thus, analysis of 20 samples generated 100 LC-MSMS data files. Limited mass range acquisitions increased the number of precursor peptide ions selected for MS-MS (34), whereas the two full mass range analyses allowed assessment of run-to-run reproducibility. Protein Identification. Proteins were identified using Turbo SEQUEST (version 2.0) (35, 36) with additional verification by BLAST database searching (37). For creating .dta files, SEQUEST parameters were as follows: start scan, 200; MW, 300-4000; mass tolerance, 1.5; grouped scans, 1; intermediate scans, 1; minimum no. of ions, 25; min TIC, 3.5 × 10-4. For creating the SEQUEST summary, the parameters were as follows: database, rat.fasta; enzyme, trypsin; amino acid modifications not specified; fragmentation ion tolerance, 0.5; peptide mass tolerance, 1.5. For identification of S-carboxymethylated proteins, an amino acid modification of cysteine +58 amu was entered. The rat.fasta database was acquired at ftp://ftp.ncbi.nih.gov/blast/db/. Advanced BLAST search criteria were as follows: program, BLASTp; database, swissprot; sequence, Fasta format; organism, rodentia; expect, 1000; filter, unchecked; matrix, PAM30. A visual inspection was performed of all SEQUEST identifications containing at least half of the theoretical fragments and an Xcorr score g1.0. For proteins identified by only one peptide or which were present only in postexposure samples, de novo interpretation of the original Xcalibur spectrum was performed. BLAST searches also were performed of the peptide

sequence with reasonable substitutions of combinations of amino acids having similar masses (for example, of glutamine for a glycine adjacent to an alanine or of tyrosine for glutamine adjacent to a glycine). When confirming the presence of an S-carboxymethylated cysteine residue (161 amu), BLAST searches were performed in which the cysteine was replaced with a tyrosine residue (163 amu) and with an unmodified cysteine (103 amu) adjacent to a glycine residue (57 amu). Amino acid sequences that did not correlate to a rat protein were excluded from the list, as were sequences for which other reasonable interpretations could be made that did not suggest identification of a rat protein.

Results 1,1-DCE Metabolites. LC-MS-MS analysis of the bile dialysate revealed the presence of both oxidized and reduced glutathione ([M + H]+ ) 308 and 613, respectively) and their sodium salts ([M + Na]+ ) 330 and 635). Three glutathione conjugates were found in bile samples collected after DCE treatment that were not present in the basal bile samples. GSCH2CO2H ([M + H]+ ) 366) was confirmed by the MS-MS spectrum, which correlated well with that of its synthetic counterpart (data not shown). A species of m/z 469 was present, the MS-MS spectrum of which corresponds to GSCOCH2-S-cysteine (Figure 4). Of particular interest in this spectrum is the signal at m/z 383, which would result from cleavage of the cysteine sulfhydryl bond. This cleavage frequently has been observed in the fragmentation of model peptides (17) and in this spectrum corresponds to the fragment [GSCOCH2SH + H]+. In addition, a species of m/z 349 appears to represent the cyclic product of the intramolecular rearrangement of ClCH2COSG as characterized by Liebler et al. (Figure 5) (16). Although the MS-MS spectrum did not contain extensive fragmentation, the signals observed corresponded well to a MS-MS spectrum of the synthetic product with common signals at 146, 218, 242, 255, 261, 275, 291, and 332 amu (data not shown).

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Figure 5. MS-MS spectrum of the intramolecular rearrangement product of ClCH2COSG, [M + H]+ ) 348, found in bile samples collected after treatment with 1,1-DCE.

Protein Fragments in Bile. Because DCE targets hepatocyte canalicular membranes and leads to elevated levels of several canalicular proteins (7), we hypothesized that fragments of damaged proteins would be excreted into the bile. Because the samples containing the metabolites had been dialyzed and were not subjected to tryptic digestion, any peptides in these samples could be protein fragments derived from damaged hepatocytes. Therefore, a SEQUEST search that specified “no enzyme” rather than trypsin was performed on the data obtained from these samples. A few peptides possibly correlating to rat proteins were detected; however, no canalicular membrane protein fragments were identified (data not shown). Biliary Protein Identification. Toxicant-evoked changes in biliary proteins were evaluated by comparing samples collected during the hour before treatment with samples collected 3 h after exposure to DCE or 6 h after exposure to diclofenac. These time points were selected to correlate, respectively, with striking alterations in canalicular ultrastructure after DCE treatment (6, 7) and intense immunohistochemical localization of adducts at the canalicular membrane after DCE damage occurs (6). Table 1 lists the proteins identified in samples obtained both before and after exposure to DCE. In agreement with previous observations (6), total biliary protein concentration decreased after exposure to DCE from approximately 2.4 mg/mL to an average of 1.3 mg/mL. However, the data in Table 1 show a marked increase in the number of different proteins in the bile after DCE treatment. A total of 22 proteins was identified in the control samples, whereas 40 were identified in the postexposure samples. Twenty-three of these proteins were present only in the postexposure samples.

Often, multiple proteins contain the same peptide sequence. In some cases, it was possible to choose the most likely match, and this protein was included in the table. For example, if one of the matches has a molecular mass that is significantly lower than the masses of the proteins contained in the band, then this choice was eliminated. Similarly, if one match corresponds to a liver protein whereas the other match corresponds to a protein that has to date only been identified in other organs, then the liver protein was selected. In some instances, in particular with immunoglobin chains, the proteins containing the peptide sequence are so similar that it was impossible to distinguish the most likely match. The proteins identified in bile of untreated animals consisted largely of serum proteins and immunoglobulins, both of which are normal biliary constituents (38, 39). The multidrug resistance protein 1a, which is apparently a canalicular membrane protein (40), was identified in the control sample. In contrast to the many changes after DCE, only six additional proteins were identified in the samples obtained after treatment with diclofenac (Table 2). The most intriguing of these was the presence of DPP IV in bands 1, 2, and 4 (correlating to approximately 200, 100, and 50 kDa, respectively). This observation is intriguing because DPP IV was identified by Hargus et al. as an apparent target for covalent modification by diclofenac in rat liver in vivo (32). The proteins identified in the basal and in the posttreatment exposure samples were very similar to the proteins identified in the basal bile obtained prior to treating animals with DCE. Protein Adducts. Several peptides identified only in the samples collected after DCE treatment contained a cysteine mass modification of +58 amu (Table 3). Cysteine thiol alkylation with iodoacetamide during standard

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Table 1. Proteins Identified in Bile of Rat Prior to and 3 h after Treatment with DCE preexposure

postexposurea

R-1-inhibitor III precursor albumin (serum albumin precursor) complement component 3 polymeric immunoglobulin receptor precursor probable GTPase-activating protein SPA-1 hemopexin serine proteinase inhibitor 1 transferrin (precursor) γ-2a immunoglobulin heavy chain R-1-inhibitor III multidrug resistance protein 1a Ig κ-chain (one of several possible variants) haptoglobin precursor Ig R-chain cyclin A2 Ig κ-chain V region (tentative sequence) Ig light chain mast cell protease 9 R-2µ-globulin transthyretin, chain A, B, C, or D hemoglobin, R-1 hemoglobin, β

accession no.b

MWc

band(s)d

R-1-inhibitor III precursor albumin (serum albumin precursor)

gi|112893 gi|19705431

163 68

2 2-5, 7-9

apolipoprotein A-I probable GTPase-activating protein SPA-1 putative RNA binding protein 1

gi|113997 gi|7514049

30 203

2 2

gi|2039348

42

2

gi|8393024 gi|130201

186 85

3 3, 4

gi|1854476 gi|11559947

76 103

3 3

gi|204503

26

3

gi|19424170

53

3

101

3

gi|16758014 gi|92743 gi|18426812 gi|13162326

51 45 39 76

4 4 4 4

gi|6978579 gi|203763

263 58

4 4

gi|11968144

99

4

gi|13492975

64

4

gi|1620510 gi|68791

190 47

4 4

gi|8393746

49

4

gi|13592027 gi|16758474 gi|13928718 gi|1220488 gi|204720 gi|112889 gi|4185807 gi|13929056 gi|57233 gi|123513 gi|125142

78 197 61 50 15 46 53 67 45 38 26

4 4 4 5, 6 5 5, 6 5 5 5 6 7

gi|125735

12

7, 8

gi|204698 gi|8393197 gi|2119648 gi|3212532 gi|6981010

17 25 21 13 15

7 7 9 9, 10 10

complement component 3 polymeric immunoglobulin receptor precursor transferrin (precursor) calcium/calmodulin-dependent serine protein kinase glutathione S-transferase Yb-1 subunit matrix metalloproteinase 3 rat DNA-binding protein AGIE-BP1 hemopexin serine proteinase inhibitor 1 adenosine deaminase bile acid CoA ligase

gi|399019

calcium channel r-1A cytochrome p-450d, IA1, IA2, c, or MC 10-formyltetrahydrofolate dehydrogenase FTZ-F1 β-1 or β-2 protein laminin-5 r-3 chain major acute phase r-1 protein precursor or T-kininogen MEK5 r-2 or MAP kinase 5 or MAP/ERK kinase MEK5 protein kinase C-E Ser-Thr protein kinase UDP-glucuronosyltransferase γ-2a immunoglobulin heavy chain Ig R-chain r-1-antitrypsin precursor fatty acid transport protein lutheran antigen serine protease inhibitor 3 haptoglobin precursor Ig κ-chain (one of several possible variants) Ig κ-chain V region (tentative sequence) Ig light chain C reactive protein R-2µ-globulin transthyretin, chain A, B, C, or D hemoglobin, R-1

a Proteins listed in boldface were not present in basal bile sample. b GeneInfo Sequence identification number. c MW (to the nearest 10 kDa) taken from database entry. d Gel location of identified protein (see Figure 4).

in-gel digestion protocols results in addition of +57 amu to cysteine; however, this alkylation step was not part of our protocol here. Because S-carboxymethylation is not known to occur endogenously, these modifications likely result from alkylation by reactive DCE intermediates (Figure 1). Figure 6 shows the MS-MS spectrum of [M + H]+ ) 1076.4, which correlates to the S-carboxymethylated rat serum albumin precursor peptide C*PYEEHIK. Searches also were performed for GSCOCH2-S-cysprotein adducts. These adducts previously had been identified in vitro and their MS-MS fragmentation patterns were characterized extensively (16, 17). A search of the data for these characteristic fragmentation patterns using the pattern recognition algorithm SALSA (41)

did not reveal the presence of any peptides containing this modification. Similarly, SALSA searches of cysteinecontaining peptides in DPP IV, serum albumin, and polymeric immunoglobin receptor precursor failed to detect diclofenac-containing modifications. Spectra were identified, however, that indicated the presence of a cysteine modified by 161 amu. This is thought to correspond to the addition of S-acetylcysteine (i.e., alkylation of the target peptide by ClCH2COSG, followed by cleavage of the glutamate and glycine residues, as shown in Figures 1 and 4). The spectrum shown in Figure 7 corresponds to LC*PTHADSLNNLANIK, which is found in the rat liver protein UDP-N-acetylglucosaminyltransferase.

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Table 2. Proteins Identified in Bile of Rat Prior to and 6 h after Treatment with Diclofenac postexposurea

accession no.b

MWc

probable GTPase-activating protein SPA-1 R-1-inhibitor III Precursor complement component 3

gi|7514049 gi|112893 gi|8393024

203 163 186

DPP IV

gi|6978773

83

1, 2, 4

calpain

gi|9506461

76

4

albumin (serum albumin precursor) polymeric immunoglobulin receptor precursor hemopexin serine proteinase inhibitor 1 transferrin (precursor) dihydropyrimidinase

gi|19705431 gi|130201 gi|16758014 gi|92743 gi|1854476 gi|2143701

68 85 51 45 76 57

2, 4 1-5 3, 4 4 4 4

apoptosis-inducing factor

gi|25742626

66

4, 5

γ-2a immunoglobulin heavy chain r-1-antitrypsin precursor vitamin D binding protein prepeptide Ig R-chain haptoglobin precursor Ig κ-chain (one of several possible variants)

gi|1220488 gi|112889 gi|203927 gi|204720 gi|123513 gi|125142

50 46 53 15 38 15

5 5 5 5, 6 6 7, 8

Ig light chain 60S ribosomal protein L4 R-2µ-globulin transthyretin, chain A, B, C, or D hemoglobin, R-1 hemoglobin, β

gi|204698 gi|1710511 gi|2119648 gi|3212532 gi|6981010 gi|17985949

14 17 12 17 26 21

7 7 8 8, 9 9 9

preexposure R-1-inhibitor III precursor complement component 3 probable GTPase-activating protein SPA-1 albumin (serum albumin precursor) polymeric immunoglobulin receptor precursor calpain hemopexin serine proteinase inhibitor 1 transferrin (precursor) serine proteinase inhibitor 1 γ-2a immunoglobulin heavy chain Ig κ-chain (one of several possible variants) haptoglobin precursor Ig R-chain Ig κ-chain V region Ig light chain R-2µ-globulin transthyretin, chain A, B, C, or D hemoglobin, R-1 hemoglobin, β

band(s)d 1 1 1, 2

a Proteins listed in boldface were not present in basal bile sample. b GeneInfo Sequence identification number. c MW (to the nearest 10 kDa) taken from database entry. d Gel location of identified protein (see Figure 2).

Table 3. S-Carboxymethyl and S-(Cysteinylacetyl) Protein Adducts Detected in Rat Bile Following DCE Treatment sequencea

protein identification

m/zb

Xcorrc

SQC*LAETEHDNIPADLPSIAADFVEDK C*PYEEHIK QVQVEIC*EFELKK EGDFGTC*IK C*VLKISDR MPFGC*VTKGDK C*QLEKLALK

S-carboxymethyl adducts serum albumin precursor serum albumin precursor golgi-associated protein GCP360 glial glutamate transporter (ajuba protein) aldolase C, fructose biphosphate vesicle-associated calmodulin-binding protein flavohemoprotein

1494.0 (+2) 1076.3 1375.5 1028.1 993.2 614.1 (+2) 1104.2

2.66 1.73 1.76 1.01 1.16 1.6 1.64

943.4 (+2)

2.11

LC*PTHADSLNNLANIK

S-(cysteinylacetyl) protein adduct UDP-N-acetylglucosaminyltransferase

a

Adducted peptide sequence. Asterisk (*) marks the adducted cystein residue. b Measured m/z value. Doubly charged peptide adduct ions are indicated in parentheses. c Sequest Xcorr score for sequence to spectrum correlation.

Discussion Proteomic profiling of biofluids has attracted great interest as a means of detecting markers of injury and disease states (34, 42-44). Here, we have applied a proteomic profiling approach to compare the effects of two hepatobiliary toxicants, DCE and diclofenac. This work provides a baseline characterization of the content of the rat biliary proteome, as well as a profile of proteins released into bile after hepatotoxicant treatment. Moreover, our analyses provide confirmation of proposed DCE metabolic routes involved in the formation of covalent adducts. Metabolism of DCE in Vivo. Previous work on DCE metabolism in rats in vivo indicated the formation of S-carboxymethylcysteine and its metabolite thiodiglycolic acid, apparently as products of GSCH2CO2H and an N-acetylcysteinyl acyl derivative (8-10). In vitro studies indicated that the primary DCE metabolites ClCH2COCl and 1,1-DCE oxide react with glutathione to form ClCH2COSG, GSCH2COSG, and GSCH2CO2H (14-16). Here, we have shown that these metabolites are products of

DCE biotransformation in vivo. GSCH2CO2H was abundantly present in bile and comparison of its MS-MS spectrum with that of its synthetic counterpart confirmed its identity. GSCOCH2SG was not detected in any of the samples. In nearly all samples collected after exposure to DCE, however, a prominent species of m/z 469 was present. The MS-MS spectra of this species corresponded to S-(cysteinylacetyl)glutathione, which would result from enzymatic cleavage of glutamate and glycine residues from GSCOCH2SG (Figure 4). This is consistent with the metabolism of glutathione and its conjugates by γ-glutamyl transpeptidase and cysteinylglycine dipeptidase in the biliary epithelium (21, 45, 46). Therefore, we suggest that GSCOCH2SG is formed in vivo as previously proposed but that upon excretion into the bile, is cleaved at glutamate and then at glycine residues to form S-(cysteinylacetyl) glutathione. Detection of the cyclic DCE glutathione conjugate ([M + H]+ ) 348) in bile provides unambiguous evidence for the formation of ClCH2COSG in vivo. Intramolecular rearrangement of ClCH2COSG has been well-character-

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Figure 6. MS-MS spectrum of [M + H]+ ) 1076.4, which represents the S-carboxymethylated rat serum albumin precursor peptide C*PYEEHIK. The asterisk indicates cysteine + 58 amu.

Figure 7. MS-MS spectrum of [M + 2H]+2 ) 943.3, which correlates to an S-cysteinylacetyl adduct of a peptide found in rat UDPN-acetylglucosaminyltransferase.

ized in vitro and proceeds rapidly at pH > 7.4 (16). Thus, rearrangement of ClCH2COSG to the cyclic product could

have occurred in the slightly basic bile or during dialysis in NH4HCO3 prior to LC-MS-MS analysis. Small quanti-

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ties of a metabolite of m/z 349 were detected only in bile samples collected after treatment with DCE. The degree of MS-MS fragmentation was limited (presumably due to the cyclic structure) yet is consistent with the structure of the cyclic product (Figure 5) and with MS-MS fragmentation of the synthetic compound. Our results provide a new perspective on DCE metabolism studies carried out by us (13, 14, 16) and others nearly 25 years ago (8-10). GSCH2COSG is probably a major hepatic metabolite of DCE but undergoes rapid intrabiliary catabolism to GSCH2CO-S-Cys, which we detected in the bile. Because ClCH2COSG is the immediate precursor to GSCH2COSG, our studies clearly establish the central roles of ClCH2COSG and its precursor ClCH2COCl in DCE metabolism in vivo. This pathway is probably a major contributor to the formation of GSCH2CO2H and its in vivo end products S-carboxymethylcysteine, S-carboxymethyl-N-acetyl cysteine, and thiodiglycolic acid (8, 10). Although DCE oxide can react directly with glutathione to form GSCH2CO2H, it is not an obligatory precursor. Moreover, competing hydrolytic pathways consume the epoxide (13, 47). Finally, our demonstration that a cysteine conjugate derived from GSCH2COSG is present in bile provides a reasonable explanation for the uncharacterized “N-acetyl cysteinyl derivative” of DCE reported by Jones and Hathway (8) via catabolism of GSCH2COSG in vivo to bis-N-acetylcysteinyl derivatives. Protein Identification in Rat Bile: Analytical Considerations. Initial attempts to identify biliary proteins involved analyzing untreated bile. This proved problematic, however, due to the interference of bile salts and other lipophilic compounds with ESI. Reverse phase LC of intact bile to remove bile salts also was attempted, but we were unable to develop chromatographic conditions that satisfactorily resolved proteins. One-dimensional SDS-PAGE satisfactorily separated biliary proteins and afforded the added advantage of providing multiple molecular mass fractions to analyze. Resolution of the proteins into several bands simplified the subsequent analysis by providing a molecular mass reference for confirming protein identifications and thus enabled more reliable protein identifications. In general, the molecular masses of the detected proteins corresponded to the apparent molecular masses indicated by the standards. In some cases, discrepancies are assumed to be due to proteolytic cleavage of the corresponding precursor protein prior to sample collection. Some proteolytic cleavage was expected since hepatocyte lysosomes discharge their contents into bile (48). Several proteins, notably serum albumin, were detected in multiple bands in all gels. A one-dimensional gel of commercially prepared rat serum albumin (Sigma) exhibited a similar distribution. This reflects the tendency of some abundant proteins to “smear” across gels based on aberrant binding of SDS or the presence of insoluble protein forms. It is important to note that protein identification reported here did not rely solely on automated SEQUEST analyses of the LC-MS-MS data. Visual inspection of the SEQUEST assignments, the MS-MS data, and experience in evaluating such data were essential. This was especially true when verifying protein identities through BLAST searching. For example, in several samples, SEQUEST identified acceptable matches to the peptide sequence DPDAVR and correlated it to alkyldihydroxy-

Jones et al.

acetone phosphate synthase. Because this protein identification was based on only one peptide, we manually interpreted the original MS-MS spectrum, which revealed that an alternative sequence of VLDAVR also was possible (DP ) 211 amu and VL ) 212 amu). A BLAST search correlated this sequence with transthyretin, a thyroid hormone-binding plasma protein from which several peptides already had been identified. Admittedly, this reliance on human judgment introduces some subjectivity, in particular where spectral assignments are ambiguous. For this reason, every effort was made to err on the side of caution when deciding whether a protein actually was present. A number of good quality spectra were discounted because the assigned sequences could not be linked to rat proteins. Indeed, one of the disadvantages of using a rat model is that the rat proteome is not well-characterized and some of these spectra may correlate to proteins and genes not yet described in the rat. This disadvantage plus low abundance would account for our not finding proteins, such as lysosomal enzymes, that others have found in bile using very sensitive enzymatic assays (48). Thus, the lists of proteins reported in Tables 1 and 2 are underinclusive. Protein Identification in Bile from Untreated Controls. Our results provide the first characterization of the rat biliary proteome by LC-MS-MS. SEQUEST analysis of the MS-MS data and BLAST database searching of logical alternative sequences resulted in positive identification of 21 proteins in bile collected prior to exposure to DCE and 19 proteins in bile collected prior to exposure to diclofenac. Seventeen proteins were found in both sets of bile samples. The basal bile samples were collected more than a year apart from different animals, thus suggesting the reproducibility of the analyses. Identified biliary proteins consisted largely of immunoglobulins and serum proteins. The former reflects the role of the biliary tract in transport of immunoglobulins to the intestine (38), whereas the latter reflects the transcellular and paracellular passage of serum components into bile (39), as well as the direct secretion of hepatocytederived plasma proteins into bile (49). Many of the >30 proteins found in bile by two-dimensional gel electrophoresis have not yet been definitely identified (50). For example, Pol et al. were able to identify approximately 10 proteins in bile rat bile proteins by using gel electrophoresis (one- and two-dimensional) and Western blotting (51). Half of these were immunoglobin chains, which also represented a significant number of the proteins identified here. We are unaware of prior reports that bile contains several of the proteins identified by our proteomic approach, for example, complement component 3 and transthyretin. Two proteins (cyclin A2 and mast cell protease 9) were detected only in the basal bile samples from the DCE experiment while one protein (calpain) was detected only in basal bile samples from the diclofenac experiment. These were identified with relatively low sequence coverage. This may not necessarily reflect a real difference in the two groups of controls as much as “lucky hits” on three lower abundance proteins. This phenomenon is typically observed in analyses of complex protein mixtures, which have been described previously (52-54). Protein Identification in Bile of DCE- or Diclofenac-Treated Rats. A total of 40 proteins were identified in bile collected after DCE exposure. This total includes most of those proteins present in untreated controls plus approximately 20 others. Few, if any, of the

Proteomic Characterization in Rats

proteins present only after exposure seem to be indicative of a specific mechanism of DCE toxicity. The proteins represent a variety of biochemical functions, and most are cytosolic proteins. Whereas the decrease in total biliary protein concentration suggests impairment of canalicular membrane transport proteins, the greater number of proteins present after exposure suggests a decreased selectivity in transport. This probably represents a loss of structural integrity of the canalicular membranes of affected hepatocytes and possibly alterations in the docking sites for vesicular transport. Interestingly, no canalicular membrane proteins were identified either before or after exposure to DCE. This was unexpected based on prior observations of increased amounts of several canalicular proteins using sensitive enzymatic assays (7). If present, membrane fragments containing canalicular membrane proteins would have been solubilized in Laemmli buffer prior to one-dimensional SDS-PAGE. Canalicular membrane proteins may have been represented at significantly lower levels than were the relatively abundant immunoglobulin and serum proteins, which could have precluded their detection. Canalicular membrane-associated proteins were detected in the bile formed after diclofenac treatment (see below). Therefore, if canalicular membrane proteins are damaged by reactive DCE metabolites, such damaged proteins are either not excreted into the bile in significant quantities or are excreted as smaller protein fragments that were not detected. In contrast to the many changes found in bile collected after DCE treatment, little change in the proteomic profile was evident in the bile samples obtained from animals after treatment with diclofenac. Only six proteins were found after treatment that were not detected in basal bile (Table 2). This is consonant with the more subtle changes in canalicular membrane integrity and bile formation observed after diclofenac treatment (18).22 Most interesting of the protein changes identified after diclofenac exposure was the presence of DPP IV, which is an integral membrane protein with a homodimeric mass of 110 kDa. Hargus et al. (32) used monoclonal antibodies to confirm the presence of diclofenac adducts of this protein in hepatocytic plasma membrane fractions isolated from rats treated with diclofenac in vivo. Their results indicated two bands containing DPP IV at 110 and 200 kDa. Using a similar approach with rat bile samples, recent Western blots indicate at least three bands containing diclofenac-protein adducts between 60 and 100 kDa.2 In our work, three peptides of DPP IV were identified in bands 1, 2, and 4, which correspond to apparent molecular masses of approximately 200, 100, and 50 kDa, respectively. However, acyl glucuronides also are known to bind to albumin (29, 30, 55-57), and in our samples, albumin was identified in multiple bands with apparent molecular masses of 50-200 kDa. Thus, assignment of targets based on immunoreactivity at apparent molecular masses of proteins of interest should be interpreted cautiously. DCE-Derived Protein Adducts. Searches for modified proteins were performed with the algorithm SALSA according to the strategies we have described previously (41, 58, 59). Our approach was to apply SALSA and SEQUEST to identify MS-MS spectra that displayed 2

Kaphalia, L., Atchison, C., Hargus, S., Pohl, L., and TreinenMoslen, M. Unpublished observations.

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1315

characteristics of different DCE-derived adducts. In our previous studies, we had systematically evaluated MSMS fragmentation characteristics of peptides modified at cysteine thiols and at other amino acids by ClCH2COSG, ClCH2COCl, and S-carboxymethylation produced by treatment with iodoacetate (i.e., the modification produced by direct reaction of DCE oxide with thiols) (17). These data were used to generate SALSA search strategies to evaluate the MS-MS data. Of these possible modifications, only S-carboxymethylation was detected in MS-MS spectra of biliary proteins. The list of S-carboxymethylated proteins in Table 3 also does not appear at this time to have clear implications for the mechanism of DCE toxicity. Indeed, most of the modified peptides are from abundant proteins such as serum albumin. Their significance, however, lies in the fact that this modification offers the first direct in vivo evidence of the proposed mechanism of DCE toxicity, as this modification would not be expected to occur endogenously. In addition, the presence of an S-(cysteinylacetyl) protein adduct (on the peptide LC*PTHADSLNNLANIK from UDP-N-acetylglucosaminyltransferase, see Table 3), together with the putative identification of an S-(cysteinylacetyl) glutathione conjugate (m/z 469), suggests the formation of GSCOCH2-S-Cys-protein adducts followed by cleavage of glutamate and glycine. This observation clearly implicates ClCH2COSG as a proteinmodifying DCE metabolite in vivo and supports its previously hypothesized role in DCE-induced canalicular injury (6, 16). Diclofenac-Derived Protein Adducts. Determining the identity of diclofenac-modified proteins also is complicated by the fact that several types of modifications of various residues are possible. Protein modification by diclofenac occurs via a reactive acyl glucuronide intermediate and involves two mechanisms. The first is via direct nucleophilic replacement of the acyl glucuronide by nucleophilic cysteine, tyrosine, or lysine residues, which would add 277 amu to the nucleophilic residue (27, 55, 57). Alternatively, the acyl glucuronide moiety may be retained, resulting in addition of 455 amu (29, 30, 57, 60). In addition, Tang et al. described the presence of diclofenac-glutathione conjugates in bile (26). Through comparison to synthetic diclofenac metabolites, they confirmed the addition of 5-OH-4-GS-diclofenac and of 5-OH-6-GS-diclofenac, which would result in the addition of 309 amu.3 Because we did not have authentic standards of the different types of diclofenac adducts available, possible fragmentation characteristics in electrospray MS-MS were inferred from previous reports, which used different ionization methods and mass analyzers (29, 57). However, SALSA searches of the data with several different sets of criteria expected to be characteristic of different types of diclofenac adducts failed to identify MS-MS spectra that could be reasonably assigned to diclofenac peptide adducts. We note that with some of the reported or hypothesized adducts, particularly those containing a glucuronate-derived moiety, MS-MS fragmentation may be extensively driven by cleavages of the adduct or glucuronide structures and thus yield little information that would allow assignment of peptide sequence. The 3 Although we did not undertake a systematic analysis of diclofenac metabolites in rat bile, we did detect species corresponding to these previously reported glutathione conjugates (data not shown).

1316 Chem. Res. Toxicol., Vol. 16, No. 10, 2003

MS-MS data also were searched with SALSA for cysteinecontaining sequences found in DPP IV, serum albumin precursor, and polymeric immunoglobin receptor precursor, which also was abundantly present. However, no spectra were identified that confirmed the presence of a diclofenac-derived modification.

Jones et al.

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Acknowledgment. This work was funded by NIH Grants ES10056, ES06694, ES00267, DK34806, and DK56494. (23)

References (1) Moslen, M. T. (2001) Toxic responses of the liver. In Casarett and Doull’s Toxicology: The Basis Science of Poisons, 6th ed. (Klaassen, C. D., Ed.) pp 471-489, McGraw-Hill, New York. (2) Bohan, A., and Boyer, J. L. (2002) Mechanisms of hepatic transport of drugs: implications for cholestatic drug reactions. Semin. Liver Dis. 22, 123-136. (3) Ballatori, N., and Rebbeor, J. F. (1998) Roles of MRP2 and oatp1 in hepatocellular export of reduced glutathione. Semin. Liver Dis. 18, 377-387. (4) Keppler, D., and Konig, J. (2000) Hepatic secretion of conjugated drugs and endogenous substances. Semin. Liver Dis. 20, 265272. (5) Suzuki, H., and Sugiyama, Y. (1998) Excretion of GSSG and glutathione conjugates mediated by MRP1 and cMOAT/MRP2. Semin. Liver Dis. 18, 359-376. (6) Moslen, M. T., Dunsford, H. A., Karnasuta, C., Chieco, P., and Kanz, M. F. (1989) Histochemical and immunocytochemical evidence of early, selective bile canaliculi injury after 1,1dichloroethylene in rats. Am. J. Pathol. 134, 1099-1112. (7) Woodard, S. H., and Moslen, M. T. (1998) Decreased biliary secretion of proteins and phospholipids by rats with 1,1-dichloroethylene-induced bile canalicular injury. Toxicol. Appl. Pharmacol. 152, 295-301. (8) Jones, B. K., and Hathway, D. E. (1978) The biological fate of vinylidene chloride in rats. Chem.-Biol. Interact. 20, 27-41. (9) Reichert, D., Werner, H. W., Metzler, M., and Henschler, D. (1979) Molecular mechanism of 1,1-dichloroethylene toxicity: excreted metabolites reveal different pathways of reactive intermediates. Arch. Toxicol. 42, 159-169. (10) McKenna, M. J., Zempel, J. A., Madrid, E. O., Braun, W. H., and Gehring, P. J. (1978) Metabolism and pharmacokinetic profile of vinylidene chloride in rats following oral administration. Toxicol. Appl. Pharmacol. 45, 821-835. (11) Greim, H., Bonse, G., Radwan, Z., Reichert, D., and Henschler, D. (1975) Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxiran formation. Biochem. Pharmacol. 24, 2013-2017. (12) Costa, A. K., and Ivanetich, K. M. (1982) Vinylidene chloride: its metabolism by hepatic microsomal cytochrome P-450 in vitro. Biochem. Pharmacol. 31, 2083-2092. (13) Liebler, D. C., and Guengerich, F. P. (1983) Olefin oxidation by cytochrome P-450: evidence for group migration in catalytic intermediates formed with vinylidene chloride and trans-1phenyl-1-butene. Biochemistry 22, 5482-5489. (14) Liebler, D. C., Meredith, M. J., and Guengerich, F. P. (1985) Formation of glutathione conjugates by reactive metabolites of vinylidene chloride in microsomes and isolated hepatocytes. Cancer Res. 45, 186-193. (15) Dowsley, T. F., Forkert, P. G., Benesch, L. A., and Bolton, J. L. (1995) Reaction of glutathione with the electrophilic metabolites of 1,1-dichloroethylene. Chem.-Biol. Interact. 95, 227-244. (16) Liebler, D. C., Latwesen, D. G., and Reeder, T. C. (1988) S-(2Chloroacetyl)glutathione, a reactive glutathione thiolester and a putative metabolite of 1,1-dichloroethylene. Biochemistry 27, 3652-3657. (17) Jones, J. A., and Liebler, D. C. (2000) Tandem MS Analysis of Model Peptide Adducts from Reactive Metabolites of the Hepatotoxin 1,1-Dichloroethylene. Chem. Res. Toxicol. 13, 1302-1312. (18) Sallustio, B. C., and Holbrook, F. L. (2001) In vivo perturbation of rat hepatocyte canalicular membrane function by diclofenac. Drug Metab. Dispos. 29, 1535-1538. (19) Seitz, S., Kretz-Rommel, A., Oude Elferink, R. P., and Boelsterli, U. A. (1998) Selective protein adduct formation of diclofenac glucuronide is critically dependent on the rat canalicular conjugate export pump (Mrp2). Chem. Res. Toxicol. 11, 513-519. (20) Wade, L. T., Kenna, J. G., and Caldwell, J. (1997) Immunochemical identification of mouse hepatic protein adducts derived from

(24)

(25)

(26)

(27) (28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38)

the nonsteroidal antiinflammatory drugs diclofenac, sulindac, and ibuprofen. Chem. Res. Toxicol. 10, 546-555. Hargus, S. J., Amouzedeh, H. R., Pumford, N. R., Myers, T. G., McCoy, S. C., and Pohl, L. R. (1994) Metabolic activation and immunochemical localization of liver protein adducts of the nonsteroidal antiinflammatory drug diclofenac. Chem. Res. Toxicol. 7, 575-582. Atchison, C. R., West, A. B., Balakumaran, A., Hargus, S. J., Pohl, L. R., Daiker, D. H., Aronson, J. F., Hoffmann, W. E., Shipp, B. K., and Treinen-Moslen, M. (2000) Drug enterocyte adducts: possible causal factor for diclofenac enteropathy in rats. Gastroenterology 119, 1537-1547. Kaphalia, L., Atchison, C., West, A. B., Pohl, L. R., and Moslen, M. T. (2001) Localization of diclofenac adducts at the canalicular membrane is associated with some alterations in biliary solutes. Toxicologist 60, 1676. Kumar, S., Samuel, K., Subramanian, R., Braun, M. P., Stearns, R. A., Chiu, S. H., Evans, D. C., and Baillie, T. A. (2002) Extrapolation of diclofenac clearance from in vitro microsomal metabolism data: role of acyl glucuronidation and sequential oxidative metabolism of the acyl glucuronide. J. Pharmacol. Exp. Ther. 303, 969-978. Poon, G. K., Chen, Q., Teffera, Y., Ngui, J. S., Griffin, P. R., Braun, M. P., Doss, G. A., Freeden, C., Stearns, R. A., Evans, D. C., Baillie, T. A., and Tang, W. (2001) Bioactivation of diclofenac via benzoquinone imine intermediates-identification of urinary mercapturic acid derivatives in rats and humans. Drug Metab. Dispos. 29, 1608-1613. Tang, W., Stearns, R. A., Bandiera, S. M., Zhang, Y., Raab, C., Braun, M. P., Dean, D. C., Pang, J., Leung, K. H., Doss, G. A., Strauss, J. R., Kwei, G. Y., Rushmore, T. H., Chiu, S. H., and Baillie, T. A. (1999) Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites. Drug Metab. Dispos. 27, 365-372. van Breemen, R. B., and Fenselau, C. (1985) Acylation of albumin by 1-O-acyl glucuronides. Drug Metab. Dispos. 13, 318-320. Ruelius, H. W., Kirkman, S. K., Young, E. M., and Janssen, F. W. (1986) Reactions of oxaprozin-1-O-acyl glucuronide in solutions of human plasma and albumin. Adv. Exp. Med. Biol. 197, 431441. Ding, A., Ojingwa, J. C., McDonagh, A. F., Burlingame, A. L., and Benet, L. Z. (1993) Evidence for covalent binding of a.cyl glucuronides to serum albumin via an imine mechanism as revealed by tandem mass spectrometry. Proc. Natl. Acad. Sci. U.S.A 90, 3797-3801. Smith, P. C., Benet, L. Z., and McDonagh, A. F. (1990) Covalent binding of zomepirac glucuronide to proteins: evidence for a Schiff base mechanism. Drug Metab. Dispos. 18, 639-644. Seitz, S., and Boelsterli, U. A. (1998) Diclofenac acyl glucuronide, a major biliary metabolite, is directly involved in small intestinal injury in rats. Gastroenterology 115, 1476-1482. Hargus, S. J., Martin, B. M., George, J. W., and Pohl, L. R. (1995) Covalent modification of rat liver dipeptidyl dipeptidase IV (CD 26) by the nonsteroidal antiinflammatory drug diclofenac. Chem. Res Toxicol. 8, 993-996. Kanz, M. F., Whitehead, R. F., Ferguson, A. E., Kaphalia, L., and Moslen, M. T. (1992) Biliary function studies during multiple time periods in freely moving rats. A useful system and set of marker solutes. J. Pharmacol. Toxicol. Methods 27, 7-15. Spahr, C. S., Davis, M. T., McGinley, M. D., Robinson, J. H., Bures, E. J., Beierle, J., Mort, J., Courchesne, P. L., Chen, K., Wahl, R. C., Yu, W., Luethy, R., and Patterson, S. D. (2001) Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest. Proteomics 1, 93-107. Eng, J. K., McCormack, A. L., and Yates, J. R. (1994) An Approach to Correlate Tandem Mass-Spectral Data of Peptides with AminoAcid-Sequences in A Protein Database. J. Am. Soc. Mass Spectrom. 5, 976-989. Yates, J. R., Eng, J. K., McCormack, A. L., and Schieltz, D. (1995) Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal. Chem. 67, 1426-1436. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389-3402. Reynoso-Paz, S., Coppel, R. L., Mackay, I. R., Bass, N. M., Ansari, A. A., and Gershwin, M. E. (1999) The immunobiology of bile and biliary epithelium. Hepatology 30, 351-357.

Proteomic Characterization in Rats (39) Kloppel, T. M., Brown, W. R., and Reichen, J. (1986) Mechanisms of secretion of proteins into bile: studies in the perfused rat liver. Hepatology 6, 587-594. (40) Kullak-Ublick, G. A., Beuers, U., and Paumgartner, G. (2000) Hepatobiliary transport. J. Hepatol. 32 (Suppl. 1), 3-18. (41) Hansen, B. T., Jones, J. A., Mason, D. E., and Liebler, D. C. (2001) SALSA: a pattern recognition algorithm to detect electrophileadducted peptides by automated evaluation of CID spectra in LCMS-MS analyses. Anal. Chem. 73, 1676-1683. (42) Davis, M. T., Spahr, C. S., McGinley, M. D., Robinson, J. H., Bures, E. J., Beierle, J., Mort, J., Yu, W., Luethy, R., and Patterson, S. D. (2001) Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. II. Limitations of complex mixture analyses. Proteomics 1, 108-117. (43) Petricoin, E. F., Ardekani, A. M., Hitt, B. A., Levine, P. J., Fusaro, V. A., Steinberg, S. M., Mills, G. B., Simone, C., Fishman, D. A., Kohn, E. C., and Liotta, L. A. (2002) Use of proteomic patterns in serum to identify ovarian cancer. Lancet 359, 572-577. (44) Liotta, L. A., Kohn, E. C., and Petricoin, E. F. (2001) Clinical proteomics: personalized molecular medicine. J. Am. Med. Assoc. 286, 2211-2214. (45) Ballatori, N., Jacob, R., and Boyer, J. L. (1986) Intrabiliary glutathione hydrolysis. A source of glutamate in bile. J. Biol. Chem. 261, 7860-7865. (46) Hinchman, C. A., Matsumoto, H., Simmons, T. W., and Ballatori, N. (1991) Intrahepatic conversion of a glutathione conjugate to its mercapturic acid. Metabolism of 1-chloro-2,4-dinitrobenzene in isolated perfused rat and guinea pig livers. J. Biol. Chem. 266, 22179-22185. (47) Cai, H. L., and Guengerich, F. P. (1999) Mechanism of aqueous decomposition of trichloroethylene oxide. J. Am. Chem. Soc. 121, 11656-11663. (48) Coleman, R. (1987) Biochemistry of bile secretion. Biochem. J. 244, 249-261. (49) Saucan, L., and Palade, G. E. (1992) Differential colchicine effects on the transport of membrane and secretory proteins in rat hepatocytes in vivo: bipolar secretion of albumin. Hepatology 15, 714-721. (50) Stark, M., Jornvall, H., and Johansson, J. (1998) Identification of hydrophobic bile proteins. J. Protein Chem. 17, 551-552. (51) Pol, A., Ortega, D., and Enrich, C. (1997) Identification and distribution of proteins in isolated endosomal fractions of rat

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1317 liver: involvement in endocytosis, recycling and transcytosis. Biochem. J. 323, 435-443. (52) Wolters, D. A., Washburn, M. P., and Yates, J. R. (2001) An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73, 5683-5690. (53) Washburn, M. P., Ulaszek, R., Deciu, C., Schieltz, D. M., and Yates, J. R., III (2002) Analysis of quantitative proteomic data generated via multidimensional protein identification technology. Anal. Chem. 74, 1650-1657. (54) Gygi, S. P., Corthals, G. L., Zhang, Y., Rochon, Y., and Aebersold, R. (2000) Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc. Natl. Acad. Sci. U.S.A. 97, 9390-9395. (55) Zia-Amirhosseini, P., Ding, A., Burlingame, A. L., McDonagh, A. F., and Benet, L. Z. (1995) Synthesis and mass-spectrometric characterization of human serum albumins modified by covalent binding of two nonsteroidal antiinflammatory drugs: tolmetin and zomepirac. Biochem. J. 311 (Pt. 2), 431-435. (56) Ding, A., Zia-Amirhosseini, P., McDonagh, A. F., Burlingame, A. L., and Benet, L. Z. (1995) Reactivity of tolmetin glucuronide with human serum albumin. Identification of binding sites and mechanisms of reaction by tandem mass spectrometry. Drug Metab. Dispos. 23, 369-376. (57) Qiu, Y., Burlingame, A. L., and Benet, L. Z. (1998) Mechanisms for covalent binding of benoxaprofen glucuronide to human serum albumin. Studies By tandem mass spectrometry. Drug. Metab. Dispos. 26, 246-256. (58) Liebler, D. C., Hansen, B. T., Davey, S. W., Tiscareno, L., and Mason, D. E. (2002) Peptide sequence motif analysis of tandem MS data with the SALSA algorithm. Anal. Chem. 74, 203-210. (59) Badghisi, H., and Liebler, D. C. (2002) Sequence mapping of epoxide adducts in human hemoglobin with LC-tandem MS and the SALSA algorithm. Chem. Res. Toxicol. 15, 799-805. (60) Kretz-Rommel, A., and Boelsterli, U. A. (1994) Mechanism of covalent adduct formation of diclofenac to rat hepatic microsomal proteins. Retention of the glucuronic acid moiety in the adduct. Drug Metab. Dispos. 22, 956-961.

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