The Journal of Toxicological Sciences, 213 Vol.30, No.3, 213-227, 2005

IDENTIFICATION OF OXIDATIVE STRESS-RELATED PROTEINS FOR PREDICTIVE SCREENING OF HEPATOTOXICITY USING A PROTEOMIC APPROACH

Toshinori YAMAMOTO, Rie KIKKAWA, Hiroshi YAMADA and Ikuo HORII

Worldwide Safety Sciences, Pfizer Global Research & Development, Nagoya Laboratories, Pfizer Inc., 5-2 Taketoyo, Aichi 470-2393, Japan

(Received January 15, 2005; Accepted April 19, 2005)

ABSTRACT — We investigated the effects of three hepatotoxicants, acetaminophen (APAP), amio- darone (AD) and tetracycline (TC), on protein expression in primary cultured rat hepatocytes with toxi- coproteomic approach, which is two-dimensional gel electrophoresis (2DE) and mass spectrometry. The objectives of this study were to search for alternative toxicity biomarkers which could be detected with high sensitivity prior to the appearance of morphological changes or alterations of analytical conventional biomarkers. The related proteins in the process of cell degeneration/necrosis such as cell death, lipid metabolism and lipid/carbohydrate metabolism were mainly affected under exposure to APAP, AD and TC, respectively. Among the differentially expressed proteins, several oxidative stress-related proteins were clearly identified after 24-hr exposure, even though they were not affected for 6-hr exposure. They were glutathione peroxidase (GPX) as a down-regulated protein as well as peroxiredoxin 1 (PRX1) and peroxiredoxin 2 (PRX2) as up-regulated proteins, which are known to serve as antioxidative in cells. These findings suggested that the focused proteins, GPX and PRXs, could be utilized as biomarkers of hepatotoxicity, and they were useful for setting high throughput screening methods to assess hepato- toxicity in the early stage of drug discovery.

KEY WORDS: Rat hepatocyte, Proteomics, Oxidative stress, Glutathione peroxidase, Peroxiredoxin, Biomarker

INTRODUCTION the early stage of drug discovery. Several approaches for the estimation of gene expression have been made Recent advances in new technologies such as to search for the appropriate genomic biomarkers combinatorial chemistry and high throughput pharma- (Jessen et al., 2003; Boess et al., 2003; Longueville et cological assay require high throughput screening al., 2003). However, there have been only a few reports procedures on safety evaluations in the early stage of on the protein expression related to hepatotoxicity drug discovery. In this context, the use of in vitro cyto- (Fountoulakis et al., 2002; Ruepp et al., 2002). In this toxicity assays to predict general toxicity in vivo is study, we have investigated protein expression as a reli- usually requested (Fry et al., 1990; Paillard et al., 1999; able biomarker in primary cultured rat hepatocytes Bugelski et al., 2000; Luber-Narod et al., 2001). exposed to several hepatotoxicants. We used acetami- The liver is one of the important organs for toxi- nophen (APAP), amiodarone (AD) and tetracycline cological evaluation from the aspects of the major (TC), whose mechanisms of hepatotoxicity are well metabolism site of xenobiotics as a primary target of known [APAP: hepatic necrosis is mediated by the drugs (Fautrel et al., 1991; Alden et al., 1999). More toxic metabolite, N-acetyl-p-benzoquinone imine sensitive and reliable endpoints as toxicologically (NAPQI) (Holme et al., 1984; Nelson, 1990); AD: responsible biomarkers were required for setting the phospholipidosis caused by phospholipid accumulation high throughput screening system of hepatotoxicity in (Dake et al., 1985; Sirajudeen et al., 2002); TC: steato-

Correspondence: Toshinori YAMAMOTO

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T. YAMAMOTO et al. sis induced by accumulation of fat in the liver (Breen et (Eugene, OR, USA). HPLC grade solvents and 3-[(3- al., 1975; Deboyser et al., 1989)]. cholamidopropyl) dimethylammonio]-1-propane- In this paper, we have investigated the effects of sulphonate (CHAPS) were purchased from Wako Pure the hepatotoxicants on protein expression in the pri- Chemicals. Sequencing grade-modified trypsin was mary cultured rat hepatocytes. Among the differentially obtained from Promega (Madison, WI, USA). An LC- µ expressed proteins, we have focused on oxidative Packings PepMap C18 -precolumn cartridge and a stress-related proteins, and discussed the feasibility of PepMap C18 capillary column were obtained from these protein biomarkers for application to high Dionex (San Francisco, CA, USA). throughput screening systems of hepatotoxicity. As a result, it is suggested that the focused proteins would Preparation of primary cultures of rat hepatocytes be utilized as biomarkers of hepatotoxicity for setting Primary hepatocytes were isolated from Sprague- high throughput screening methods to assess hepato- Dawley male rats (Charles River Japan, Yokohama, toxicity in the early stage of drug discovery. Japan) using the two-step collagenase perfusion method (Seglen, 1976; Kikkawa et al., 2005). Rat MATERIALS AND METHODS hepatocytes were cultured in Williams’ E culture medium (WEM, Invitrogen, Carlsbad, CA, USA) sup- Experimental design plemented with 10 mM 2-[4-(2-hydroxyethyl)-1- Primary cultured rat hepatocytes were exposed to piperazinyl]ethanesulfonic acid (HEPES, Invitrogen) three hepatotoxicants, APAP, AD and TC, for 6 and 24 buffer,pH7.4,10%fetalbovineserum(FBS,Invitro- hr. Concentrations of the test compounds (APAP: 10 gen), 100 U/mL penicillin, 100 µg/mL streptomycin mM, AD: 50 µM, TC: 500 µM) were determined based (Invitrogen), and insulin-transferring-selenium-A sup- on our previous cytotoxicity study in primary rat hepa- plement (Invitrogen). Rat hepatocytes were seeded on tocytes (Kikkawa et al., 2005). Protein expression was six-well type-I collagen coated plates (BD Bio- examined by two-dimensional gel electrophoresis sciences, Bedford, MA, USA) at a density of 105 cells/ (2DE), and proteins that expressed differentially in cm2. Prior to treatment with test compounds, hepato- ° control and treated cells were identified by mass spec- cytes were incubated at 37 Cundera5%CO2/95% air- trometric analysis following In-gel tryptic digestion. humidified atmosphere for 3 hr to attain cell attach- Eventually, our goal was to address several proteins ment. Viability of cultured rat hepatocytes was tested that were affected in common among the treatments of by LDH leakage (Roche Diagnostics GmbH, Man- compounds, because these proteins were potential can- nheim, Germany), mitochondrial respiration ability didates as hepatotoxicity markers. [water-soluble tetrazolium salts (WST-1) reduction assay, Roche Diagnostics GmbH] and morphological Test compounds observations. Acetaminophen (APAP) was purchased from Sigma (St. Louis, MO, USA). Amiodarone hydrochlo- Treatment conditions ride (AD) was obtained from ICN Biomedicals Inc. The test compounds were dissolved in dimethyl (Aurora, OH, USA). Tetracycline hydrochloride (TC) sulfoxide (DMSO, Wako Pure Chemicals), and the was purchased from Wako Pure Chemicals (Osaka, final concentration of DMSO in medium (FBS(−)) was Japan). adjusted to 1% for test solutions. After pre-incubation, the medium was replaced with a medium containing Chemicals and reagents test compounds, and hepatocytes cultured in 6-well Electrophoresis grade reagents [acrylamide, aga- plates were exposed to test compounds for 6 and 24 hr. rose, ammonium persulphate (APS), bromophenol Based on the results of our previous cytotoxicity study blue (BPB), glycerol, N,N,N',N'-tetramethylethylene- (Kikkawa et al., 2005), the concentrations of the test diamine (TEMED), sodium dodecyl (SDS) and compounds were set at 10 mM, 50 µM and 500 µMfor urea] and Immobiline DryStrip (pH3-10 L, 24 cm) APAP, AD and TC, respectively. At the end of the were purchased from Amersham Biosciences exposure periods, hepatocytes were harvested using a (Uppsala, Sweden). Pharmalyte (3-10) and TRIZMA clean cell scraper and recovered into 100 mM HEPES base were obtained from Sigma. SyproRuby protein buffer. They were frozen and stored in liquid nitrogen gel stain was purchased from Molecular Probes until protein extraction for 2DE experiments.

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Two-dimensional gel electrophoresis (2DE) They were then rehydrated in 20 ng/mL of TPCK- Total rat hepatocytes proteins were precipitated modified trypsin at 4°C. In-gel tryptic digestion was with 10% trichloroacetic acid (TCA)/acetone, and then carried out at 37°C overnight, and digested peptides proteinpelletswereresolubilizedin2D-lysisbuffer[7 were then recovered into 5%(v/v) aqueous formic acid M urea, 2 M thiourea (Invitrogen), 4%(v/v) CHAPS, (Wako Pure Chemicals). DeStreak reagent, 1%(v/v) Pharmalyte (pH3-10) and trace BPB] after completely washing with ice-cold MS/MS analysis and protein identification acetone. The hepatocytes lysates from individual wells Tryptic peptides from 2D-gel protein gel plugs (n=3) were pooled, and then their protein concentra- were subjected to peptide sequencing tags (PSTs) for tion was determined with a modified Lowry’s method protein identification using MS/MS spectra derived (Lowry et al., 1951) using RC DC Protein Assay Kit from capillary liquid chromatography coupled to a (Bio-Rad Laboratories, Hercules, CA, USA). One hun- hybrid quadrupole orthogonal acceleration time-of- dred micrograms of proteins was applied to immobilized flight mass spectrometer (CapLC and Q-TOF Ultima pH gradient gels (pH 3-10 L, 24 cm) in triplicate using API, Micromass, Manchester, UK) equipped with a the sample cup at the acidic end. Isoelectric focusing nano-ESI ion source. Peptides injected to the liquid was performed via step-wise voltage increments from chromatograph were first trapped and desalted isocrat- µ 250 V to 8000 V until the total volt-hours reached 69 ically on an LC-Packings PepMap C18 -precolumn kVhrs using an Ettan IPGphor Isoelectric Focusing cartridge (5 µm, 100Å, 300 µmid× 5 mm) and then Unit (Amersham Biosciences). Before performing the separated on an analytical PepMap C18 capillary col- second dimensional SDS-polyacrylamide gel electro- umn (3 µm, 100Å,75µmid× 15 cm) at 200 nL/min phoresis (SDS-PAGE), focused strips were subjected using a 60 min isocratic gradient of 5-80% acetonitrile to a two-step equilibration in equilibration buffer containing 0.1% formic acid. Product ion spectra were including 50 mM Tris-HCl (pH8.8), 6 M urea, 39%(v/ automatically acquired by data-dependent exact prod- v) glycerol, 2%(v/v) SDS, BPB supplemented with uct ion analysis at the criteria of intensity and charges 1%(w/v) dithiothreitol (DTT, Sigma) and 2.5%(w/v) (+2and+3) of precursor ions. The datasets of the MS/ iodoacetamide (IAA, Sigma), respectively. Following MS spectra, including peptides sequence information, equilibration, IPG strips were transferred onto self-cast were searched against the SWISS-PROT (GeneBio, 12.5%T SDS-polyacrylamide gels (20 cm × 26 cm) Geneva, Switzerland) database using Mascot Daemon and resolved in the second dimension at 20 W/gel at (Matrix Science, London, UK) as a client attached to 25°C using an Ettan DALTtwelve separation unit the Mascot search protocol. (Amersham Biosciences). Protein spots on 2D-gels were visualized with SyproRuby protein gel stain. The Statistical analysis images of fluorescence-stained 2D-gels were digitally The data were expressed as the mean values or scanned with Typhoon 9200 fluorescence image ana- the mean ± SD of 3 gels for fold-changes of normal- lyzer (Amersham Biosciences). Protein expression was ized spot volumes. Statistical analyses were made by assessed in triplicate using the Progenesis Workstation means of Student’s t-test (Gad and Weil, 1994), and analysis system (Nonlinear Dynamics, Cuthbert values with p<0.05 were considered to be statistically House, UK). Protein spots that varied more than two- significant. fold and were unique were identified as differentially expressed spots and subjected to further protein identi- RESULTS AND DISCUSSION fication analysis. Determination of exposure levels of hepatotoxicants In-gel tryptic digestion based on cytotoxicity of primary cultured rat hepa- Protein spots of interest were excised from the tocytes gels using ProXCISION proteomics gel-cutting robot We have already reported cytotoxicity in primary (PerkinElmer, Boston, MA, USA). The proteins in the cultured rat hepatocytes exposed to APAP, AD and TC gel plugs were subjected to reduction in 10 mM DTT in regard to morphology, LDH release and WST-1 and then alkylation in 25 mM IAA in 25 mM ammo- reduction assay. Based on the results of our previous nium bicarbonate. Gel plugs were then dehydrated in study, the dose levels and exposure term were deter- acetonitrile and dried in a vacuum centrifuge SPD2010 mined as a high dose (over the EC50 value) with the Speed-Vac (ThermoSavant, Waltham, MA, USA). exposures of 6 and 24 hr, which would be expected to

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T. YAMAMOTO et al. cause the toxicities concerned (Kikkawa et al., 2005). protein identification were subjected to peptide In terms of the usability of primary cultures of rat sequence tag analysis using product ion spectra derived hepatocytes, the advantages have been reported in from nano-ESI Q-Tof hybrid mass spectrometer fol- comparison with a cell line screening system such as lowing In-gel tryptic digestion. From the above HepG2 (Fry et al., 1990; Paillard et al., 1999; Wang et number of spots, 12, 26, 39, 63, 75 and 52 spots were al., 2002). Furthermore, it is said that primary cultured positively identified, and resulted in an indication of rat hepatocytes preserve intact cellular functions such several remarkable proteins. Table 2 summarizes the as Phase I (CYP mediated oxidation, etc.) and Phase II results of protein identification showing protein defini- metabolism (UGT mediated glucuronidation, etc.) tion and their fold-changes. (Ichihara et al., 1980; Kilberge, 1982; Vandenberghe et al., 1988). Functional analysis of the differentially expressed proteins Effects on protein expressions in primary cultured Among the identified proteins, expression rat hepatocytes changes of cellular assembly/organization-categorized Total proteins in cultured hepatocytes were sepa- proteins were detected by the treatment with almost all rated by 2DE, and the protein spots on 2D-gels were the hepatotoxicants, such as fructose-bisphosphate visualized following fluorescent staining. Total hepato- aldolase B (SWISS-PROT Accession number: cytes proteins lysed in 7 M urea/2 M thiourea- P00884), Keratin 8 (Q10758), transthyretin (P02767), containing buffer were clearly separated as approxi- calreticulin (P18418) and thiosulfate mately 3,000 protein spots, which were greater than (P24329). One of these proteins, fructose-bisphosphate those lysed in 8 M urea-containing buffer (about 2,000 aldolase B (liver-type aldolase), is known to play an protein spots, data not shown). Differential proteome important role in the assembly of the actin cytoskeleton maps, which were the overlaid gels images of both through its binding ability to actin (O’Reilly and control and treated group, showed the expression alter- Clarke, 1993; Kao et al., 1999). Since they are funda- ations of several protein spots by treatment with APAP, mental components of the living cells, these protein AD and TC. The number of proteins showing expres- expressions would be related to the cascading process sion changes of more than 2-fold and unique to a given through cell degenerative/necrotic changes to cell compound is shown in Table 1. The results of compar- death. isons revealed the effects on protein expression as Functional analysis of the differential protein follows (APAP: 15 up-regulated and 25 down-regu- expression profiles revealed the specified effects on lated after 6-hr exposure, 19 up-regulated and 54 protein expression by each hepatotoxicant. The pro- down-regulated after 24-hr exposure; AD: 12 up-regu- files of protein expression changes by APAP exposure lated and 55 down-regulated after 6-hr exposure, 30 were related to the process of cell death [α-enolase up-regulated and 81 down-regulated after 24-hr expo- (P04764), peroxiredoxin 1 (Q63716), regucalcin sure; TC: 54 up-regulated and 51 down-regulated after (Q03336), thioredoxin (P11232), glutathione peroxi- 6-hr exposure, 18 up-regulated and 52 down-regulated dase (P04041), etc.] and amino acid metabolism after 24-hr exposure). The specialized spots for further [methionine sulfoxide reductase A (Q923M1), and

Table 1. Number of differentially expressed spots on 2D-gels of proteins from primary cultured rat hepatocytes treatedwithAPAP(10mM),AD(50µM) and TC (500 µM)for6and24hr. Number of differentially expressed spot 1) Treatment Periods Up-regulated Down-regulated Total Increased Unique Decreased Unique APAP 6 hr 8 7 13 12 40 24 hr 13 6 33 21 73 AD 6 hr 9 3 30 25 67 24 hr 18 12 42 39 111 TC 6 hr 47 7 28 23 105 24 hr 13 5 34 18 70 1) The expressions in treated cells were compared to control cells.

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Table 2. Differentially expressed proteins successfully identified in primary cultured rat hepatocyte treated with 10 mM APAP, 50 µM AD and 500 µM TC for 6 and 24 hr. Spot No. 1) Definition Accession No. 2) Difference 3) APAP-6 hr 482 Pyruvate carboxylase, mitochondrial precursor P52973 2.7 1757 Fumarate hydratase, mitochondrial precursor P14408 2.2 3621 Pyruvate kinase, isozymes R/L P12928 Unique on Treated 2626 Triosephosphate P48500 Unique on Control 1481 Aldehyde dehydrogenase, mitochondrial precursor P11884 Unique on Control 2926 Transthyretin precursor P02767 Unique on Control 2670 Peptide methionine sulfoxide reductase Q923M1 −2.0 2346 Alcohol dehydrogenase A chain P06757 −2.2 2346 Aspartate aminotransferase, mitochondrial precursor P00507 −2.2 1247 60kDa heat shock protein, mitochondrial precursor P19226 −2.2 2061 Fructose-bisphosphate aldolase B P00884 −2.6 2643 Glutathione S- Yb-1 P04905 −6.9

APAP-24 hr 3628 Senescence marker protein-30 Q03336 6.1 2526 3-mercaptopyruvate sulfurtransferase P97532 5.7 1431 Glutamate dehydrogenase, mitochondrial precursor P10860 2.1 1431 UDP-glucose 6-dehydrogenase O70199 2.1 3029 Peptidyl-prolyl cis-trans isomerase B precursor P24368 2.0 3687 Peroxiredoxin 1 Q63716 Unique on Treated 3730 Peroxiredoxin 1 Q63716 Unique on Treated 3599 Triosephosphate isomerase P48500 Unique on Control 3217 Thioredoxin P11232 Unique on Control 2848 Glutathione S-transferase Yb-1 P04905 Unique on Control 2535 DOPA/tyrosine P52847 Unique on Control 1990 Keratin, type I cytoskeletal 19 (Fragment) Q63279 Unique on Control 1606 α enolase P04764 Unique on Control 3221 D-dopachrome tautomerase P80254 −2.1 2989 II β precursor Q9QZK9 −2.3 2476 Thiosulfate sulfurtransferase P24329 −2.3 1831 Keratin, type I cytoskeletal 19 (Fragment) Q63279 −2.3 1508 Keratin, type II cytoskeletal 8 Q10758 −2.4 2966 Glutathione peroxidase P04041 −2.4 578 Major vault protein Q62667 −2.6 2910 Catechol O-methyltransferase, membrane-bound form P22734 −2.6 2089 Fructose-bisphosphate aldolase A P05065 −2.7 2353 Thiosulfate sulfurtransferase P24329 −2.7 1410 UDP-glucose 6-dehydrogenase O70199 −2.8 3649 Voltage-dependent anion-selective channel protein 2 P81155 −3.6 2346 Thiosulfate sulfurtransferase P24329 −4.6 3665 Keratin, type II cytoskeletal 8 Q10758 −7.2 3665 Actin, cytoplasmic 1 P60711 −7.2

AD-6 hr 2646 Senescence marker protein-30 (Regucalsin) Q03336 5.6 1285 Serum albumin precursor P02770 2.3 833 Programmed cell death 6 interacting protein (Fragment) Q9QZA2 2.2 1048 Far upstream element binding protein 2 Q99PF5 2.2

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Table 2 Continued Spot No. 1) Definition Accession No. 2) Difference 3) 1048 Serotransferrin precursor P12346 2.2 1056 Liver 10 precursor P16303 2.0 4165 Peroxisomal multifunctional type 2 P97852 Unique on Treated 1269 Acyl- oxidase 2, peroxisomal P97562 Unique on Control 1376 Phosphoglucomutase P38652 Unique on Control 1801 Hsp90 co-chaperone Cdc37 Q63692 Unique on Control 2145 α-2-macroglobulin receptor-associated protein precursor Q99068 Unique on Control 2281 Catalase P04762 Unique on Control 2289 Keratin, type II cytoskeletal 8 Q10758 Unique on Control 2363 3-oxo-5-β-steroid 4-dehydrogenase P31210 Unique on Control 2764 Electron transfer flavoprotein α-subunit, mitochondrial precursor P13803 Unique on Control 2935 14-3-3 protein zeta/delta P35215 Unique on Control 3059 Apolipoprotein A-I precursor P04639 Unique on Control 4295 Cytochrome c oxidase polypeptide Va, mitochondrial precursor P11240 Unique on Control 4392 Retinol-binding protein I, cellular P02696 Unique on Control 2127 Keratin, type I cytoskeletal 19 Q63279 −2.1 1094 Moesin O35763 −2.1 1897 26S protease regulatory subunit 7 Q63347 −2.2 2587 Aryl sulfotransferase P17988 −2.2 2369 Aflatoxin B1 aldehyde reductase member 2 Q8CG45 −2.2 2677 Estrogen sulfotransferase, isoform 1 P52844 −2.2 2423 3-oxo-5-β-steroid 4-dehydrogenase P31210 −2.2 2423 Ornithine carbamoyltransferase, mitochondrial precursor P00481 −2.2 2535 Glycerol-3-phosphate dehydrogenase [NAD+], cytoplasmic O35077 −2.3 2535 Arginase 1 P07824 −2.3 3230 ATP synthase D chain, mitochondrial P31399 −2.3 4226 Cytochrome c oxidase polypeptide Vb, mitochondrial precursor P12075 −2.3 4413 Glutathione transferase oemga 1 Q9Z339 −2.4 2938 3-mercaptopyruvate sulfurtransferase P97532 −2.4 3042 Peroxiredoxin 6 O35244 −2.5 1554 26S protease regulatory subunit 4 P62193 −2.6 4408 Glutathione S-transferase Yb-1 P04905 −2.6 2386 3-oxo-5-β-steroid 4-dehydrogenase P31210 −2.9 2386 Ornithine carbamoyltransferase, mitochondrial precursor P00481 −2.9 1161 Protein disulfide isomerase A4 precursor P38659 −3.0 1161 Long -chain-fatty-acid CoA , liver isozyme P18163 −3.0 4284 Endoplasmic reticulum protein ERp29 precursor P52555 −3.5 2262 Farnesyl pyrophosphate synthetase P05369 −3.7 2262 Fructose-bisphosphate aldolase B P00884 −3.7 4400 Pyruvate carboxylase, mitochondrial precursor P52873 −4.1 2971 Adenylate kinase isoenzyme 2, mitochondrial P29410 −4.9

AD-24 hr 4247 Apolipoprotein E precursor P02650 3.8 2050 Arginase 1 P07824 3.4 961 Serum albumin precursor P02770 2.7 1288 Liver carboxylesterase 10 precursor P16303 2.6 2024 Fructose-1,6-bisphosphatase P19112 2.6 1881 Apolipoprotein A-IV precursor P02651 2.5

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Table 2 Continued Spot No. 1) Definition Accession No. 2) Difference 3) 985 Serum albumin precursor P02770 2.5 778 Serotransferrin precursor P12346 2.4 2365 Voltage-dependent anion-selective channel protein 1 Q9Z2L0 2.4 891 Long-chain-fatty-acid-CoA ligase, liver isozyme P18163 2.3 1715 Keratin, type I cytoskeletal 19 Q63279 2.2 1229 Catalase P04762 2.2 4177 78kDa glucose-regulated protein precursor P06761 2.2 1796 Serum albumin precursor P02770 2.1 4238 Peroxiredoxin 2 P35704 Unique on Treated 4237 Peroxiredoxin 1 Q63716 Unique on Treated 4203 Peroxiredoxin 1 Q63716 Unique on Treated 4072 Voltage-dependent anion-selective channel protein 1 Q9Z2L0 Unique on Treated 4297 Guanine nucleotide-binding protein β subunit-like protein 12.3 P25388 Unique on Treated 4301 Keratin, type I cytoskeletal 21 P25030 Unique on Treated 1696 Keratin, type I cytoskeletal 19 Q63279 Unique on Control 510 Keratin, type I cytoskeletal 21 P25030 Unique on Control 3167 UK114 P52759 Unique on Control 2408 ARF-related protein Q63055 Unique on Control 2408 DOPA/tyrosine sulfotransferase P52847 Unique on Control 2350 Short chain 3-hydroxyacyl-CoA dehydrogenase, mitochondrial precursor Q9WVK7 Unique on Control 2249 Thiosulfate sulfurtransferase P24329 Unique on Control 2117 Hydroxyacid oxidase 3 Q07523 Unique on Control 2017 Fructose-bisphosphate aldolase B P00884 Unique on Control 2012 Fructose-bisphosphate aldolase B P00884 Unique on Control 1970 Fructose-1,6-bisphosphatase P19112 Unique on Control 1937 Keratin, type II cytoskeletal 8 Q10758 Unique on Control 1937 Serum / 1 P55159 Unique on Control 1916 Alcohol dehydrogenase A chain P06757 Unique on Control 1916 Aspartate aminotransferase, mitochondrial precursor P00507 Unique on Control 1887 Betaine--homocysteine S-methyltransferase O09171 Unique on Control 1867 Acyl-CoA dehydrogenase, medium-chain specific, mitochondrial precursor P08503 Unique on Control 1688 Keratin, type II cytoskeletal 8 Q10758 Unique on Control 1017 T-plastin Q63598 Unique on Control 947 Aminopeptidase B O09175 Unique on Control 601 Glycogen phosphorylase, liver form P09811 Unique on Control 1538 α enolase P04764 Unique on Control 2159 Glyceraldehyde 3-phosphate dehydrogenase P04797 −2.0 4171 Actin, cytoplasmic 1 P60711 −2.1 1847 Betaine--homocysteine S-methyltransferase O09171 −2.1 2879 Insulin receptor substrate-1 P35570 −2.1 4211 60S acidic ribosomal protein P0 P19945 −2.1 1386 Adenylyl cyclase-associated protein 1 Q08163 −2.2 2136 Alcohol dehydrogenase [NADP+] P51635 −2.2 1039 Leukotriene A-4 P30349 −2.2 324 Catalase P04762 −2.2 1138 Catalase P04762 −2.4 1135 Rab GDP dissociation inhibitor α P50398 −2.4 326 ATP-citrate synthase P16638 −2.6 429 Heat shock cognate 71kDa protein P08109 −2.6

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Table 2 Continued Spot No. 1) Definition Accession No. 2) Difference 3) 2627 Glutathione S-transferase Yc-1 P04904 −2.8 1152 Long-chain-fatty-acid-CoA ligase, liver isozyme P18163 −2.8 1695 Trifunctional enzyme β subunit, mitochondrial precursor Q60587 −2.8 2721 Glutathione S-transferase Ya-1 P00502 −2.9 4305 Thiosulfate sulfurtransferase P24329 −3.3 2574 Proteasome subunit α type 4 P21670 −3.4 1150 Acyl-CoA dehydrogenase, very-long-chain specific, mitochondrial precursor P45953 −3.7 2333 Thiosulfate sulfurtransferase P24329 −3.9 2830 Glutathione peroxidase P04041 −4.6 1864 Keratin, type I cytoskeletal 19 Q63279 −5.2 2214 Thiosulfate sulfurtransferase P24329 −6.6 2313 Senescence marker protein-30 (Regucalcin) Q03336 −7.4

TC-6 hr 2678 Glutathione S-transferase Ya-1 P00502 5.1 2691 Glutathione S-transferase Ya-1 P00502 4.2 2327 Estrogen sulfotransferase, isoform 1 P52844 3.3 1667 26S protease regulatory subunit 8 P62198 3.2 2016 Serine/threonine protein PP1-α catalytic subunit P62138 3.1 1947 Farnesyl pyrophosphate synthetase P05369 3.0 3494 Tubulin β-5 chain P05218 3.0 1313 Keratin, type II cytoskeletal 8 Q10758 3.0 1660 Pyruvate dehydrogenase E1 component α subunit, somatic form, P26284 2.9 mitochondrial precursor 1516 Kynureninase P70712 2.8 3464 Dipeptidyl-paptidase III O55096 2.8 2349 Estrogen sulfotransferase, isoform 6 P49890 2.6 1342 Tubulin β chain P04691 2.5 2187 3-α-hydroxysteroid dehydrogenase P23457 2.5 3468 Cytochrome c oxidase subunit IV isoform 1, mitochondrial precursor P10888 2.5 2636 Glutathione S-transferase Yb-1 P04905 2.5 2663 Glutathione S-transferase Yb-3 P08009 2.5 2644 Glutathione S-transferase Yb-2 P08010 2.5 2782 ATP synthase D chain, mitochondrial P31399 2.4 2982 Actin, cytoplasmic 1 P60711 2.3 2205 N-hydroxylamine sulfotransferase P50237 2.3 3453 14-3-3 protein zeta/delta P35215 2.3 1403 Aldehyde dehydrogenase, mitochondrial precursor P11884 2.3 2533 Enoyl-CoA hydratase, mitochondrial precursor P14604 2.2 1051 Peroxisomal multifunctional enzyme type 2 P97852 2.2 2638 Glutathione S-transferase Yb-1 P04905 2.2 2586 Glutathione S-transferase Yc-1 P04904 2.2 2193 3-α-hydroxysteroid dehydrogenase P23457 2.2 1291 Propionyl-CoA carboxylase β chain, mitochondrial precursor P07633 2.1 1291 D-3-phosphoglycerate dehydrogenase O08651 2.1 1291 Pyruvate kinase, isozymes R/L P12928 2.1 2432 α-tocopherol transfer protein P41034 2.1 1347 Keratin, type II cytoskeletal 8 Q10758 2.1 1347 Tubulin β chain P04691 2.1

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Table 2 Continued Spot No. 1) Definition Accession No. 2) Difference 3) 2046 3-oxo-5-β-steroid 4-dehydrogenase P31210 2.1 2385 Histone H1.2 P15865 2.1 2493 Chloride intracellular channel protein 4 Q9Z0W7 2.0 1484 Phenylalanine-4-hydroxylase P04176 2.0 1484 α enolase P04764 2.0 2216 Aryl sulfotransferase P17988 2.0 3429 Nonspecific lipid-transfer protein, mitochondrial precursor P11915 Unique on Treated 3478 Tropomyosin 1 α chain P04692 Unique on Treated 3489 60S ribosomal protein L18 P12001 Unique on Treated 3495 Phenylalanine-4-hydroxylase P04176 Unique on Control 3495 Protein disulfide isomerase A6 precursor Q63081 Unique on Control 1194 Amine oxidase [flavin-containing] B P19643 Unique on Control 2974 Transthyretin precursor P02767 Unique on Control 2957 Transcription elongation factor B polypeptide 2 Q63529 Unique on Control 2711 Membrane associated progesterone receptor component 1 P70580 Unique on Control 2657 Growth factor receptor-bound protein 2 P29354 Unique on Control 2625 3-hydroxyacyl-CoA dehydrogenase type II O70351 Unique on Control 2501 Guanidinoacetate N-methyltransferase P10868 Unique on Control 2374 Ketohexokinase Q02974 Unique on Control 2149 Pyridoxal kinase O35331 Unique on Control 2090 Ornithine carbamoyltransferase, mitochondrial precursor P00481 Unique on Control 1827 Acetyl-CoA acetyltransferase, mitochondrial precursor P17764 Unique on Control 1827 Alcohol dehydrogenase class II Q64563 Unique on Control 1827 Cystathionine γ- P18757 Unique on Control 1749 4-hydroxyphenylpyruvate dioxygenase P32755 Unique on Control 1749 Adenosylhomocysteinase P10760 Unique on Control 1749 Acyl-CoA dehydrogenase, long-chain specific, mitochondrial precursor P15650 Unique on Control 1700 Pyruvate dehydrogenase E1 component α subunit, somatic form, P26284 Unique on Control mitochondrial precursor 1445 Aldehyde dehydrogenase, mitochondrial precursor P11884 Unique on Control 884 Argininosuccinate synthase P09034 Unique on Control 601 Elongation factor 2 P05197 Unique on Control 2376 Keratin, type II cytoskeletal 8 Q10758 Unique on Control 1280 Propionyl-CoA carboxylase β chain, mitochondrial precursor P07633 Unique on Control 2868 Translocon-associated protein, delta subunit precursor Q07984 −2.0 1599 Nonspecific lipid-transfer protein, mitochondrial precursor P11915 −2.0 479 Elongation factor 2 P05197 −2.0 479 Glycogen phosphorylase, liver form P09811 −2.0 765 Thimet oligopeptidase P24155 −2.1 461 Endoplasmin precursor Q29092 −2.1 1893 Fructose-1,6-bisphosphatase P19112 −2.4 3465 Protein disulfide isomerase A4 precursor P38659 −2.5 920 Serum albumin precursor P08835 −2.5 2805 Plasma retinol-binding protein precursor P04916 −2.7 1416 ATP synthase β chain, mitochondrial precursor P10719 −2.7 1416 Ca(2+)/calmodulin-dependent protein kinase phosphatase Q9WVR7 −2.7 777 Long-chain-fatty-acid-CoA ligase, liver isozyme P18163 −2.7 918 Serum albumin precursor P08835 −2.9 2082 Aflatoxin B1 aldehyde reductase member 2 Q8CG45 −2.9

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Table 2 Continued Spot No. 1) Definition Accession No. 2) Difference 3) 2249 Senescence marker protein-30 (Regucalcin) Q03336 −3.2 2962 ATP synthase delta chain, mitochondrial precursor P35434 −3.2 3149 Calreticulin precursor P18418 −6.6

TC-24 hr 2263 Senescence marker protein-30 Q03336 3.8 2602 Cytochrome b5 P00173 3.8 1255 Keratin, type II cytoskeletal 8 Q10758 3.5 1255 Calreticulin precursor P18418 3.5 2518 Peroxiredoxin 1 Q63716 3.1 1256 Keratin, type II cytoskeletal 8 Q10758 2.3 1256 Calreticulin precursor P18418 2.3 541 Endoplasmin precursor Q29092 2.2 3904 Peroxiredoxin 2 P35704 Unique on Treated 4016 60S ribosomal protein L18 P12001 Unique on Treated 1841 Transaldolase Q9EQS0 Unique on Treated 4044 10-formyltetrahydrofolate dehydrogenase P28037 Unique on Treated 776 Heat shock protein HSP 90-β P34058 Unique on Control 776 Serotransferrin precursor P12346 Unique on Control 858 Dynein intermediate chain 2, cytoplasmic Q62871 Unique on Control 1201 Hydroxymethylglutaryl-CoA synthase, cytoplasmic P17425 Unique on Control 1206 Formimidoyltransferase-cyclodeaminase O88618 Unique on Control 1215 Calreticulin precursor P18418 Unique on Control 1326 Calreticulin precursor P18418 Unique on Control 1604 Keratin, type II cytoskeletal 8 Q10758 Unique on Control 1813 Keratin, type II cytoskeletal 8 Q10758 Unique on Control 1893 Eukaryotic translation initiation factor 2 subunit 1 P05199 Unique on Control 3744 Hydroxymethylglutaryl-CoA synthase, mitochondrial precursor P22791 Unique on Control 3752 Keratin, type I cytoskeletal 19 Q63279 Unique on Control 3762 Fructose-1,6-bisphosphatase P19112 Unique on Control 4042 Aldehyde dehydrogenase 1A1 P51647 Unique on Control 3693 Dynein internediate chain 2, cytoplasmic Q62871 −2.0 2484 Keratin, type I cytoskeletal 21 P25030 −2.0 815 Serotransferrin precursor P12346 −2.1 979 Serum albumin precursor P02770 −2.1 1481 26S protease regulatory subunit 6B Q63570 −2.1 1481 Protein disulfide isomerase A6 precursor Q63081 −2.1 588 α-actinin 4 Q9QXQ0 −2.1 3840 Pyruvate carboxylase, mitochondrial precursor P52873 −2.1 3743 Keratin, type II cytoplasmic 19 Q63279 −2.2 568 10-formyltetrahydrofolate dehydrogenase P28037 −2.2 2260 Carbonyl reductase [NADPH] 1 P47727 −2.3 3765 Apolipoprotein A-IV precursor P02651 −2.3 956 Serum albumin precursor P08835 −2.4 3764 Acyl-CoA dehydrogenase, short/branched chain specific, mitochondrial P70584 −2.4 precursor 1770 Fructose-bisphosphate aldolase A P05065 −2.4 3901 Carnitine O-palmitoyltransferase II, mitochondrial precursor P18886 −2.4 3901 Glucokinase regulatory protein Q07071 −2.4

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Table 2 Continued Spot No. 1) Definition Accession No. 2) Difference 3) 3868 Tubulin β chain P04691 −2.4 445 ATP-citrate synthase P16638 −2.6 1328 Keratin, type II cytoplasmic 8 Q10758 −2.8 1328 Aldehyde dehydrogenase, mitochondrial precursor P11884 −2.8 952 Serum albumin precursor P08835 −3.0 3872 NADPH-cytochrome P450 reductase P00388 −3.2 3872 Heat shock 70 kDa protein 1/2 Q07439 −3.2 944 Serum albumin precursor P02770 −3.3 266 Elongation factor 2 P05197 −3.4 1694 Keratin, type I cytoskeletal 19 Q63279 −3.5 674 Heat shock protein HSP 90-β P34058 −3.7 2576 Plasma retinol-binding protein precursor P04916 −4.1 2600 Ferritin heavy chain P19132 −4.3 745 Keratin, type I cytoskeletal 21 P25030 −4.3 2603 Ferritin light chain P02793 −5.4 1) Spot no. was a unique number on 2D-gels for individual groups (no references). 2) Accession number from SWISS-PROT. 3) Positive values indicate up-regulation, whereas negative values indicate down-regulation by APAP, AD and TC exposures. transthyretin (P02767)]. These findings were well cor- tive enzyme that scavenges various peroxides and related with the known mechanism of APAP toxicity, prevents apoptotic cell death (Hockenbery et al., 1993; namely, glutathione (GSH) depletion and cell death by Fujii and Taniguchi, 1999). The decrease in GPX apoptosis (Mitchell et al., 1973; Nelson, 1990; Ray et expression results in an increased susceptibility of cells al., 1996; Ray and Jena, 2000). AD exposure revealed to oxidative damage. In addition, the down-regulation the effects on the pathway of lipid metabolism [apoli- of GPX was considered to be related to GSH depletion poprotein A-I (P04639), IV (P02651) and E (P02650), in hepatocytes, which has been shown in the case of and acyl-CoA synthetase (P18163), α-2-macroglobu- acetaminophen toxicity (Adamson and Harman, 1989; lin receptor-associated protein (Q99068), etc.], which Noriega et al., 2000). Other differentially expressed would lead to the phospholipidosis (Kasim et al., 1987, proteins, peroxiredoxin 1 (Q63716, PRX1) and perox- 1990; Honegger et al., 1993; Sirajudeen et al., 2002). iredoxin 2 (P35704, PRX2), were up-regulated by 24- TC exposure mainly had an effect on lipid/carbohy- hr exposure of all the hepatotoxicants. PRX1 and drate metabolism [ATP citrate lyase (P16638), aldehyde PRX2 are known as intracellular antioxidant proteins dehydrogenase 2 (P11884), transaldolase 1 (Q9EQS0), that protect cells against oxidative stress through redox etc.], which would induce the steatosis (Porokhniak et regulations (Hoffmann et al., 2002), by participation in al., 1987; Deboyser et al., 1989). the signaling transduction cascades and modulation of cell proliferation/differentiation (Kang et al., 1998). It Expression changes of oxidative stress-related pro- was also reported that PRX proteins could protect cells teins from apoptosis through the inhibition of c-Abl (Wen Toxicant-induced alterations of stress-related and Van Etten, 1997) and c-Myc (Mu et al., 2002) and proteins have been previously reported (Witzmann et the regulation of NF-κB(Jinet al., 1997) and TNF-α al., 1995, 1996). In the present study, several oxidative (Kang et al., 1998), and with a mechanism similar to stress-related proteins were affected by more than one that of Bcl-2 (Zhang et al., 1997). PRXs were presum- hepatotoxicant. The expression of glutathione peroxi- ably up-regulated by toxicant-induced stress to protect dase (P04041, GPX) was significantly decreased by hepatocytes. The decreased expression of GPX protein more than 2-fold after treatment with APAP and AD and the increased expression of PRX proteins observed for 24 hr (Fig. 1), whose expression change by APAP in this study were in agreement with previous findings; exposure was also detected in our previous study namely, the changes of both GPX and PRX1 gene reported (Kikkawa et al., 2005). GPX is an antioxida- expression by bromobenzene hepatotoxicity (Heijne et

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T. YAMAMOTO et al. al., 2003), and the changes of PRX2 protein expression Application of oxidative stress-related proteins to causing oxidative stress (Rabilloud et al., 2002). More- high throughput screening system of hepatotoxicity over, we observed effects on the expression of other The objective of this study was to address the oxidative stress-related proteins, such as the 60kDa alternative toxicity markers that would be more sensi- heat shock protein (P19226, HSP60), catalase (P04762), tive and reliable in comparison with conventional peroxiredoxin 6 (O35244), 78kDa glucose-regulated markers in order to apply the sufficient high through- protein (P06761, GRP78), heat shock cognate 71kDa put screening system of hepatotoxicity. The widely protein (P08109) and heat shock protein HSP90-β available conventional endpoints such as LDH release (P34058, HSP84), even though the changes were intended to show the degree of cell damage by loss of compound-specific. membrane integrity, and the change of oxidative stress markers with WST-1 assay would result from the direct responses derived from the cellular functions. Oxida- tive stress was generally induced by exposure to

Fig. 1. Relative changes of glutathione peroxidase (GPX) expression in primary cultured rat hepatocytes exposed to APAP (10 mM), AD (50 µM) and TC (500 µM) for 6 and 24 hr. Data are shown as means ± SD (N=3). *Statistically significant compared to control using Student’s t-test (p<0.05).

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Oxidative stress-related proteins for hepatotoxicity screening in rat hepatocytes. toxicants as well as by the external environment such Breen, K.J., Schenker, S. and Heimberg, M. (1975): as ROS (Reactive Oxygen Species), and resulted in cell Fatty liver induced by tetracycline in the rat. damage. Therefore, oxidative stress was probably Dose-response relationships and effect of sex. given to the effect to cellular functions before toxico- Gastroenterology, 69, 714-723. logical changes caused by compounds occurred. The Bugelski, P.J., Atif, U., Molton, S., Toeg, I., Lord, P.G. identified oxidative stress-related proteins, GPX, and Morgan, D.G. (2000): A strategy for pri- PRX1 and PRX2, were considered to be potential tox- mary high throughput cytotoxicity screening in icity biomarkers of hepatotoxicity that would be more pharmaceutical toxicology. Pharm. Res., 17, sensitive, reliable and mechanism-based. Recently, 1265-1272. PRX1 was shown to be induced in coordination with Dake, M.D., Madison, J.N., Manatogomery, C.K., heme oxygenase-1 (HO-1), which was also called heat Shellitoo, J.E., Hinchcliffe, W.A., Winkler, M.L. shock protein 32 (HSP32), under stress conditions in and Baindton, D.F. (1985): Electron micro- various cells and tissues (Immenschuh et al., 1995, scopic demonstration of lysosomal inclusion 2003; Nakaso et al., 2000; Nakahira et al., 2003; Bauer bodies in lung, liver, lymph nodes and blood et al., 2003). We did not identify HO-1 as differentially leucocytes of patients with amiodarone pulmo- expressed proteins within the successfully identified nary toxicity. Am. J. Med., 78, 506-512. proteins, but HO-1 should also be paid attention to as Deboyser, D., Goethals, F., Krack, G. and Roberfroid, an oxidative stress marker of hepatotoxicity. Further M. (1989): Investigation into the mechanism of quantitative confirmation of expression changes by tetracycline-induced steatosis: Study in isolated means of Western blotting and/or immunohistochemi- hepatocytes. Toxicol. Appl. Pharmacol., 97, cal analysis is needed, and also comparative investigations 473-479. of these proteins expression with in vivo study would Fautrel, A., Chesne, A., Guillouzo, G., Sousa, G., be required. In conclusion, the estimation of protein Placid, M., Rahmani, R., Braut, F., Pichon, J., biomarkers would be of great value for setting a high Hoellinger, H., Vintezou, P., Diarte, I., Melcion, throughput screening system to evaluate hepatotoxicity C., Cordier, A., Lorenzon, G., Benicourt, M., in the early stage of drug discovery. Vannier, B., Fournex, R., Peloux, A.F., Bichet, N., Gouy, D. and Cano, J.P. (1991): A multicen- REFERENCES tre study of acute in vitro cytotoxicity in rat liver cell. Toxicol. In Vitro, 5, 543-547. Adamson, G.M. and Harman, A.W. (1989): A role for Fountoulakis, M., de Vera, M.-C., Crameri, F., Boess, the glutathione peroxidase/reductase enzyme F., Gasser, R., Albertini, S. and Suter, L. (2002): system in the protection from paracetamol tox- Modulation of gene and protein expression by icity in isolated mouse hepatocyte. Biochem. carbon tetrachloride in the rat liver. Toxicol. Pharmacol., 38, 3323-3330. Appl. Pharmacol., 183, 71-80. Alden, C.L., Sagartz, J.E., Smith, P.F., Wilson, A.G., Fry, J.P., Garle, M.J., Hammond, A.H. and Hatfield, A. Bunch, R.T. and Morris, D.L. (1999): The (1990): Correlation of acute lethal potency with pathologist and toxicologist in pharmaceutical in vitro cytotoxicity. Toxicol. In Vitro. 4, 175- product discovery. Toxicol. Pathol., 27, 104- 178. 106. Fujii, J. and Taniguchi, N. (1999): Down regulation of Bauer, I., Rensing, H., Florax, A., Ulrich, C., Pistorius, superoxide dismutases and glutathione peroxi- G., Redl, H. and Bauer, M. (2003): Expression dase by reactive oxygen and nitrogen species. pattern and regulation of heme oxygenase-1/ Free Rad. Res., 31, 301-308. heat shock protein 32 in human liver cells. Gad, S.C. and Weil, C.S. (1994): Statistics for toxicol- Shock., 20, 116-122. ogists. In Principles and Methods of Toxicology Boess,F.,Kamber,M.,Romer,S.,Gasser,R.,Muller, (Hayes, A.W., ed.), 3rd ed., pp. 221-274, Raven D., Albertini, S. and Suter, L. (2003): Gene Press, New York. expression in two hepatic cell lines, cultured pri- Heijne, W.H.M, Stierum, R.H., Slijper, M., van mary hepatocytes, and liver slices compared to Bladeren,P.J.andvanOmmen,B.(2003):Toxi- the in vivo liver gene expression in rats: Possible cogenomics of bromobenzene hepatotoxicity: A implications for toxicogenomics use of in vitro combined transcriptomics and proteomics systems. Toxicol. Sci., 73, 386-402. approach. Biochem. Pharmacol., 65, 857-875.

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