Ih

AMa

b

c

d

e

f

a

ARA

KZHIq

1

gKcTap

ri(emrmi

0h

Experimental and Toxicologic Pathology 65 (2013) 809– 816

Contents lists available at SciVerse ScienceDirect

Experimental and Toxicologic Pathology

jo ur nal homepa ge: www.elsev ier .de /e tp

dentification of proteins related to early changes observed in Humanepatocellular carcinoma cells after treatment with the mycotoxin Zearalenone

mel Chatti Gazzaha,b, Luc Camoinc,d,e,f, Salwa Abida, Chayma Bouaziza,oncef Ladjimib, Hassen Bachaa,∗

Laboratory of Research on Biologically Compatible Compounds, Faculty of Dentistry, Rue Avicenne, Monastir, 5000, TunisiaLaboratory of Genetic and Cellular Biology, CNRS, UMR 8159, Versailles St-Quentin University, 45 Avenue des Etats-Unis, Versailles 78035, FranceInserm,U1068, CRCM, Marseille Protéomique, Marseille, F-13009, FranceInstitut Paoli-Calmettes, Marseille, F-13009, FranceAix-Marseille Univ, F-13284, Marseille, FranceCNRS, UMR7258, CRCM, Marseille, F-13009, France

r t i c l e i n f o

rticle history:eceived 9 July 2012ccepted 22 November 2012

eywords:earalenone (ZEA)

a b s t r a c t

Zearalenone (ZEA) is a mycotoxin produced by some Fusarium species. ZEA often occur as a contaminantin cereal grains and animal feeds. Human exposure occurs by ingestion of mycotoxin-contaminatedproducts and can cause serious health problems. It was established that this mycotoxin have an hepato,haemato, immuno and genotoxic properties (Maaroufi et al., 1996; Lioi et al., 2004). While most ZEAtoxic effects have been quite well investigated, more studies are required to elucidate its mechanisms

epatocarcinoma cells (HepG2)sobaric tagging for relative and absoluteuantification (iTRAQ)

of toxicity. In order to better understand the molecular mechanisms involved in ZEA toxicity, we used aproteomic approach, to assess the early changes in protein expression initiated by ZEA in HepG2 cells.Our results showed that, after 8 h of exposure, cells were still viable and showed a significant change in anumber of proteins involved in diverse cellular processes. These changes may provide the early affectedfunctions and yield further insight into mechanisms underlying the involvement of mycotoxin-induceddiseases.

© 2012 Elsevier GmbH. All rights reserved.

. Introduction

Mycotoxins are secondary metabolites produced by three mainenera of fungi (Aspergillus, Fusarium and Penicillium) (Bennett andlich, 2003). Human exposure occurs by ingestion of mycotoxin-ontaminated products and can lead to serious health problems.he pathologies in humans and animals as well as economical lossre important. Therefore, mycotoxins have become a worldwidereoccupation (CAST, 2003).

Zearalenone (ZEA) is one of the most widely distributed Fusa-ium mycotoxins which are encountered at high incidence in manymportant corps intended for human and animal consumptionsScudamore and Patel, 2000; Engelhardt et al., 2006; MacDonaldt al., 2005; Tabuc et al., 2009). ZEA is a non-steroidal, estrogenicycotoxin and its role as a mammalian endocrine disrupter is being

ecognized with effects in both males and females of different ani-al species (Ryu et al., 2002). Moreover, ZEA was found hepatotoxic

t induces adverse liver lesions (Maaroufi et al., 1996; Conkova et al.,

∗ Corresponding author. Tel.: +216 73 42 55 50; fax: +216 73 42 55 50.E-mail address: hassen.bacha@fmdm.rnu.tn (H. Bacha).

940-2993/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.etp.2012.11.007

2001). It is equally haemato-toxic and causes several alterations ofimmunological parameters (Maaroufi et al., 1996). The mechanismswhereby all these effects are induced are still not totally understood

Global techniques such as proteomics provide effective strate-gies and tools for toxicological studies and are regarded as apowerful tool to investigate the cellular responses to toxicants(Dowling and Sheehan, 2006). In contrast to conventional bio-chemical methods, the proteomic approach offers great potentialin identifying proteins involved in the response of organismsto contaminants through massive comparison of protein expres-sion profiles and helps to identify novel and unbiased biomarkersrelated to toxicity. The proteomic approaches have proved to bevaluable in identifying early responses to this toxin and, con-comitantly, identifying the mechanisms of toxicity involved in theeffects of mycotoxin on organisms including humans.

Recently, in our laboratory, several studies have shown thatZEA is cytotoxic through inhibition of cell viability in different cul-tured cell lines (Abid-Essefi et al., 2004; Hassen et al., 2005; El

Golli et al., 2006). A further set of experiments has demonstratedthat ZEA induced several genotoxic effects in vitro and in vivo(Ghédira-Chékir et al., 1998; Abid-Essefi et al., 2004; Ouanes et al.,2003, 2005; Hassen et al., 2007; Ayed-Boussema et al., 2007). This

8 Toxico

ctZ

dptrioTtrbn(

utet

fZari2mc

cep

2

2

wT1S

mcvma

2

bccu(5swetd

10 A.C. Gazzah et al. / Experimental and

onsiderable cytotoxic and genotoxic potential could be related tohe substantial degree of intracellular oxidative stress generated byEA.

It is well known that in response to genotoxic stress and DNAamage, the cell may undergo an intricate network of multipleathways including the arrest of the cell cycle progression andhe activation of repair mechanisms. Then, in case of efficient DNAepair, cells activate the cycle and take up normal cell growth,f not, cell activate the apoptotic cascade leading to cell deathr enhance neoplastic transformation (Zhou and Elledge, 2000).hus, the apoptotic machinery is an essential element, protectinghe integrity of multicellular organisms and allowing the selectiveemoval of damaged cells. In addition, the induction of apoptosisy DNA damage agent is an important protective mechanism fromeoplastic transformation, thus escaping the risk of carcinogenesisSitailo et al., 2002).

It should be noted that a large number of studies have beenndertaken to investigate the induction of apoptosis by the myco-oxin ZEA both in vivo and in vitro (Abid-Essefi et al., 2004; Bouazizt al., 2009). However, other investigations have shown no apop-otic activity in cultured cells (Lioi et al., 2004).

The human hepatoma HepG2 cells line was chosen as a modelor multiple reasons. Hepatocytes constitute a target system forEA toxicity and the liver is a prominent site for metabolizationnd for intense oxidative processes in the body. HepG2 cells areeported to retain many of the properties of primary liver cells,ncluding the metabolic activation (Lu and Huang, 1994; Feng et al.,002). Furthermore, it has been widely described that ZEA and itsetabolites competitively bind to estrogen receptors in different

ell models including HepG2 cells (Breithofer et al., 1998).In this study, we used a proteomic approach, to assess the early

hanges in protein expression initiated by ZEA in HepG2 cells. Wemployed the fast and reliable iTRAQ technique, which is based oneptide labeling with stable heavy isotopes.

. Methods

.1. Cell cultures and treatment

HepG2 cells, derived from a human hepatocellular carcinomaere cultured in Dulbecco’s modified Eagle’s medium (DMEM).

he medium was supplemented with 10% foetal calf serum (FCS),% l-glutamine (200 mM), 1% of mixture Penicillin (100 IU/ml) andtreptomycin (100 �g/ml) at 37 ◦C in an atmosphere of 5% CO2.

ZEA were obtained from Sigma Chemical Co. (St. Louis, MO), thisycotoxin was dissolved in pure ethanol. To obtain the studied

oncentrations in the cell culture media, the mycotoxin treatmentolume was negligible and represents about 0.025% of the totaledium volume. For this reason, we have chosen untreated cells

s control.

.2. Viability assay

For the determination of cell viability, we have measuredoth concentrations and time exposure effects. HepG2 cells wereultured in 24 well multidishes and treated with different con-entrations of ZEA for 24 h. Then other cells were treated with anique ZEA concentration (100 �M), at different treatment times2, 4, 8, 24, 30, 48 and 60 h) separately. Then, they were incubated

min at 37 ◦C with 0.2 �g/ml fluorescein diacetate (FDA) (Poly-ciences), a non fluorescent compound which becomes fluorescent

hen cleaved by esterases in living cells as described by Rincheval

t al. (2002). Finally cells were analyzed with XL3C flow cytome-er (Beckman-coulter, France), living cells are FDA positive whereasead cells are FDA negative.

logic Pathology 65 (2013) 809– 816

2.3. Apoptosis status

Flow cytometric measurements were performed using a XL3Cflow cytometer (Beckman-Coulter). Fluorescence was induced bythe blue line of an argon ion laser (488 nm) at 15 mW. Analyseswere performed on 106 HepG2 cells placed in 24-multiwell plates,incubated with ZEA (100 �M), at the different treatment times (2,4, 8, 24, 30, 48 and 60 h). To distinguish apoptotic versus necroticcells, propidium iodide (PI, Sigma) staining was also performed. PIspecifically penetrates into necrotic cells after loss of their plasmamembrane integrity. After mycotoxin treatments, the media fromthe culture dishes containing late apoptotic cells were kept incentrifuge tubes. The adherent HepG2 cells (containing living andearly apoptotic cells) were detached using trypsin, pooled with thecorresponding media, centrifuged and resuspended in completemedium at a time of 1 × 106 cells/ml. Cells were then loaded with0.1 �M DiOC6(3) by incubation for 30 min at 37 ◦C. The mitochon-drial membrane potential (�� m) was assessed by the retentionof DiOC6(3) (Molecular Probes, Cergy-Pontoise, France). After thisincubation, we add 5 �g/ml PI for 5 min prior to flow cytometryanalysis. Apoptotic cells correspond to low DiOC6(3) and low PIstaining cells (noted DiOC6(3)/PI-).

2.4. Determination of caspase-3 activity

The cells were cultured (106 cells/ml) in 25 cm2 flasks, totalvolume 5 ml of medium per flask, in the absence or the pres-ence of ZEA (100 �M) at 37 ◦C at different treatment times (0, 2,4, 8, 24, 30, 48 and 60 h). Cells were harvested and centrifugedat 5000 g and the pellet was incubated in lysing buffer (Hepes0.5 M, 0.5% Nonidet-P40, 1 mM PMSF, 1 mg/ml aprotinin, 2 mg/mlleupeptin, pH 7.4) for 10 min and then centrifuged at 5000 g for20 min. Supernatants were retrieved and aliquots corresponding to50 mg total protein along with acetylated tetrapeptide (Ac-DEVD)substrate labeled with the chromophore p-nitrianiline (pNA) wereadded in a 96-well microplate. In the presence of active caspase-3, cleavage and release of pNA from the substrate occurs. FreepNA produces a yellow color detected by a spectrophotometerat 405 nm. A standard curve was realized in order to determinethe correspondence between OD and pNA concentration, then theresults were expressed as caspase-3I specific activity (pmol pNAper h/mg protein) calculated as indicated by the manufacturers(Promega, France).

2.5. Protein preparation for iTRAQ experiments

In this study, the iTRAQ method was used to identify changein protein in hepatocarcinoma cells after treatment with ZEA. Thehepatocarcinoma cells (HepG2) were chosen as a model system. Todetect early changes at the proteome level, concentration (100 �M)of this mycotoxin were added to the cells and an incubation periodof 8 h was chosen to avoid an apoptotic effect with longer treat-ment.

Cells at 70% confluency were incubated for 8 h with mediumalone (control) or in addition to 100 �M ZEA. After incubation, cellswere washed with ice-cold PBS, scrapped and centrifuged (for 5 minat 1000 × g). The cell pellet was resuspended in a cell lysis buffercontaining 250 mM NaCl, 5 mM EDTA, 50 mM Hepes, 0.1% NP40,pH = 7. The lysate was centrifuged at 6000 × g for 10 min, followedby BioRad Protein assay for measuring protein concentration. The

volume corresponding to 100 �g of protein were precipitated using4 volume of acetone, and incubated overnight at −200 C. The Pro-tein content was recovered by centrifugation at 6000 × g for 5 minand stored at −80 ◦C before use.

Toxico

2

w((satcfl1v

2

acperEfMWrdatBF1apatv

2

(p(PsaaDasd15t

lfMftm

A.C. Gazzah et al. / Experimental and

.6. Peptide labeling

Precipitate corresponding to 100 �g of protein for each sampleas dissolved in 25 �l of 500 mM tri-ethyl ammonium bicarbonate

Sigma) including 0.1% SDS. Samples were then reduced using (tris2-carboxyethyl) phosphine), alkylated with methyl methanethio-ulfonate, digested with trypsin, and labeled with iTRAQ reagentsccording to the iTRAQ Reagents Application Kit (Applied Biosys-ems). The ratio enzyme/substrate was 1/10 and the pH washecked to assure a complete digestion. Samples were labeled asollows with a different isobaric tag. The controls samples wereabeled with 114, 115 and the two “treated samples” with 116 and17 labels. Labeled samples were then combined and dried in aacuum concentrator.

.7. Strong cation exchange fractionation

In order to remove SDS, excess unbound iTRAQ reagent,nd to decrease sample complexity before reversed-phase nano-hromatography and tandem mass spectrometry (nLC–MS/MS),eptides were washed and fractionated using a strong cationxchange chromatography. In brief, dried peptides (400 �g) wereesuspended in 1 ml of 5 mM KH2PO4 and 20% acetonitrile (Carlorba), pH 2.8 (buffer A). 80% of the peptide mixture was then pre-ractionated using a Poly SULFOETHYL A column (PolyLC, Columbia,

D) 5 �m of 200 mm length × 2.1 mm I.D., 300 A pore size, on aaters model 625 HPLC system (Waters) with a constant flow

ate of 0.2 mL/min. The column was first ran under isocratic con-itions with 100% Buffer A for 30 min. Then a 19 min gradient waspplied to raise the concentration of Buffer B to 63%, followed byhe following steps: a plateau at 63% during 5 min, and 100% inuffer A for 5 min. The chromatogram was monitored at 210 nm.ractions were collected every 30 s and pooled together to obtain0 final fractions with approximately equal amounts of peptidesccording to the average UV absorbance intensity of fractions (sup-osedly 35 �g per pool). For high salt concentration fractions, andditional step was used to desalt fractions using C18-Sep-Pak car-ridge (Waters). These resulting pooled fractions were dried in aacuum concentrator and stored at −20 ◦C prior to nLC–MS/MS.

.8. Reverse-phase nano liquid chromatography fractionation

Each dried SCX peptide fraction was redissolved in 0.1% TFAFluka) and 10% acetonitrile (Carlo Erba), and an evaluated 2 �gortion was injected twice to an Ultimate 3000 nano-HPLCDionex). Peptides were purified and first concentrated on a C18ep Map pre-column from Dionex (0.3 mm I.D. × 5 mm, 100 A poreize, 3 �m particle size) at a flow rate of 30 nl/min in 0.1% TFAnd 2% acetonitrile (buffer A). Subsequently, peptides were sep-rated on a C18 PepMap 100 analytical reverse phase column fromionex (75 �m I.D. × 150 mm, 100 A pore size, 3 �m particle size)t a flow rate of 300 nl/min. After equilibration in 7% buffer B (20%olution A mixed vol/vol with 80% acetonitrile) a multi slope gra-ient started 3 min after the injection signal as follows: 16% B at4 min post injection, 19% at 22 min, 23% at 25 min, 32% at 51 min,0% at 65 min and a 95% plateau from 69 to 79 min before a returno initial conditions for 16 min.

Fractionation was done using the Probot automated fraction col-ector (Dionex). Fraction collection started after an 18 min delayollowing the injection signal and was performed directly on the

ALDI target (blank plate). Fractions were collected every 10 sor a total of 384 spots per fraction. Eluent and matrix solu-ion were mixed on target cyano-4-hydrocinnamic acid (CHCA)

atrix was dissolved at 2 mg/ml in 70% acetonitrile containing

logic Pathology 65 (2013) 809– 816 811

0.1% TFA and 110 �M glu-fibrinopeptide-B for internal calibration(m/z = 1570.677).

2.9. MS spectrum acquisition

Offline mass spectrometry analyses were done using a 4800MALDI-TOF-TOF mass spectrometer (Applied Biosystems). Spectraacquisition and processing were performed using the 4000 seriesexplorer software (Applied Biosystems) version 3.5.28193 build1011 in positive reflectron mode at fixed LASER fluency with lowmass gate and delayed extraction. External plate calibration wasperformed using 4 calibration points spotted throughout the plate,additional internal calibration of spectra was obtained with theconcomitant measurement of the co-deposited Glu-fibrinopeptide.For each fraction, steps of 50 spectra in the range of 850–4000 Dawere acquired at a 200 Hz LASER shot frequency. Five hundred spec-tra per sample were summed and processed to obtain monoisotopicvalues from isotopes clusters with a raw spectra signal to noise ratioof 20.

2.10. Tandem MS and data-dependant tandem MS

In each MS spectrum generated from each nLC fraction, the8 most abundant peaks were selected for fragmentation startingwith the least abundant. A cut off was applied at a minimum s/n of15. Neighboring precursors within 200 resolution were excluded.1000 MS/MS spectra per precursor were summed by increment of50 and were subjected to baseline subtraction and Savitsky-Golaysmoothing (with a polynomial order of 4 and 24 point across peak).

2.11. Protein identification

All acquisitions were combined and protein identification andquantification were carried out using the ProteinPilot software ver-sion 2.0.1 (Applied Biosytems, MDS-Sciex, Foster City, CA). Thesearch was performed against the Human protein database fromthe International Protein Index (IPI) version 3.38 concatenatedwith a contaminant protein database. Data was processed with thefollowing criteria; trypsin cleavage specificity, carbamidomethy-lated cysteins, biological modifications for the ID focus settings and“thorough” search effort. A protein was considered to be signifi-cantly identified when 2 or more high confidence (>95%) uniquepeptides were assigned, the protein identification had to have a95% confidence (unused protein score >1.3), and the iTRAQ quan-tification fold difference p value<0.05. Each protein reported wasidentified on the basis of several peptides.

3. Results

3.1. Effect of ZEA on HepG2 cells

Exposure of HepG2 cells to ZEA leads to reduced viability. Thuscytotoxic effect of ZEA on HepG2 cells was measured by FDAassay. Results of this measurement indicate that mycotoxin treat-ment causes a marked decrease of cell viability in a dose andtime-dependent manner. IC50 value determined after 24 h of celltreatments was about 250 �M (Fig. 1A). When, HepG2 cells wereexposed to 100 �M of ZEA for different treatment times (2, 4, 8,24, 30, 48 and 60 h), we observed an inhibition of cell proliferationin a time-dependent manner (Fig. 1B). A clear increase of percent-age of apoptotic cells was observed 30 h after treatment indicatingapoptosis at 100 �M of ZEA treatment (Fig. 2A).

Therefore, the whole cell lysates were prepared and the activityof caspase-3, which is a key executioner of apoptosis. An increasein caspase-3 activity was found in HepG2 cells as compared to thecontrol treated cells in response to ZEA. This activity increased

812 A.C. Gazzah et al. / Experimental and Toxicologic Pathology 65 (2013) 809– 816

Fig. 1. (A) Percentage of cell viability (FDA+) after exposure of HepG2 cells to ZEA atthe indicated concentrations for 24 h as measured by flow cytometry. (B) Percentageof cell viability (FDA+) after exposure to 100 �M ZEA at different times points (0, 2,4o

f9

3

wet

Fig. 2. (A) Percentages of apoptotic HepG2 cells (Dioc6(3)-/pi-cells), after ZEA treat-ment (100 �m) at different exposure times as assessed by flow cytometry. Thisgraph is representative of three independent experiments as assessed by stu-dent’s test (p < 0.05). (B) Measurement of caspase-3 (DEVD-pNa cleavage) activity in

TD

, 8, 24, 30, 48 and 60 h) as measured by flow cytometry. This graph is representativef three independent experiments. Data are expressed as the mean ± S.D.

rom 0.083 ± 0.026 pmol pNA/h/�g of protein in control cells to.6 ± 2.56 at 30 h of exposure to ZEA (Fig. 2B).

.2. Analysis of ZEA-induced changes of the HepG2 proteome

To elucidate these effects at the protein level, crude extractsere prepared from ZEA treated and untreated HepG2 cells. Both

xtracts were then digested with trypsin and the generated pep-ides were labeled with the iTRAQ reagent. This highly complex

able 1istribution of the 67 identified proteins in HepG2 according to their biological function.

Function Molecules

Cellular function and maintenance HSPD1, MSN, HSPA1A, ALDOA, CKEG:4869), DSP, METAP2

Cellular compromise MSN, HSPD1, GPD2, HSP90AA1, HSUMO2, HSPA1A,TAGLN2, S100A4(includes EG:6741)

Post-translational modification HSPD1, P4HB, SUMO2, APOA1, PRDEG:10988), SLC3A2, ERP29, YWHA

DNA replication, recombination and repair RAN, TUBB,RAN, METAP2, NPM1, HCellular assembly and organization MSN, SCAMP3, CAP1, CKB, HSPA1B

BASP1, PFN1, HSPA5, KRT9, PLEC1(includes EG:10988),YWHAZ, SERP

Molecular transport MSN, SCAMP3, APOA1,GPD2, PRDXEG:4869), ARCN1, PPP1CA, ERP29,

Cell morphology HSPD1, CAP1, TPM3, HSP90AA1, APFN1, TMSB4X

Cellular growth and proliferation HSPD1, PKM2, ILF2 (includes EG:3HSPA5, PLEC1, APOA1, TPM3, SFRSS100A6

Cell cycle TUBB, DARS, NPM1 (includes EG:4RAN, CSE1L

Lipid metabolism SLC3A2, APOA1, STIP1, MAP4, PRDCarbohydrate metabolism PPP1CA, APOA1, GPD2, ALDOA, PG

HepG2 cells exposed to ZEA (100 �M) at different treatments times. These resultscorrespond from at least three independent experiments and were expressed asmeans ± S.D.

peptide mixture was analyzed by MALDI technique. The detectionand the identification of proteins by ProteinPilot were further fil-tered as follows: minimum of two peptides for the calculation ofiTRAQ ratio; ratio with p-values lower than 0.05.

The cellular distribution of the 67 different proteins identified is

depicted in Table 1. The differentially expressed proteins were func-tionally categorized, based on their known biological functions intoten categories namely Cellular Function and Maintenance, CellularCompromise, Post-Translational Modification, Cellular Assembly

p-Value

B, HSP90AA1, HSP90AB1, HSPA1B, TUBB, NPM1 (includes 8.28E−07

SP90AB1, HSPA1B,TUBB, PPP1CA, MAP4, KRT18, GPSN2, PLEC1,, PRDX6, ANXA5,SUB1, STIP1,SERPINH, RAN,GOLPH3, SSB

4.37E−07

X6, HSP90AA1, HSP90AB1, PPP1CA, METAP2 (includesZ, PRDX4, CCT4, HSPBP1,HSPA5

2.64E−06

SPA1A, HSPA1B, RAD23B, NCL, NCL, RAD23B, SUB1 6.35E−05, TUBB, DSP, NPM1(includesEG:4869), TMED10, MAP4, KRT18,

, APOA1, HSPA1A, TPM3, ALDOA, S100A4, ANXA5, METAP2INH1, RAN, SEC23A, M6PRBP1,TMSB4X

1.9E−04

5, SLC16A3, PRDX6, SLC16A1, ANXA5, NPM1 (includesTMED10, SLC3A2, YWHAZ, RAN, SEC23A

3E−04-4

NXA5, PPP1CA, TMED10, YWHAZ, SERPINH1, MAP4, SEC23A, 9.33E−04-

608),EBNA1BP2, NPM1 (includes EG:4869), DSP, PPP1CA, PFN1,1, PGK1, S100A4, LDHA, LDHB, CDC37, SLC3A2, SERPINH1,

8.01E−03

869), METAP2 (includes EG:10988), HSPA1A, MAP4, S100A4, 4.68E−03

X6, HSP90AB1, IDI1, GPSN2, ANXA5 2.2E−03K1, PRDX6, PKM2, PGAM1, PPA1, ANXA5 2.85E−04

A.C. Gazzah et al. / Experimental and Toxicologic Pathology 65 (2013) 809– 816 813

Table 2Regulated proteins for cells treated with 100 �M.

Gene name Proteins name Accession number Fold change p value Fold change

ALDOA 45 kDa protein IPI00796333 0.018 1.06CDC37 Hsp90 co-chaperone Cdc37 IPI00013122 0.451 1.12DSP 40S ribosomal protein S3 IPI00744851 0.020 1.09HSP90AA1 Heat shock protein 90 kDa alpha (cytosolic), class A member 1

isoform 1IPI00382470 0.000 1.06

HSP90AB1 Heat shock protein HSP 90-beta IPI00414676 0.003 1.04HSPA5 HSPA5 protein IPI00003362 0.027 1.04HSPA1B; HSPA1A Heat shock 70 kDa protein 1A IPI00845339 0.000 1.19IDI1 Isopentenyl-diphosphate delta isomerase IPI00220014 0.032 1.11LDHA Isoform 1 of l-lactate dehydrogenase A chain IPI00217966 0.000 1.08LDHB l-lactate dehydrogenase B chain IPI00219217 0.038 1.06PFN1 Profilin-1 IPI00216691 0.014 1.07PGK1 Phosphoglycerate kinase 1 IPI00169383 0.015 1.08STIP1 cDNA FLJ76863, highly similar to Homo sapiens

stress-induced- phosphoprotein 1 (Hsp70/Hsp90-organizingprotein) (STIP1), mRNA

IPI00871856 0.010 1.05

TMSB4X TMSB4X protein (Fragment) IPI00816008 0.001 1.21YWHAZ 14-3-3 protein zeta/delta IPI00021263 0.039 1.071ANXA5 Uncharacterized protein ANXA5 (Fragment) IPI00872379 0.006 1.095CAP1 Adenylyl cyclase-associated protein IPI00639931 0.001 1.191MSN Uncharacterized protein MSN (Fragment) IPI00872814 0.000 1.099NPM1 Isoform 1 of Nucleophosmin IPI00549248 0.003 1.061P4HB Protein disulfide-isomerase precursor IPI00010796 0.024 1.041S100A4 Protein S100-A4 IPI00032313 0.000 1.139PLEC1 Plectin 1 isoform 6 IPI00186711 0.007 1.168PRDX6 Peroxiredoxin-6 IPI00220301 0.001 1.132HSPA1A Heat shock 70 kDa protein 1A IPI00845339 0.0000 1.29RAN 26 kDa protein IPI00795671 0.007 1.083S100A6 Protein S100-A6 IPI00027463 0.035 1.064SFRS1 Isoform ASF-1 of Splicing factor, arginine/serine-rich 1 IPI00215884 0.032 1.166SSB Lupus La protein IPI00009032 0.030 1.081SUB1 Activated RNA polymerase II transcriptional coactivator p15 IPI00221222 0.000 1.119TAGLN2 24 kDa protein IPI00647915 0.015 1.127TPM3 Isoform 2 of Tropomyosin alpha-3 chain IPI00218319 0.002 1.111CCT4 cDNA FLJ77660 IPI00873222 0.013 1.064S100A4 Protein S100-A4 IPI00032313 0.000 1.139TPI1 Isoform 1 of Triosephosphate isomerase IPI00465028 0.008 1.067PPP1CA Serine/threonine-protein phosphatase PP1-alpha catalytic

subunitIPI00550451 0.006 1.124

EBNA1BP2 Probable rRNA-processing protein EBP2 IPI00745955 0.024 1.194MAP4 Microtubule-associated protein 4 isoform 1 variant (Fragment) IPI00745518 0.012 1.121PPA1 Inorganic pyrophosphatase IPI00015018 0.033 1.163DSP 40S ribosomal protein S3 IPI00744851 0.049 0.107SLC16A3 Monocarboxylate transporter 4 IPI00006666 0.003 1.128TUBB Tubulin beta chain IPI00011654 0.022 1.142BASP1 Brain acid soluble protein 1 IPI00299024 0.042 1.081APOA1 Apolipoprotein A-I precursor IPI00021841 0.008 1.167GOLPH3 Golgi phosphoprotein 3 IPI00005490 0.009 1.240M6PRBP1 Isoform B of Mannose-6-phosphate receptor-binding protein 1 IPI00303882 0.017 1.162SLC16A1 Monocarboxylate transporter 1 IPI00024650 0.003 1.194ARS Aspartyl-tRNA synthetase, cytoplasmic IPI00216951 0.006 −1.17HSPD1 60 kDa heat shock protein, mitochondrial precursor IPI00784154 0.020 −1.06PGAM1 HCG 2015138 Phosphoglycerate mutase 1 IPI00549725 0.047 −1.12PKM2 Isoform M2 of Pyruvate kinase isozymes M1/M2 IPI00479186 0.010 −1.07PRDX4 Protein IPI00639945 0.031 −1.10CSE1L Isoform 3 of Exportin-2 IPI00219994 0.049 −1.14IPO5 Importin-5 IPI00217564 0.005 −1.05KRT18 Keratin, type I cytoskeletal 18 IPI00554788 0.000 −1.13SERPINH1 Serpin H1 precursor IPI00032140 0.039 −1.07CKB Creatine kinase B-type IPI00022977 0.000 −1.09METAP2(includes EG:10988) Methionine aminopeptidase IPI00789396 0.037 −1.40SUMO2 10 kDa protein IPI00790142 0.000 −1.16HSPD1 60 kDa heat shock protein, mitochondrial precursor IPI00784154 0.020 −1.06SEC23A Protein transport protein Sec23A IPI00017375 0.007 −1.95TMED10 Transmembrane emp24 domain-containing protein 10

precursorIPI00028055 0.013 −1.27

ERP29 Endoplasmic reticulum protein ERp29 precursor IPI00024911 0.040 −1.16GPSN2 Isoform 1 of Synaptic glycoprotein SC2 IPI00100656 0.008 −1.10ILF2 Interleukin enhancer binding factor 2 variant (Fragment) IPI00872107 0.007 −1.14GPD2 Isoform 1 of Glycerol-3-phosphate dehydrogenase,

mitochondrial precursorIPI00017895 0.030 −1.23

SLC3A2 solute carrier family 3 (activators of dibasic and neutral aminoacid transport), member 2 isoform c

IPI00604710 0.031 −1.06

DARS Aspartyl-tRNA synthetase, cytoplasmic IPI00216951 0.006 −1.17

8 Toxico

altus

sFiH

ocH

rMlcisciucl

4

rTttmtohs

o(d

cc4piwaTm

p(tctlmr(to

14 A.C. Gazzah et al. / Experimental and

nd Organization, Molecular Transport, Cell Morphology and Cel-ular Growth and Proliferation (Table 1). It was clearly noticedhat 46 proteins out of those 67 differentially expressed werep-regulated; however 21proteins were down-regulated. Table 2hows up- and down-regulated proteins.

Our results showed that after 8 h exposure to ZEA, cells weretill viable with significant changes in protein level. The Cellularunction and Maintenance-related proteins that were up regulatedn the ZEA-treated cells included a number of chaperones proteinsp70 (HspA1B) and Hsp90 (Hsp90AA1 and Hsp90AB1).

Simultaneously, we found that the anti-apoptotic protein nucle-phosmin (NPM) and protein 14-3-3 zêta/delta (YWHAZ)), wereo-up-regulated, together with the decrease of other members ofSP family (Hspd1), which are considered to be anti-apoptotic.

The alterations in the expression levels of some cytoskeletonegulatory proteins in this study such as profilin, tropomyosin,icrotubule-associated protein 4 (MAP4), TMSB4X and tubu-

in (TUBB) are associated with the structure/functioning of theytoskeleton. Another observation corresponding to others proteinnvolved in the regulation of various important cellular functionsuch as cell growth, cell–cell communication, energy metabolism,ontraction, and cell motility. Meanwhile, many enzymes involvedn cell metabolism such as glycolysis and lipidic metabolism werep regulated in ZEA treated HepG2 cells such as the important gly-olytic enzyme phosphoglycerate kinase (PGK) and sub-units ofactate dehydrogenase (LDHA/B)

. Discussion

This is the first study which investigated the early proteomicesponse of hepatic cells (HepG2) in response to ZEA exposure.he protein profiles investigation may help to identify new geneshat are altered by ZEA treatment and provide new data abouthe toxic mechanisms of mycotoxin-induced diseases. Risk assess-

ent of mycotoxin can be evaluated by its biological response;his response allows us to provide a theoretical basis for researchn the mechanism of action of this toxin. A biomarker may alsoelp to detect mycotoxin contamination more accurately andpecifically.

In this study, cells have been exposed for 8 h to 100 �M ZEA (95%f viable cells). Our results showed a clear increase of apoptotic cellsDiOC6(3)-/IP-) after 30 h of treatment, indicating a late apoptoticeath.

A total of 982 proteins were detected with a limitation to 95%onfidence. More specifically, 67 proteins were modified in HepG2ells treated with mycotoxin compared to control. Among them6 are up-regulated and 21 are down-regulated (Table 2). Theseroteins were implicated in different biological functions. Follow-

ng analysis with ingenuity pathway software, several proteinsere found to be involved in a variety of cellular processes, such

s cellular compromise, cellular function and maintenance, Post-ranslational Modification, cellular assembly and organization, cellorphology and Molecular transport.Our study showed the deregulation of a number of chaperones

rotein. Indeed, a significant induction of Hsp70 (HspA1B), Hsp90Hsp90AA1 and Hsp90AB1) and Hsps in HepG2 cells after exposureo ZEA. We have demonstrated that Hsp70 was over expressed asonfirmed by Chatti Gazzah et al. (2010). It have been demonstratedhat, a maximum Hsp70 induction is often related to early cellu-ar toxicity (Aït-Aïssa et al., 2003). In addition, these chaperones

ay assist the folding of proteins into appropriate conformations,

e-fold misfolded proteins, and previously aggregated proteinsGlover et al., 1998; Hendrick and Hartl, 1993). Furthermore, post-ranslational modifications, proper folding and oligomerizationf the secretory proteins in the endoplasmic reticulum (ER) are

logic Pathology 65 (2013) 809– 816

essential prerequisites for their recruitment into the transport vesi-cles heading toward the cell exterior. It has been reported thatstress induced generation of misfolded proteins constitutes thetrigger signal that up regulates the heat shock response (Hendrickand Hartl, 1993). The magnitude of the ER reorganization uponmassive accumulation of unfolded proteins invokes not only ER-specific but also general cellular stress mechanisms (Bush et al.,1997; Wyttenbach et al., 2000). The misfolded and aggregatedproteins then trigger the unfolded protein response (UPR). Underless severe situations; UPR results in the reduction of generalprotein synthesis and selectively activates the expression of pro-teins facilitating the chaperone function. Indeed, the heat shockresponse is induced in a protective mechanism in response togeneration of abnormal proteins and alteration of cellular func-tions. It should be considered as a factor facilitating survival ofcells with damage (Frydman, 2001; Young et al., 2004). Damag-ing signals allow Hsp70 to take on new roles to provide defensein the cell. It has been showed that Hsp70 induction by certainenvironmental chemicals is generally correlated with early cyto-toxic events and is a secondary consequence of damages that affectcellular integrity (Singh et al., 2009). This suggest that Hsp maybe an important mediator in maintaining survival in “stressed”cells.

Hsp90AA1 and Hsp90AB1 are others members of up regulatedproteins in this study. The essential functions of Hsp90 changeaccording to the needs of the cell. Hsp90 can facilitate the proteinsynthesis and early folding events. It was found to facilitate proteinsecretion and transport, as well as cell cycle regulation. HSP90 mayact in many aspects of vesicular transport and protein traffickinginvolving the ordered assembly and disassembly of large multi-subunit complexes. Because proliferation is critically dependenton membrane trafficking, impairment of this process would inhibitcell growth. Furthermore, Hsp90 interacts with a variety of proteinkinases and transcription factors important for growth and devel-opment. Our finding confirmed that treatment of HepG2 cells with100 �M ZEA for 8 h keep the cells alive and in proliferative state.

Others members of HSP family are generally considered to beanti-apoptotic due to their role in the inhibition of the activationof caspases (Beere, 2004). However, the role of Hspd1 remainsenigmatic in apoptosis; nevertheless reports involve Hspd1 inaccelerating the activation of caspase 3 during apoptosis, as wellas over expression of Hspd1 preventing apoptosis in cardiac cells(Harn-Shen et al., 2009). The role of Hspd1 may be dependent onthe type of cell and the time of expression. In the present study, weobserved regulation of this protein in the treated cell compared tothe controls. This data confirmed that ZEA significantly inhibitedcell apoptosis at early time of exposure.

Other protein over expressed by ZEA at 100 �M, is NPM. Thisprotein is a multifunctional protein frequently over expressed inactively proliferating cells. It has also been reported that overexpression of NPM promotes the survival after exposure to DNAdamage and oxidative stress (Li et al., 2006). It suggests that NPMhas a role in reducing the susceptibility of chromosomal DNA todamage rather than promoting DNA damage repair (Li et al., 2006).In another study, it was shown that NPM over expression inducesrapid entry of hematopoietic stem cells into the cell cycle and sup-presses the expression of several negative cell cycle regulators thatare associated with G1-to-S transition. Indeed NPM elevates exac-erbates stress-induced cell cycle arrest. It has been reported thatactivation of NPM contributes to the cellular response to geno-toxic stress (Yang et al., 2002). In addition, NPM protects cells fromapoptotic cell death induced by diverse stresses through a mecha-

nism involving inhibition of the p53 tumor suppressor protein (Liet al., 2004a,b). Other studies demonstrated that NPM is essentialfor the maintenance of genomic stability and cell survival (Grisendiet al., 2005; Colombo et al., 2005). This data confirmed that

Toxico

ot

(3�lcm1iiaabrevob(otu

lba(alwDpfidtpSeS2stischmemaiaittdews

asta

A.C. Gazzah et al. / Experimental and

ver-expression of NPM also promotes the survival of cells exposedo ZEA.

We also have shown the up-regulation of anti-apoptotic proteinYWHAZ) at the early time-point. It is well-established that 14-3-

proteins, which consists of seven isoforms in human cells (�, �,, �, �, �, ) (Martin et al., 1993), play crucial roles in many bio-ogical processes including control of cell proliferation, response ofells to DNA damage, prevention of apoptosis, regulation of chro-atin structure, and gene expression. One important function of

4-3-3 proteins is to support cell survival by binding and sequester-ng proteins which otherwise activate signaling pathways that arenvolved in initiating apoptosis. It has been reported that YWHAZ,s antiapoptotic factors in cells by interacting with a number ofpoptosis regulatory proteins, such as Bad (Datta et al., 2000). It haseen shown that interaction with Bad induces complex formationesulting in inhibition of Bad: it is a Bcl-XL-mediated apoptosis (Zhat al., 1996; Datta et al., 2000); consequently promoting cell sur-ival (Yang et al., 1995; Jiang et al., 2007). The early up-regulationf this 14-3-3 protein could also point to an inhibition of apoptosisy altering Bcl2 family members, leading to the survival of the cellsD’Hertog et al., 2008). Our observation confirms that at this timef exposure and at this concentration of ZEA (100 �M), this myco-oxin is able of both down-regulate pro-apoptotic factor Bax andp-regulate anti-apoptotic factor Bcl-2 (Chatti Gazzah et al., 2010).

We also reported in this study an increase in S100A6 and S100A4evels in HepG2 cells exposed to ZEN. These proteins are mem-ers of the S100 protein family of small Ca2+-binding proteinsssociated with a variety of cellular and extracellular processesMazzanti et al., 2004; Cerutti et al., 2004). The two isoforms aressociated with a range of biological functions (e.g., enzyme regu-ation, cytoskeleton organization, transcriptional regulation) and

ith putative involvement in many diseases (e.g., Alzheimer’s,own syndrome, multiple sclerosis, cancer, ventricular hypertro-hy, heart failure, rheumatoid arthritis, chronic bronchitis, cysticbrosis, and psoriasis) (Marenholz et al., 2004). Many studiesemonstrated that S100A6 interacts with several proteins. Indeed,he S100A6 interaction with Sgt1, involved in ubiquitin-mediatedrotein degradation of the known oncogene �-catenin, suggests100A6 involvement in regulation of cell proliferation (Nowotnyt al., 2002). It has also been reported that up-regulation of100A6 plays a role in the cellular stress response (Orre et al.,007). Furthermore the colocalization of S100A6 with tropomyosintress fibers indicates that S100A6 is involved in regulation ofhe cytoskeleton in response to stress. The S100A4, protein wasnvolved in the regulation of various important cellular functionsuch as cell growth, cell–cell communication, energy metabolism,ontraction, and cell motility (Lu et al., 2005). Orre et al. (2007)ave demonstrated alteration of S100A6 and S100A4 in the redoxodifications and in the cellular response to genotoxic stress (Orre

t al., 2007). These data inderlines S100 protein answer to ZEA treat-ent and the genotoxic stress generated by this mycotoxin. We

lso observed in our study up regulation by ZEA of several proteinsnvolved in the DNA replication, recombination, and repair suchs Rad23, and HSP70. Indeed, treatment by ZEA (100 �M) for 8 h,nduced DNA damage as demonstrated by the high level of RAD23,his protein was suggested to play a role in DNA damage recogni-ion (Jansen et al., 1998). Recently it was demonstrated that the DNAamage leads to mitotic spindle defects. Furthermore, HSP70 overxpression was considered as a factor facilitating survival of cellsith damaged mitotic spindles and aberrantly segregated chromo-

omes (Glowala et al., 2002).Our study clearly demonstrated up-regulation of RAN protein

t early time-point. RAN is a member of the Ras superfamily ofmall G-proteins, and play an important role in nucleo-cytoplasmicransport and other cellular processes, including mitotic spindlessembly and post-mitotic nuclear envelope assembly (Wilde et al.,

logic Pathology 65 (2013) 809– 816 815

2001). This illustrates the mechanism by which the transport regu-lation participates to the control of cell cycle progression. RAN hasalso been shown to be involved in the regulation of cell cycle tran-sitions. These results are perfectly in coherence with our resultswitch underlined the cell cycle arrest that occurred as early eventinduced by ZEA at 100 �M after 8 h of exposure (Chatti Gazzah et al.,2010). Another study also reported that, this cell cycle arrest wasdue to the stabilization of the microtubule network and tubulindimers, and the inhibition of microtubule disassembly (Mollinedoand Gajate, 2003).

In our study, we identified a number of proteins which areinvolved in the regulation of Cell Morphology, accompanied bycytoskeleton arrangement. Indeed, we observed an alteration inthe expression levels of some cytoskeleton regulatory proteins,such as profilin, tropomyosin, Microtubule-associated protein 4(MAP4), TMSB4X and tubulin (TUBB). As reported in several study,these proteins are associated with structure and functioning of thecytoskeleton (Suzuki et al., 1998). MAP4 regulates assembly level ofmicrotubule. TMSB4X is an actin sequestering protein which alsoplays a role in regulation of actin polymerization. Tubulins, wereobserved to be involved in diverse cellular functions such as cellmobility, cell adhesion, cytoskeleton structure, mitosis, cell growth,proliferation, differentiation and apoptosis (Suzuki et al., 1998).

In addition, this analysis allowed the observation of manyenzymes involved in cells metabolism such as the glycolysis andthe lipid metabolism. The important glycolytic enzymes PGK andLDHA/B were up regulated in ZEA treated HepG2 cells. LDHA andLDHB are sub-units of lactate dehydrogenase (LDH) and are associ-ated to generate five tetrameric forms of LDH isoenzymes. LDHA isinvolved in metabolic activities, particularly the redox reaction atthe end of glycolysis (Sharon et al., 2009). PGK1 (phosphoglyceratekinase), which is involved in the glycolytic pathway, is regulated byHIF-1a (Semenza et al., 1994). This suggests that ZEA causes severealterations of energy metabolism that could lead to a reducedoxidative phosphorylation of substrates and an enhanced energygeneration via glycolysis.

Lipid metabolism was identified as the main biological functionaffected. The up-regulation of apolipoprotein (APOA1) may indicatethe perturbation of lipid metabolism by ZEA, as APOA1 promotescholesterol efflux from tissues to the liver for excretion.

5. Conclusion

In conclusion, the iTRAQ approach coupled to two-dimensionalchromatography and MALDI-MS is a suitable tool for the evaluationof proteome change in cell lysates. We found several differentialproteins at this early time of treatment with ZEA (100 �M). Wefound a small change in proteome. 67 proteins were regulated onthe 981 proteins identified. These changes may provide the earlyaffected functions and yield further insight into mechanisms under-lying the involvement of mycotoxin-induced diseases.

References

Abid-Essefi S, Ouanes Z, Hassen W, Baudrimont I, Creppy E, Bacha H. Cytotoxicity,inhibition of DNA and protein syntheses and oxidative damage in cultured cellsexposed to zearalenone. Toxicology In Vitro 2004;18:467–74.

Aït-Aïssa S, Pandard P, Magaud H, Arrigo AP, Thybaud E, Porcher JM. Evaluation ofan in vitro hsp70 induction test for toxicity assessment of complex mixtures:comparison with chemical analyses and ecotoxicity tests. Ecotoxicology andEnvironmental Safety 2003;54(1):92–104.

Ayed-Boussema I, Ouanes Z, Bacha H, Abid S. Toxicities induced in cultured cellsexposed to zearalenone: apoptosis or mutagenesis? Journal of Biochemical and

Molecular Toxicology 2007;21(3):136–44.

Bouaziz C, Martel C, Sharaf el dein O, Abid-Essefi S, Brenner C, Lemaire C, et al.Fusarial toxin-induced toxicity in cultured cells and in isolated mitochondriainvolves PTPC-dependent activation of the mitochondrial pathway of apoptosis.Toxicological Sciences 2009;110:363–75.

8 Toxico

B

BB

B

C

C

C

C

C

D

D

E

E

F

F

G

G

G

J

H

H

H

H

J

L

L

L

L

L

L

16 A.C. Gazzah et al. / Experimental and

ush KT, Goldberg AL, Nigam SK. Proteasome inhibition leads to a heat-shockresponse, induction of endoplasmic reticulum chaperones, and thermotoler-ance. The Journal of Biological Chemistry 1997;272:9086–92.

ennett JW, Klich M. Mycotoxins. Clinical Microbiology Reviews 2003;16:497–516.eere HM. The stress of dying: the role of heat shock proteins in the regulation of

apoptosis. Journal of Cell Science 2004;117:2641–51.reithofer GK, Sciiihitano MS, Karthanasis SK, Butt TR, Jungbauer A. Regulation of

human estrogen receptor by phytoestrogens in yeast and human cells. The Jour-nal of Steroid Biochemistry and Molecular Biology 1998;67:421–9.

AST. Mycotoxins risks in plant, animal and human systems, Task Force Report, No.139. Ames, IA: Council for Agricultural Science and Technology; 2003. p. 1–191.

hatti Gazzah A, El Golli Bennour E, Bouaziz C, Abid S, Ladjimi M, Bacha H. Sequentialevents of apoptosis induced by zearalenone in cultured hepatocarcinoma cells.Mycotoxin Research 2010;26(3):187–97.

erutti JM, Delcelo R, Amadei MJ. A preoperative diagnostic test that distinguishesbenign from malignant thyroid carcinoma based on gene expression. The Journalof Clinical Investigation 2004;113:1234–42.

olombo E, Bonetti P, Lazzerini DE, Martinelli P, Zambonis R, Marine JC, et al. Nucle-ophosmin is required for DNA integrity and p19Arf protein stability. Molecularand Cellular Biology 2005;25:8874–86.

onkova E, Laciakov A, Pastonova B, Seidel H, Kovac G. The effect of zearalenone onsome enzymatic parameters in rabbits. Toxicology Letters 2001;121:145–9.

atta SR, Katsov A, Hu L, Petros A, Fesik SW, Yaffe MB, et al. 14-3-3 proteins andsurvival kinases cooperate to inactivate BAD by BH3 domain phosphorylation.Molecular Cell 2000;6:41–51.

owling VA, Sheehan D. Proteomics as a route to identification of toxicity targets inenvironmental toxicology. Proteomics 2006;6:5597–604.

ngelhardt G, Barthel J, Sparrer D. Fusarium mycotoxins and ochratoxin A in cerealsand cereal products: results from the Bavarian Health and Food Safety Authorityin 2004. Molecular Nutrition & Food Research 2006;50:401–5.

l Golli E, Hassen W, Bouslimi A, Bouaziz C, Ladjimi M, Bacha H. Induction of Hsp 70in Vero cells in response to mycotoxins cytoprotection by sub-lethal heat shockand by vitamin E. Toxicology Letters 2006;166:122–30.

eng AS, Narins PM, Xu C-H. Vocal acrobatics in a Chinese frog Amolops tormotus.Naturwissenschaften 2002;89:352–6.

rydman J. Folding of newly translated proteins in vivo: the role of molecular chap-erones. Annual Review of Biochemistry 2001;70:603–47.

risendi S, Bernardi R, Rossi M, Cheng K, Khandker L, Manova K, et al. Roleof nucleophosmin in embryonic development and tumorigenesis. Nature2005;437:147–53.

lowala M, Mazurek A, Piddubnyak V, Fiszer-Kierzkowska A, Michalska J, KrawczykZ. Identification of nucleolin and nucleophosmin as genotoxic stress-responsiveRNA-binding proteins. Nucleic Acids Research 2002;30(10):2251–60.

hédira-Chékir L, Maaroufi K, Zakhama A, Ellouz F, Dhouib S, Creppy EE, et al.Induction of a SOS repair system in lysogenic bacteria by zearalenone and itsprevention by vitamin E. Chemico-Biological Interactions 1998;13(1–1):15–25.

iang P, Du W, Wu M. p53 and Bad: remote strangers become close friends. CellResearch 2007;17:283–5.

assen W, El Golli E, Baudrimont I, Théophile Mobio A, Ladjimi M, Creppy EE,et al. Cytotoxicity and HSP70 induction in Hep G2 cells in response to Zear-alenone and cytoprotection by sub-lethal heat shock. Toxicology 2005;207(2):293–301.

assen W, Ayed-Boussema I, Azqueta Oscoz A, De Cerain Lopez A, Bacha H. The roleof oxidative stress in zearalenone-mediated toxicity in Hep G2 cells: oxidativeDNA damage, gluthatione depletion and stress proteins induction. Toxicology2007;232:294–302.

endrick JP, Hartl FD. Molecular chaperone functions of heat shock proteins. AnnualReview of Biochemistry 1993;62:349–84.

arn-Shen C, Tzu-En W, Chi-Chang J, Hong-Da L. Myocardial heat shock protein60 expression in insulin-resistant and diabetic rats. Journal of Endocrinology2009;200:151–7.

ansen LE, Verhage RA, Brouwer J. Preferential binding of yeast Rad4. Rad23 complexto damaged DNA. The Journal of Biological Chemistry 1998;273:33111–4.

i J, Zhang X, Sejas DP, Bagby GC, Pang Q. Hypoxia-induced nucleophosmin pro-tects cell death through inhibition of p53. The Journal of Biological Chemistry2004a;279:41275–9.

i J, Seja s DP, Rani R, Koretsky T, Bagby GC, Pang Q. Nucleophosmin regulates cellcycle progression and stress response in hematopoietic stem/progenitor cells.Journal of Biological Chemistry 2006;281(24):16536–45.

ioi MB, Santoro A, Barbieri R, Salzano UMV. Ochratoxin and zearalenone: a compar-ative study on genotoxic effects and cell death induced in bovine lymphocytes.Mutation Research 2004;557:19–24.

u HH, Zhou L, Haydon RC, Deyrup AT, Montag AG, Huo D, et al. Increased expres-sion of S100A6 is associated with decreased metastasis and inhibition of cellmigration and anchorage independent growth in human osteosarcoma. CancerLetters 2005;29:135–48.

u SC, Huang HY. Comparison of sulphur amino acid utilization for GSH synthesis

between HepG2 cells and cultured rat hepatocytes. Biochemical Pharmacology1994;47:859–69.

i J, Zhang X, Sejas DP, Bagby GC, Pang Q. Hypoxia-induced nucleophosmin pro-tects cell death through inhibition of p53. Journal of Biological Chemistry2004b;279:41275–9.

logic Pathology 65 (2013) 809– 816

Maaroufi K, Chekir L, Creppy EE, Ellouz F, Bacha H. Zearalenone induces mod-ifications of haematological and biochemical parameters in rats. Toxicon1996;34:535–40.

MacDonald SJ, Anderson S, Brereton P, Wood R, Damant AJ. Determination of zear-alenone in barley, maize and wheat flour, polenta, and maize-based baby food byimmunoaffinity column cleanup with liquid chromatography: interlaboratorystudy. Journal of AOAC International 2005;88:1733–40.

Martin H, Patel Y, Jones D, Howell S, Robinson K, Aitken A. Antibodies against themajor brain isoforms of 14-3-3 protein. An antibody specific for the N-acetylatedaminoterminus of a protein. FEBS Letters 1993;331:296–303.

Mazzanti C, Zeiger MA, Costouros NG. Using gene expression profiling todifferentiate benign versus malignant thyroid tumors. Cancer Research2004;64:2898–903.

Marenholz I, Heimann CW, Fritz G. S100 proteins in mouse and man: from evo-lution to function and pathology (including an update of the nomenclature).Biochemical and Biophysical Research Communications 2004;322:1111–22.

Mollinedo F, Gajate C. Microtubules, microtubule-interfering agents and apoptosis.Apoptosis 2003;8:413–50.

Nowotny M, Spiechowicz M, Jastrzebska B, Filipek A, Kitagawa K, Kuznicki J. Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins.The Journal of Biological Chemistry 2003;278:26923–8.

Ouanes Z, Abid S, Ayed I, Anane R, Mobio T, Creppy E, et al. Induction of micronucleiby zearalenone in Veromonkey kidney cells and in bonemarrow cells of mice:protective effect of vitamin E. Mutation Research 2003;538(1–2):63–70.

Ouanes Z, Ayed-Boussema I, Baati T, Creppy EE, Bacha H. Zearalenone induces chro-mosome aberrations in mouse bone marrow: preventive effect of 17�-estradiol,progesterone and Vitamin E. Mutation Research/Genetic Toxicology and Envi-ronmental Mutagenesis 2005;565(2/3):139–49.

Orre LM, Pernemalm M, Lengqvist J, Lewensohn R, Lehtio J. Up-regulation,modification, and translocation of S100A6 induced by exposure to ionizingradiation revealed by proteomics profiling. Molecular & Cellular Proteomics2007;6:2122–31.

Rincheval V, Renaud F, Lemaire C, Godefroy N, Trotot P, Boulo V, et al. Bcl-2can promote p53-dependent senescence versus apoptosis without affectingthe G1/S transition. Biochemical and Biophysical Research Communications2002;298(2):282–8.

Ryu D, Jackson LS, Bullerman LB. Effects of processing on zearalenone. Advances inExperimental Medicine and Biology 2002;504:205.

Scudamore KA, Patel S. Survey for ochratoxin A, zearalenone and fumonisins inmaize imported into the United Kingdom. Food Additives and Contaminants2000;17:407–16.

Sharon KH, Marlene M, Darfler MB, Nicholl JY, Kerry G, Tony J, et al. LC/MS-basedquantitative proteomic analysis of paraffin-embedded archival melanomasreveals potential proteomic biomarkers associated with metastasis. PLoS ONE2009;42:4430.

Semenza GL, Roth PH, Fang HM, Wang G. Transcriptional regulation of genes encod-ing glycolytic enzymes by hypoxia-inducible factor 1. The Journal of BiologicalChemistry 1994;269:23757–63.

Singh MP, Reddy MM, Mathur N, Saxena DK, Chowdhuri DK. Induction of hsp70,hsp60, hsp83 and hsp26 and oxidative stress markers in benzene, toluene andxylene exposed Drosophila melanogaster: role of ROS generation. Toxicologyand Applied Pharmacology 2009;235:226–43.

Sitailo LA, Tibudan SS, Denning MF. Activation of caspase-9 is required for UV-induced apoptosis of human keratinocytes. Journal of Biological Chemistry2002;277:19346–52.

Suzuki H, Nagata H, Shimada Y, Konno A. Decrease in gamma-actin expression,disruption of actin microfilaments and alterations in cell adhesion systemsassociated with acquisition of metastatic capacity in human salivary gland ade-nocarcinoma cell clones. International Journal of Oncology 1998;12:1079–84.

Tabuc C, Marin D, Guerre P, Sesan T, Bailly JD. Molds and mycotoxin content of cerealsin southeastern Romania. Journal of Food Protection 2009;72: 662–5.

Wyttenbach A, Carmichael J, Swartz J, Furlong RA, Narain Y, Rankin J, et al.Effects of heat shock, heat shock protein 40 (HDL-2), and proteasome inhi-bition on protein aggregation in cellular models of Huntington’s disease.Proceedings of the National Academy of Sciences of the United States of America2000;97:2898–903.

Wilde A, Lizarraga SB, Zhang L, Wiese C, Gliksman NR. Ran stimulates spindle assem-bly by changing microtubule dynamics and the balance of motor activities.Nature Cell Biology 2001;3:221–7.

Yang C, Maiguel DA, Carrier F. Identification of nucleolin and nucleophosminas genotoxic stress-responsive RNA-binding proteins. Nucleic Acids Research2002;30(10):2251–60.

Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ. Bad, a heterodimericpartner for Bcl-XL and Bcl-2 displaces Bax and promotes cell death. Cell1995;80:285–91.

Young JC, Agashe VR, Siegers K, Hartl FU. Pathways of chaperone-mediated proteinfolding in the cytosol. Nature Reviews Molecular Cell Biology 2004;5:781–91.

Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phosphorylation of deathagonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell 1996;87:619–28.

Zhou BB, Elledge SJ. DNA damage response: putting checkpoints in perspective.Nature 2000;408:433–9.