Deoxynivalenol

Caffeine (CAF), Cytotoxicity Detection Kit (LDH) Roche, D-mannitol (MAN), D-Mannitol Colorimetric Assay Kit, disodium fumarate, deoxynivalenol (DON), L-glutamate, lucifer yellow (LY), and sodium pyruvate were obtained from Sigma Aldrich (St. Louis, MO, USA). All inorganic salts required for preparing Krebs Bicarbonate Buffer (KRB), ethanol, and glucose were purchased from Avantor (Gliwice, Poland). Amoxicillin trihydrate (AMX) and doxycycline hyclate (DOX) were generously donated by the pharmaceutical company Biofaktor Sp. z o.o. (Skierniewice, Poland) The quality of antibiotics was consistent with the monographs of the European Pharmacopoeia and corresponded to the quality of active substances used in the production of veterinary drugs.
Tissue transportation, preparation, and incubation were performed in Krebs Bicarbonate Buffer (KRB) containing 108 mM NaCl, 4.7 mM KCl, 1.8 mM Na₂HPO₄, 0.4 mM KH₂PO₄, 15 mM NaHCO₃, 1.2 mM MgSO₄, 1.25 mM CaCl₂, 11.5 mM glucose, 4.9 mM L-glutamate, 5.4 mM disodium furmate, and 4.9 mM sodium pyruvate at pH 7.4, and saturated with oxygen using a 95%/5% O₂/CO₂ mixture by gassing for 60 min [37 (link)].
For HPLC and LC-MS/MS analysis, acetonitrile, methanol, and formic acid (HPLC grade) were obtained from Avantor Chemicals (Radnor, PA, USA), trichloracetic acid and heptafluorobutyric acid were obtained from Sigma Aldrich (St. Louis, MO, USA).

HPLC grade solvents (hexane, methanol (MeOH) and acetonitrile (AcN)) were purchased from Merck (Darmstadt, Germany). Deionized water (<18 MΩ cm resistivity) was obtained in the laboratory using a Milli-Q SP Reagent Water System (Millipore, Bedford, MA, USA).
Standards of mycotoxins including (aflatoxins (B1, B2, G1 and G2); enniatins (A, A1, B and B1); fumonisins (B1 and B2); nivalenol (NIV); deoxynivalenol (DON); 15-acetyldeoxynivalenol (15-ADON); 3-acetyldeoxynivalenol (3-ADON); zearalenone (ZEA); beauvericin (BEA); fusarenon X (FUS-X)) were purchased from Sigma Aldrich (Madrid, Spain). The T-2 and HT-2 toxins were provided from BiopureReferenzsubstanzenGmBH (Tulln, Austria). Finally, fumonisin B3 was provided by the Research Program “PROMEC” (Tygerberg) in South Africa. All stock solutions of mycotoxins standards were stored in glass-stoppered bottles in darkness at −20 °C until the final analysis. For the derivatization, the reagent composed of BSA (N,O-bis (trimethylsilyl) acetamide)/TMCS (trimethylchlorosilane)/TMSI (N-trimethylsilyimidazole) (3:2:3) was obtained from Supelco (Bellefonte, PA, USA).

Aflatoxin B₁ (AFB₁), zearalenone (ZEN), deoxynivalenol (DON), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,6-dimethoxy phenol (DMP), syringaldazine (SGZ), and methyl syringate were purchased from Sigma-Aldrich (St. Louis, MO, USA). FB₁ (fumonisin B₁) and OTA (ocharatoxin A) were purchased from Pribolab (Beijing, China). DNA polymerase, T4 ligase, acetonitrile, and trifluoroacetic acid were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Vanillin, p-coumaric, syringic acid, syringaldehyde, caffeic acid, 1-hydroxybenzotriazole (HBT), gallic acid, isopropyl-β-D-thiogalactoside (IPTG), and kanamycin were purchased from Solarbio (Beijing, China). Ni-NTA agarose was purchased from QIAGEN (Hilden, Germany). The fungal laccase from Ganoderma sp. was purchased from Sunson (Yinchuan, Ningxia, China). Plant extracts from E. brevicornu, C. sativus L., L. angustifolia, A. officinalis, and S. tenuifolia were purchased from Ciyuan Biotech (Xi’an, Shanxi, China). All other chemicals were of analytical grade or chromatographically pure, and were commercially available.

Deoxynivalenol and zearalenone standards were purchased from Sigma Aldrich (Vienna, Austria). The stock solutions standards were prepared in methanol:water (1:1) at a concentration of 1 μg/mL.
All reagents used in the following analyses were analytical and LC/MS-MS grade. For quality assurance in the mycotoxin analyses, wheat flour certified reference materials (CRM) (Trilogy, Washington) were used. The concentrations for DON and ZEN were 700 ± 100 μg/kg and 454.2 ± 37.6 μg/kg, respectively.

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.

As protease inhibitors, EDTA-2Na (purity: >99.5%) and PMSF (purity: >98.5%), were purchased from Dojindo Laboratories (Kumamoto, Japan) and Sigma-Aldrich Japan (Tokyo, Japan), respectively. Acetonitrile (ACN, LC/MS grade, purity: 99.8%), methanol (MeOH, LC/MS grade, purity: 99.7%), ethanol (EtOH, special grade, purity: 99.5%), formic acid (FA, LC/MS grade, purity: 99.5%), acetic acid (AcOH, LC/MS grade, purity: 99.5%), trifluoroacetic acid (TFA, special grade, purity: 98.0%), ammonium carbonate (special grade), and 28% ammonia solution (NH₄OH, special grade) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Water used for the preparation of all the solutions was purified using a Milli-Q apparatus (Millipore, Billerica, MA, USA); LC/MS analysis used ultrapure water from Wako. The mycotoxins (ochratoxin A, fumonisin B1, zearalenone, deoxynivalenol, patulin) were purchased from Sigma (St. Louis, MO, USA). Drugs with amide, ester, and sulfonamide bonds and amino acid-containing compounds were obtained from the following companies: Aldrich and Sigma Japan (Tokyo, Japan), Nacalai Tesque (Kyoto, Japan), Tokyo Chemical Industry (Tokyo, Japan), and Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

In this study, the mycoparasitic biocontrol Sphaerodes mycoparasitia SMCD 2220-01 strain deposited in IDAC under accession number 301008-01 (Public Health Agency of Canada—International Depositary Authority of Canada Collection, Winnipeg, Canada) has been used for decomposition and detoxification of Fusarium mycotoxins. Zearalenone (ZEN), deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), and 15-acetyl-deoxynivalenol (15-ADON) shown in Fig. 1 were purchased from Sigma-Aldrich Canada Ltd., Oakville, ON. HPLC grade organic solvents were purchased from Fisher Scientific. The stock solutions of each of mycotoxins were prepared by dissolving each mycotoxin in acetonitrile. Potato dextrose broth (PDB, BD Difco) and agar (PDA) were used for maintaining SMCD 2220-01 and biodegradation experiments.Fig. 1

Chemical structure of the mycotoxin Zearalenone (ZEN), deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), and 15-acetyl-deoxynivalenol (15-ADON)