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Proteomics 2013, 13, 3175–3188 3175DOI 10.1002/pmic.201300015
RESEARCH ARTICLE
Proteomic changes in chicken primary hepatocytes
exposed to T-2 toxin are associated with oxidative
stress and mitochondrial enhancement
Peiqiang Mu1∗, Ming Xu1∗, Lei Zhang2, Kaixin Wu1, Jun Wu1, Jun Jiang1, Qingmei Chen1,Lijuan Wang1, Xianqing Tang1 and Yiqun Deng1
1 Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College ofLife Sciences, South China Agricultural University, Guangzhou, Guangdong, P. R. China
2 Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou,Guangdong, P. R. China
T-2 toxin is a mycotoxin that is toxic to plants, animals, and humans. However, its molecularmechanism remains unclear, especially in chickens. In this study, using 2D electrophoresiswith MALDI-TOF/TOF-MS, 53 proteins were identified as up- or downregulated by T-2 toxinin chicken primary hepatocytes. Functional network analysis by ingenuity pathway analysisshowed that the top network altered by T-2 toxin is associated with neurological disease, cancer,organismal injury, and abnormalities. Most of the identified proteins were associated with oneof eight functional classes, including cell redox homeostasis, transcriptional or translationalregulation, cell cycle or cell proliferation, stress response, lipid metabolism, transport, carbo-hydrate metabolism, and protein degradation. Subcellular location categorization showed thatthe identified proteins were predominantly located in the mitochondrion (34%) and interest-ingly, the expression of all the identified mitochondrial proteins was increased. Further cellularanalysis showed that T-2 toxin was able to induce the ROS accumulation and could lead to anincrease in mitochondrial mass and adenosine 5′-triphosphate content, which indicated thatoxidative stress and mitochondrial enhancement occurred in T-2 toxin-treated cells. Overall,these results characterize the global proteomic response of chicken primary hepatocytes to T-2toxin, which may lead to a better understanding of the molecular mechanisms underlying itstoxicity.
Keywords:
Chicken / Mitochondrial enhancement / Oxidative stress / Proteome / T-2 toxin
Received: January 10, 2013Revised: August 5, 2013
Accepted: August 9, 2013
� Additional supporting information may be found in the online version of this article atthe publisher’s web-site
Correspondence: Professor Yiqun Deng, Guangdong ProvincialKey Laboratory of Protein Function and Regulation in AgriculturalOrganisms, College of Life Sciences, South China AgriculturalUniversity, Guangzhou, Guangdong 510642, P. R. ChinaE-mail: [email protected]: +86-20-38604967
Abbreviations: ATP, adenosine 5′-triphosphate; DCFH-DA, 2′, 7′-dichlorofluorescin diacetate; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; IPA, ingenuity pathway analysis;PI, propidium iodide
1 Introduction
T-2 toxin is a mycotoxin that belongs to the group of type A tri-chothecenes. It is produced by several fungal genera that caninfect crops in the field or during storage and subsequentlycontaminate animal feed and human food [1,2]. Many reportsworldwide have described an association between T-2 toxinand agricultural damage and adverse effects on animals [3–6].
∗These authors contributed equally to this work.
Colour Online: See the article online to view Figs. 1, 2, and 4 incolour.
C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
3176 P. Mu et al. Proteomics 2013, 13, 3175–3188
The consumption of T-2 toxin can lead to different toxiceffects, such as fatal alimentary toxic aleukia disease in hu-mans and vomiting in pigs [7]; therefore, the assessment ofT-2 toxin levels in foodstuffs, especially in chicken, whichserve as a major human food source, is important. Chick-ens are at particular risk of T-2 toxin exposure because manychicken feed mainly contain cereal grains. Previous reportsshowed that the average T-2 toxin in cereal grains rangesbetween 0.03 mg/kg and 0.155 mg/kg [8, 9]. The intake ofT-2 toxin-contaminated feed can lead to decreased weightgain and egg production in chickens and egg hatching isalso impaired [10–13]. T-2 toxin can also cause feather alter-ations in chickens [14]. At the molecular level, exposure to T-2toxin causes a significant reduction in serum total protein andcholesterol levels and an increase in serum uric acid and lac-tate dehydrogenase levels [15]. Furthermore, DNA fragmenta-tion has been observed in chicken spleen leukocytes, and sup-plementation with nucleotides was able to reduce the degreeof this damage [16]. Cell viability, alkaline phosphatase activ-ity, and glutathione content in primary cultures of chickentibial growth plate chondrocytes were all markedly decreasedupon exposure to T-2 toxin [17]. A T-2 toxin-containing dietwas associated with significant decreases in antioxidant con-centrations [18]. However, the exact molecular mechanism ofT-2 toxicity in chickens is unknown.
Several experiments to investigate the toxicity of T-2toxin have already been performed in various cell lines,especially cancer cells, and a variety of mechanisms havebeen proposed. The inhibition of elongation of proteinbiosynthesis in eukaryotic cells and apoptosis are generallyconsidered the main cytotoxic effects of T-2 toxin [1, 3]. T-2toxin-induced apoptosis has been observed in various celltypes, such as HL60, Jurkat, U937 [19], Vero cells [20], andhuman hepatoma cells [21]. Apoptosis has also been reportedin vivo in thymic and splenic lymphocytes, bone marrow,and intestinal epithelial cells [22] and in the skin [23], kid-ney [24], and brain [25] in mice. In addition to the inhi-bition of protein biosynthesis and apoptosis, the inhibitionof DNA and RNA synthesis by T-2 toxin has also been ob-served in several cell lines [26–28]. Other activities of T-2toxin have also been observed, such as inducing single-strandbreaks in the DNA of lymphoid cells [29], impairing antibodyproduction [30], altering membrane functions [31], reducinglymphocyte proliferation [32], and altering the maturation ofdendritic cells [33]. However, the in vitro–in vivo and inter-species extrapolation of the action of T-2 toxin has not beenclearly clarified.
Understanding the mechanism of action of T-2 toxin isessential for predicting its potential deleterious effects anddeveloping countermeasures. Therefore, this study was in-tended to evaluate the actions of T-2 toxin in chickens andexplore its potential molecular mechanisms at the proteomelevel. Primary hepatocytes have been successfully used inmany toxicity studies because they reflect the in vivo re-sponse to some degree and are easily manipulated [34, 35].In this study, we investigated the proteomic changes caused
by T-2 toxin exposure in primary cultures of chicken hepato-cytes and analyzed T-2 toxicity at the cellular and molecularlevels.
2 Materials and methods
2.1 Chemicals and reagents
T-2 toxin was purchased from Sigma-Aldrich (St. Louis, MO,USA). Williams’ E medium, Medium 199, Dulbecco’s modi-fied Eagle’s medium and FBS were obtained from Invitrogen(Carlsbad, CA, USA). All other chemicals including CHAPS,DTT, and PMSF, were obtained from GE Healthcare (Upp-sala, Sweden) or Merck (Darmstadt, Germany), unless oth-erwise indicated. All primers were synthesized by InvitrogenBiotechnology, Guangzhou (Guangzhou, China). All otherchemicals and reagents were of the highest analytical gradeavailable.
2.2 Chicken primary hepatocytes isolation, culture,
and T-2 toxin treatment
Eighteen-day-old chicken embryos were purchased fromthe Institute of Animal Science, Guangdong Academyof Agricultural Sciences (Guangzhou, China). Hepatocyteswere isolated as described previously [36]. Chicken em-bryo primary hepatocytes were cultured in Williams’ Emedium (Sigma-Aldrich) containing 8% FBS, 100 U/mLpenicillin/streptomycin, 10 nM insulin, and 10 nMdexamethasone (Sigma-Aldrich) for 5 h and then cultured inMedium 199 (Gibco BRL, Carlsbad, CA, USA) containing 8%FBS, 100 U/mL penicillin/streptomycin, 10 �g/mL insulin,1 �g/mL dexamethasone, and 2 mM L-glutamate (Invitro-gen) for 30 h. The medium was then changed to medium199 containing 8% FBS, 100 U/mL penicillin/streptomycin,10 �g/mL insulin, 1 �g/mL dexamethasone, 2 mML-glutamate, and various concentrations of T-2 toxin andcultured as described in the following experimental pro-cedures, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Giemsa staining, 2DE,quantitative real-time RT-PCR, flow cytometry assay, singlecell gel electrophoresis, mitochondrial mass determination,and adenosine 5′-triphosphate (ATP) content determination.All cells were maintained in a humidified incubator at 37�Cwith 5% CO2. All experimental procedures were approved bythe Institute of Animal Care and Use Committee of SouthChina Agricultural University, and adhered to the ChineseGuidelines for the Proper Conduct of Animal Experimentsfor the use of laboratory animals.
2.3 MTT assay and Giemsa staining
Cell viability was measured using the MTT assay [37].Chicken primary hepatocytes were seeded onto 96-well plates
C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
Proteomics 2013, 13, 3175–3188 3177
(1 × 105 cells per well) and treated for 48 h with different con-centrations of T-2 toxin (0–320 ng/mL), referred to the MTTassay of porcine primary hepatocytes under T-2 toxin treat-ment [34]. Subsequently, 0.5 mg/mL MTT (Sigma-Aldrich)was added to each well, and the plates were incubated at 37�Cfor an additional 4 h. The medium containing MTT was thenremoved, and 200 �L DMSO (Sigma-Aldrich) was added toeach well to dissolve the formazan crystals. The absorbanceat 570 nm was measured using a microplate reader (Bio-Radlaboratories, Hercules, CA, USA). All experiments were per-formed in triplicate.
For Giemsa staining, chicken primary hepatocytes weretreated with 0.05, 0.1, or 0.2 �g/mL T-2 toxin for 48 h. Thecells were subsequently washed in Dulbecco’s PBS, fixed with50% methanol for 2 min, fixed with absolute methanol for10 min, stained with Giemsa solution for 2 min and thenstained with 1/10 Giemsa solution for 2 min. Finally, thecells were washed with ultra-pure water and observed by lightmicroscopy (Olympus IX70-S1F, Tokyo, Japan).
2.4 Two-dimensional gel electrophoresis
The chicken primary hepatocytes from the control and0.05 �g/mL T-2 toxin treatment groups were homogenizedin 0.1 mL of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS,30 mM Tris, 40 mM DTT, 2% IPG buffer, and 1 mM PMSF).The homogenates were centrifuged at 20 000 × g for 20 minat 4�C. The 2D Clean-Up Kit (GE Healthcare) was used toremove ionic interfering components from the protein ex-traction. The protein concentration in the urea-containingprotein samples was quantified using a 2D Quant Kit (GEHealthcare). A 250 �g sample of protein was diluted to450 �L with rehydration solution (8 M urea, 2% CHAPS,40 mM DTT, and 0.5% IPG buffer) and applied to immobi-lized dry IPG strips (linear pH gradient from pH 4–7, 24 cm)and rehydrated passively for 14 h. The samples were thenfocused for 30 min at 100 V, 30 min at 200 V, 1 h at 500 V,30 min gradient at 1000 V, and 4 h gradient at 8000 V, for atotal of 50 000 V h, using the IPGphor III system (AmershamBiosciences, Piscataway, NJ, USA). Focused IPG strips wereequilibrated in equilibration solution I containing 6 M urea,2% SDS, 30% glycerol, 50 mM Tris–HCl, pH 6.8, and 1% DTTfor 15 min and then equilibrated for an additional 15 min inequilibration solution II that was identical to equilibrationsolution I, except that the DTT was replaced by 2.5% iodoac-etamide. After equilibration, 12% SDS-PAGE was performedon an Ettan DALT II system (Amersham Biosciences). The2DE gel was then stained with silver nitrate [38]. The 2DE gelof the control and T-2 toxin-treated groups were replicatedthree times biologically.
2.5 2DE image analysis
The stained 2DE gels were scanned with MagicScan soft-ware on an Imagescanner (Amersham Biosciences) and an-
alyzed using ImageMaster V 5.0 software (GE Healthcare,Piscataway, NJ, USA) according to the manufacturer’s pro-tocol. Spot intensities were calculated by the spot volume(after normalizing the image by the total spot volume nor-malization method) multiplying the total area of all the spots.The change index was described as the ratio between thespot percentages relative to the spot volumes in the controland T-2 toxin-treated chicken primary hepatocytes. Proteinswere classified as being differentially expressed between thecontrol and T-2 toxin-treated groups when the spot intensityshowed a ≥1.3-fold difference between the control and T-2toxin-treated groups. Differences in protein expression levelswere evaluated with the Student’s t-test and considered to besignificant when p < 0.05.
2.6 Protein identification by MALDI-TOF/TOF
analysis
The spots of interest were manually excised from the silver-stained gels and subjected to in-gel trypsin digestion. Afterdigestion, the peptides were then extracted twice using 0.1%TFA in 50% ACN. The extracts were pooled and dried com-pletely using a Speed Vac vacuum concentrator. The mix-tures of peptides were redissolved in 0.1% TFA. Then, 0.4 �Lof matrix (CHCA in 30% ACN, 0.1% TFA) was added into0.8 �L of peptide solution before spotting on the target plate.
Protein identification was performed on an AB SCIEXMALDI TOF-TOFTM 5800 Analyzer (AB SCIEX, Foster,CA, USA) equipped with a neodymium: yttrium-aluminum-garnet laser (laser wavelength was of 349 nm) according to ourpreviously described method [34] and listed in the supportinginformation.
2.7 Bioinformatics analysis of differentially
expressed proteins
The functional networks were identified by the ingenuitypathway analysis (IPA) according to the biological functionsand/or diseases (Ingenuity Systems, Redwood, CA, USA).The statistically significance score for each network were gen-erated based on the fit of the network to the set of genesdefined by the user. The score is derived from the nega-tive log of p-value, which indicates the likelihood that thefocus genes would be found together in a network by ran-dom chance. If the score of a functional network is 20 orhigher, the network is not being generated by random chancealone with at least 99% confidence. In addition, to obtain amore detailed and precise view of the biological significanceof the altered proteins, these differently expressed proteinswere further categorized according to their main molecu-lar functions and subcellular localizations by searching theGene Ontology (http://www.geneontology.org/) and PubMed(http://www.ncbi.nlm.nih.gov) databases.
C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
3178 P. Mu et al. Proteomics 2013, 13, 3175–3188
2.8 Quantitative real-time RT-PCR
Total RNA was isolated from 0.05 �g/mL T-2 toxin-treatedchicken primary hepatocytes or controls using Trizol reagent(Invitrogen) according to the manufacturer’s instruction.RNA samples (3 �g) were reverse transcribed by M-MLVreverse transcriptase (Promega, Madison, WI, USA) withOligo d (T) (MBI Fermentas, Newington, NH, USA) andrandom primers. PCR primers specific for the genes encod-ing the proteins identified in MALDI-TOF/TOF-MS were de-signed based on expressed sequence tags from Gallus gallus(Supporting Information Table S2). All primers were synthe-sized by Invitrogen (Guangzhou, China). The GAPDH waschosen as an internal control and used for normalization.Each RT-PCR assay was replicated three times. Fold differ-ences in expression levels were calculated using the 2−��Ct
method [39].
2.9 Flow cytometry assay
The chicken primary hepatocyte cells were exposed to0.05 �g/mL of T-2 toxin for 12, 24, 48, or 72 h. Then, theROS content and level of apoptosis were examined with BDFACSCaliburTM flow cytometer (BD Biosciences, FranklinLakes, NJ, USA) using CellQuest 3.1 (BD Biosciences) andFlowJo software (Tree Star, Ashland, OR, USA).
Intracellular ROS were detected using an oxidation-sensitive fluorescent probe 2′, 7′-dichlorofluorescin diacetate(DCFH-DA) (Beyotime, Shanghai, China). After treatmentwith T-2 toxin (0.05 �g/mL for 12, 24, 48, and 72 h),chicken primary hepatocytes cells were washed twice withPBS and incubated with 10 �M DCFH-DA solution at 37�C for20 min. The fluorescence intensity was then measuredby BD FACSCaliburTM flow cytometry at an excitationwavelength of 488 nm and emission wavelength of535 nm.
To examine apoptosis, an apoptosis detection kit (KeyGEN,Nanjing, China) based on annexin V-FITC was used. AnnexinV specifically binds to phosphatidylserine, which is exposedto the plasma membrane surface during the early stages ofcell apoptosis, and propidium iodide (PI) is a vital dye la-beling the nucleus in dying cells. Therefore, the viable cellsare not stained by annexin V or PI, and the early apoptoticcells are stained with annexin V-FITC only, whereas the lateapoptotic cells and necrotic cells are double-stained with bothannexin V-FITC and PI. Cells were treated with or without0.05 �g/mL T-2 toxin for 12, 24, 48, and 72 h, resuspended inbinding buffer at a final cell concentration of 1 × 106 cells/mL,and then incubated with both annexin V-FITC and PI for10 min in the dark. The fluorescence intensity was analyzedby BD FACSCaliburTM flow cytometry at an excitation wave-length of 425 nm, FITC fluorescence detection wavelengthof 525 nm, and PI fluorescence detection wavelength of630 nm.
2.10 Single cell gel electrophoresis
Oxidative damage to the DNA of chicken primary hepatocyteswas determined by the single cell gel electrophoresis (cometassay) according to Olive [40]. T-2 toxin-treated or -untreatedcells were harvested by trypsin digestion, and the cell densitywas adjusted to 1 × 106 cells/mL before electrophoresis. Theelectrophoresis slides were precoated in 0.5% normal-meltingpoint agarose by dipping; then, a 10 �L cell sample was mixedwith 70 �L of 0.7% low-melting point agarose and layeredon the precoated slide. Finally, the cell layer was covered by75 �L of 0.7% low-melting point agarose and lysed at 4�C forat least 1 h in lysing solution (2.5 M NaCl, 100 mM EDTA,10 mM Tris-base, 1% Triton X-100, and 10% DMSO) andthen washed in PBS twice. The lysed slides were then dippedinto alkaline buffer (300 mM NaOH, 1 mM EDTA, pH>13)for 30 min to allow the unwinding of the DNA and the ex-pression of alkali-labile damage. Single cell electrophoresiswas carried out in a prechilled alkaline solution at 25 V and300 mA for 30 min. After electrophoresis, the cells were neu-tralized in 0.4 mM Tris-HCl, pH 7.5 for 15 min at 4�C; theneutralization procedure was repeated three times. Then, thecells were stained with PI (50 �g/mL) for 10 min. To visualizethe DNA damage, the PI-stained DNA was observed using a20× objective lens on a fluorescence microscope (OlympusIX70-S1F). A minimum of 100 comets on each slide was an-alyzed using CASP CometScore software. The tail moment([percent of DNA in the tail]×[tail length]) was used to indicatethe degree of DNA damage.
2.11 Mitochondrial mass and ATP content
determination
The ATP content in the samples was analyzed using theATP bioluminescent somatic cell assay (FLASC) Kit (Sigma-Aldrich), which is based on the production of light throughthe reaction of ATP with added luciferase and luciferin, asrecommended in the kit manual. Briefly, 0.1 mL of ATP As-say Mix Working Solution was added to a 1.5 mL Eppendorftube and allowed to stand at room temperature for 3 min.Then, the ATP of T-2 toxin-treated or -controlled cells (1 ×106 cells/mL) was released by adding 0.1 mL of 1× somaticcell ATP releasing reagent with 0.05 mL ATP standard so-lution (0.5 �g/mL) in another new tube. Finally, 0.1 mL ofthe ATP-released cell samples were mixed with the preparedATP Assay Mix Working Solution, and the light emitted bythe reaction of ATP with luciferase and luciferin was im-mediately measured with a GLOMAX 20/20 luminometer(Promega).
The mitochondria mass was determined by using Mi-toTracker red (Molecular Probes, Eugene, OR, USA),a mitochondria-selective membrane potential-independentdye. The cells was grown on 12 mm diameter coverslips andincubated with 0.05 �g/mL T-2 toxin for 48 h. And then,the cells were incubated with 200 nM MitoTracker red for
C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
Proteomics 2013, 13, 3175–3188 3179
Control 0.05 μg/ml T-2
0
20
40
60
80
100
120
0.00 0.10 0.20 0.30 0.40
T-2 concentration (μg/ml)
IC50=0.06 μg/ml
A B
Cel
l via
bilit
y (c
ontr
ol %
)
0.1 μg/ml T-2 0.2 μg/ml T-2
Figure 1. Viability of T-2 toxin-treated primary hepatocytes. (A) Cell viability was assessed using the MTT assay, measuring absorbanceat 570 nm. The viability of T-2 toxin-treated cell is expressed as a percentage of the value in control cells. Each point represents themean ± SD of three duplicate experiments. (B) Representative light micrographs of Giemsa staining under an optical microscope areshown for each treatment group.
30 min, washed three times with 1 × PBS, fixed by 5%paraformalclehyde for 10 min and then stained the nucleus by4′, 6-diamidino-2-phenylindole for 2 min. The red and bluefluorescence intensity was examined under a Zeiss AxioObserver D1 fluorescence microscope (Zeiss, Gottingen,Germany) using 63× magnification and was counted [41].
3 Results
3.1 The cytotoxicity of T-2 toxin in chicken primary
hepatocytes
To assess the toxicity of T-2 toxin in chicken primary hep-atocytes, the cell viability, and cell morphology of the T-2toxin-treated cells were analyzed. As expected, the viability ofthe chicken primary hepatocyte cells was inversely related tothe concentration of T-2 toxin after a 48-h treatment. The cal-culated IC50 of T-2 toxin was 0.062 �g/mL (Fig. 1A), demon-strating its obvious toxicity in chicken primary hepatocytes.The toxicity of T-2 toxin was further analyzed by Giemsastaining for morphological changes, which demonstrated thatthe T-2 toxin-treated cells rounded up and decreased in size,exhibiting increased intercellular spaces (Fig. 1B). The celldensity decreased distinctly with increasing of T-2 toxin con-centration (Fig. 1B). These results indicated that T-2 toxin hasobvious toxicity in chicken primary hepatocytes.
3.2 Proteomic profiles of chicken primary
hepatocytes regulated by T-2 toxin
To further understand the toxicity of T-2 toxin in primarychicken hepatocytes from a molecular perspective, the pro-
teomic profile changes induced by T-2 toxin were investigatedby comparative 2DE proteomic analysis. A total of 92 pro-tein spots from chicken primary hepatocytes that exhibited agreater than 1.3-fold difference in spot intensity after treat-ment with 0.05 �g/mL of T-2 toxin for 48 h were detectedin three replicate gels from three independent experiments(marked with numbers and crosses with lines in Fig. 2A). Ofthese 92 proteins, 53 were identified by MALDI-TOF/TOF-MS analysis, including 34 that were upregulated (≥1.3-fold)and 19 that were downregulated (≥1.3-fold) by T-2 toxin. Theaccession number, protein score, experimental mass, andpI, sequence coverage, fold change, molecular function, andsubcellular location of each differentially regulated proteinare shown in Table 1. The full name and enlarged spots ofthe identified proteins were shown in Supporting Informa-tion Table S1. Furthermore, the genes encoding 23 of theseproteins were randomly selected for further analysis by qRT-PCR, and 20 of them showed changes in expression that wereconsistent with the 2DE analysis (Supporting InformationFig. S1).
3.3 Functional networks, molecular functions, and
subcellular localizations of T-2 toxin-regulated
proteins
To further interpret these data in a biological context, theproteins from Table 1 were analyzed using IPA tool. Thethree top networks of proteins that were up- or downregu-lated by T-2 toxin treatment in chicken primary hepatocytes(Table 2, Supporting Information Fig. S2–S4) were: (i) neuro-logical disease, cancer, organismal injury, and abnormalities,
C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
3180 P. Mu et al. Proteomics 2013, 13, 3175–3188
pH4 7MW (KDa) 4 7
200116
97.266.444.3
29.0
20.114.3
6.5
Control T-2 toxin
Transcriptional or translational
regulation13%
Cell cycle or cell proliferation
15%
Cell redoxhomeostasis
15%
Others14%
Protein degradation
7%
Carbohydrate metabolism
5%Transport
9%Lipid
metabolism11%
Stress response
11%
A
Cytoplasm24%
Mitochondrion34%
100% up
0% down
Golgi apparatus
1%
Peroxisome3%Extracellular
space6%
Plasma membrane
6%
Cytosol7%
Nucleus15%
Ribosomes4%
B C
Figure 2. Representative 2DE gel of T-2 toxin-treated or untreated chicken primary hepatocytes, molecular functions, and subcellularlocalizations of identified proteins. (A) Representative 2DE gel. Numbers and crosses with lines indicate protein spots that changed bymore than twofold compared to the control. (B) The annotated molecular functions of the identified proteins. (C) The subcellular localizationsof the identified proteins.
(ii) protein trafficking, auditory disease, cellular compromise,and (iii) amino acid metabolism, molecular transport, smallmolecule biochemistry. The top functional network regulatedby T-2 toxin indicates that chickens may have a high risk ofneurological disease when exposed to T-2 toxin.
Analysis of the molecular function annotations of the pro-teins regulated by T-2 toxin revealed an enrichment in cellredox homeostasis (15%), cell cycle or cell proliferation reg-ulation (15%), transcriptional or translational (13%), stressresponse (11%), lipid metabolism (11%), transport (9%), pro-tein degradation (7%), and carbohydrate metabolism (5%)(Fig. 2B). The greatest number of identified proteins wereinvolved in cell redox homeostasis, which suggested that T-2toxin might trigger an oxidative stress response in chickenprimary hepatocytes.
Subcellular localization analysis indicated that 34% of theaffected proteins were mitochondrial, 24% were cytoplasmic,15% were nuclear, 7% were cytosolic, 6% localized to theplasma membrane, 6% localized to the extracellular space,4% localized to the ribosome, 3% localized to the peroxisome
and 1% localized to the Golgi apparatus (Fig. 2C). Theseresults suggest that the T-2 toxin may induce mitochondrialchanges in chicken primary hepatocytes.
3.4 T-2 toxin induces oxidative stress and
mitochondrial enhancement
To examine whether T-2 toxin induces oxidative stress andmitochondrial changes in chicken primary hepatocytes at thecellular level, the ROS content, mitochondrial mass, and cel-lular ATP levels of T-2 toxin-treated or -untreated chickenprimary hepatocytes were determined. As expected, the pro-portion of ROS-positive cells and intensity of the ROS signalin 0.05 �g/mL T-2 toxin-treated cells increased over time,compared to the untreated cells (Fig. 3). After treatmentfor 72 h, the proportion of ROS-positive cells increased to85% (Fig. 3). Interestingly, the mitochondrial mass and cel-lular ATP level increased significantly after T-2 toxin treat-ment. After a 48-h treatment with 0.05 �g/mL T-2 toxin, the
C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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on
,pla
sma
mem
bra
ne
80T
XN
L1g
i|508
0660
849
955/
8.72
;40
849/
5.11
476
296
−2.1
±0.
3Pr
ote
ind
isu
lfid
eox
ido
red
uct
ase
acti
vity
Cyt
op
lasm
,nu
cleu
s
Cel
lcyc
leo
rce
llp
rolif
erat
ion
5N
PM
1g
i|345
3078
8038
128/
4.87
;17
136/
5.36
162
184
−2.8
±0.
3H
isto
ne
bin
din
g,R
NA
bin
din
gN
ucl
eolu
s
18A
RH
GD
IBg
i|507
2856
822
929/
5.08
;21
162/
5.07
176
263
−4.8
±0.
4G
TPa
seac
tiva
tor
acti
vity
Cyt
op
lasm
,cyt
oso
l25
CD
V3
gi|7
1896
619
2160
0/4.
95;5
318
4/5.
1213
835
6−4
.2±
0.3
Cel
lpro
lifer
atio
nC
yto
pla
sm,n
ucl
eus
58S
EP
T2
gi|3
2692
9511
3938
0/6.
24;5
105
8/7.
6310
936
6−2
.5±
0.3
GT
Pb
ind
ing
Cyt
op
lasm
62P
SM
B7
gi|2
2414
4619
2803
2/5.
51;1
793
1/7.
7941
866
16−2
.5±
0.3
Pro
tein
po
lyu
biq
uit
inat
ion
Cyt
oso
l77
RA
NG
AP
1g
i|575
2516
016
424/
5.63
;34
454/
5.12
168
5716
−3.6
±0.
3G
TPa
seac
tiva
tor
acti
vity
Cyt
op
lasm
,nu
cleu
s78
RA
NB
P1
gi|4
3142
223
739/
5.15
;26
838/
5.10
156
163
−2.1
±0.
1G
TPa
seac
tivi
tyC
yto
pla
sm,n
ucl
eus
Tran
scri
pti
on
alo
rtr
ansl
atio
nal
reg
ula
tio
n7
NP
M3
gi|1
1809
2933
1474
6/4.
29;1
715
8/5.
2820
940
8−2
.8±
0.3
Nu
clei
cac
idb
ind
ing
Nu
cleo
lus
16S
ET
gi|3
9536
1724
348/
4.97
;20
996/
5.10
187
397
−4.8
±0.
4Pr
ote
inp
ho
sph
atas
ein
hib
ito
rac
tivi
tyE
nd
op
lasm
icre
ticu
lum
,n
ucl
eus
23W
DR
61g
i|945
3681
933
426/
5.24
;35
503/
5.08
279
5112
−4.2
±0.
3H
isto
ne
H3-
K4
trim
ethy
lati
on
Cyt
op
lasm
,nu
cleu
s
26H
NR
NP
Cg
i|620
8863
422
044/
9.44
;50
635/
5.12
193
53
−2.5
±0.
3m
RN
Ab
ind
ing
Nu
cleu
s61
MR
PS
23g
i|507
5841
422
130/
6.66
;18
056/
9.01
222
205
−2.5
±0.
3S
tru
ctu
ralc
on
stit
uen
to
fri
bo
som
eM
ito
cho
nd
rio
n
74M
RP
L12
gi|1
1809
9877
2094
0/9.
66;1
593
3/5.
5727
535
6−3
.6±
0.3
Str
uct
ura
lco
nst
itu
ent
of
rib
oso
me
Mit
och
on
dri
on
90H
NR
PD
Lg
i|718
9674
131
590/
9.01
;42
858/
9.86
149
255
−2.1
±0.
1R
NA
bin
din
gN
ucl
eus,
cyto
pla
sm92
TS
FMg
i|622
8664
523
940/
9.33
;37
054/
5.67
286
5212
−2.8
±0.
3Tr
ansl
atio
nel
on
gati
on
fact
or
acti
vity
Intr
acel
lula
r,m
ito
cho
nd
rio
n,n
ucl
eus
C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
3182 P. Mu et al. Proteomics 2013, 13, 3175–3188
Ta
ble
1.
Co
nti
nu
ed
Sp
ota)
No
.Pr
ote
inn
ame
Acc
.No
.b)
Th
eo.;
Ob
s.M
r/p
Ic)Pr
ote
insc
ore
d)
Seq
cov.
(%)e)
Pep
tid
esf)
Fold
g)
Mo
lecu
lar
fun
ctio
nh
)Lo
cati
on
i)
Tran
spo
rt4
TIM
M8A
gi|5
0745
652
1090
7/5.
13;1
110
5/5.
1530
068
158.
9±
0.8
Pro
tein
targ
etto
mit
och
on
dri
on
Mit
och
on
dri
on
14P
RB
P4
gi|1
3240
321
3975
.44;
1651
3/5.
5223
271
164
±0.
4Tr
ansp
ort
erac
tivi
ty,
reti
no
l-b
ind
ing
pro
tein
,E
xtra
cellu
lar
reg
ion
19S
NA
P23
gi|3
1385
1076
2387
4/4.
9;22
602/
5.15
102
63
4.1
±0.
2Pr
ote
intr
ansp
ort
Cel
lula
rm
emb
ran
e31
NA
PG
gi|5
0736
711
3538
7/5.
48;4
495
3/5.
4917
341
102.
2±
0.1
Intr
acel
lula
rp
rote
intr
ansp
ort
Mit
och
on
dri
on
66C
LIC
4g
i|118
1015
8735
857/
5.61
;25
194/
5.38
345
304
−4.1
±0.
2Vo
ltag
e-ga
ted
chlo
rid
ech
ann
elac
tivi
tyC
yto
pla
sm,i
ntr
acel
lula
r,m
ito
cho
nd
rio
n,n
ucl
eus,
cyto
sol
Str
ess
resp
on
se32
CN
PY
3g
i|363
7314
5325
232/
7.78
;40
483/
5.63
128
399
2.2
±0.
1In
nat
eim
mu
ne
resp
on
seE
nd
op
lasm
icre
ticu
lum
36M
PS
Tg
i|507
2878
833
486/
5.80
;33
062/
5.85
598
6718
2±
0.3
Th
iosu
lfat
esu
lfu
rtra
nsf
eras
eac
tivi
tyC
yto
pla
sm,m
ito
cho
nd
rio
n
48A
NX
A2
gi|4
5382
533
3890
1/6.
92;3
744
5/8.
3832
151
122.
9±
0.3
Ph
osp
ho
lipas
ein
hib
ito
rac
tivi
tyE
xtri
nsi
cto
pla
sma
mem
bra
ne
54G
LUL
gi|4
5382
781
4274
6/6.
38;4
934
5/9.
8061
636
72.
7±
0.2
Glu
tam
ate-
amm
on
ialig
ase
acti
vity
Mit
och
on
dri
on
,Go
lgi
app
arat
us
56G
LUL
gi|4
5382
781
4274
6/6.
38;5
067
7/8.
7028
429
72.
3±
0.3
Glu
tam
ate-
amm
on
ialig
ase
acti
vity
Mit
och
on
dri
on
65H
SP
25g
i|580
3755
819
086/
6.31
;21
282/
5.37
238
318
4±
0.5
Mo
lecu
lar
chap
ero
nes
Peri
nu
clea
rre
gio
no
fcy
top
lasm
Lip
idm
etab
olis
m37
EC
H1
gi|3
6374
7010
2281
3/11
.0;2
616
8/7.
0725
59
42.
7±
0.1
Iso
mer
ase
acti
vity
Mit
och
on
dri
on
38E
CH
1g
i|363
7470
1022
813/
11.0
;25
161/
6.91
122
43
3.7
±0.
3Is
om
eras
eac
tivi
tyM
ito
cho
nd
rio
n43
EC
HS
1g
i|326
9232
5723
924/
5.28
;24
025/
9.24
463
4915
6.5
±0.
3E
no
yl-C
oA
hyd
rata
seac
tivi
tyM
ito
cho
nd
rio
n
45E
CH
DC
2g
i|507
5161
630
801/
9.21
;24
704/
8.59
207
6515
3.1
±0.
5Ly
ase
acti
vity
Mit
och
on
dri
on
46E
CI2
gi |5
0734
079
3391
4/9.
03;2
580
5/8.
9220
746
123.
1±
0.3
Iso
mer
ase
acti
vity
Mit
och
on
dri
on
64A
PO
A1
gi|2
2701
628
790/
5.45
;22
870/
5.39
410
7216
2.3
±0.
2P
ho
sph
olip
idb
ind
ing
Ext
race
llula
rre
gio
n,p
lasm
am
emb
ran
eC
arb
ohy
dra
tem
etab
olis
m44
PG
AM
1g
i|718
9598
529
051/
7.03
;24
309/
8.79
403
6217
2.4
±0.
3B
isp
ho
sph
og
lyce
rate
mu
tase
acti
vity
Cyt
oso
l
51R
NF1
9Ag
i|118
1018
0138
672/
6.41
;39
583/
8.74
348
5010
8±
1.3
UD
P-g
luco
se4-
epim
eras
eac
tivi
tyC
yto
sol
64A
PO
A1
gi|2
2701
628
790/
5.45
;22
870/
5.39
410
7216
2.3
±0.
3P
ho
sph
olip
idb
ind
ing
Ext
race
llula
r,p
lasm
am
emb
ran
e
C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
Proteomics 2013, 13, 3175–3188 3183
Ta
ble
1.
Co
nti
nu
ed
Sp
ota)
No
.Pr
ote
inn
ame
Acc
.No
.b)
Th
eo.;
Ob
s.M
r/p
Ic)Pr
ote
insc
ore
d)
Seq
cov.
(%)e)
Pep
tid
esf)
Fold
g)
Mo
lecu
lar
fun
ctio
nh
)Lo
cati
on
i)
Pro
tein
deg
rad
atio
n20
PS
MF1
gi|7
1896
121
2935
2/5.
19;2
693
2/5.
1319
147
10−2
.3±
0.2
Pro
teas
om
ein
hib
ito
rC
yto
pla
sm62
PS
MB
7g
i|224
1446
1928
032/
5.51
;17
744/
8.76
418
6616
−2.5
±0.
1Pr
ote
inp
oly
ub
iqu
itin
atio
nC
yto
sol
70C
LPP
gi|5
6269
518
2307
8/8.
76;2
230
8/6.
1029
636
62.
2±
0.1
Ser
ine-
typ
een
do
pep
tid
ase
acti
vity
Mit
och
on
dri
on
75C
TS
Bg
i|461
9545
538
475/
5.74
;20
374/
5.11
350
344
−2.7
±0.
1C
yste
ine-
typ
een
do
pep
tid
ase
acti
vity
Ext
race
llula
rsp
ace
Oth
er22
EFH
D1
gi|7
2535
161
2696
7/5.
07;2
675
6/5.
0722
549
104.
1±
0.3
Neu
ron
pro
ject
ion
dev
elo
pm
ent
Mit
och
on
dri
alin
ner
mem
bra
ne
27V
IMg
i|572
4009
030
092/
4.75
;60
913/
5.13
336
6815
-21
±2.
3Pr
ote
inb
ind
ing
,tr
ansc
rip
tio
nfa
cto
rP
lasm
am
emb
ran
e,cy
toso
l
28P
PA2
gi|1
1809
0079
5218
0/8.
91;4
529
1/5.
5821
65
44.
8±
0.4
Ino
rgan
icd
iph
osp
hat
ase
acti
vity
Mit
och
on
dri
on
,cyt
op
lasm
30S
TOM
L2g
i|118
1036
3932
061/
6.14
;53
317/
5.55
259
4716
3.7
±0.
3R
ecep
tor
bin
din
gM
ito
cho
nd
rio
n,c
yto
pla
sm34
SE
PH
S1
gi|2
5576
0030
4340
8/5.
64;4
314
8/5.
6412
820
51.
3±
0.2
Sel
enid
e,w
ater
dik
inas
eac
tivi
tyPe
roxi
som
e
50E
SD
gi|3
2691
4131
3200
1/6.
14;3
870
2/8.
8024
928
83.
4±
0.4
S-f
orm
ylg
luta
thio
ne
hyd
rola
seac
tivi
tyC
yto
pla
sm,n
ucl
eus
63V
IMg
i|572
4009
030
092/
4.75
;62
194/
5.10
357
7218
−8.5
±1.
1Pr
ote
inb
ind
ing
,tr
ansc
rip
tio
nfa
cto
rP
lasm
am
emb
ran
e,cy
toso
l
71N
CL
gi|1
1959
1368
5857
6/4.
57;5
809
0/6.
2734
427
4−5
.8±
1.3
Nu
clei
cac
idb
ind
ing
Cyt
op
lasm
,nu
cleu
s
a)T
he
po
siti
on
so
fth
ese
spo
tsar
ed
isp
laye
din
the
gel
sin
Fig
.2.
b)
Th
eG
enb
ank
iden
tifi
cati
on
nu
mb
ero
fth
eid
enti
fied
pro
tein
s.c)
Th
eth
eore
tica
lan
do
bse
rved
rela
tive
mo
lecu
lar
mas
san
dis
oel
ectr
icp
oin
to
fid
enti
fied
pro
tein
s.d
)T
he
MA
SC
OT
sco
reo
fid
enti
fied
pro
tein
s.e)
Th
ese
qu
ence
cove
rag
eo
fp
rote
ins
inp
erce
nta
ge
ob
tain
edb
yM
ALD
I-TO
F/TO
F-M
Sid
enti
fica
tio
n.
f)N
um
ber
of
un
iqu
ep
epti
des
mat
ched
toth
ep
rote
inse
qu
ence
.g
)Po
siti
veva
lues
rep
rese
nt
up
reg
ula
tio
naf
ter
T-2
toxi
ntr
eatm
ent;
neg
ativ
eva
lues
rep
rese
nt
do
wn
reg
ula
tio
naf
ter
T-2
toxi
ntr
eatm
ent.
h)
Th
eid
enti
fied
pro
tein
sar
eg
rou
ped
acco
rdin
gto
thei
rfu
nct
ion
sb
yse
arch
ing
the
Gen
eO
nto
log
y(h
ttp
://w
ww
.gen
eon
tolo
gy.
org
/)an
dPu
bM
ed(h
ttp
://w
ww
.ncb
i.nlm
.nih
.gov
)d
atab
ases
.i)
Th
esu
bce
llula
rlo
caliz
atio
ns
are
cate
go
rize
db
yse
arch
ing
the
Gen
eO
nto
log
y(h
ttp
://w
ww
.gen
eon
tolo
gy.
org
/).
C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
3184 P. Mu et al. Proteomics 2013, 13, 3175–3188
Table 2. Functional networks generated by IPA analysisa)
Associated network functions Molecules in network Score
Neurological disease, cancer,organismal injury, andabnormalities
Actin, AKR1B1, Alpha catenin, ANXA2, APOA1, ARHGDIB, caspase, caveolin, CD3,Ck2, CLIC4, creatine kinase, CRYAB, CTSB, GLUL, histone h3, HNRNPC, LDL,Mapk, NAPG, NCL, NFkB (complex), NPM1, NPM3, P38 MAPK, PGAM1, Pkc(s),PSMF1, RANBP1, Ras, RBP4, SNAP23, TCR, Vegf, VIM
47
Protein trafficking, auditorydisease, cellular compromise
ADRM1, C11orf58, CDV3, ECH1, EFHD1, ESD, GRHPR, HIBADH, IDE, JOSD1, MPST,MRPS23, MSTN, PPA2 (includes EG:27068), PSMB7, PSME1, RNF19A, SEPHS1,SNCAIP, STOML2, SUFU, TIMM9, TIMM10, TIMM44, TIMM23B, TIMM8A,TIMM8B, TOMM7, TOMM22, UBC, UBE2L3, UBE2L6, UCKL1, USP30, WDR61
37
Amino acid metabolism,molecular transport, smallmolecule biochemistry
26s Proteasome, Akt, APP, BSG (includes EG:12215), C18-ceramide, CNPY3, CRYM,ECHS1, ERK1/2, GLRX5, GPI, Hsp27, HTT, IDE, IL6, MRAS, MRPL12, NADHdehydrogenase, NADH2 dehydrogenase (ubiquinone), ND2 (includesEG:140532), NDUFB10, NDUFS8, PARK7, PDGF-AA, PSME1, PTEN, SEPT2, SET,SYN1, taurine, trehalose, TSC1, TXNL1, UTP14A, VHL
20
a) The double or single underline in the table indicates up- or downregulation by T-2 toxin, respectively.
mitochondrial mass and ATP levels of the chicken primaryhepatocytes increased by threefold, compared to untreatedcells (Fig. 4), which might indicate mitochondrial functionwas enhanced in the T-2 toxin-treated chicken primary hepa-tocytes.
Because high ROS content always causes DNA damageand induces cell apoptosis [35], and increasing ATP content isalso associated with apoptosis [42, 43], the DNA damage wasexamined and the apoptosis was examined. The comet assayshowed that the DNA tail length and DNA content in thetail increased with time after T-2 toxin treatment, suggestingan increase in DNA breakage due to T-2 toxin treatment(Supporting Information Fig. S5). PI-FITC double stainingrevealed that T-2 toxin induced cell apoptosis in chickenprimary hepatocytes, and the proportion of late apoptotic andnecrotic cells increased over time after 0.05 �g/mL T-2 toxintreatment (Supporting Information Fig. S6). In summary, T-2toxin induced oxidative stress and mitochondrial enhance-ment in chicken primary hepatocytes, which may be causedby the alteration of proteins related to cell redox homeostasisand significant changes in the mitochondrion proteome.
4 Discussion
The trichothecene mycotoxins produced by Fusarium speciesare frequent contaminants of cereal grains and products in-tended for animal consumption. T-2 toxin is the most toxictrichothecene; animals suffer from several pathological con-ditions after consuming of foodstuffs contaminated with tri-chothecenes [4].
Proteomic analyses of the effects of T-2 toxin have been per-formed previously to study the metabolic pathways in porcineprimary hepatocytes [34]. Here, we mainly focused on the toxi-city of T-2 toxin in chicken using proteomic analysis. Through2DE and MALDI-TOF/TOF-MS, we identified 53 T-2 toxin-regulated proteins, which were mainly involved in cell redoxhomeostasis, cell cycle or cell proliferation, transcriptional ortranslational regulation, stress response, lipid metabolism,
transport, carbohydrate metabolism, or protein degradation.The proteins in these eight groups are generally consistentwith the previously reported toxic effects of T-2 toxin, such asoxidative stress and the inhibition of protein synthesis. Theinduction of oxidative stress by T-2 toxin has been reportedin many cell types, including rat ovarian granulosa cells [44],THP-1 monocytes [45], and chicken growth plate chondro-cytes [17]. These results indicate that oxidative stress may bea common effect of T-2 toxin. Another well-known effect ofT-2 toxin is the inhibition of protein synthesis through bind-ing to peptidyl transferase, which is an integral component ofthe 60S ribosomal subunit [7,46]. In this study, another threetranslation-related enzymes, TSFM, MRPS23, and MRPL12,were also found to be regulated by T-2 toxin (Table 1). Thesethree enzymes may also be related to the inhibition of proteinsynthesis by T-2 toxin. Apart from translation, three proteinsrelated to transcription, including SET, HNRNPC, and NPM3were identified (Table 1), which may be related to the inhibi-tion of RNA synthesis by T-2 toxin.
In addition to these proteins that may be related with thosewidely considered action of T-2 toxin, two subunits of mito-chondrial complex I, NDUFB10 and NDUFS8, were foundto be regulated by T-2 toxin in chicken primary hepatocytes(Table 1). In particular, NDUFB10 was upregulated morethan 20-fold by T-2 toxin (Table 1). Similarly, some mem-bers of mitochondrial complex I are regulated by T-2 toxinin porcine primary hepatocytes [34]. Mitochondrial complexI has been shown to be related to many neurological dis-eases in humans, such as Parkinson’s disease and Leighsyndrome [47, 48]. In the present study, the top functionalnetworks of T-2 toxin-regulated proteins generated by IPAanalysis were neurological disease, cancer, organismal in-jury, and abnormalities (Table 2, Supporting InformationFig. S2). Hence, chicken feed containing T-2 toxin may bea high risk factor for neurological disease. However, the hy-pothesis that the correlation of the function of proteins iden-tified in chicken primary hepatocytes with the action of T-2toxin, such as inhibiting protein, DNA or RNA synthesis andneurological disease requires further information.
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Proteomics 2013, 13, 3175–3188 3185
Figure 3. ROS production in T-2 toxin-treated or untreated chicken primary hepatocytes. Chicken primary hepatocytes were untreated(Control) or treated with 0.05 �g/mL of T-2 toxin for 12, 24, 48, and 72 h and exposed to DCFH-DA, followed by flow cytometry analysis.The data shown are representative dot-plot graphs of different groups of cells from three independent experiments. The M1 and M2 zonesindicate the fluorescence intensity of ROS-negative and -positive cells, respectively. Data are expressed as the mean% ± SEM of fluorescentcells in different groups from three independent experiments.
Most of the identified proteins under T-2 toxin treatmentwere localized to the mitochondrion, and all the identified mi-tochondrial proteins were upregulated by T-2 toxin (Fig. 2C,Table 1) that inspired our interest in the function of themitochondria in T-2 toxin-treated cells. The major role ofmitochondrion is to supply power to the cell through theaerobic metabolism of acetyl-CoA by the tricarboxylic acid cy-cle and oxidative phosphorylation, which produces ATP [49].ATP is used for many biological processes, such as mus-cle contraction, the synthesis, and degradation of biologicalmolecules, membrane transport, and apoptosis [50]. Interest-ingly, the mitochondrial mass and ATP content of chicken pri-mary hepatocytes increased significantly after treatment with0.05 �g/mL T-2 toxin (Fig. 4). It was reported that oxida-tive stress could induce mitochondrion mass increase, whichsuggested being a feedback mechanism that compensatedfor defects in mitochondria harboring mutated mitochon-drial DNA and a defective respiratory system [51]. In thisstudy, the T-2 toxin can induce oxidative stress to chicken pri-mary hepatocytes. Hence, we propose that the mitochondrionmass and ATP increase may be also a compensational mech-anism for the defects, such as DNA damage which brought
by T-2 toxin. Changes in mitochondrial protein expressionhave been proposed to alter the coupling efficiency of mito-chondrial oxidative phosphorylation, which influences ATPgeneration, and ROS generation [52]. The ROS content of T-2toxin-treated cells increased significantly compared to the un-treated cells (Fig. 3), which may also be partly due to the largevariation in mitochondria protein expression. The other ma-jor role of mitochondrion is maintaining oxidation–reduction(redox) balance [49]. Mitochondria are the major sites of cel-lular ROS production, which occurs through the reduction ofmolecular oxygen to superoxide by Complexes I and III of theelectron transport chain [53, 54]. Two subunits of Complex Iwere upregulated by T-2 toxin in chicken primary hepatocytes(Table 1), which may trigger ROS production and thereby in-duce ROS accumulation.
In summary, the toxicity of T-2 toxin in chicken pri-mary hepatocytes was investigated by proteomic analysis. Itwas found that T-2 toxin-induced oxidative stress and mi-tochondrion enhancement in chicken primary hepatocytes,which may be caused by changes in the expression of pro-teins related to cell oxidative stress and the mitochondrialproteome.
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3186 P. Mu et al. Proteomics 2013, 13, 3175–3188
Figure 4. Mitochondrial mass and ATP content changes inducedby T-2 toxin. (A) Representative MitoTracker (red) and nuclear(blue) staining of untreated (Control) and 48 h treated chicken pri-mary hepatocytes by 0.05 �g/mL T-2 toxin (T-2 toxin). (B) Ratio ofthe intensity of MitoTracker/nuclear staining in chicken primaryhepatocytes treated with 0.05 �g/mL T-2 toxin for 48 h. (C) ATPcontent of 0.05 �g/mL T-2 toxin treated chicken primary hepato-cytes relative to control.
This work was supported by the National Basic Research Pro-gram of China (973 Program) [Grant 2009CB118802]; the Na-tional Natural Science Foundation of China [Grant 31172087];the Specialized Research Fund for the Doctoral Program of HigherEducation of China [Grant 20114404110010]; the Guang-dong Natural Science Foundation [Grant S2012040007589]; theChina Postdoctoral Science Foundation [Grant 2012M511577];
The authors have declared no conflict of interest.
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