MTBE (cat. no. 1634-04-4; 99.9% purity) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Chinese hamster ovary (CHO) cells were purchased from the cell bank at the Chinese Academy of Science (Shanghai, China). RPMI-1640 medium, fetal bovine serum (FBS), penicillin-streptomycin and trypsin were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit to measure cell viability was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Test kits for LDH, MDA, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). CyDye DIGE fluorescent dyes were obtained from GE Healthcare Life Sciences (Chalfont, UK). Hspa9 antibodies (cat. no. ab171089) were obtained from Abcam (Cambridge, UK). Hspa8 (cat. no. sc-7298) and β-actin (cat. no. sc-47778) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All other chemicals were of analytical grade.

CHO cells were cultured in RPMI-1640 media containing 10% FBS, 100 µg/ml penicillin and 100 µg/ml streptomycin in 5% CO₂ at 37°C. MTBE was administered at concentrations of 0, 0.5, 5.0, 25.0, 50.0 or 100.0 mM when cell confluence reached 80%, and the cells were treated for 6, 12 and 24 h. MTBE concentrations ranging between 0.5 and 100.0 mM were relatively high compared with expected environmental exposure (~0.45 µM) (19,22). However, 100 mM MTBE is a lower concentration compared with that used in previous studies focusing on MTBE toxicity (210 mM) (23).

CHO cell viability was assessed using an MTT assay, as described previously with minor modifications (24). Briefly, cells (100 µl) were seeded into 96-well culture plates (Corning Incorporated, Corning, NY, USA) at a density of 8.0×10⁻³ cells/well, and the cells were treated with MTBE at the stated concentrations and incubated at 37°C in an atmosphere of 5% CO₂ for 6, 12 and 24 h. Folloiwng treatment, 20 µl MTT working solution (5 mg/ml) was added to each well and the plates were incubated at 37°C for 4 h. The MTT solution was discarded and 150 µl dimethyl sulfoxide was added to each well. The optical density was measured on a microplate reader at 490 nm. Exposure to MTBE to 12 h resulted in the greatest inhibition of cell viability, and was subsequently used for further biochemical analysis.

The fraction of total LDH activity in the cell supernatants was used as an indicator of membrane leakage or cell lysis. Cytosolic LDH activity was estimated, as previously described (25). Following 12 h exposure to MTBE at 37°C in a 5% CO₂ incubator, the medium was collected and the level of LDH released by cells was determined using an assay kit (Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's protocol. Following the reaction, the absorbance of each sample was measured at a wavelength of 450 nm with an Infinite M1000 Microplate Reader (TECAN Group, Ltd., Männedorf, Switzerland).

Lipid peroxidation was assessed by determining the levels of thiobarbituric acid reactive substances, using the method previously described by Mihara and Uchiyama (26). CHO cells were seeded into 6-well plates (1.0×10⁶ per well) and were allowed to attach for 24 h. The medium was replaced with RPMI-1640 medium along with 0, 0.5, 5.0, 25.0, 50.0 or 100.0 mM MTBE for 12 h. The treated cells were fragmented and centrifuged at 4,000 × g at 37°C for 10 min, and the supernatants were measured using the MDA test kit (Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's instructions. The absorbance was determined using an Infinite M1000 Microplate Reader (TECAN Group, Ltd.) at wavelength of 532 nm. MDA content was calculated, according to a standard curve and expressed as nmol/mg protein. The protein content was determined using a Bradford assay, with FBS used as the standard (27).

The enzyme activity of SOD, CAT and glutathione GSH-Px were examined to investigate the effect of MTBE-induced oxidative stress. These indexes were detected with corresponding assay kits (Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's protocol. CHO cells were seeded into 6-well plates (1.0×10⁶ per well) and were allowed to attach for 24 h. The medium was replaced with RPMI-1640 medium along with 0, 0.5, 5.0, 25.0, 50.0 or 100.0 mM MTBE for 12 h. The medium was subsequently removed and the cells were pooled in a 1.5 ml tube. The cells were centrifuged at 3,000 × g at 4°C for 10 min, and the pellet was resuspended with 1,000 µl PBS and centrifuged at 10,000 × g at 4°C for 15 min. The supernatant was collected for use in SOD, CAT and GSH-Px assays. The activities of SOD, CAT and GSH-Px were calculated according to a standard curve and expressed as U/mg protein. Protein content was determined using the Bradford assay with FBS as the standard (27).

Treated and untreated samples were minimally labeled (25 µg proteins per 200 pmol dyes) with cyanine (Cy)2, Cy3 or Cy5 fluorescent dyes, according to the manufacturer's protocol (GE Healthcare Life Sciences). A reference design was used, in which a 50.0 mM MTBE exposure condition and control was labeled once with Cy3 and once with Cy5, with the reference sample labeled with Cy2 to generate an internal standard for normalization. By including the internal standard on each gel, the abundance of each protein spot of the individual biological samples was measured relative to its corresponding spot in the internal standard present on the same gel.

For 2-D DIGE, immobilized pH gradient (IPG) strips were dehydrated in an Ettan IPGphor 3 isoelectric focusing system (GE Healthcare Life Sciences). Later samples were focused until a total of 8,000 Vh was achieved. Each strip was embedded on top of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and separated on an Ettan DALT electrophoresis system (GE Healthcare Life Sciences). A loading of 1,000 mg unlabeled proteins was performed in parallel for spots picking and in-gel digestion.

Following 2-D DIGE, the gels were scanned using a Typhoon Trio Variable Mode Imager (GE Healthcare Life Sciences) at excitation/emission wavelengths of 532(blue)/580, 532(green)/425 and 633(red)/425 nm for Cy2, Cy3 and Cy5, respectively. The resultant maps were analyzed using Decyder 2D v6.5 (GE Healthcare Life Sciences). Gel images were imported into the software, and the protein spots were analyzed with the differential in-gel analysis and biological analysis modules. To compare protein expression levels, the spot volume of the Cy3 and Cy5 samples was normalized to the corresponding spot volume of the Cy2 internal standard samples from the same gel. Student's t-test and one-way analysis of variance (ANOVA) were used to calculate the statistical significance. The spots exhibiting statistically significant differences (P<0.05) were further analyzed.

The spots of interest detected by Decyder software analysis were manually excised from the 2-D DIGE gel and stained using Coomassie Brilliant Blue G-250 staining solution (0.12% Coomassie Brilliant Blue G-250, 20% ethanol, 10% phosphoric acid and 10% ammonia sulfate). The protein spots were washed three times with Milli-Q Ultrapure water (Merck Millipore, Darmstadt, Germany) and were subsequently destained with destaining solution (25 mM ammonium bicarbonate/50% acetonitrile). Gel pieces were dehydrated with 100% acetonitrile and incubated at 37°C for 30 min. Each gel piece was digested overnight at 37°C with 0.03 µg sequencing-grade trypsin (Promega, Madison, WI, USA) in 15 ml of 25 mM ammonium bicarbonate buffer. The tryptic peptides were used for MALDI-TOF-MS/MS analysis.

All mass spectrums were acquired on an AutoFlex MALDI-TOF/TOF with LIFT technology (Bruker Corporation, Billerica, MA, USA). Protein identification by PMF and MS/MS spectra was performed using the MASCOT search engine 2.2 (Matrix Science, Inc., Boston, MA, USA) and BioTools software (version 3.2 SR4; Bruker Corporation). Search parameters were set as follows: Taxonomy: Mus musculus; enzyme: Trypsin; missed cleavage: 1; fixed modification: Carbamidomethyl (C); variable modification: Oxidation (M); peptide tolerance: ±100 ppm; mass tolerance: ±0.3 kDa; ions: [M+H]. Confident matches in the SwissProt database (www.ebi.ac.uk/uniprot) were defined by the MASCOT score, the sequence coverage by matching peptides and statistical significance (P<0.05). Protein ontology classification was performed by importing proteins into the Protein Analysis Through Evolutionary Relationships (PANTHER) classification system (http://www.pantherdb.org/) (28). The proteins were grouped according to the association with molecular functions and biological processes.

The MTBE-induced effects on Hspa9 and Hspa8 protein expression levels were confirmed by western blot analysis. CHO cells underwent 12 h exposure to 0, 0.5, 5.0, 25.0, 50.0 or 100.0 mM MTBE and were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and collected by scraping. Protein concentration was determined using the Bradford assay, with FBS used as the standard. The resultant whole lysates (20 µg/lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrically transferred onto a polyvinylidene fluoride membrane (GE Healthcare Life Sciences). The membranes were blocked in 5% milk dissolved in Tris-buffered saline with 0.04% Tween-20 (TBST), and were subsequently incubated with Hspa9 (1:1,000), Hspa8 (1:1,000) and β-actin antibodies (1:2,000) at 4°C overnight. Following primary antibody incubation, the membranes were washed with TBST buffer three times and incubated at room temperature for 2 h with goat anti-mouse IgG-HRP (cat. no. sc-2031) or goat anti-rabbit IgG-HRP (cat. no. sc-2054) secondary antibodies at 1:4,000 dilutions (Santa Cruz Biotechnology, Inc.). The blots were incubated in Super signal^®West Dura Extended Duration Substrate (Thermo Fisher Scientific, Inc.) and detected using the ECL™ western blotting detection system (Image-Quant™ RT; GE Healthcare Life Sciences).

All experiments were replicated ≥3 independent times and the data are expressed as the mean ± standard deviation. The data were analyzed using one-way ANOVA tests followed by Dunnett's tests to determine the statistical difference between treatment groups. These tests were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) and P<0.05 was considered to indicate a statistically significant difference.

To study the cytotoxic effect of MTBE exposure in CHO cells, cell viability was assessed using an MTT assay following treatment with 0, 0.5, 5.0, 25.0, 50.0 or 100.0 mM MTBE for different durations: 6, 12 and 24 h. Incubation of CHO cells for 6 h with 50.0 and 100.0 mM of MTBE resulted in a significant decrease in cell viability compared with the control cells (P=0.0086 and P=0.001, respectively; Fig. 1). When CHO cells were incubated with MTBE for 12 h cell viability significantly decreased at concentrations ≥5.0 mM compared with control cells (5.0 mM, P=0.036; 25.0 mM, P=0.042; 50.0 mM, P=0.023; 100.0 mM, P=0.026; Fig. 1). When CHO cells were incubated with MTBE for 24 h, cell viability significantly decreased at 50.0 and 100.0 mM compared with the control cells (P=0.028 and P=0.025, respectively; Fig. 1). By comparing cell viability rates at the different exposure times, 12 h MTBE exposure was selected for further analysis of MTBE-induced cytotoxicity and oxidative stress in CHO cells as it resulted in the highest inhibition of cell viability. The concentration of 50.0 mM was selected for 2DE analysis, and 5.0 and 50.0 mM were selected for western blot analysis.

Plasma membrane damage was assessed by monitoring LDH release following 12 h exposure to 0, 0.5, 5.0, 25.0, 50.0 or 100.0 mM MTBE. MTBE exposure induced a significant increase in LDH leakage at concentrations ≥25.0 mM compared with the control group (25.0 mM, P=0.001; 50.0 mM, P=0.002; 100.0 mM, P=0.042; Fig. 2).

The effect of 0, 0.5, 5.0, 25.0, 50.0 or 100.0 mM MTBE on lipid peroxidation, as indicated by MDA content, was estimated. MDA is one of the end products of lipid peroxidation and the MDA assay has previously been used for assessing oxidative damage (29). Exposure to MTBE for 12 h induced a significant increase in MDA levels in CHO cells, at all concentrations ≥5.0 mM, compared with control cells 5.0 mM, P<0.001; 25.0 mM, P<0.001; 50.0 mM, P<0.001; 100.0 mM, P<0.001; Fig. 3), indicating an increase in lipid peroxidation.

SOD, CAT and GSH-Px are three important antioxidative enzymes, and their activity levels following MTBE exposure were assessed. Exposure to 50.0 mM or 100.0 mM MTBE for 12 h induced a significant increase of SOD activity compared with the control cells (P=0.035 and P=0.002, respectively; Fig. 4). Exposure to 25.0, 50.0 or 100.0 mM MTBE induced a significant increase of CAT activity in CHO cells compared with control cells (P<0.001, P<0.001 and P<0.001, respectively; Fig. 5) and a significant increase of GSH-Px activity compared with control cells (P<0.001, P<0.001 and P<0.001, respectively; Fig. 6).

To assess the effect of MTBE exposure on protein profiles, a quantitative proteomic analysis by 2DE was performed between 50.0 mM MTBE-treated CHO cells and the untreated control cells. Comparative analysis of the protein profiles was performed between the four test groups using DeCyder software (version 6.5; Fig. 7). The matching of protein spots was corrected by inserting landmarks. With this labeling strategy, it is possible to analyze all biological replicates in a single experimental run. Following 2-D DIGE analysis, protein spots demonstrating ≥1.2-fold differential expression were selected for MS/MS identification (30).

A total of 27 protein spots demonstrated statistically significant differences between the 50.0 mM MTBE-exposed CHO cells and untreated control cells, with 19 spots were downregulated and 8 spots were upregulated in 50.0 mM MTBE-exposed CHO cells compared with the control cells (P<0.05; Table I; Fig. 8). The spots were digested with trypsin and MALDI-TOF/TOF mass spectrometery was applied to obtain high-resolution peptide mass fingerprints and peptide fragment fingerprints. The proteins were subsequently identified by searching the SwissProt database. Of the upregulated spots, 2 were identified as ATP synthase subunit β (Atp5b; Table I), 2 were identified as heat shock protein family A (Hsp70) member 8 (Hspa8; Table I) and 2 were identified as β-actin (Actb; Table I).

The 24 identified proteins were classified with respect to their molecular functions (Fig. 9A) and biological processes (Fig. 9B), according to PANTHER (28). The most dominant function that the identified proteins were involved in was catalytic activity (32.1%), followed by binding (28.6%) and structural molecule activity (17.9%; Fig. 9A). The most dominant biological processes included metabolic processes (30.2%), cell processes (18.9%) and localization (15.1%).

Hspa9 and Hspa8 were selected as representative proteins and subjected to western blot analysis to confirm the differential expression levels (Fig. 10A). The protein expression levels of Hspa9 and Hspa8 were significantly upregulated in CHO cells following 12 h MTBE exposure (50.0. mM) compared with the control cells (P<0.01 and P<0.01, respectively, Fig. 10B and C, respectively). These results were consistent with the 2DE data (Table I).