B6C3F1 male mice (8 weeks old) and Wistar male rats (7 weeks old) were purchased from Charles River (Japan) and they were acclimatized to the SPF mouse and rat rooms for 1 week. The mice and rats were fed commercial γ-irradiated pellets (Purina, Korea) and UV sterilized water ad libitum. Each animal room was maintained at 23 ± 2℃ (relative humidity 50 ± 10%) and a 12-h light/dark cycle. The animal housing and the experiment were performed according to the Code of Laboratory Animal Welfare Ethics, National Veterinary Research and Quarantine Service, Korea.

DON, aflatoxin B1 (AFB1) and zearalenone (ZEA) were purchased from Sigma-Aldrich (USA) and these were dissolved in distilled water for DON and in corn oil for AFB1 and ZEA. In the experiment of mice treated with DON, DON was diluted to doses of 0.83, 2.5 and 7.5 mg/kg body weight (bw) and these doses were administered orally at 10 ml/kg bw via gavage once per day for 8 days to the mice. In the experiment of rats treated with DON, AFB1 or ZEA, rats were orally administered with DON 7.5, AFB1 1.0 or ZEA 240 mg/kg bw via gavage once per day for 8 days. The next day of the last administration, the mice or rats were anesthetized with diethylether and their blood was collected via abdominal vein and it was transferred to a vessel for the serum and to an EDTA-containing vessel for the plasma. The serum and plasma were separated by centrifugation at 12,000 × g for 15 min and they stored at -80℃ until performing the protein profiling and Igs assay.

The blood plasma of the mice was diluted with lysis buffer (Urea 9.5 M, CHAPS 2% and DTT 1%) and its protein content was adjusted to 5 mg/ml. For the hydrophobic protein profiling, binding of the proteins onto the surface of chip (H50; Ciphergen Biosystems, USA) was conducted in a deep-well type assembly (Bioprocessor assembly; Ciphergen Biosystems, USA). The chip surface was activated twice by 50 µl of 50% acetonitrile for 5 min each time and then twice for 5 min each time by 150 µl of binding buffer (0.1% trifluoroacetic acid in 10% acetonitrile). Twenty µl of the plasma (5 mg protein/ml) was mixed with 80 µl of binding buffer and then this was applied to each spot on a H50 chip. The chip was incubated for 30 min with shaking at room temperature. Each spot was washed three times with 150 µl of binding buffer and once with 150 µl of distilled water for 5 min. For the profiling assay of the copper immobilized proteins, the anionic proteins, the strong or weak cationic proteins and the normal phased proteins, copper immobilized (IMAC30), anionic (Q10), cationic (CM-high and CM-low) and a normal phase (NP20) protein chip (Ciphergen Biosystems, USA) was used, respectively, and each activation and the binding buffer that were used for each protein chip were applied with following the same steps as for the hydrophobic protein profiling. All of the chips were washed after the sample reacted and they were completely air-dried and then treated twice with 1 µl saturated sinapinic acid in 50% acetonitrile and 0.5% trifluoroacetic acid. After complete air-drying, the chip was inserted into the Protein Biology Mass Specrtometry System (SELDI-TOF/MS; Ciphergen Biosystems, USA) for determining the mass peaks (time of flight) at a laser intensity of 215 and a detected ion sensitivity of 9. All the data was normalized by the total ion current and then significant mass peaks compared to those of vehicle control were selected by biomarker wizard programme (version 3.0; Ciphergen Biosystems, USA) and the height or area of the selected mass peaks was compared between each group.

The blood plasma was diluted to 5 mg protein/ml with adsorption buffer (1 M ammonium sulfate and 50 mM sodium phosphate, pH 7.0). Before the application of the plasma sample, a column (Methyl Ceramic HyperD F column; Ciphergen Biosystems, USA) was equilibrated twice with 200 µl of adsorption buffer. 500 µl of the diluted plasma sample (5 mg protein/ml) was added to the column and hydrophobic protein in the sample was allowed to bind for 30 min at room temperature. After the column was spun in a microcentrifuge, 500 µl of elution buffer (50 mM sodium phosphate pH 7.0) was applied for 10 min for four times each and the fraction of hydrophobic protein was eluted by centrifugation and the eluted fraction obtained at each centrifugation was combined in one vessel. 2 µl of the eluted fractions was applied to a gold chip to confirm that the targeted protein was fractionated by the mass peak analysis via SELDI-TOF/MS. Two ml of the eluted fraction was concentrated 20-fold to 100 µl with using a spin column that was designed for collecting materials with a mass range of 10,000~30,000 and then desalting them (VivaSpin6; VivaScience, Germany). Two µl of the concentrated fraction was then applied to a gold chip in order to reconfirm that the candidate protein with a targeted mass peak was collected by SELDI-TOF/MS analysis. The concentrated fraction of the protein was separated by 12% SDS-PAGE at 80 volts and it was visualized by Coomassie-blue staining [37]. The band of SDS-PAGE gel containing the target protein was excised and then the protein was digested with trypsin. The digested polypeptides were analyzed by LC/Q-TOF (Thermo, USA) and a MALDI-TOF mass spectrometer (Applied Biosystems, USA) and then the candidate protein was identified with the Mascot and ProFound protein web search engine (Matrix Science , USA).

Twelve µl of the concentrated fraction obtained after column separation of the control mouse plasma or the DON-administered mouse plasma was mixed with 3 µl of sample buffer (0.5 M Tris-HCl pH6.8, 10% glycerol, 10% SDS, 5% 2-mercaptoethanol and 1% bromophenol blue), and this was heated at 95℃ for 5 min and then loaded on the 12% SDS-PAGE. After electrophoretic running at 80 volts, the protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane for 2 h at 100 volts and the membranes were blocked with blocking buffer (PBS buffer containing 0.05% Tween 20 and 7% fat free skim milk). The PVDF membrane was incubated for 2 h in 5 µl of chicken polyclonal primary antibody to haptoglobin diluted 1 : 1,400 along with 7 ml of blocking buffer. After three washes with washing buffer containing 0.05% Tween 20 in PBS for 5 min each time, the membrane was incubated for 1 h in 2 µl of secondary antibody conjugated to alkaline phosphatase diluted 1 : 3,500 in 7 ml of blocking buffer. After five washes, the specific plasma protein was visualized by adding 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as substrate for alkaline phosphatase.

The amount of haptoglobin in the blood plasma of the mice was quantified with using a mouse haptoglobin ELISA kit (Immunology Consultants, USA). The sample was diluted 1 : 10,000 in diluent (PBS containing BSA, 0.25% Tween and 0.1% Proclin 300). 100 µl of the diluted sample or each dose standard was added to each well that was coated with purified anti-mouse haptoglobin and this was incubated for 15 min at 22℃. Following four aspirations and washings with washing buffer (PBS containing 0.5% Tween), 100 µl of diluted (1 : 100) anti-mouse haptoglobin antibodies conjugated with horseradish peroxidase was added to each well and this was incubated at room temperature for 15 min. After four washes, 100 µl of chromogenic substrate solution containing 3,3',5,5'-tetramethylbenzidine (TMB) and hydroperoxide in citric acid buffer (pH 3.3) was added to each well. After incubation at room temperature for 10 min, the concentration of haptoglobin was measured at 450 nm.

To confirm if haptoglobin is a specific biomarker for DON intoxication and exposure as compared with the other mycotoxins AFB1 and ZEA, the level of haptoglobin in the plasma of male rats (8 weeks old, Wistar; Charles River, Japan) that were orally administered AFB1 1.0, ZEA 240 or DON 7.5 mg/kg bw for 8 days was measured by using a mouse haptoglobin ELISA kit (Immunology Consultants, USA).

The levels of immunoglobulins in the serum of the mice and rats were quantified with using an ELISA Quantitation Kit (Bethyl Lab, USA). 100 µl of goat anti-mouse or goat anti-rat IgG affinity purified antibody that was diluted 1 : 100 with coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) was coated onto each well for 60 min at room temperature. Each well was washed three times with washing solution (50 mM Tris, 0.14 M NaCl and 0.05% Tween 20, pH 8.0) and the wells were blocked with blocking (postcoat) solution (50 mM Tris, 0.14 M NaCl and 1% BSA, pH 8.0) for 30 min at room temperature. The serum sample was diluted (1 : 1,000 for IgA and IgM and 1 : 10,000 for IgG) in sample diluent (50 mM Tris, 0.14 M NaCl, 1% BSA and 0.05% Tween 20, pH 8.0). 100 µl of the diluted or non-diluted serum sample and each dose standard were then added into each well and this was incubated for 60 min at room temperature. After five aspirations and washes, 100 µl of the goat anti-mouse or goat anti rat Ig antibodies conjugated with horseradish peroxidase and diluted (1 : 50,000 for IgG, 1 : 40,000 for IgA, 1 : 100,000 for IgM and 1 : 20,000 for IgE) with the conjugate diluent (50 mM Tris, 0.14 M NaCl, 1% BSA and 0.05% Tween 20, pH 8.0) was added to each well and this was incubated for 60 min at room temperature. Following five washings, 100 µl of the substrate solution containing the TMB peroxidase substrate and peroxidase solution was added to each well and this was incubated for 30 min at room temperature. To stop the TMB reaction, 100 µl of 2 M H₂SO₄ was applied to each well and the levels of the Igs were measured at 450 nm.

The data is expressed as the mean ± SD of six individual animals. Statistical analysis was performed using ANOVA and then Duncan's test. A p value < 0.05 was judged to be significant and a p value < 0.01 was highly significant compared to vehicle control group.

The protein profiles of the plasma of the mice that were administered with DON were acquired by the protein chip arrays of the CM-high, CM-low, Q10, H50, IMAC30 and NP20 ProteinChips. The 9.7 kDa copper-immobilized protein and the 11.7 kDa hydrophobic protein were significantly increased and the 17.4 kDa weak cationic protein was decreased by DON in a dose-dependent manner (Fig. 1). Among those proteins, the 11.7 kDa hydrophobic protein captured on the H50 chip was the most highly expressed (Fig. 2). The average mean peak intensity of the 11.7 kDa protein in the plasma of the mice that were administered with DON 7.5 mg/kg bw was 20 times higher as compared with that of the control.

The 11.7 kDa hydrophobic protein that was highly expressed in the blood plasma of mice that were administered DON 7.5 mg/kg bw was purified by a Methyl Ceramic HyperD F column. The column optimizing buffer and binding buffer were 1 M ammonium sulfate and 50 mM sodium phosphate pH 7.0, respectively, and the elution buffer was 50 mM sodium phosphate pH 7.0. The binding and elution was monitored by spotting the eluates of each step on the gold chip and performing SELDI-TOF/MS analysis (Fig. 2). The 11.7 kDa protein that was purified by column fractionation and SDS-PAGE separation (Fig. 3) was identified by performing LC/Q-TOF/MS and MALDI-TOF/MS along with using the Mascot and ProFound protein search engine programs. The search results showed that the top matching protein was haptogloin with 148 top score (p < 0.05) and this protein showed 7% sequence coverage, as determined by LC/Q-TOF/MS, and the haptogloin precursor had a 0.65 z score and 17% sequence coverage, as determined by MALDI-TOF/MS, respectively.

To confirm the identification result of the haptoglobin precursor for the 11.7 kDa hydrophobic protein, western blotting analysis was performed using polyclonal antibody against chicken haptoglobin. The band of 11.7 kDa protein was purified by using a Methyl Ceramic HyperD F column, it was separated by SDS-PAGE and then the protein was transferred to a PVDF membrane. This was reacted with haptoglobin antibody and the protein was expressed at a higher density compared to that of the control (Fig. 4).

In order to quantify the amount of total haptoglobin in the plasma, ELISA was performed with using polyclonal antibody against mouse haptoglobin. The blood plasma of the mice that were orally administered DON 0.83, 2.5 and 7.5 mg/kg bw and vehicle control (D.W.), respectively, were applied to an ELISA kit. The amount of haptoglobin in the plasma was increased in a dose-dependent manner by DON (Fig. 5). That is, the normal mean value of the haptoglobin was 7,762.82 ng/ml, but it was increased to 8,399.92, 12,252.95 and 22,298.18 ng/ml by DON 0.83, 2.5 and 7.5 mg/kg bw, respectively.

To confirm the potential specificity of haptoglobin as a biomarker for DON intoxication, we compared the expressions of haptoglobin in the rats with DON, AFB1 and ZEA intoxication. The amount of haptoglobin in the blood plasma of the rats that were orally administered DON 7.5, AFB1 1.0 and ZEA 240 mg/kg bw, respectively, for 8 days was measured with a mouse ELISA kit. The normal mean value of the haptoglobin in the blood plasma of a rat was 209 ng/ml, and this was approximately 40 times lower than that of the mouse. The mean value of the haptoglobin in the blood plasma of the rats that were administered DON 7.5 mg/kg bw was 1.3 times higher compared to that of the control, while the haptoglobin was completely decreased by AFB1 1.0 mg/kg bw and was slightly decreased by ZEA 240 mg/kg bw (Fig. 6).

In order to quantify the level of Igs in the serum of the mice, we performed ELISA using goat anti-mouse antibodies against IgG, IgA, IgM and IgE. The serum of the mice that were orally administered DON 0.83, 2.5 and 7.5 mg/kg bw, respectively and the vehicle control (D.W.) was applied to an ELISA kit. The amounts of IgG and IgM in the serum were decreased by DON in a dose-dependent manner and the IgA was decreased at 7.5 mg/kg bw DON without any change of the IgE (Fig. 7).

To compare the expression of Igs between DON, AFB1 and ZEA intoxication, the amounts of Igs in the serum of rats that were orally administered DON 7.5, AFB1 1.0 or zearalenone 240 mg/kg bw for 8 days were measured with a rat ELISA kit. IgG and IgA were decreased by DON, but the IgG, IgA, IgM and IgE were all increased by AFB1. No changes in the Igs were observed by ZEA administration (Fig. 8).