Toxicology 324 (2014) 55–67

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Mitochondrial proteomic analysis reveals the molecular mechanisms underlying reproductive toxicity of zearalenone in MLTC-1 cells Yuzhe Li a , Boyang Zhang a , Kunlun Huang a , Xiaoyun He a , YunBo Luo a , Rui Liang a , Haoshu Luo b , Xiao Li Shen a , Wentao Xua a, * a Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China b Department of Animal Physiology, College of Biological Sciences, China Agricultural University, Beijing, People’s Republic of China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 13 May 2014 Received in revised form 3 July 2014 Accepted 18 July 2014 Available online 22 July 2014

Zearalenone (ZEA), a Fusarium mycotoxin that contaminates cereal crops worldwide, has been shown to affect the male reproductive system and trigger reactive oxygen species (ROS) generation. However, the mechanisms of its toxicity have not been fully understood. Because mitochondrion is a key organelle involved in producing ROS and generating metabolic intermediates for biosynthesis, an iTRAQ-based mitoproteomics approach was employed to identify the molecular mechanism of zearalenone toxicity using mitochondria of mouse Leydig tumor cells (MLTC-1). A total of 2014 nonredundant proteins were identified, among which 1401 proteins (69.56%) were overlapped. There were 52 differentially expressed proteins in response to ZEA, and they were primarily involved in energy metabolism, molecular transport and endocrine-related functions. Consistent with mitochondrial proteomic analysis, the ATP and intracellular Ca2+ levels increased after ZEA treatment. The results suggest that lipid metabolism changed significantly after low-dose ZEA exposure, resulting in two alterations. One is the increase in energy production through promoted fatty acid uptake and b-oxidation, along with excessive oxidative stress; the other is an inhibition of steroidogenesis and esterification, possibly resulting in reduced hormone secretion. A hypothetical model of ZEA-induced mitochondrial damage is proposed to provide a framework for the mechanism of ZEA toxicity. ã 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Zearalenone Reproductive toxicity Mitoproteomics Fatty acid oxidation Endocrine disorder Lipid metabolism Mouse Leydig tumor cells

1. Introduction Zearalenone (ZEA), a mycotoxin produced by several species of Fusarium, is a frequent contaminant of cereal crops worldwide. It is a non-steroidal estrogen that causes endocrine disruptions in estrogen-responsive tissues of mammals. Animals and humans are exposed to ZEA by consuming cereals and their by-products. ZEA has been shown to affect the male reproductive system. ZEA caused testicular germ cell depletion (Kim et al., 2003), altered

Abbreviations: Acetyl-CoA, acetyl coenzyme A; AGC, automatic gain control; EF, error factor; FBS, fetal bovine serum; HCD, higher energy C-trap dissociation; hCG, human chorionic gonadotropin; IC50, concentration of inducing 50% of cell death; MEHP, mono-ethylhexyl phthalate; MLTC-1, mouse Leydig tumor cells; MMTS, methyl methanethiosulfonate; PBS, phosphate-buffered saline; PDH, pyruvate dehydrogenase; PPARs, peroxisome proliferator-activated receptors; PPP, pentose phosphate pathway; ROS, reactive oxygen species; TBST, Tris-buffered saline containing 0.1% Tween-20; TCA, tricarboxylic acid cycle; TCEP, Tris-(2-carboxyethyl) phosphine; WST-8, water-soluble tetrazolium salt; ZEA, zearalenone; Dcm, mitochondrial membrane potential. * Corresponding author. Tel.: +86 10 6273 7786; fax: +86 10 6273 7786. E-mail addresses: [email protected], [email protected] (W. Xua). http://dx.doi.org/10.1016/j.tox.2014.07.007 0300-483X/ ã 2014 Elsevier Ireland Ltd. All rights reserved.

testis morphological parameters (Filipiak et al., 2009), reduced serum testosterone concentrations (Yang et al., 2007), and disturbed fertility (Koraichi et al., 2013). The majority of ZEA toxicity is attributed to its estrogenic activity (Ryu et al., 2002; Shier et al., 2001). However, the low binding affinity of ZEA towards estrogen receptors has been confirmed as less than 1/10 that of 17b-estradiol (Takemura et al., 2007). Thus, recent reports argue that the overall ZEA toxicity is not solely caused by its estrogenicity (El Golli Bennour et al., 2009; Hassen et al., 2007), and other mechanisms that target general cellular compartments could be involved. Oxidative damage is likely to be evoked as one of the main pathways of ZEA toxicity both in vivo and in vitro (Bouaziz et al., 2008; El Golli-Bennour and Bacha 2011; Hassen et al., 2007; Zourgui et al., 2008). ZEA induces significant alterations in all tested oxidative stress markers of Balb/c mice (Zourgui et al., 2008). A study on fission yeast also demonstrates ZEA toxicity via the accumulation of reactive oxygen species (ROS) and the regulation of antioxidant enzyme activities (Mike et al., 2013). Because the major endogenous source of cellular ROS is the mitochondrial electron transport chain, mitochondria are particularly susceptible to oxidative damage.

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ZEA reportedly induced cell apoptosis via a mitochondrial pathway involving mitochondrial alterations including ROS production (Bouaziz et al., 2008). Additionally, Salwa Abid-Essefi reported that the apoptotic effects and genotoxicity of ZEA were largely prevented by the antioxidant vitamin E, which was consistent with the toxic effects of ZEA being induced by oxidative stress (Abid-Essefi et al., 2003; Ghedira-Chekir et al., 1998). These results point to a central role for mitochondria in the toxic effects induced by ZEA, but the way in which ZEA toxicity might be regulated by mitochondria is still unclear. At present, knowledge about the ZEA toxicity mechanism, especially at the molecular level, is insufficient (Banjerdpongchai et al., 2010; Gazzah et al., 2013; Heneweer et al., 2007). Furthermore, to our knowledge, no study has undertaken global protein expression profiling to investigate the reproductive toxicity mechanisms of ZEA at the mitochondrial level. It is well known that Leydig cells play a crucial role in regulating the process of spermatogenesis and synthesizing testosterone. Alteration of Leydig cell function can lead to adverse effects on testicular functions. The aim of the present study was to apply a proteomic approach to assess the changes in mitochondrial protein expression initiated by ZEA in mouse Leydig tumor cells (MLTC-1). The iTRAQ-based proteomics method makes it possible to perform global protein expression analyses and to detect quantitative changes accurately. Because the steroidogenesis of MLTC-1 cells can be stimulated by human chorionic gonadotropin (hCG), and the mitochondrion is a key organelle involved in steroidogenesis, hCG was also used in the present study to explore the effect that ZEA exerts on mitochondria during steroidogenesis and testosterone secretion. 2. Materials and methods

trypsinized. The fluorescence intensity was then determined by FACSCalibur (BD Biosciences, USA). At least 1 104 cells in gate were collected for flow cytometry analysis. 2.4. Measurement of mitochondrial membrane potential (DC m) The mitochondrial membrane potential (DCm) was monitored with rhodamine 123 (Beyotime, PRC), a fluorescence probe that is selectively retained in mitochondria with intact membrane potentials. The uptake of rhodamine 123 by mitochondria is proportional to the DCm. In brief, cells were treated as above, washed once with PBS and incubated for 30 min at 37  C with 1 mM rhodamine 123, followed by two PBS washes to remove the fluorescence probe. The fluorescence intensity was then determined by FACSCalibur (BD Biosciences, USA). At least 1 104 cells in gate were collected for flow cytometry analysis. 2.5. Mitochondria isolation Mitochondria were isolated by using a Cell Mitochondria Isolation Kit (Beyotime, PRC) according to the manufacturer’s instructions (Shen et al., 2013). In brief, MLTC-1 cells were treated with different ZEA concentrations, then washed with PBS, trypsinized and centrifuged to collect the cells. The cells were resuspended in ice-cold PBS and centrifuged at 600  g, 4  C for 5 min. The precipitate was resuspended in mitochondria isolation reagent (2  107 cells/ml) supplemented with 1 mM PMSF on ice for 15 min. The resuspension was homogenized until Trypan Blue stained approximately 50% of the cells blue. To extract highly pure mitochondria, the homogenate was centrifuged at 1000  g at 4  C for 10 min. The supernatant was then centrifuged in a new tube at 3500  g at 4  C for 10 min. The precipitate from this process was mitochondria.

2.1. Cell culture and treatment MLTC-1 cells were grown in 1640 medium supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA), 100 U/ml penicillin, 100 mg/ml streptomycin, and 250 ng/ml amphotericin B (Macgene, PRC) at 37  C in 5% CO2 and 95% saturated atmospheric humidity. The base medium, RPMI-1640, contains 2 g/l glucose without any galactose. MLTC-1 cells at approximately 90% confluence were divided into four experimental groups as follows: treated with serum-free medium as a control (group control); treated with 5 mM ZEA (sigma) for 24 h (group ZEA); treated with serum-free medium for 24 h and then stimulated with 0.1 U/ml hCG (sigma) for 2 h (group hCG) and treated with 5 mM ZEA for 24 h and then stimulated with 0.1 U/ml hCG for 2 h (group ZhCG). 2.2. Cell viability assay Cell viability was determined by using Cell Counting Kit-8 according to the manufacturer’s instructions. In brief, 1 104 cells/well were seeded in a 96-well flat-bottomed plate, grown at 37  C for 24 h, treated with different concentrations of ZEA for 24 h, and washed once with phosphate-buffered saline (PBS). Subsequently, 10 ml of water-soluble tetrazolium salt (WST-8) dye and 100 ml of PBS were added to each well, and the cells were incubated at 37  C for 1 h. Finally, the dye absorbance of reduced WST-8 was determined at 450 nm in a plate reader. 2.3. Determination of intracellular ROS The cells were cultured in a 6-well plate at a density of 2  105 cells/well. After 24 h of ZEA exposure, the cells were then loaded with 10 mM DCFH-DA (Beyotime, China) and incubated at 37  C for 20 min. The cells were washed twice with serum-free medium and

2.6. ITRAQ labeling, mass spectrometry identification and mitoproteome data analysis 2.6.1. Sample preparation The samples were ground in liquid nitrogen. One milliliter of lysis buffer (7 M urea, 2 M thiourea, and 1  Protease Inhibitor Cocktail (Roche Ltd., Basel, Switzerland)) was added to each sample, followed by sonication on ice and centrifugation at 13,000 rpm for 10 min at 4  C. The supernatant was transferred to a fresh tube and stored at 80  C until needed. 2.6.2. ITRAQ labeling and protein digestion The proteins in each sample were precipitated with ice-cold acetone, and they were then redissolved in dissolution buffer (0.5 M triethylammonium bicarbonate and 0.1% SDS). The proteins were then quantified by BCA protein assay, and 100 mg of protein was tryptically digested; the resulting peptide mixture was labeled using chemicals from the iTRAQ reagent kit (Applied Biosystems, California, USA). Disulfide bonds were reduced in 5 mM Tris-(2-carboxyethyl) phosphine (TCEP) for 1 h at 60  C, and then the cysteine residues were blocked in 10 mM methyl methanethiosulfonate (MMTS) for 30 min at room temperature before digesting with sequence-grade modified trypsin (Promega, Madison, WI). For labeling, each iTRAQ reagent was dissolved in 50 ml of isopropanol and added to the respective peptide mixture. Proteins were labeled with the iTRAQ tags as follows: control-117 isobaric tag, ZEA-118 isobaric tag, hCG-119 isobaric tag, and ZhCG-121 isobaric tag. The labeled samples were combined and dried in vacuo. A SepPac C18 cartridge (1 cm3/50 mg, Waters Corporation, Milford, MA) was used to remove the salt buffer and was then dried in a vacuum concentrator for the next step.

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2.6.3. High pH reverse phase separation The peptide mixture was redissolved in buffer A (buffer A: 20 mM ammonium formate in water, pH 10.0, adjusted with ammonium hydroxide), and then fractionated by high pH separation by using ACQUITY UPLC system (Waters Corporation, Milford, MA) connected to a reverse phase column (XBridge C18 column, 2.1 mm  150 mm, 3.5 mm, 300 Å, Waters Corporation, Milford, MA). A high pH separation was performed using a linear gradient beginning at 5% B and increasing to 35% B in 40 min (B: 20 mM ammonium formate in 90% ACN, pH 10.0, adjusted with ammonium hydroxide) (Gilar et al., 2005). The column was re-equilibrated at initial conditions for 15 min. The column flow rate was maintained at 200 ml/min and the column temperature was maintained at room temperature. Twelve fractions were collected, and each fraction was dried in a vacuum concentrator for the next step. 2.6.4. Low pH nano-HPLC–MS/MS analysis The peptides were resuspended in 80 ml of solvent C (C: water with 0.1% formic acid; D: ACN with 0.1% formic acid), separated by nanoLC and analyzed by on-line electrospray tandem mass spectrometry. The experiments were performed on a nano-ACQUITY UPLC system (Waters Corporation, Milford, MA) connected to an LTQ Orbitrap XL mass spectrometer (Thermo Electron Corp., Bremen, Germany) equipped with an online nanoelectrospray ion source (Michrom Bioresources, Auburn, USA). Eighteen microliter peptide samples were loaded onto the Thermo Scientific Acclaim PepMap C18 column (100 mm  2 cm, 3 mm particle size) with a flow of 10 ml/min for 5 min and subsequently separated on the analytical column (Acclaim PepMap C18, 75 mm  15 cm) with a linear gradient from 5% D to 45% D in 165 min. The column was re-equilibrated at initial conditions for 15 min. The column flow rate was maintained at 300 nl/min and the column temperature was maintained at 35  C. An electrospray voltage of 1.9 kV versus the inlet of the mass spectrometer was used. LTQ Orbitrap XL mass spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra (m/z 400–1600) were acquired in the Orbitrap with a mass resolution of 30,000 at m/z 400, followed by five sequential HCD–MS/MS scans. The automatic gain control (AGC) was set to 500,000 ions to prevent the over-filling of the ion trap. The minimum MS signal for triggering MS/MS was set to 1000. In all cases, one microscan was recorded. MS/MS scans were acquired in the Orbitrap with a mass resolution of 7500. The dissociation mode was HCD (higher energy C-trap dissociation). Dynamic exclusion was used with two repeat counts, a 10 s repeat duration, and the m/z values triggering MS/MS were put on an exclusion list for 120 s. For MS/MS analysis, precursor ions were activated using 40% normalized collision energy and an activation time of 30 ms. 2.6.5. Database searching and criteria The protein identification and quantification for the iTRAQ experiment was performed with ProteinPilot software version 4.0 (Applied Biosystems, California, USA). The database was the Mouse UniProtKB/Swiss-Prot database (Release 2012_12_27, with 16,409 sequences) The Paragon Algorithm of the ProteinPilot software was used for peptide identification and isoform-specific quantification. The data search parameters were set up as follows: a trypsin (KR) cleavage with two missed cleavages was considered; fixed modification of cysteines by MMTS, iTRAQ modification of peptide N terminal, methionine oxidation and iTRAQ modification of lysine residues were set as variable modification. To minimize false positive results, a strict cutoff for protein identification was applied with the unused ProtScore 1.3, which corresponds to a confidence limit of 95%, and at least two peptides with the 95% confidence were considered for protein quantification. The

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resulting data set was auto bias-corrected to remove any variations imparted by unequal mixing when combining different labeled samples. For iTRAQ quantitation, the peptide for quantification was automatically selected by Pro Group algorithm (at least two peptides with 99% confidence) to calculate the reporter peak area, error factor (EF), and P-value. 2.6.6. Analysis of proteomic data Differentially expressed proteins were classified according to annotations from the UniProt knowledge base (Swiss-Prot/ TrEMBL, http://www.uniprot.org/) and the GO database (http:// www.geneontology.org/). The David database (http://david.abcc. ncifcrf.gov/) was used to elucidate biological process and molecular function. Pathways were elucidated according to KEGG_PATHWAY and PANTHER_PATHWAY associated with each differentially expressed protein. The STRING database (version 9.0) (http://string-db.org/) was used to predict and visualize the protein–protein interaction networks among differentially expressed proteins. 2.7. Verification of proteomic data by western blot analysis The isolated mitochondria were lysed on ice in RIPA lysis buffer containing 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and a complete protease inhibitor cocktail (sodium orthovanadate, sodium fluoride, EDTA, and leupeptin) (Beyotime, PRC) supplemented with 1 mM PMSF. The cells were then homogenized using a 1 ml syringe (Shen et al., 2013). Cellular lysates were centrifuged at 13,000  g for 10 min at 4  C. The supernatant proteins were collected and quantified with a BCA Protein Assay Kit (Cwbiotech, PRC). Equal amounts (40 mg) of protein from each sample were loaded on 4–16% tricine-SDS-PAGE gels and blotted onto nitrocellulose membranes for 1.5 h at 80 V. Nonspecific binding was blocked by incubating the samples in blocking buffer (1% BSA and Tris-buffered saline containing 0.1% Tween-20 (TBST)) for 1 h, and the membranes were incubated for 1 h with one of the following primary antibodies: rabbit anti-CPT2 (Santa Cruz Biotech, sc-20671; 1:1000), rabbit anti-AIF (Cell Signaling, 5318; 1:1000), rabbit anti-prohibitin 2 (Santa Cruz Biotech, sc-67045; 1:1000), rabbit anti-cytochrome c (Cell Signaling, 4280; 1:1000), and mouse anti-GAPDH (Cali-Bio CB100127; 1:1000). After washing the membranes with TBST 3 times for 5 min each time, the membranes were probed with AP-labeled goat antirabbit or anti-mouse antibody. After washing the membranes as described above, specific bands were detected with BCIP/NBT (Merck-Calbiochem). The total gray values of each band were digitized with BandScan V4.3. The relative expression level of each protein was normalized with a reference protein (GAPDH), and the resulting ratios in the CK group were normalized to 1. 2.8. Verification of ATP activation in MLTC-1 cells ATP levels in whole-cell lysates were measured using a firefly luciferase-based ATP assay kit (Beyotime, PRC) according to the manufacturer’s instructions (Li et al., 2012). MLTC-1 cell culture and ZEA exposure were performed as indicated in Section 2.1. After rinsed with PBS, the cells were schizolysised by solution and then centrifuged at 12,000  g at 4  C for 5 min and the supernatant was collected. In a 1.5 ml tube, 100 ml of the supernatant was mixed with 100 ml of ATP detection solution. Luminance was immediately measured using a Synergy H1 multidetection microplate reader (Bio-Tek USA). Standard quantification curves were also generated by using known amounts of an ATP standard, and sample ATP concentrations were normalized to cell equivalents. The ATP content was expressed as a percentage of the ATP content in cells of the control group.

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2.9. Verification of Ca2+ turbulence in MLTC-1 cells The intracellular Ca2+ level in MLTC-1 cells was measured using the fluorescence Ca2+ indicator Fluo-3 AM (Che et al., 2012). The cell culture and ZEA exposure was performed as indicated above (Section 2.1). After 24 h of ZEA exposure, the treated and control cells were collected, rinsed twice with PBS, and stained with 5 mg/ml of Fura-3 AM for 30 min. After staining, the cells were rinsed twice with PBS and incubated for another 10 min at 37  C before fluorescence-intensity detection by FACSCalibur (BD Biosciences, USA). 3. Results 3.1. Cell viability assay To analyze the effect of zearalenone on MLTC-1 cell viability, the cells were treated with different doses (from 0 to 50 mM) of zearalenone for 24 h. As shown in Fig. 1, zearalenone caused cell death in a dose-dependent manner as its concentration increased. We observed that when the cells were treated with zearalenone at concentrations over 5 mM, the cell viability decreased significantly compared with the control (P < 0.05). 3.2. Determination of ROS production ROS are generated in and around mitochondria; thus, we further explored whether zearalenone has an effect on the intracellular ROS levels of MLTC-1 cells. ROS generation was investigated using DCFH-DA fluorescent probe, which detects peroxide radicals and various other active oxygen radicals. Upon interaction with ROS, the fluorochrome DCFH-DA is converted to fluorescent DCF. Therefore, the level of intracellular ROS can be evaluated by analyzing DCF fluorescence. As shown in Fig. 2, the DCF fluorescence of MLTC-1 cells treated with zearalenone was statistically enhanced in a dose-dependent manner compared with the control, implying that zearalenone treatment results in an increase of intracellular ROS in MLTC-1 cells. As shown in Fig. 2, the mean fluorescence intensity of DCF was significantly increased with 5, 7.5, 10 and 20 mM zearalenone treatments (P < 0.05)

Fig. 2. ZEA-induced ROS production. MLTC-1 cells were incubated with various concentrations of ZEA (0–50 mM) for 24 h. The experiment was repeated for at least three times in three replicates. The data shown are the means  SD. The data were analyzed by one-way ANOVA. “*” and “**” indicate a significant difference compared with the control group (*, P < 0.05; **, P < 0.01).

in Fig. 3, the Rh123 fluorescence of MLTC-1 cells statistically decreased in a dose-dependent manner as the concentration of zearalenone increased from 5 mM to 50 mM, implying that zearalenone leads to the loss of mitochondrial transmembrane potential in MLTC-1 cells. 3.4. Determination of the concentration of zearalenone for mitoproteome analysis

We also tested whether zearalenone caused changes in the mitochondrial transmembrane potential of MLTC-1 cells. As shown

In studies of the toxicity mechanisms of ZEA in vitro, a concentration of inducing 50% of cell death (IC50) was often selected (Ayed et al., 2011) and varied between different cell lines (Abid-Essefi et al., 2003). In the current study, we found that three physiological symptoms associated with mitochondrial function, increased ROS, decreased mitochondrial membrane potential, and the death of cells are all significantly modulated when the ZEA concentration is approximately 5 mM or above. Moreover, Yang et al. (Yang et al., 2012) reported that mono-ethylhexyl phthalate (MEHP) induced mitochondrial damage and dysfunction as the cell viability decreased by approximately 10%. Mitochondria damage occurs before cell death, so it is reasonable to use a ZEA concentration with mild cytotoxicity to study the molecular events involved in ZEA-induced mitochondria damage. Therefore, the cells were collected after 5 mM ZEA treatment for mitochondrial protein extraction, and the proteomic analysis was then investigated by iTRAQ.

Fig. 1. Cytotoxicity of ZEA. MLTC-1 cells were incubated with various concentrations of ZEA (0–50 mM) for 24 h. The effect of ZEA on cell viability was measured by a CCK-8 assay. The values are the means  SD. The experiment was repeated for at least three times in five replicates. The data were analyzed by one-way ANOVA. “*” and “**” indicate a significant difference compared with the control group (*, P < 0.05; **, P< 0.01).

Fig. 3. ZEA-induced loss of mitochondrial membrane potential (DCm). MLTC-1 cells were incubated with various concentrations of ZEA (0–50 mM) for 24 h. The data are the means  SD. The experiment was repeated for at least three times in three replicates. The data were analyzed by one-way ANOVA. “**” indicates a significant difference compared with the control group (P < 0.01).

3.3. Determination of mitochondrial membrane potential (DC m)

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Zinedine reviewed the occurrence of zearalenone in foods and feeds, providing overwhelming evidence of global contamination of cereals and animals with zearalenone (Zinedine et al., 2007). For most crops around the world, zearalenone contamination range from 0.001 to 1.0 mg/kg, and in some area certain crops are highly contaminated as up to 600 mg/kg. In vivo, the tissue distribution of zearalenone in rats was characterized after intravenous or oral administration of zearalenone at different doses. The steady-state zearalenone concentration is about 100–2000 ng/g in various tissues, and 200–500 ng/g in the serum (Shin et al., 2009). A study regarding pigs fed with mycotoxin-contaminated oats demonstrated that the concentration of zearalenone in liver is about 1.0–3.1 ng/g (Zollner et al., 2002). Past studies have suggested that long-term consumption of low, but biologicallyactive levels of zearalenone post potential health risk to human (Kuiper-Goodman et al., 1987). Though the zearalenone concentration tested in the current study is relatively higher than the real world most exposure levels, it was still possible to obtain scientific evidence for the assumption of the mechanism of zearalenone toxicity. 3.5. Data analysis of the mitoproteome 3.5.1. Differentially expressed mitoproteome The differentially expressed mitoproteome with ZEA and/or hCG treatment was quantified using an iTRAQ-based proteomics approach. A total of 22,510, 24,081 and 23,764 distinct peptides were identified from 53,586, 55,580 and 53,699 spectra in three biological replicates, respectively. The numbers of identified proteins with unused score >1.3 were 1655, 1729 and 1707, respectively. Among these proteins, the number of identified proteins with peptides 2 was 1179, 1254 and 1239, respectively. We identified a total number of 2014 nonredundant proteins for three biological samples, among which 1401 proteins (69.56%) overlapped between the three biological samples (Fig. 4A). If we considered the proteins identified by at least two distinct peptides

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and ratios with P values  0.05 in all three biological replicates, 52 proteins with differential expression regulated by ZEA were identified (Table 1). Under hCG treatment and using the above criteria, we identified 15 proteins that were differentially expressed following ZEA exposure (Table 2). These two groups of proteins were used for further analysis. 3.5.2. Classification of differentially expressed mitoproteome In total, 52 proteins in response to ZEA were identified. These proteins were assigned to different functional categories, such as carbohydrate metabolism, molecular transport, lipid metabolism, endocrine system function, protein metabolism, and redox homeostasis. 3.5.2.1. Carbohydrate metabolism. ZEA treatment induced 52 differentially expressed proteins, and 11 proteins were involved in carbohydrate metabolism (Fig. 4B and Table 1). Most of the proteins are key enzymes of the tricarboxylic acid cycle, the pentose phosphate pathway, glycolysis and the glycolytic pathway. Among them, 5 proteins (IDH3A, MDHM, PYC, ODPA, and DLDH) are essential enzymes in the citrate cycle (TCA cycle), and they are all up-regulated after ZEA exposure. Specifically, isocitrate dehydrogenase 3 alpha (IDH3A) is responsible for the rate-limiting step of the TCA cycle. ODPA and DLDH are members of the pyruvate dehydrogenase (PDH) complex, which is a key regulator of TCA cycle flux. The PDH complex catalyzes the conversion of pyruvate into acetyl coenzyme A (acetyl-CoA), and thus, the up-regulation of the PDH complex would promote the entry of glucose-derived carbons into the TCA cycle (Schulze and Downward, 2011). Therefore, the up-regulation of these 5 proteins suggested that the TCA cycle was activated. Three enzymes (6PGD, TALDO, and TKT) involved in the pentose phosphate pathway (PPP) are down-regulated. 6-Phosphogluconate dehydrogenase (6PGD) is the third enzyme of the pentose phosphate pathway. This enzyme catalyzes the oxidative decarboxylation of

Fig. 4. An overview of the differentially expressed mitoproteome. (A) A Venn diagram depicting the overlap of proteins identified by iTRAQ measurements with three biological replicates. (B) Functional classification and pathways of identified, significantly different mitochondrial proteins in response to ZEA versus the control. The protein– protein interaction networks are shown in (B) according to the String database. The representative networks were profiled according to GO biological processes, molecular functions, or KEGG pathways and were integrated with the String database. The interactions among proteins that are strongly supported by previous work are linked by lines. The gene names of corresponding proteins are displayed in the networks.

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Table 1 The mitochondrial proteins that were differentially expressed in response to ZEA treatment as quantified by iTRAQ. Protein name

Gene name

Accession number

No. of unique peptides

Unused protein score

%Cov (95)

118(O):117 mean  SD

MDHM PYC IDH3A ODPA

P08249 Q05920 Q9D6R2 P35486

26 50 9 8

39.36 90.61 18.03 17.65

67.75 40.83 27.05 20.51

1.304  0.010 1.179  0.023 1.213  0.063 1.289  0.042

DLDH PCKGM PGK1 ALDOC TKT TALDO 6PGD

O08749 Q8BH04 P09411 P05063 P40142 Q93092 Q9DCD0

10 14 36 11 18 7 14

19.84 25.07 58.82 11.05 38.33 12.15 29.92

22.2 22.34 69.3 32.78 32.91 18.1 36.02

1.215  0.045 1.245  0.017 0.733  0.010 0.769  0.026 0.804  0.012 0.790  0.034 0.792  0.036

AT5F1 ETFD

Q9CQQ7 Q921G7

8 29

15.78 54.26

28.52 50.65

1.353  0.073 1.246  0.023

ETFB CYC QCR2 CHCH3

Q9DCW4 P62897 Q9DB77 Q9CRB9

14 5 14 3

29.52 9.69 28.68 7.94

54.12 54.29 34.88 13.66

1.209  0.057 1.460  0.019 1.218  0.062 1.214  0.068

SAM50 IMMT TOM70 STML2 TOM40

Q8BGH2 Q8CAQ8 Q9CZW5 Q99JB2 Q9QYA2

13 26 4 10 4

26.79 60.79 9.29 20.33 7.21

34.75 37.91 5.73 36.54 10.25

1.237  0.016 1.218  0.017 1.226  0.054 1.141  0.020 1.247  0.068

Lipid metabolism Aspartate aminotransferase, mitochondrial Perilipin-1 Carnitine O-palmitoyltransferase 2, mitochondrial Acyl-CoA synthetase family member 2, mitochondrial Alcohol dehydrogenase class 4 mu/sigma chain Lanosterol 14-alpha demethylase Acetyl-CoA acetyltransferase, cytosolic

AATM PLIN1 CPT2 ACSF2 ADH7 CP51A THIC

P05202 Q8CGN5 P52825 Q8VCW8 Q64437 Q8K0C4 Q8CAY6

16 6 17 15 9 18 7

29.56 11.59 35.2 30.5 16.93 36.31 13.72

39.77 15.47 32.67 26.67 20.32 35.59 22.67

1.285  0.034 1.304  0.012 1.204  0.046 1.149  0.031 0.814  0.035 1.279  0.015 0.788  0.020

Endocrine system development and function Prolyl endopeptidase Keratin, type I cytoskeletal 10 Peptidyl-prolyl cis-trans isomerase FKBP4 Prohibitin-2 Heat shock protein 75 kDa, mitochondrial Heat shock 70 kDa protein 4

PPCE K1C10 FKBP4 PHB2 TRAP1 HSP74

Q9QUR6 P02535 P30416 O35129 Q9CQN1 Q61316

6 5 4 19 14 6

13.65 8.49 9.42 36.38 24.46 12.89

9.3 10 7.42 61.2 19.12 9.63

0.773  0.028 0.270  0.039 0.841  0.016 1.209  0.033 1.221  0.041 0.822  0.046

Protein synthesis and modification Alanine—tRNA ligase, cytoplasmic Aspartate—tRNA ligase, cytoplasmic Arginine—tRNA ligase, cytoplasmic T-complex protein 1 subunit epsilon

SYAC SYDC SYRC TCPE

Q8BGQ7 Q922B2 Q9D0I9 P80316

16 14 4 11

33.75 29.4 6.27 19.77

17.67 28.34 6.06 19.22

0.806  0.011 0.856  0.017 0.830  0.034 1.174  0.032

Redox homeostasis Aldose reductase-related protein 2 Alanine aminotransferase 1 MOSC domain-containing protein 2, mitochondrial

ALD2 ALAT1 MOSC2

P45377 Q8QZR5 Q922Q1

12 4 9

16.58 9.53 18.22

32.28 8.27 26.04

0.799  0.011 0.798  0.018 1.287  0.026

Ca2+homeostasis Annexin A7 Apoptosis-inducing factor 1, mitochondrial Synaptic vesicle membrane protein VAT-1 homolog Annexin A3

ANXA7 AIFM1 VAT1 ANXA3

Q07076 Q9Z0X1 Q62465 O35639

8 16 8 6

13.98 31.15 17.66 9.83

17.28 29.08 22.41 23.22

0.851  0.020 1.222  0.027 1.246  0.004 0.789  0.028

Other Monofunctional C1-tetrahydrofolate synthase, mitochondrial Dihydropyrimidinase-related protein 2 Multifunctional protein ADE2 Prohibitin Plasminogen activator inhibitor 1 RNA-binding protein Ribonuclease inhibitor

C1TM DPYL2 PUR6 PHB PAIRB RINI

Q3V3R1 O08553 Q9DCL9 P67778 Q9CY58 Q91VI7

16 14 13 12 5 9

32.72 27.02 25.54 25.75 7.28 17.72

20.16 35.49 32.71 46.69 14.5 22.37

1.189  0.019 0.814  0.007 0.845  0.011 1.209  0.036 0.776  0.058 0.802  0.009

Carbohydrate metabolism Malate dehydrogenase, mitochondrial Pyruvate carboxylase, mitochondrial Isocitrate dehydrogenase (NAD) subunit alpha, mitochondrial Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial Dihydrolipoyl dehydrogenase, mitochondrial Phosphoenolpyruvate carboxykinase (GTP), mitochondrial Phosphoglycerate kinase 1 Fructose-bisphosphate aldolase C Transketolase Transaldolase 6-Phosphogluconate dehydrogenase, decarboxylating Molecular transport ATP synthase subunit b, mitochondrial Electron transfer flavoprotein-ubiquinone oxidoreductase, mitochondrial Electron transfer flavoprotein subunit beta Cytochrome c, somatic Cytochrome b-c1 complex subunit 2, mitochondrial Coiled-coil-helix-coiled-coil-helix domain-containing protein 3, mitochondrial Sorting and assembly machinery component 50 homolog Mitochondrial inner membrane protein Mitochondrial import receptor subunit TOM70 Stomatin-like protein 2 Mitochondrial import receptor subunit TOM40 homolog

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6-phosphogluconate to ribulose 5-phosphate with concomitant production of NADPH (Tetaud et al., 1999). Transketolase (TKT) and transaldolase (TALDO) are key enzymes of the non-oxidative branch of PPP determining its efficiency (Pacal et al., 2011). The down-regulation of the three essential enzymes in PPP implies that the pentose phosphate pathway activity was repressed after ZEA treatment. In addition, two glycolytic enzymes (PGK1 and ALDOC) were down-regulated in response to ZEA treatment. Phosphoglycerate kinase-1 (PGK1) is an ATP-generating glycolytic enzyme that forms part of the glycolytic pathway (Shi et al., 2010). One rate-controlling enzyme of gluconeogenesis (Nyirenda et al., 2006), namely phosphoenolpyruvate carboxykinase (PCKGM), was up-regulated in response to ZEA treatment. Therefore, increased TCA cycle activity and gluconeogenesis activity with the inhibition of PPP and glycolysis activity all indicated that carbohydrate metabolism was disrupted by ZEA treatment. 3.5.2.2. Molecular transport. Of the ZEA-induced differentially expressed proteins (Fig. 4B and Table 1), 9 were identified as being involved in transport processes, and all of them were up-regulated. Among these proteins, 5 were involved in the oxidative phosphorylation pathway, namely ATP synthase subunit b (AT5F1) and 4 proteins from the electron transport chain (ETFD, ETFB, QCR2, and CYC). The up-regulation of these proteins indicated that mitochondrial respiration would be activated by ZEA treatment because proteins involved in mitochondrial electron transport complex III (QCR2) and complex V (AT5F1) were activated, along with the activation of cytochrome c (CYC) that transfers electrons between complexes III and IV. Importantly, two of these proteins, namely electron transfer flavoprotein subunit beta (ETFB) and electron transfer flavoproteinubiquinone oxidoreductase (ETFD), are primarily involved in the oxidation of fatty acids (Isackson et al., 2013). These proteins play an essential role by transferring the electrons coming from acyl-CoA dehydrogenases to the respiratory chain at the coenzyme Q level (Wanders et al., 2010). Most mitochondrial proteins are encoded in the nucleus, synthesized as precursor forms in the cytosol and imported into mitochondria with help of protein translocases (Qiu et al., 2013). The translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly machinery (SAM) are two of the key protein translocases on the mitochondrial outer membrane. Three of the identified proteins (TOM40, TOM70, and SAM50) are

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essential components of protein translocases that play a direct role in mitochondrial protein precursor channeling. Additionally, three transport-related proteins are also up-regulated. Mitofilin (IMMT) interacts with the TOM complex and promotes protein import (von der Malsburg et al., 2011). Coiled-coil-helix-coiled-coil-helix domain-containing protein 3 (CHCH3) may act as a scaffolding protein that stabilizes protein complexes involved in protein import and it interacts with Sam50 (Darshi et al., 2011). STML2 up-regulation could also lead to an increased rate of protein transport into the mitochondria (Christie et al., 2011). Collectively, the concurrent increased expression of the six proteins indicates that the transport of precursors is activated; thus, ATP synthesis may be activated and the translocases provide cells with more metabolites (Dudek et al., 2013). 3.5.2.3. Lipid metabolism. Another highly enriched pathway is lipid metabolism, with 8 proteins involved (Fig. 4B and Table 1). Among lipid metabolism-related proteins, three proteins (ACSF2, AATM and CPT2) involved in mitochondrial fatty acid b-oxidation were up-regulated in the present study. Acyl-CoA synthase family member 2 (ACSF2) catalyzes the initial reaction in fatty acid metabolism by forming a thioester with CoA. After activation, the carnitine shuttle transports the long-chain acyl-CoAs into the mitochondrial matrix. An important member of the carnitine shuttle called carnitine palmitoyltransferase II (CPT2) is also up-regulated. In addition, fatty acid-binding protein (FABP-1) facilitates cellular uptake of long-chain free fatty acids (Zhou et al., 1995). Therefore, the mitochondrial fatty acid b-oxidation seems to be stimulated. PLIN1 is normally located on the lipid droplet surface and acts to control both basal and stimulated lipolysis (Brasaemle et al., 2009). Mitochondria and lipid droplets closely abut each other, and they interact to regulate lipid metabolism (Greenberg et al., 2011). When the cells are under certain treatments, more lipid droplets can be found in direct connection with mitochondria (Tarnopolsky et al., 2007). Thus, our observation of increased PLIN1 in isolated mitochondria may indicate that neutral lipid transfer and lipolysis are promoted. The up-regulated PHB and PHB2 are members of the PHB complex, which is involved in lipid partitioning (Osman et al., 2009). Therefore, it is proposed that fatty acid trafficking and accumulation are also regulated. In contrast to stimulated fatty acid oxidation, steroidogenesis seems to be disturbed. The cholesterol synthetic enzyme, P450 lanosterol 14 a-demethylase (CYP51A1) (Debeljak et al., 2003), is up-regulated. However, the enzyme responsible for cholesterol

Table 2 Mitochondrial proteins were differentially expressed in response to ZEA treatment after hCG stimulation as quantified by iTRAQ. Protein name

Gene name Accession number No. of unique peptides Unused protein score %Cov (95)

118(O):117 mean  SD

Calreticulin Protein disulfide-isomerase Platelet glycoprotein 4 Synapse-associated protein 1 Acid sphingomyelinase-like phosphodiesterase 3b Estradiol 17-beta-dehydrogenase 11 Sodium/potassium-transporting ATPase subunit alpha-1 Monoglyceride lipase Talin-1 Programmed cell death 6-interacting protein Synaptobrevin homolog YKT6 D-3-phosphoglycerate dehydrogenase Activator of 90 kDa heat shock protein ATPase homolog 1 Dynamin-1-like protein Actin-related protein 3

CALR PDIA1 CD36 SYAP1 ASM3B DHB11 AT1A1

P14211 P09103 Q08857 Q9D5V6 P58242 Q9EQ06 Q8VDN2

13 31 4 6 7 11 38

22.2 43.3 8.92 12.24 14.45 23.75 66.26

45.21 65.14 8.69 16.44 16.89 38.93 37.44

0.813  0.049 0.814  0.059 0.814  0.059 0.826  0.016 0.827  0.005 0.881  0.010 0.891  0.010

MGLL TLN1 PDC6I YKT6 SERA AHSA1

O35678 P26039 Q9WU78 Q9CQW1 Q61753 Q8BK64

43 18 12 12 14 6

70.84 47.14 25.66 20.19 27.85 10.88

60.71 9.09 16 63.64 32.08 23.37

0.895  0.009 1.107  0.009 1.131  0.032 1.149  0.022 1.176  0.029 1.222  0.035

DNM1L ARP3

Q8K1M6 Q99JY9

6 8

12.59 15.88

7.95 25.12

1.226  0.064 1.236  0.033

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Fig. 5. Protein expression of MLTC-1 cells varied in response to ZEA and/or hCG. MLTC-1 cells were treated in four groups as follows: treatments with serum-free medium as a control (group control); treatments with 5 mM ZEA for 24 h (group ZEA); treatments with serum-free medium for 24 h and then stimulated with 0.1 U/ml hCG for 2 h (group hCG); and treatments with 5 mM ZEA for 24 h and then stimulated with 0.1 U/ml hCG for 2 h (group ZhCG). The western blot analysis results (A) and relative expression (B) of each protein are shown. Different lowercase letters indicate significant differences (P < 0.05). The experiment was repeated for at least three times in two replicates. The data shown in (B) are the means  SD.

esterification (ACAT2) (Parini et al., 2004) is down-regulated. Furthermore, the detoxifying enzyme ADH7, which is involved in the metabolism of steroids and lipid peroxidation products, is also down-regulated (Hoog et al., 2003; Yin et al., 1999). Thus, cholesterol homeostasis may be disrupted by the addition of ZEA. 3.5.2.4. Endocrine system development and function. The downregulation of PPCE may affect the meiosis of spermatocytes, differentiation of spermatids, and sperm motility in mice (Kimura et al., 2002). Keratin 10 plays an important role in supporting testicular structure and function, including spermatogenesis and testicular cell morphology, through cytoskeleton reorganization (Bakshi et al., 2010). Four other proteins are involved in steroid receptor function. FKBP4 contributes to steroid receptor activation (Charpentier et al., 2000; Li et al., 2011a). PHB2 is a repressor of estrogen receptor activity (DeFranco et al., 1998). The decreased FKBP4 along with the increased PHB2 seems to affect the sensitivity of steroid receptors. Furthermore, TRAP1 and HSP74 belong to the heat shock protein 90 family and heat shock protein 70 family, respectively. The two heat shock protein families play essential roles in the maturation of the steroid receptor in achieving a hormone-binding competent state in addition to regulating receptor cytoplasmic-nuclear trafficking (DeFranco et al., 1998). 3.5.2.5. Protein synthesis and modification. A group of proteins that covered diverse functions in protein synthesis and folding are regulated. The reduced expression of SYAC, SYRC and SYDC implied an inhibition of protein translation and biosynthesis, but TCPE with the protein folding assistance function is up-regulated. 3.5.2.6. Redox homeostasis. Three redox homeostasis-associated proteins (MOSC2, ALT1 and ALD2) are regulated differently. Up-regulated MOSC2 is involved in the regulation of nitric oxide synthesis (Kotthaus et al., 2011). ALT1 deletion suppresses cytochrome c oxidase subunit 2 expression, ultimately generating reactive oxygen species (Yu et al., 2013). Testis-specific aldose reductase-related protein 2 (ALD2), which is a component of the cellular antioxidant defense mechanism (Martin and Maser, 2009), is also down-regulated. The result is in accordance with the increased ROS triggered by ZEA. 3.5.2.7. Ca2+ homeostasis. Among the 52 differentially expressed proteins induced by ZEA, 8 were related with calcium signaling (Fig. 4B and Table 1). Three of them (IMMT, STML2 and ANXA7) play a role in regulating cellular Ca2+ homeostasis, and 5 of them

(AIF, CYC, ALD2, VAT-1 and ANXA3) are modulated by the perturbation of intracellular Ca2+. The Ca2+ signal is a key mediator in regulating the release of AIF and CYC from mitochondria into the cytosol (Andreyev and Fiskum, 1999; Yu et al., 2009). Aldose reductase-related protein (ALD2) is reported to be involved in calcium signaling (Das et al., 2012). VAT-1 is a calcium-controlled molecule and is possibly involved in specific signaling cascades (Koch et al., 2003). According to UniProtKB annotations, ANXA3 is a calcium-dependent phospholipid-binding protein. 3.5.2.8. HCG stimulation partially reversed the effect of ZEA on mitochondria. Following hCG stimulation, most of the alterations of mitochondrial proteins by ZEA treatment were reversed. Only 15 differentially expressed proteins were identified, and only 2 of them are mitochondria-specific proteins (Table 2). Additionally, none of the differentially expressed mitochondrial protein in the ZEA/control comparison appeared in the ZhCG/hCG comparison. This finding indicates that hCG stimulation can partly reverse the mitochondrial alterations caused by ZEA. 3.6. Verification of the proteomic data by western blot analysis Four representative antibodies (CPT2, PHB2, AIF and CYC) were involved in fatty acid oxidation, endocrine function, stress response and transport, which were selected to verify the mitoproteomics results. The western blot analysis results revealed similar up-regulation change trends with the iTRAQ approach (Fig. 5A and Table 1). By comparing with the control, CPT2, PHB2, AIF and CYC were up-regulated by ZEA treatment, which was increased by 40.4%, 26.8%, 23.7%, and 63.6% with western blot analysis (Fig. 5B), and by 20.4%, 20.9%, 22.2% and 50.0% with the iTRAQ approach (Table 1), respectively. 3.7. Verification of ATP activation in MLTC-1 cells exposed to 5 mM ZEA Although the proteomics data showed alterations in enzymes of many metabolic pathways, the proteomics data alone do not provide information about flux through these pathways. Because of the apparent complexity of the interacting metabolic pathways, possible “cross-talking” among enzymes, and the presence of various feedback loops, two of the end products/participants in these metabolic pathways were selected for verifications. Because many pathways involve alterations in ATP generation, we measured the intracellular ATP as a direct verification of the proteomics result. As shown in Fig. 6, we found a slightly but

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significantly higher ATP generation after ZEA treatment. The increase would be consistent with our observation of enzymes up-regulated in TCA cycle, fatty acid oxidation and oxidative phosphorylation pathway. 3.8. Verification of Ca2+ turbulence in MLTC-1 exposed to 5 mM ZEA Some of the ZEA-induced differentially expressed proteins are involved in calcium homeostasis, and thus we examined the intracellular Ca2+ levels in fluo-3-loaded MLTC-1 cells. Fig. 7 shows that the intracellular Ca2+ levels were elevated after ZEA treatment. This increase in intracellular Ca2+ confirmed a toxic perturbation of the intracellular Ca2+ homeostasis caused by ZEA exposure. 3.9. Statistical analysis The experiments were repeated at least three times, with at least three replicates. Microsoft Excel 2003 and SPSS 13.0 were used for the statistical analyses. Data were subjected to an analysis of variance (ANOVA), and a comparison of means was carried out by Duncan’s multiple-range test. Differences were considered to be significant for P < 0.05. 4. Discussion This study aimed to identify the ZEA toxicity mechanisms as regulated by the mitochondria using an iTRAQ-based mitoproteomics approach. There were 52 significantly different proteins expressed in response to ZEA treatment, and representative proteins from the mitoproteomics profile are discussed in detail. 4.1. Zearalenone activated energy metabolism In the current study, our proteomic data showed the up-regulation of a set of proteins associated with energy production. These include fatty acid oxidation, the TCA cycle, the respiratory electron transport chain, and Ca2+-mediated signal transduction. The concomitant effects of these proteomic alterations were seen with increased levels of ATP and total Ca ions (Figs. 6 and 7). Mitochondrial fatty acid b-oxidation is an important source of energy. We identified up-regulated proteins associated with the first step of fatty acid degradation. Enrichment of these proteins

Fig. 6. Intracellular ATP production of MLTC-1 cells varied in response to ZEA and/or hCG. MLTC-1 cells were treated in four groups as follows: treatments with serumfree medium as a control (group control); treatments with 5 mM ZEA for 24 h (group ZEA); treatments with serum-free medium for 24 h and then stimulated with 0.1 U/ ml hCG for 2 h (group hCG); and treatments with 5 mM ZEA for 24 h and then stimulated with 0.1 U/ml hCG for 2 h (group ZhCG). The ATP level of each group is shown. Different lowercase letters indicate significant differences (P < 0.05). The experiment was repeated for at least three times in three replicates. The data shown are the means  SD.

Fig. 7. Intracellular Ca2+ concentration of MLTC-1 cells varied in response to ZEA and/or hCG. MLTC-1 cells were treated in the following four groups: treatments with serum-free medium as a control (group control); treatments with 5 mM ZEA for 24 h (group ZEA); treatments with serum-free medium for 24 h and then stimulated with 0.1 U/ml hCG for 2 h (group hCG); and treatments with 5 mM ZEA for 24 h and then stimulated with 0.1 U/ml hCG for 2 h (group ZhCG). The Ca2+ level of each group is shown. Different lowercase letters indicate significant differences (P < 0.05). The experiment was repeated for at least three times in three replicates. The data shown are the means  SD.

may promote an increased acyl-CoA flux into the mitochondrial matrix. Concomitantly, two electron transfer proteins, specifically the transporting electrons coming from fatty acid b-oxidation directly to the respiratory chain, are also up-regulated. The evidence indicates that more fatty acids are burning to provide energy. Five essential enzymes in the TCA cycle are all up-regulated after ZEA exposure, including the rate-limiting enzyme IDH3A, which is closely related to an increased capacity for ATP production. In addition, ZEA exposure can elevate intracellular Ca2+ level as is verified. The elevated Ca2+ content potentially leads to an increased rate of pyruvate oxidation and citric acid cycle flux (Denton et al., 1987; Ristow et al., 2000). Thus, all the evidence above indicates up-regulated TCA cycle activity. However, we noted a decreased level of glycolytic enzymes. This finding suggests that the activated TCA cycle is most likely not consuming more acetyl-CoA from glycolysis, but from fatty acid b-oxidation. Acetyl-CoA from oxidation of hydrolyzed fatty acids can also enter the TCA cycle, where the acetate is further oxidized to CO2 and H2O, yielding NADH, FADH2, and ATP. Collectively, most of these enzymes were up-regulated, possibly indicating a general alteration in mechanisms involved in the fatty acid b-oxidation, TCA cycle and the oxidative phosphorylation pathway, activating energy production. STML2, PHB, PHB2, QCR2 (complex III) and AT5F1 (complex V) are up-regulated by ZEA, and there is a strong relationship among them. The proposed function of STML2 is to recruit prohibitins (PHB and PHB2) to cardiolipin to form cardiolipin-enriched microdomains in which electron transport complexes (complexes III and V) are optimally assembled. STML2 then regulates mitochondrial biogenesis and function, most likely through the prohibitin functional interactome (Christie et al., 2011). Therefore, the up-regulation of STML2 expression might be associated with increased oxygen consumption and oxidative phosphorylation, leading to increased ATP stores in the cell. In most mammalian cells, ATP is mainly formed through mitochondrial oxidative phosphorylation. In the present study, complex III (QCR2), complex V (AT5F1), TOM40, TOM70, and SAM50 were activated by ZEA treatment (Table 1). The majority of respiratory chain subunits are encoded by nuclear genes, the protein products of which are translated on cytosolic ribosomes and imported into mitochondria by the import machineries of the translocase of the outer membrane (TOM) and the translocase of the inner membrane (Mick et al., 2011). Therefore, the up-regulation of

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TOM40, TOM70, and SAM50 with ZEA treatment probably stimulated the import of complexes III and V. It has been reported that mitochondrial electron transport chain is the major endogenous source of cellular ROS (Azad et al., 2009). In this study, complex III (QCR2) was significantly increased by ZEA, and this disruption resulted in the increase of ROS, which is consistent with observed ROS levels (Fig. 2). A concomitant increase of intracellular Ca2+ was observed in our study, since the imported Ca2+ may also contribute to the stimulated mitochondrial ROS production (Norberg et al., 2010). Because ROS are produced primarily in mitochondria, it appears that mitochondria are a primary target for the destructive action of ROS. This mitochondrial damage triggered by excessive ROS plays an important role in the toxic effect of ZEA. As indicated by the pathway analysis, we further noticed that many ZEA up-regulated proteins, such as ACAT2, CPT2, CYP51A1, GOT2, GPT, PC PLIN1 and TALDO1, involved in energy metabolism, especially lipid metabolism, are associated with peroxisome proliferator-activated receptors (PPARs). PPARs are transcription factors that are members of the superfamily of nuclear hormone receptors (Kersten et al., 2000), and all these proteins are involved in a network with PPARa isoform as a key node. In hepatic cells, PPARa acts as a master regulator of lipid catabolism by inducing the expression of numerous genes involved in mitochondrial and peroxisomal fatty acid oxidation, as well as other lipid-related pathways, inflammatory pathways, and glucose metabolism (Rakhshandehroo et al., 2010). PPARa activation has been shown to stimulate fatty acid utilization in the heart through the transcriptional control of genes responsible for cardiac fatty acid uptake, esterification and b-oxidation (Kalinowska et al., 2009). Taken together, there is considerable evidence that PPARa activation appears to be a key regulator of ZEA toxicity. In another way, the activation of PPARa may be the result of altered intracellular fatty acids levels, because fatty acids and their analogs have been shown to activate PPARs (Georgiadi and Kersten, 2012). The ZEA-activated PPARs may function as part of a feed-forward mechanism aimed at promoting oxidation of incoming fuels and thereby preventing the intracellular accumulation and consequent lipotoxicity of fatty acids by stimulating their oxidation. The activation of PPAR by fatty acids

may protect against lipotoxicity by inhibiting hydrolysis of circulating triglycerides and consequent uptake of fatty acids (Georgiadi et al., 2010). The changes in mitochondrial behavior, mitochondria–lipid droplets interaction and energy metabolism provide new insight for further understanding the molecular mechanisms of zearalenone. Though we used Leydig cell as the material, maybe the result suggests a common primary mechanism for ZEA toxicity. Certainly the speculation needs more experiments to verify, for example in other cells or in vivo. 4.2. Zearalenone disturbed steroid synthesis ZEA induced the down-regulation of essential enzymes in the PPP. Although PPP occurs exclusively in the cytoplasm of mammals, it is found to be most active in lipid- and steroid-synthesizing tissues, such as gonads (Riganti et al., 2012). Thus, their presence in isolate Leydig cell mitochondria might be inevitable due to the tissue-dependent high levels of the PPP enzymes. The pentose phosphate pathway is a process that oxidizes glucose and generates NADPH and pentoses. However, its primary role is anabolic rather than catabolic, and it plays a crucial role in steroid biosynthesis. The decreased expression of PPP enzymes indicates an inhibition of PPP and thus affects the steroid synthesis. Leydig cells are the predominant source of the male sex steroid hormone testosterone (Akingbemi, 2005). Steroidogenic cells store cholesteryl esters in intracellular lipid droplets as precursors for steroid hormone synthesis. Cholesteryl ester-rich lipid droplets’ mobilization is the preferred initial source of cholesterol for steroidogenesis. These lipid droplets are known to be coated by PLIN1 (Servetnick et al., 1995). As shown in the results, PLIN1 expression is significantly up-regulated in response to ZEA, so we hypothetically speculate that the communication between mitochondria and the lipid droplets are promoted. Additionally, the up-regulation of PHB and PHB2 makes the hypothesis more convincing, because the prohibitins located in the mitochondria have been found to directly interact with lipids (Artal-Sanz and Tavernarakis, 2009). The cholesterol trafficking between lipid droplets and mitochondria seems to be enhanced.

Fig. 8. A hypothetical model of the toxicity mechanism for ZEA-induced mitochondrial damage. The proteins up-regulated by ZEA are shown in red color. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Then what kind of effect is ZEA actually exerting on cholesteryl ester synthesis needs be identified. Proteomic analysis data suggests that the basic steroid homeostasis has been disturbed by ZEA. The enzyme responsible for cholesterol synthesis (CYP51A1) was up-regulated while the enzyme responsible for cholesterol esterification (ACAT2) was down-regulated. In combination with the observed PPP inhibition, ZEA treatment affects basic hormone secretion. Moreover, these findings at the cellular level are consistent with in vivo observations of suppressed testosterone secretion after ZEA exposure (Yang et al., 2007). It is reported that excessive oxidative stress may be the cause of reduced steroidogenesis (Abidi et al., 2008). The accumulated evidence indicates that ZEA might disturb steroid homeostasis through mitochondria dysfunction. The toxicity of zearalenone has been studied for decades, and plenty of evidence showed that zearalenone can disrupt steroidogenesis on different levels. Frizzell used the special cell line for endocrine disruptor investigations, the H295R cell, to explore the effect of zearalenone on steroidogenesis. Results showed that zearalenone altered production of progesterone, estradiol, testosterone and cortisol hormones (Frizzell et al., 2011). Ranzenigo demonstrated that zearalenone altered FSH-induced estradiol and progesterone production in porcine granulosa cells (Ranzenigo et al., 2008). In the experiments mentioned above, zearalenone usually had biphasic effects on certain type of steroid hormone production, exhibiting opposite effects on different doses. Yang et al. found that zearalenone intraperitoneal injection treatment reduced mice serum testosterone concentrations at all doses in a dose-dependent manner (Yang et al., 2007). Based on the previous studies, it seems convincing that zearalenone has steroidogenesis disrupting effects. Our results provided information of mechanism of zearalenone steroidogenesis toxicity based on the mitochondria proteomic analysis. What we find valuable is that the cholesteryl ester-rich lipid droplets’ mobilization, indicating that the cholesterol trafficking between lipid droplets and mitochondria are promoted. Furthermore, we found that many proteins in the mitochondria modulated by PPARa were regulated after zearalenone treatment. PPARa regulates the metabolism of cholesterol (Marrapodi and Chiang, 2000), the precursor of sex hormone synthesis. Therefore, the activation of PPARa could possibly impact sex hormone synthesis. Some xenobiotic compounds may disrupt testosterone biosynthesis by lowering the delivery of cholesterol into the mitochondria and decreasing the conversion of cholesterol to pregnenolone and androstandione in the testis (Li et al., 2011b). Zearalenone was proved to cause oxidative stress by inducing lipid peroxidation in Caco-2 cells (Kouadio et al., 2007) and cultured Vero cells (Othmen et al., 2008). In Vero cells, from green monkey kidney, zearalenone induces oxidative stress (El Golli et al., 2006), causes DNA damage, increases MDA formation (Abid-Essefi et al., 2004) and induces apoptosis, and these effects can be partially protected by vitamin E (Abid-Essefi et al., 2003). This is consistent with the toxic effects of zearalenone being burning more fatty acid and causing oxidative stress in the current study, and finally leading to apoptosis. Vitamin E pretreatment restores about 40% of apoptosis induced by zearalenone in HepG2 cells (El Golli Bennour et al., 2009). Vitamin E would be acting as anti-oxidant, especially in the lipid system. The fact that vitamin E alleviates the toxicity of zearalenone suggests that lipid peroxidation is crucial to zearalenone toxicity. In summary, a hypothetical model for the toxicity mechanism of ZEA-induced mitochondria damages is proposed in Fig. 8. However, more work is yet to be done to validate the center position of lipid peroxidation. More studies are currently underway to determine how zearalenone affects ATP and intracellular calcium and their kinetic analysis.

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5. Conclusion Our mitochondria proteomic analysis suggests that significant changes occurred in lipid metabolism after low dose ZEA exposure, resulting in two alterations. One is the increase in energy production through promoted fatty acid uptake and b-oxidation along with the excessive oxidative stress; the other is the inhibited steroidogenesis and esterification possibly resulting in reduced hormone secretion. The result highlighted that disturbance in lipid metabolism might be the key element of ZEA toxicity. Further studies in other tissues and animal models will be needed to elucidate the contribution of the described lipid metabolism events to the toxicity of ZEA. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgments This work was funded by the Fundamental Research Funds for the Central Universities (Grant Nos. 2012QJ151 and 2013QJ036). The funders have no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Abid-Essefi, S., Baudrimont, I., Hassen, W., Ouanes, Z., Mobio, T.A., Anane, R., Creppy, E.E., Bacha, H., 2003. DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by vitamin E. Toxicology 192, 237–248. Abid-Essefi, S., Ouanes, Z., Hassen, W., Baudrimont, I., Creppy, E., Bacha, H., 2004. Cytotoxicity, inhibition of DNA and protein syntheses and oxidative damage in cultured cells exposed to zearalenone. Toxicol. In Vitro 18, 467–474. Abidi, P., Zhang, H., Zaidi, S.M., Shen, W.J., Leers-Sucheta, S., Cortez, Y., Han, J., Azhar, S., 2008. Oxidative stress-induced inhibition of adrenal steroidogenesis requires participation of p38 mitogen-activated protein kinase signaling pathway. J. Endocrinol. 198, 193–207. Akingbemi, B.T., 2005. Estrogen regulation of testicular function. Reprod. Biol. Endocrinol. 3, 51. Andreyev, A., Fiskum, G., 1999. Calcium induced release of mitochondrial cytochrome c by different mechanisms selective for brain versus liver. Cell Death Differ. 6, 825–832. Artal-Sanz, M., Tavernarakis, N., 2009. Prohibitin and mitochondrial biology. Trends Endocrinol. Metab. 20, 394–401. Ayed, Y., Ayed-Boussema, I., Ouanes, Z., Bacha, H., 2011. In vitro and in vivo induction of chromosome aberrations by alpha- and beta-zearalenols: comparison with zearalenone. Mutat. Res. 726, 42–46. Azad, M.B., Chen, Y., Gibson, S.B., 2009. Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment. Antioxid. Redox Signal. 11, 777–790. Bakshi, H., Sam, S., Rozati, R., Sultan, P., Islam, T., Rathore, B., Lone, Z., Sharma, M., Triphati, J., Saxena, R.C., 2010. DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by crocin from kashmiri saffron in a human pancreatic cancer cell line. Asian Pac. J. Cancer Prev. 11, 675–679. Banjerdpongchai, R., Kongtawelert, P., Khantamat, O., Srisomsap, C., Chokchaichamnankit, D., Subhasitanont, P., Svasti, J., 2010. Mitochondrial and endoplasmic reticulum stress pathways cooperate in zearalenone-induced apoptosis of human leukemic cells. J. Hematol. Oncol. 3, 50. Bouaziz, C., Sharaf El Dein, O., El Golli, E., Abid-Essefi, S., Brenner, C., Lemaire, C., Bacha, H., 2008. Different apoptotic pathways induced by zearalenone, T-2 toxin and ochratoxin A in human hepatoma cells. Toxicology 254, 19–28. Brasaemle, D.L., Subramanian, V., Garcia, A., Marcinkiewicz, A., Rothenberg, A., 2009. Perilipin A and the control of triacylglycerol metabolism. Mol. Cell. Biochem. 326, 15–21. Charpentier, A.H., Bednarek, A.K., Daniel, R.L., Hawkins, K.A., Laflin, K.J., Gaddis, S., MacLeod, M.C., Aldaz, C.M., 2000. Effects of estrogen on global gene expression: identification of novel targets of estrogen action. Cancer Res. 60, 5977–5983. Che, T., Sun, H., Li, J., Yu, X., Zhu, D., Xue, B., Liu, K., Zhang, M., Kunze, W., Liu, C., 2012. Oxytocin hyperpolarizes cultured duodenum myenteric intrinsic primary

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