Proteomic Identification of Potential Susceptibility Factors in Drug

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Chemical Profiles Proteomic Identification of Potential Susceptibility Factors in Drug-Induced Liver Disease Kevin D. Welch,*,†,‡ Bo Wen,‡,§ David R. Goodlett,§,| Eugene C. Yi,| Hookeun Lee,| Timothy P. Reilly,†,⊥ Sidney D. Nelson,§ and Lance R. Pohl† Molecular and Cellular Toxicology Section, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, Department of Medicinal Chemistry, University of Washington, Seattle, Washington, 98195 and Institute for Systems Biology, Seattle, Washington 98103 Received January 19, 2005

Drug-induced liver disease (DILD) causes significant morbidity and mortality and impairs new drug development. Currently, no known criteria can predict whether a drug will cause DILD or what risk factors make an individual susceptible. Although it has been shown in mouse studies that the disruption of key regulatory factors, such as cyclooxygenase-2 (COX2), interleukin (IL)-6, and IL-10, increased susceptibility to DILD caused by acetaminophen (APAP), no single factor seems to be absolute. As an approach to better understand the multifactorial basis of DILD, we compared the hepatic proteome of mice that are resistant (SJL) and susceptible (C57Bl/6) to APAP-induced liver disease (AILD), using solution-based isotope-coded affinity tag (ICAT) liquid chromatography mass spectrometry. Several novel factors were identified that were more highly expressed in the livers of SJL mice, including those involved in stress response, cell proliferation and tissue regeneration, and protein modification, implicating these proteins as potential hepatoprotective factors. There was also a selective loss of several mitochondrial proteins from the livers of the susceptible C57Bl/6 mice, suggesting that the loss of functional mitochondria may indeed play a role in AILD. These findings indicate that comparative hepatic proteomic analyses of susceptible and resistant mouse strains may provide a global approach for identifying potential risk factors and mechanistic pathways responsible for DILD.

Introduction (DILD)1

Drug-induced liver disease is often life threatening and is one of the major reasons new drugs never reach the market or are withdrawn postmarketing (1). Most cases of DILD are classified as idiosyncratic reactions due in large part to their unpredictability and to insufficient knowledge of susceptibility factors and mechanisms of toxicity. The relatively low incidence and idiosyncratic nature of most cases of DILD suggests that susceptibility might be determined by multiple risk * To whom correspondence should be addressed. Tel: 301-451-2497. Fax: 301-480-4852. E-mail: [email protected]. † National Institutes of Health. ‡ These authors contributed equally to this work. § University of Washington. | Institute for Systems Biology. ⊥ Current address: Immunotoxicology Department, Drug Safety Evaluation, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Syracuse, NY 13057. 1 Abbreviations: ALT, alanine aminotransferase; AILD, APAPinduced liver disease; APAP, acetaminophen; COX, cyclooxygenase; DILD, drug-induced liver disease; GST, glutathione S-transferase; HO1, heme oxygenase; HSP, heat shock protein; IL, interleukin; IGFBP1, insulin-like growth factor binding protein 1; NAPQI, N-acetyl-pbenzoquinone imine; E1, ubiquitin-activating enzyme E1; ICAT, isotope-coded affinity tag; ESI MS/MS, electrospray tandem mass spectrometry.

factors. In this regard, it has been postulated that factors affecting drug metabolism and reactive metabolite formation, such as CYP and N-acetyltransferase polymorphisms, drug interactions, gender, and extrahepatic diseases, may have contributed to the susceptibility of individuals to a variety of DILDs (2). Studies with acetaminophen (APAP) in mice have suggested that multiple factors unrelated to drug metabolism may also affect susceptibility to DILD. These include the hepatoprotective factors interleukin (IL)-10 (3), IL-6 (4), and cyclooxgenases-2 (5) and the protoxicant factors lipopolysaccharide (6), interferon-γ (7), and macrophage migration inhibitory factor (8). Global genomic and proteomic expression approaches have been used to identify additional factors that may play a role in liver injury caused by APAP and other drugs. Although genomic studies have led to the identification of several genes that may be involved in DILD (9-13), a disadvantage of this method is that mRNA and protein levels do not always correlate (14), consequently requiring extensive confirmatory protein expression studies. This problem is circumvented by proteomic approaches that directly focus on the identification and quantification of proteins, as illustrated in several mecha-

10.1021/tx050011b CCC: $30.25 © 2005 American Chemical Society Published on Web 06/01/2005

Proteomic Identification of Susceptibility Factors

nistic studies of APAP toxicity. For example, in one study, the hepatic proteomes of wild-type mice and GST π knock-out mice were compared to elucidate factors that may explain the increased resistance of the GST π knockout mice to APAP-induced liver disease (AILD) (15). Sixteen proteins were found with at least a 2-fold difference in expression between the two strains of mice. Most notable were the increased expressions of peroxiredoxins 1, 2, and 6 (also referred to as 1-cys peroxiredoxin or antioxidant protein 2) and a protein disulfide isomerase in the GST π deficient mice, possibly as a compensatory protective response to the loss of GST π (15). In another study, researchers compared the hepatic proteomes of mice treated with APAP to that of its nontoxic isomer N-acetyl-m-aminophenol (AMAP) (16). Perhaps the most toxicologically important finding of this investigation was the apparent loss of a number of mitochondrial proteins from the livers of the APAPtreated mice. Additionally, mass spectrometry has been used to identify hepatic protein targets of N-acetyl-pbenzoquinone imine (NAPQI), the toxic reactive metabolite of APAP (17). These proteomic studies were performed using variations of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with mass spectrometry-based techniques to identify the proteins (18). Although this approach has led to important findings, it does have some limitations. It discriminates against hydrophobic membrane proteins, very large and small proteins, extremely basic and acidic proteins, and lower abundant proteins such as transcription factors that are difficult to detect when total cell lysates are analyzed (19). In addition, the relative expression levels of a given protein can be difficult to quantify by this method due to the possibility that a singly stained spot on a 2D-PAGE gel may contain multiple proteins. Such problems in pairwise protein quantification can often be overcome by another approach that relies on the use of differential labeling of protein samples with stable isotopes prior to solution-based, peptide fractionation by liquid chromatography and protein identification and quantification by mass spectrometry. While there are several methods for the use of stable isotope labeling to measure pairwise changes in proteomes, the method that we have employed in the current study is based on the use of isotope-coded affinity tag (ICAT) reagents (19). In this study, we have identified for the first time wildtype mice that are susceptible (C57Bl/6) and resistant (SJL) to AILD and compared their hepatic proteomes. Analysis of the proteomics data has led to the identification of several novel factors that may function as hepatoprotective factors in liver injury models. Accordingly, deficiencies in these factors may predispose mice and possibly humans to DILD.

Experimental Procedures Animal Handling and Drug Treatment. SJL and C57Bl/6 (B6) male mice (20-25 g) were obtained from commercial sources (Jackson Laboratories, Bar Harbor, ME; Taconic Farms, Terrytown, NY, respectively). Mice were acclimated to a 12 h light/dark cycle in a temperature- and humidity-controlled, specific pathogen-free environment in autoclaved, microisolator cages for at least 6-7 days according to National Institutes of Health standards. Mice were allowed free access to autoclaved food and water until experimental use. Before each experiment, food was withheld from the animals overnight (>16 h) to deplete

Chem. Res. Toxicol., Vol. 18, No. 6, 2005 925 hepatic glutathione stores uniformly as previously described (20). APAP (300 mg/kg, dissolved in warm saline) or saline was then administered intraperitoneally whereupon food supplies were restored. Six hours after treatment, blood samples were taken by retro-orbital puncture, and preselected mice were killed to obtain liver tissues for histological and proteomic analyses. The remaining mice were monitored for 48 h for the occurrence of APAP-induced deaths. Assessment of Liver Injury. Liver damage was initially evaluated by measuring the serum activity of alanine aminotransferase (ALT) using a microtiter plate adaptation of a commercially available kit (Sigma, St. Louis, MO). Liver sections that had been fixed in buffered formalin were processed by standard histological techniques, stained with hematoxylin and eosin (H/E), and examined for histopathological evidence of liver injury. Immunoblot Analysis. Sections of liver were homogenized in buffer (100 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 250 mM sucrose, pH 7.5) containing a protease inhibitor cocktail (Complete, Roche, Mannheim, Germany). Equivalent amounts of protein were then diluted in sample buffer under reducing conditions (125 mM Tris-HCl, pH 6.8, 0.5% SDS, 20% glycerol, 40 mM dithiothreitol, and bromophenol blue), boiled, and resolved on 10% acrylamide gels. After transfer to nitrocellulose (BioRad, Hercules, CA), nonspecific binding was blocked with 5% nonfat dried milk, and blots were probed with peroxiredoxin 1 and 6 antibodies (a kind gift from Dr. Sue Goo Rhee, NHBLI/ NIH), anti-E1 ubiquitin activating enzyme (Sigma), anti-murine cyclooxygenase (COX)-1 (Cayman Chemical, Ann Arbor, MI), anti-human GST M1-1 (Oxford Biomedical Research Inc, Oxford, MI), anti-rat IGF binding protein-1 (Upstate Biotechnology, Lake Placid, NY), or rabbit anti-APAP protein adduct serum (provided by Drs. Neil R. Pumford and Jack A. Hinson, University of Arkansas, AR) followed by incubation with an appropriate peroxidase-conjugated secondary antibody (Chemicon International, Temecula, CA). Protein signals were visualized with the use of SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnologies, Inc.; Rockford, IL) and Kodak Image Station IS440CF imager and software (Eastman Kodak, Rochester, NY). Preparation of ICAT Reagent-Labeled Peptides. The methods used for the ICAT analysis of liver tissues have been recently described (21). Approximately 50 mg of liver from a saline- or APAP-treated B6 or SJL mouse was placed in a 1.75 mL eppendorf tube. One milliliter of Mammalian Protein Extraction Reagent (M-PERTM; Pierce) containing 50 mM PMSF was added to each tube followed by sonication on ice (Branson Sonifier 250, 10 s, 10 cycles). The homogenized samples were centrifuged at 16000g for 15 min at 4 °C, and the supernatants were transferred to clean eppendorf tubes and dried to near dryness by vacuum centrifugation at room temperature. Proteins were reconstituted in 200 µL of ICAT-labeling buffer (200 mM Tris-HCl buffer, pH 8.3, containing 0.05% SDS, 5 mM EDTA, and 6 M urea), and protein concentrations were determined using the BCA protein assay (Pierce) with bovine BSA as a standard protein. For quantitative protein analysis, 1.6 mg of proteins was reduced by the addition of Tris(2carboxyethyl) phosphine (TCEP; Sigma) to a 5 mM final concentration, followed by adjustment of the pH to 8.3 with 200 mM Tris-HCl buffer (pH 8.7). Reduced proteins were labeled with d0- or d8-ICAT reagent (Applied Biosystems, Foster City, CA) with a ratio of approximately 0.5 nmol ICAT/µg of protein. The samples were mixed well and incubated while gently shaking in the dark for 90 min at room temperature. The labeling reaction was stopped by the addition of a 5-fold molar excess of dithiothreitol over the ICAT reagent. Pair samples each containing 500 µg of liver protein were combined, and proteins were digested with trypsin (trypsin:protein ratio, 1:50, w/w) at 37 °C overnight. The reactions were quenched by the addition of glacial acetic acid to pH 3.0, and tryptic peptides were separated by strong cation exchange (SCX) chromatography on a PolySULFOETHYL A column (2.1 mm × 20 cm, 5 µM

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particles, 300 Å pore size; PolyLC Inc., Columbia, MD) used with a Shimadzu LC-10AD VP liquid chromatography system (Columbia, MD). A binary gradient from 10 to 40% buffer B was applied over 1 h (buffer A: 10 mM K2HPO4, 25% acetonitrile, pH 3.0; buffer B: 350 mM KCl, 10 mM K2HPO4, 25% acetonitrile, pH 3.0). ICAT-labeled peptides were then purified by affinity chromatography of each SCX fraction using Ultralink monomeric avidin (Pierce Biotechnology Inc.) as described previously (22, 23). Purified SCX fractions of ICAT-labeled peptides were dried by vacuum centrifugation and dissolved in 12 µL of 0.1% formic acid. Microcapillary Liquid Chromatography. ICAT-labeled peptides were analyzed by µLC-electrospray tandem mass spectrometry (ESI MS/MS) as described previously (24). For each purified SCX fraction, a sample volume of 4 µL was loaded onto a C18 precolumn (100 µm × 2 cm) with 5% solvent B (100% acetonitrile) at an unsplit flow rate of 5 µL/min in 5 min (21). After sample loading and cleanup, peptides were separated by reversed-phase chromatography using a 75 µm × 15 cm selfpacked Magic C18AQ (Michrom BioResources, Inc., Auburn, CA) column at a flow rate of 250 nL/min using binary solvent composition gradients with solvent A (99.9% H2O, 0.1% HCO2H) and solvent B (100% acetonitrile). Linear binary gradients of 5-32% solvent B were generated over 150 min, followed by isocratic elution at 70% solvent B for 4 min. The total analysis time for a single µLC-MS/MS run was 178 min from injection to end of isocratic wash. The above procedure including sample loading, valve switching, and solvent composition gradient elution is fully automated and controlled from an ITMS computer. Mass Spectrometric Analysis and Data Analysis. Peptide fragmentation by collision-induced dissociation (CID) was carried out in an automated fashion using the dynamic exclusion option on a ThermoFinnigan LCQ DECA XP ion trap mass spectrometer (Thermo Electron Corporation, San Jose, CA). Eluting peptides were selected for CID during a procedure that alternated between a MS scan over the m/z range 400-1800 Da and a MS/MS scan in which the single most abundant peptide ion was subjected to CID. Each scan cycle lasted an average of ∼1.6 s. The specific m/z value of the peptide fragmented by CID was excluded from reanalysis for 3 min. Automated data processing for protein identification was achieved utilizing SEQUEST, a computer program that performs the correlation of experimental data with theoretical spectra generated from a known protein sequence (25). Tandem mass spectra of peptides having a probability score of at least 0.5 were considered correctly identified (26). Peptide quantification was performed using the XPRESS program as described (22). Quantification of the ratio of each protein (isotopically heavy vs light) was made using an in house program called ASAPRatio (27). Using a second program, ProteinProphet (28, 29), the assigned peptides were grouped according to corresponding protein. The probability of a correct protein assignment was computed by ASAPRatio. Proteins that were represented by numerous peptides, high percentage sequence coverage, or extremely strong single ion elution profiles were classified as abundant. Information on the freely available ISB software tools used for this study may be found on line at http://www.systemsbiology.org/Default.aspx?pagename)proteomicssoftware. To quantify the protein ratio between samples, the XPRESS program was used to calculate the ICAT ratio (22). Protein abundance ratios greater than 2.0 or less than 0.5 were set as a threshold indicating significant changes of potential biological interest based on the distribution of values for all proteins quantified in this study. Data are presented as means ( SD when appropriate. Statistical Analysis. Statistical comparisons between two groups were made using either Student’s t-test or Fisher’s exact test. Differences were considered significant when p < 0.05.

Results During preliminary toxicity studies, we found that SJL mice were comparatively resistant while C57Bl/6 (B6)

Welch et al.

Figure 1. Comparison of susceptibility differences between SJL and B6 strains of mice to APAP-induced hepatic injury and lethality. Following 300 mg/kg of APAP, liver injury was assessed by (A) measuring serum ALT activity after 6 h (n ) 16 per strain) and (B) deaths after 48 h (n ) 10 per strain). Results shown represent the means ( SEM. Significance was determined using Student’s t-test for data shown in panel A and Fisher’s exact test for data shown in panel B; *p < 0.05.

mice were highly susceptible to AILD. The levels of serum ALT released into the blood from injured hepatocytes were significantly higher in B6 than SJL mice 6 h following treatment with APAP (Figure 1A). Histological examination of liver tissue sections confirmed these biochemical findings by showing minor injury in SJL mice as compared to extensive bridging perivenous necrosis in B6 mice (Figure 2). There was also a significant difference in mortality between the strains. Within 48 h after APAP treatment, 50% of the B6 mice had died whereas all of the SJL mice survived (Figure 1B). These marked differences in susceptibility of B6 and SJL mice to AILD did not appear to be due to a disparity in the metabolism of APAP into its reactive NAPQI metabolite, because total levels of APAP-protein adducts formed in the liver 3 h following APAP treatment did not differ significantly between the strains (Figure 3A,B). However, there did appear to be differences in the expression levels of protein adducts of approximately 30 and 50 kDa between the strains. The significance of this observation is not known at this time. We used ICAT reagent-based quantitative proteome analysis as a global approach to uncover factors that may have roles in determining the susceptibility differences of B6 and SJL mice to AILD. Four different pairwise comparisons were used in the study. They included (i) SJL saline-treated vs B6 saline-treated mice, (ii) SJL APAP-treated vs B6 APAP-treated mice, (iii) SJL APAPtreated vs SJL saline-treated mice, and (iv) B6 APAP-


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Figure 2. Histological comparison of liver injury in SJL and B6 strains following treatment with APAP. Photomicrographs (10×, objective lens) of H&E stained liver sections from SJL and B6 mice 6 h after treatment with 300 mg/kg APAP or saline. Results shown are representative of each group (n ) 4 per group).

treated vs B6 saline-treated mice. Peptides were considered correctly identified when a probability score was at least 0.5 (26), and for this study, differences in protein expression in the pairwise comparisons were judged significant when they differed by at least 2-fold. Typical mass spectra obtained for the identification of an ICATlabeled peptide by CID in the mass spectrometer operated in the MS/MS mode and the relative quantitative MS of light (B6 mouse) and heavy (SJL mouse) ICATlabeled peptides are shown in Figure 4A,B, respectively. Inherent differences in protein expression levels between SJL and B6 mice were determined by comparing their hepatic proteomes after saline treatment. Overall, the pattern of liver protein expression was similar between SJL and B6 mice. Of the 1236 proteins identified, 121 were significantly differentially expressed in the two strains of mice with an equal number of proteins more abundant in each strain (see Supporting Information). After extensive review of the literature, several of the proteins that were more highly expressed in the liver of the SJL mouse that might contribute to the resistance of this strain to AILD are shown in Table 1. In comparison of hepatic proteomes of SJL and B6 mice following APAP treatment, a 6 h time point was chosen to uncover early response, hepatoprotective factors that would have roles in limiting injury progression after initiation of toxicity by NAPQI and its covalent adducts. Sixteen hundred thirty-two proteins were identified of which 247 were expressed at significantly different levels in the two strains of mice with 161 of these proteins being more abundant in the SJL mouse (see Supporting Information). After reviewing the literature, we have listed several of the proteins (Table 2) that may have roles in protecting the SJL mice from AILD. The reason a number of proteins were more highly expressed in the pairwise comparison of the SJL mouse with the B6 mouse following APAP treatment appeared

Figure 3. Immunochemical detection of APAP-protein adducts in mouse liver homogenates. Three hours after APAP treatment, livers were obtained and homogenates were prepared and used for analysis of APAP-protein adducts formation. (A) Immunoblot (50 µg/lane, from individual mice) showing comparable APAP-protein adducts in B6 and SJL mice. Molecular mass marker (kDa) migrations are shown on the right. (B) Densitometry determination of adduct levels shown in panel A. Results shown represent the means ( SD.

to be due in part to their selective loss from the liver of the B6 mouse as determined in the pairwise comparisons of the APAP-treated vs saline-treated studies (Table 3). Several mitochondrial proteins followed this trend highlighting the potential importance of mitochondrial damage in AILD. We confirmed the differences in protein expression levels between the APAP-treated mice for several proteins using immunoblotting techniques because only one liver was used per treatment group for ICAT analysis. Despite some minor differences in the relative magnitude of change in protein expression within and between animal groups, the results were consistent with the proteomics data, indicating that the results from the ICAT analyses are representative of the proteomic differences between the strains (Figure 5).

Discussion Recent technological developments in genomics and proteomics have dramatically increased the ability to study complex pathological events, including episodes of drug toxicity. In the current study, we compared the hepatic proteomes of SJL and B6 mice to uncover factors that might explain the differences in susceptibility of

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Welch et al. Table 2. Comparative Expression Levels of Proteins Liver Homogenates of SJL and B6 Mice Following APAP Treatment protein name

Figure 4. Peptide identification and relative abundance quantification of peroxiredoxin 1 between SJL strain (heavy) and B6 strain (light) after APAP treatment. (A) Tandem mass spectrum of a peroxiredoxin 1 peptide HGEVC* PAGWKPGSDTIKPDVN. C* designates cysteine labeled with the heavy (d8) form of ICAT reagent. Peptide fragment ions of y and b series show a high PeptideProphet probability score of 1.00 to the SEQUEST-predicted fragmentation ions of peptide HGEVC*PAGWKPGSDTIKPDVN. (B) Quantitative MS indicating the relative abundance of peroxiredoxin 1 between two strains and the calculated d0 (light) to d8 (heavy) ratio obtained using the XPRESS program. Table 1. Comparative Expression Levels of Proteins in Liver Homogenates of SJL and B6 Mice Following Saline Treatment protein name lactoferrin galectin-1 tripeptidyl-peptidase II proteasome subunit β-type 1 DnaJ homologue subfamily A member 1

ratio (SJL:B6)a

Swiss-Prot ID

HUGO symbol

3.9 ( 0.3 3.9 ( 0.1 2.5 ( 0.0 2.2 ( 0.5

P08071 P16045 Q64514 O09061

Ltf Lgals1 Tpp2 Psmb1

2.0 ( 0.0

P54102

Hsj2

a Results are expressed as a ratio of protein expression in SJL vs B6 mice.

these two strains of mice to AILD. When the hepatic proteomes of saline-treated mice were compared, several potential hepatoprotective proteins were more highly expressed in the liver of SJL mice that might allow this strain to respond more effectively to the early stages of liver injury caused by APAP as compared to the B6 mice (Table 1). DnaJ homologue subfamily A member 1 is a member of the heat shock protein (HSP) 40 family. HSP40 proteins have been shown to bind to HSP70 leading to enhanced chaperone function of HSP70 by up-regulating its ATPase activity (30). The chaperone activity of the HSP70/40 complex is cytoprotective, at least in part,

ratio Swiss-Prot HUGO ID symbol (SJL:B6)a

ubiquitin-like 2 activating 10.0 ( 0.1 enzyme E1B complement C5 precursor 7.1 ( 0.0 prostaglandin G/H synthase 5.5 ( 0.0 1 precursor (COX-1) peroxiredoxin 1 5.1 ( 0.0 170 kDa glucose-regulated 4.4 ( 0.0 protein (GRP170) or Hyou1 protein Hsp70 binding protein 4.3 ( 0.0 GST µ-2 3.2 ( 0.1 senescence marker protein-30 3.1 ( 0.1 or regucalcin thioredoxin, mitochondrial 2.9 ( 0.2 precursor proteasome subunit R-type 1 2.5 ( 0.2 methylcrotonyl-CoA carboxylase 2.5 ( 0.2 R-chain, mitochondrial biliverdin reductase B [flavin 2.4 ( 0.1 reductase (NADPH)] dual specificity mitogen-activated 2.4 ( 0.0 protein kinase kinase 2 succinate dehydrogenase 2.3 ( 0.1 cytochrome b560 subunit GST µ-6 2.3 ( 0.0 E1 2.2 ( 0.1 proteasome subunit R-type 3 2.1 ( 0.0 peroxiredoxin 6 2.1 ( 0.1 GST θ-1 2.1 ( 0.1 GST µ-1 2.1 ( 0.1 microsomal GST 2.0 ( 0.1 26S protease regulatory 2.0 ( 0.1 subunit 7 BAG family molecular 2.0 ( 0.0 chaperone regulator-3 ornithine carbamoyltransferase, 2.0 ( 0.2 mitochondrial precursor IGFBP-1 precursor 0.5 ( 0.1 thioredoxin reductase 2, 0.2 ( 0.1 mitochondrial precursor

Q9Z1F9

Uble1b

P06684 P22437

Hc Ptgs1

P35700 Q8VCI2

Prdx1 Hyou1

Q9CYV8 Hspabp P15626 Gstm2 Q64374 Rgn P97493

Txn2

Q9R1P4 Psma1 Q99MR8 Mccc1 Q923D2

Blvrb

Q63932

Map2k2

Q9CZB0

Sdhc

O35660 Q02053 O70435 Q9QWP4 Q64471 P10649 Q9CQ57 P46471

Gstm6 Ube1x Psma3 Prdx6 Gstt1 Gstm1 Mgst1 Psmc2

Q9JLV1

Bag3

P11725

Otc

P47876 Q9JLT4

Igfbp1 Txnrd2

a Results are expressed as a ratio of protein expression in SJL vs B6 mice.

because it facilitates the proper folding of nascent as well as denatured proteins that result from oxidative modifications (31-34) and other environmental stresses including possible covalent modifications due to reactive metabolites of drugs (35). Denatured proteins may cause cellular injury when they aggregate and/or lose a vital physiologic activity (36). Indeed, denatured proteins have been implicated in the etiology of liver injury caused by APAP in mice (35) and CCl4 in rats (37), and HSPs, including HSP40, are thought to be hepatoprotective in these models (4, 8, 35, 38). The elevated levels in SJL mice of proteasome subunit β-type 1, also known as proteasome component 5, suggest that potentially harmful altered proteins formed in the liver of this strain might be rendered harmless by being degraded in the proteasome system more rapidly than those formed in the liver of the B6 mice. In support of this idea, it has recently been shown that decreased proteasome activity is associated with increased severity of liver injury and oxidative stress in experimental alcoholic liver disease (39). Similarly, tripeptidyl peptidase II, a very large aminopeptidase with endoproteolytic activity that was more highly expressed in SJL mice, could act in combination with or independent of the proteasome system to



Table 3. Effect of APAP Treatment on Expression Levels of Proteins in Liver Homogenates of B6 and SJL Mice

protein name

APAP (SJL:B6)a

B6 (APAP: saline)b

SJL (APAP: saline)c

Swiss-Prot ID

HUGO symbol

peroxiredoxin 1 GRP170 precursor senescence marker protein-30 or regucalcin thioredoxin, mitochondrial 60S ribosomal protein L28 methylcrotonyl-CoA carboxylase R-chain, mitochondrial δ-aminolevulinic acid dehydratase succinate dehydrogenase cytochrome b560 subunit 60S ribosomal protein L24 cytochrome P450 3A11 ornithine carbamoyltransferase, mitochondrial precursor

5.1 ( 0.0 4.4 ( 0.0 3.1 ( 0.1 2.9 ( 0.2 2.6 ( 0.1 2.5 ( 0.2 2.3 ( 0.1 2.3 ( 0.1 2.3 ( 0.1 2.1 ( 0.0 2.0 ( 0.2

0.6 ( 0.2 0.1 ( 0.0 0.6 ( 0.1 0.4 ( 0.1 0.5 ( 0.2 0.5 ( 0.2 0.4 ( 0.1 0.5 ( 0.0 0.5 ( 0.2 0.4 ( 0.0 0.6 ( 0.2

1.5 ( 0.3 0.8 ( 0.2 1.1 ( 0.2 0.9 ( 0.0 1.8 ( 0.0 1.3 ( 0.0 1.2 ( 0.1 2.7 ( 0.2 1.6 ( 0.0 0.9 ( 0.2 1.6 ( 0.5

P35700 Q8VCI2 Q64374 P97493 P41105 Q99MR8 P10518 Q9CZB0 P38663 Q64459 P11725

Prdx1 Hyou1 Rgn Txn2 Rpl28 Mccc1 Alad Sdhc Rpl24 Cyp3a11 Otc

a Results are expressed as a ratio of protein expression in SJL vs B6 strains in APAP-treated mice. b Results are expressed as a ratio of protein expression in APAP- vs saline-treated B6 mice. c Results are expressed as a ratio of protein expression in APAP- vs salinetreated SJL mice.

Figure 5. Confirmation of ICAT quantitative proteomic analysis by immunoblot analysis. Immunochemical detection of peroxiredoxins 1 and 6, E1 ubiquitin activating enzyme, COX1, GST µ-1, and insulin-like growth factor binding protein 1 (IGFBP-1) in liver homogenates from SJL and B6 mice 6 h after treatment with 300 mg/kg APAP.

degrade a variety of potentially toxic, altered cellular proteins (40). Another potentially hepatoprotective protein found more highly expressed in the liver of saline-treated SJL mice is lactoferrin. This multifunctional iron-binding protein is widely distributed in the body and has been found in hepatocytes and hepatic nonparenchymal sinusoidal cells including vascular endothelium of patients diagnosed with chronic inflammatory liver diseases where it has been suggested to have a protective function (41). Indeed, one mechanism by which lactoferrin may protect against cellular injury is by scavenging free iron, which can accumulate in inflamed tissues and mediate the production of toxic reactive oxygen species (42). Lactoferrin also has iron-independent antiinflammatory activities including the down-regulation of several proinflammatory cytokines such as TNF-R, IL-1β, and IL-8, which is proposed to be due mainly to its neutralizing effects on inflammatory properties of bacterial lipopolysaccharides and bacterial unmethylated CpG-containing oligonucleotides (43, 44). Galectin-1 may also protect SJL mice from AILD. This protein is a member of a family of β-galactoside-binding lectins that share growth regulatory and immunomodulatory activities (45, 46). Most relevant is the finding that galectin-1 protects against concanavalin A-induced liver injury in part, by preventing increases in TNF-R and INF-γ levels (47). These cytokines may also be effector molecules in the murine models of AILD (3, 7, 48). Following APAP treatment, an even greater number of potentially hepatoprotective proteins were found to be more highly expressed in the liver of the SJL mice

including stress response proteins (Table 2). HSP70 binding protein is one of these proteins. This protein, like HSP40, is a cochaperone for HSP70, modulating its chaperone activity by affecting nucleotide exchange (49, 50). Similarly, Bcl-2-associated athanogene-3 (BAG-3), a member of a family of proteins that also regulate HSP70 activity by affecting nucleotide exchange and substrate release (51), was more abundant in the SJL mice. Another stress protein more highly expressed in the SJL mice is 170 kDa glucose-regulated protein (GRP170), a resident of the endoplasmic reticulum that, like HSPs, also has chaperone activity that can prevent aggregation of misfolded proteins in the endoplasmic reticulum (52). The SJL mice also had higher levels of components of the machinery that could dispose of denatured proteins more effectively after APAP treatment than B6 mice. These proteins included proteasome subunit R-type 1, proteasome subunit R-type 3, and 26S protease regulatory subunit 7 of the proteasome complex (Table 2). In addition, the SJL mice had higher hepatic levels of ubiquitin-activating enzyme E1 (E1) (Table 2). This enzyme catalyzes the first step in protein ubiquitination (53), a posttranslational modification that can affect the activity of proteins in a variety of ways, including tagging them for degradation by the proteasome system (54). One of the proteins with the highest comparative expression level in the SJL mice vs B6 mice was ubiquitin-like 2 activating enzyme E1B, which is also known as small ubiquitin-related modifier (SUMO)-1 activating enzyme subunit 2 (Table 2). This enzyme activates SUMO, a small protein structurally related to ubiquitin, so that it can be conjugated to lysine residues of a large number of proteins leading to the regulation of proteinprotein interactions, subcellular nuclear localization, protein-DNA interactions, and enzymatic activity (55). Many transcription factors are sumoylated, which can lead to their up-regulation or down-regulation. For example, sumoylation of heat shock factors HSF1 and HSF2 stimulates their DNA binding activity (56, 57) and may have a role in up-regulating HSPs that may have a role in protecting against AILD. On the other hand, sumoylation may also lead to the negative regulation of other transcription factors such as NF-κB, Sp3, and c-Jun (55) that may affect the response of the liver to drugs that can cause hepatotoxicity. Another protein expressed higher in the liver of SJL mice following APAP treatment was complement C5 precursor (Table 2). Recent studies have shown that

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complement proteins not only serve as mediators of innate immune defense against foreign pathogens but also modulate diverse cellular processes including liver regeneration. Complement C5 precursor is activated when it is cleaved by C5 convertase releasing C5 anaphylatoxin (C5a), which functions in the innate immune system in part by acting as a local mediator of inflammation (58, 59). Studies using C5 deficient mice and an inhibitor of the C5a receptor have shown that C5a is also essential for liver regeneration following partial hepatectomy (59). Signaling through the C5a receptor leads to the up-regulation of IL-6 and TNF-R and the activation of their downstream transcription factors STAT-3 and NF-κB, respectively, which are known to be involved in liver regeneration. Moreover, because C5a signaling has also been shown to have a role in liver regeneration following CCl4-induced liver injury (58), it is quite possible that this signaling pathway protects the liver from injury caused by APAP and other drugs. Other factors were found more highly expressed in the liver of SJL mice following APAP treatment that may protect against damage to membrane lipids, proteins, and DNA caused by reactive oxygen and nitrogen species (Table 2) (3, 60, 61). These include several members of the glutathione S-transferase (GST) family of proteins, which can catalyze the detoxification of electrophilic metabolites of xenobiotics including NAPQI (62) and inactivate endogenous electrophilic R, β-unsaturated aldehydes, quinones, epoxides, and hydroperoxides formed as secondary metabolites of oxidative stress (63). For example, it has been shown that members of the µ-1 and θ-1 family of cytosolic GSTs can protect against oxidative damage to the skin induced by exposure to ultraviolet B (64). Perhaps these and other µ and θ family members may have similar protective roles in the liver. Another class of GSTs that was more abundant in the SJL mice is microsomal GST-1 (Table 2), which exhibits glutathione peroxidase activity toward fatty acid hydroperoxides (65). The peroxidase activity associated with the microsomal GSTs (MGSTs) is referred to as nonselenium glutathione peroxidase activity and represents one of the important mechanisms that exists in cells for protection against hydroperoxides (66). On the other hand, some MGSTs may also catalyze the formation of lipid mediators of allergy and anaphylaxis, leukotrienes C4, D4, and E4 (67). In addition to the catalytic activities of GSTs, some members have also been shown to regulate mitogen-activated protein (MAP) kinase signaling pathways that can contribute to cellular fate such as survival, differentiation, and apoptosis (63, 68). In this regard, recent work suggests that the µ-1 class of GSTs can inhibit the activity of apoptosis signal-regulating kinase 1 (ASK1) through a physical interaction. ASK1 is a MAP kinase kinase kinase (MAPKKK) that can activate MAP kinases 3 and 6 leading to the activation of p38 MAP kinase or activation of MAP kinases 4 and 7 resulting in the activation of c-Jun N-terminal kinase (JNK) (69). It appears that a µ-1 class of GSTs blocks the p38 MAP kinase pathway of cell signaling, while thioredoxin inhibits JNK-mediated cell signaling (70). These inhibitors can dissociate during cellular stress resulting in apoptosis. However, high levels of the inhibitors can reverse this process and suppress the apoptotic pathway of cell death, which may explain in part how SJL mice are better protected from liver injury than are the B6 mice by GST µ class proteins. Similarly, recent work

Welch et al.

suggests that mitochondrial thioredoxin-2 can also inhibit ASK-1-mediated apoptosis initiated in the mitochondria (71). Whether this protective pathway would be more dominant in the SJL mice even though they expressed higher levels of thioredoxin-2 precursor is not clear because more hepatic mitochondrial thioredoxin reductase 2 precursor, which is required to maintain thioredoxin-2 in its active reduced state, was detected in the liver of B6 mice (Table 2). Several other antioxidant proteins more highly expressed in the liver of the SJL mice following APAPtreated might contribute to the resistance of this strain of mice to AILD (Table 2). One family of proteins that likely is hepatoprotective are the peroxiredoxins, which have been shown to play a vital role in protecting cells against reactive oxygen species (72-75). In addition to reducing hydrogen peroxide and alkyl hydroperoxides, peroxiredoxins are also efficient peroxynitrite reductases (76). Consequently, the increased levels of peroxiredoxins 1 and 6 may allow the SJL mice to more efficiently remove reactive oxygen/nitrogen species that are formed following an hepatotoxic dose of APAP (3, 77, 78). Biliverdin reductase is another potential hepatoprotectant that is associated with antioxidant activity (79, 80). This enzyme plays an important role in the heme degradation pathway. Heme oxygenase 1 (HO-1), also known as HSP32, first metabolizes heme to biliverdin. Then biliverdin reductase rapidly reduces biliverdin to bilirubin, a potent antioxidant that can be converted back to biliverdin when it is oxidized, thus establishing a potential antioxidant redox cycle (81). In this regard, biliverdin treatment of rats has been shown to protect the liver against ischemia and reperfusion injury (82) and AILD (83) where hepatoprotection was suggested to be due at least in part to the reduction of biliverdin to bilirubin. Another highly expressed hepatic protein that may protect SJL mice from AILD is senescence marker protein-30 (SMP-30) (Table 2), a cytosolic protein whose expression decreases with age (84). It is also termed regucalcin because of it calcium binding properties. Studies with primary hepatocytes from SMP-30 deficient and wild-type mice (85) and Hep G2 cells (86) suggest that SMP-30 may protect hepatocytes from apoptosis induced by TNF-R in part by enhancing the plasma membrane calcium pump activity and activating the Akt signaling pathway. Another signaling molecule more highly expressed in the SJL mice is mitogen-activated protein kinase kinase 2 (MAPKK or MEK) (Table 2), which phosphorylates and activates extracellular signalregulated kinase 1/2 (ERK 1/2). Recent work suggests that the MEK/ERK signaling pathway may have roles in hepatocyte proliferation and survival induced by hepatic growth factors (87, 88) and IL-6 (89) and thus may help explain the resistance of the SJL mice to AILD. The results of the SJL APAP-treated vs SJL salinetreated and B6 APAP-treated vs B6-saline treated comparisons (Table 3) proved to be revealing because they suggest that the elevated expression levels of some proteins in the SJL vs B6 comparison following APAP treatment (Table 2) might be due in part to their selective loss from the liver of the B6 mice. Because several of these proteins are potentially hepatoprotective and/or are localized in the mitochondria, which is a key target in AILD (16, 90-92), it is possible that their loss could


contribute to the increased susceptibility of B6 mice to AILD. In summary, the fact that multiple redundant factors protect the liver from AILD in animal studies (3-5, 93, 94) suggests that these and other factors may also have a hepatoprotective role in other DILDs and possibly contribute to the relatively low incidence of liver disease caused by most drugs in humans. As a more global approach to uncover additional hepatoprotective factors, we have compared the hepatic proteomes of two strains of mice, one resistant (SJL) and one susceptible (B6) to AILD. A number of proteins more highly expressed in the resistant SJL strain have been identified for the first time as possible hepatoprotectants in DILD. In the future, studies with knock-out mice, neutralizing antibodies, and/or siRNA approaches will be needed to establish conclusively the molecular roles of these factors in DILD. In addition, it remains to be determined whether polymorphisms that lead to low levels of expression of hepatoprotective factors result in an increased susceptibility to DILD in humans.


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Acknowledgment. We thank Drs. Neil Pumford and Jack Hinson (University of Arkansas, Little Rock, AR) for their contribution of anti-APAP serum. We also acknowledge the expert technical assistance of Jose Flores (NHLBI, Bethesda, MD). This work was supported by NIH Grant P51 RR00166 (S.D.N. and D.R.G.) and the UW NIEHS sponsored Center for Ecogenetics and Environmental Health: NIEHS P30ES07033.

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Supporting Information Available: Tables with complete listing of the results from the four pairwise proteomic comparisons. This material is available free of charge via the Internet at http://pubs.acs.org.

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