T 2 toxin

DON was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). 3-ADON, 15-ADON, the T-2 toxin, o-phenylenediamine (OPD), bovine serum albumin (BSA), chicken egg albumin (OVA), 1,1′-carbonyldiimidazole (CDI), and polyvinyl alcohol (PVA) were purchased from Sigma-Aldrich (Milwaukee, WI, USA). NX-2 was isolated from liquid cultures of F. graminearum NRRL44211. NX-3 was prepared by treating NX-2 with 0.1N NaOH. 7-hydroxy isotrichodermin was isolated from cultures of F. verticillioides expressing FgTri1 that were fed isotrichodermin [42 (link)]; 7-hydroxy isotrichodermol was prepared by treating 7-hydroxy isotrichodermin with 0.1N NaOH. 7-hydroxy 4,15-diacetoxyscirpenol was isolated from cultures of F. sporotrichioides MB1716 [43 (link)] transformants expressing Tri1 from F. graminearum NRRL29214 [44 (link)]. Peroxidase-conjugated goat anti-mouse IgG (GAMP) was purchased from Jackson Immuno Research Laboratories, Inc. (West Grove, PA, USA). Acetonitrile (ACN), methanol (MeOH), and ethanol (EtOH) were HPLC grade and purchased from major suppliers. Phosphate-buffered saline (PBS) was 0.01 M sodium phosphate and 0.145 M sodium chloride, pH 7.2. OVA-PBS was 0.1% (w/v) OVA in PBS. BSA-PBS was 1% (w/v) BSA in PBS. Tween–PBS was 0.02% (v/v) Tween-20 in PBS. PVA-PBS was 1% (w/v) PVA in PBS, pH 7.2.

The third generation of chondrocytes were seeded in a sterile 16-well plate (approximately 1 × 10⁴ cells per well). The cultured cells were adherent in 24 hours. They were randomly divided into four groups and treated with T-2 toxin (Sigma-Aldrich, St. Louis, MO, USA) at different concentrations, including 0.00 ng/mL, 0.32 ng/mL, 1.60 ng/mL, and 8.00 ng/mL. Cell viability was measured by RTCA-DP.

High-performance liquid chromatography (HPLC) grade methanol, acetonitrile, formic acid and ammonium formate (supplied by Sigma-Aldrich^®, St. Louis, MO, USA). HPLC grade Milli Q^® water was supplied by Biosciences East Central Africa (BecA), International Livestock Research Institute (ILRI), (P.O. Box 30709 Nairobi 00100, Kenya). Aflatoxin B1 was analysed by a multiple mycotoxin analysis method with mixed mycotoxins standards (Aflatoxin B1, B2, G1 and G2, Fumonisin B1 and B2, T-2 toxin, HT-2 toxin, and diacetoxyscirpenole) obtained from Sigma-Aldrich (Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands).

Acetonitrile, methanol, formic acid and 25% ammonium hydroxide (LC–MS grade) and ethyl acetate were obtained from Sigma-Aldrich, Schnelldorf, Germany. Evolute express ABN SPE columns were obtained from Biotage, Sweden. Water was purified on a Milli Q system (Millipore Corporation, USA). Aflatoxin B1, B2, G1, and G2, citrinin, cyclopiazonic acid, deoxynivalenol, enniatin A1 and B1, fumonisin B1 and B2, nivalenol, ochratoxin A, patulin, sterigmatocystin, T2 toxin and zearalenone were all obtained from Sigma-Aldrich.

All the feed was made up by the processing-workshop of feedstuff in the Animal Nutrition Institute of Sichuan Agricultural University, which meet the nutritional requirement of ROSS 308. There were no common mycotoxins, such as aflatoxins, deoxynivalenol, ochratoxin A, zearalenone and T-2 toxin, were found in this feed by the ELISA kit (Huaan Mangech Biotech, Beijing, China). Firstly, the T-2 toxin (purity ≥ 98%; Sigma Aldrich, St. Louis, MO, USA) powder was dissolved by 95% ethanol, and mixed in 1 kg feed and dry it. Then, the mixture was added into feed to get the get the target concentration (0 mg/kg, 0.5 mg/kg, 1 mg/kg, and 2 mg/kg, respectively) of T-2 toxin. At last, we used the ELISA kit (Huaan Mangech Biotech) to confirm the final concentration of toxins in the feed.

The human breast adenocarcinoma cell line MCF-7 (ATCC, HTB-22) cells were preserved in our laboratory, which were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit Haemek, Israel). The MCF-7 cells were cultured at 37 °C with a 5% CO₂ atmosphere in a constant temperature incubator. T-2 toxin, N-Acetyl-L-cysteine (NAC), Cycloheximide (CHX) and MG132 (proteasome inhibitor) were purchased from Sigma Aldrich (St. Louis, MO, USA).

ZEN (100 µg/mL, 2 mL), T-2 toxin (100 µg/mL, 1 mL), and HT-2 toxin (100 µg/mL, 1 mL) were obtained from Sigma (St Louis, MO, USA). DON (B-trichothecenes mix 2, 100 µg/mL, 5 mL), AFs (AFs mix 1, 2 µg/mL for AFB₁ and AFG₁, and 0.5 µg/mL for AFB₂ and AFG₂, 5 mL), OTA (10 µg/mL, 5 mL), and FB₁ and FB₂ (FUMs mix 3, 50 µg/mL, 5 mL) were purchased from Biopure (Getzersdorf, Austria). Phosphate-buffered saline (PBS, pH 7.4) was purchased from Sigma. HPLC-grade acetonitrile and methanol were supplied by Merck (Darmstadt, Germany). Ammonium acetate (HPLC grade, 99.0%, Netherlands) and formic acid (mass spectrometry grade, 98%, St. Louis, USA) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). For purification, a Myco6in1+ IAC column obtained from Vicam was used (Boston, MA, USA). A Myco6in1+ column is intended to simultaneous detect the multiple mycotoxins including AFs, ochratoxin, FUMs, ZEN, DON, NIV, T-2, and HT-2 toxin.