4.1. Chemicals and Reagents
All chemicals and reagents were acquired from Scharlau (Scharlau Chimie S.A., Barcelona, Spain), Carlo Erba (Carlo Erba, Val de Rueil 27, France) and Sigma Chemical Co (Sigma, Saint Quentin Fallavier, France). The Bio-Rad kit were purchased from Bio-Rad Laboratories (Biorad, München, Germany). The DC assay kit were purchased from Uptima Interchim (Interchim, Montluçon, France). Caspase 3/7 Glow assays and the cAMP-Glo Assay were purchased from Promega (Promega, Charbonnieres les bains, France). Enzymatic hydrolysis of ZEN conjugates and its metabolites was performed by the H-2 β-glucuronidase from Helix pomatia acquired from Sigma (Cat# G0875). FB1, FB2, FB3, ZEN, α-ZOL, β-ZOL, ZAN, α-ZAL, β-ZAL, DON, DON-3-glucoside, deepoxy-DON, 15-acetyl-DON, 3-acetyl-DON, fusarenone x, nivalenol, diacetoxyscirpenol, 15 monoacetoxyscirpenol, T2 toxin, HT2 toxin, T2 tetraol, verrucarol, roridin A, verrucarin A, moniliformin, tenuazonic acid, ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, ergotamine, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, ochratoxin B, alpha-ochratoxin, cyclopiazonic acid, citrinin, patulin and sterigmatocystin were purchased as standards from Biopure (Romer Labs, Getzersdorf, Austria) and Sigma. Solutions of [13C34]-FB1, [13C34]-FB2, [13C34]-FB3 and [13C18]-ZEN at certified concentrations were acquired from Biopure (Romer Labs, Tulln, Austria). Sphinganine, sphingosine and C20 sphinganine were acquired from BioValley (BioValley S. A., Marne la Vallée, France). FUMONIPREP and EASI-EXTRACT® ZEARALENONE columns were acquired from R-Biopharm Rhône Ltd. (R-Biopharm AG, Glasgow, Scotland). Pure water, acetonitrile, methanol, formic acid, ammonium acetate and acetic acid used for analysis of mycotoxins were of LC-MS analytical grade whereas other reactants were of HPLC analytical grade.
4.4. Animal Exposure
The study was conducted in agreement to the guidelines of the Declaration of Helsinki and the French National Guidelines for the care and use of animals for research purposes. Protocol was approved by the institutional review board UEPFG of INRAE (protocol code PP–5–12 approved in date of February 29, 2012). Seventy-five male mule ducks (Cairina moschata
× Anas platyrhynchos
) from the commercial lines MMG × PKL (Couvoir Ducournau, Bonnegarde, France) were reared in individual cages at the Palmipole station (Domaine d’Artiguères, Benquet, France). From 0 to 84 days of age, the ducks were fed with mycotoxin-free diets to best meet their nutritional needs. On the 84th day of age, the ducks were weighed and distributed to form five weight-homogeneous groups each containing 15 birds to be exposed to FUS. The force-feeding program lasted 12 days and followed the usual practice in the species [46
]. The corn was administered as a mixture of 26% whole corn, 36% ground corn and 38% water as follow: 250 g of corn on the first meal, followed by a progressive increase of 25 g per meal to reach a volume of 500 g of corn per meal on day 6, which was maintained until the end of the study (Table S1
). Capsules containing FB, DON and ZEN were administered in the middle of the meal to the ducks exposed to FUS while mycotoxin-free capsules were administered to the control animals not exposed to mycotoxins. Each animal received two meals per day. At the end of the study, the animals were left to fast for eight hours, then a blood sample was taken from the post-occipital sinus of the spinal vein on lithium heparin vacutainer tubes before ducks were stunned by electro narcosis and killed by exsanguination.
4.5. Sample Collection
All the animals were autopsied for macroscopic investigation of outward appearance of the eyes, digestive system (including beak and accessory glands), kidneys and the musculoskeletal, respiratory, cardiovascular and reproductive systems to identify anomalies. Organs (liver, kidneys, heart, spleen, pancreas and testes) were collected and the intestine was emptied prior to isolation of the different fractions (gizzard, duodenum, jejunum, ileum and caeca, including caeca tonsils). All organs and digestive fractions were weighed and the length of the duodenum, jejunum and ileum was measured. Tissue fractions of organs and intestine were collected and stored in neutral buffered formalin for microscopic investigation while the remaining liver and testes were stored at −80 °C for other analysis.
4.6. Biochemistry, Hematology, Histopathology and Tissue Fractions
The concentrations of proteins, cholesterol and uric acid and the activities of lactate dehydrogenase (LDH, EC 18.104.22.168), alkaline phosphatase (ALP, EC 22.214.171.124) and alanine aminotransferase (ALT, EC 126.96.36.199) were measured in plasma with a clinical chemistry analyzer KONELAB 20 (Fisher Scientific SAS, Illkirch, France) according to the manufacturer’s instructions. Concentrations in analytes were expressed in g/L or mmol/L and enzymes activities were expressed in UI/L. Malassez cells was used for manually counting of the number of white blood cell and results were expressed in %.
Fixed tissues were cut into fractions of about 1 cm and embedded in paraffin blocks. Paraffin blocks were cut into slices of 4 μm that were stained with hematoxylin, eosin and saffron (HES). Semi-quantitative analysis of lesions was measured for each tissue of each animal using the following score: absence = 0: slight = 1, moderate = 2, marked = 3.
S9 homogenates of liver were obtained by milling 5 g of liver in 15 mL of PBS as previously described [11
]. Five hundred microliters of S9 were deproteinized with 1.25 M metaphosphoric acid (vol:vol). The testis extracts were obtained by three consecutive freeze/thaw cycles in PBS. S9 fractions, deproteinized S9 and testis extracts were stored at −80 °C until analysis.
Melt rate of the liver was measured as previously described [47
]. Briefly, 60 g of liver were placed in a tin and cooked in an autoclave at 85 °C for 60 min. After refrigeration, the tins were heated to 75–80 °C, 20 min and the melting rate was measured as the percentage of melted fat in proportion to the initial weight of the liver.
4.7. Markers of Oxidative Damage and Analysis of Testis Extracts
Markers of oxidative damage in plasma and S9 liver fractions and analysis of testis extracts were investigated as previously described [11
]. Briefly, the concentration of protein in the S9 homogenate and in the testis, the extract was determined with the Bio-Rad protein kit and the DC assay kit, respectively. Malondialdehyde (MDA) was measured by formation of a pink complex with thiobarbituric acid that was extracted by butanol and fluorometrically dosed (excitation at 515 nm, emission at 548 nm). The activity of superoxide dismutase (SOD, EC 188.8.131.52) was measured in the presence of nitroblue tetrazolium by the inhibition of the formation of blue formazan by 1 mU xanthine-xanthine oxidase (EC 184.108.40.206). The reaction was monitored spectrophotometrically at 540 nm. The activity was obtained by linear regression using a standard curve performed with bovine erythrocyte SOD. The activity of catalase (CAT, EC 220.127.116.11) was measured with H2
as substrate by the formation of formaldehyde, which was spectrophotometrically dosed at 540 nm. The activity was obtained by linear regression using a standard curve performed with formaldehyde. The activity of glutathione reductase (GRed, EC 18.104.22.168) was measured by reduction of oxidized glutathione (GSSG) into glutathione (GSH) in the presence of NADPH. The activity was obtained by following the decrease in NADPH absorbance at 340 nm. The activity of glutathione peroxidase (GPx, EC 22.214.171.124) was measured in a coupled reaction with GRed. In the presence of GSH, GPx reduces cumene hydroperoxide to produce GSSG. Then, GSSG is reduced into GSH by GRed in the presence of NADPH. The activity of the enzyme was obtained by following the decrease in absorbance at 340 nm. The deproteinized S9 fraction is used to measure total glutathione (TG) using an optimized enzymatic recycling method for the determination of GSH content. GSH reacts with Ellman’s reagent to form 5-thio-2-nitrobenzoic acid (TNB) and GS-TNB. Both GSSG and GS-TNB are reduced by GRed to produce GSH, permitting the formation of more TNB. The amount of TNB produced was spectrophotometrically measured at 405 nm. The GSSG amountis obtained by a first derivatization of GSH with 2-vinylpyridine before measurement of TGs, which block the reaction of GSH.
Caspase 3/7 Glow assays were performed using the Caspase-Glo® 3/7 Assay System kit of Promega (Promega, Charbonnieres les Bains, France) according to the manufacturer’s instructions. The activity of caspase-3/7 is obtained by using a luminogenic substrate containing the sequence asp-glu-val-asp that liberates luciferin after cleavage by caspases. Luciferin is used by luciferase to generate luminescence that was expressed in relative light units normalized to 100,000 seminiferous tubules cells. The concentration of cAMP was measured using the cAMP-Glo® Assay kit of Promega as recommended by the manufacturer. The concentration of testosterone was determined by radioimmunoassay with a sensitivity of 15 pg/tube and 5.3% intra-assay coefficients of variation.
4.9. Mycotoxins in Liver
The concentration of mycotoxins in the liver was analyzed by UHPLC-MSMS after sample clean-up on immunoaffinity column and isotopic dilution, as previously described [29
]. The UHPLC MSMS system was a 1260 model, the detector was a 6410 triple quad, both from Agilent (Agilent, Santa Clara, CA, USA). Mycotoxins were separated on a Poroshell 120 column (3.0 × 50 mm, 2.7 µ). Detection was done in positive electrospray ionization mode in the following conditions: temperature at 300 °C, gas delivered at 10 L/min, nebulization at 25 psi and capillary voltage at 4000 V. Two transitions were used as qualifiers for FB1, FB2, FB3, ZEN, α-ZOL, β-ZOL, ZAN, α-ZAL and β-ZAL, whereas one qualifier was used for the internal standards. MassHunter quantitative analysis software (Agilent) was used to analyze the chromatograms. A relative standard deviation of 20% was considered acceptable for measuring accuracy. The signal suppression and enhancement (SSE) was calculated by dividing the slope of calibration curve fitted to matrix by the slope of calibration curve based on net solvent and expressed in percent. Values of SSE of 80–120% were considered acceptable. The maximum variation of the ratio of the qualifiers measured in the samples had to be less than 20% of the ratio measured in the standard solutions. The maximum variation in the retention time of the analytes in the samples had to be less than 5% of the retention time measured in the standard solutions.
The method used to measure FB in liver was previously described [29
]. Briefly, one g of liver was homogenized in 4 mL of distilled water acetonitrile/methanol (2:1:1) in presence of 5 mg of NaCl and 12.5 ng of [13
]-FB1 and [13
]-FB2 as IS. Samples were extracted 2h on a stir table, then centrifuged. Aqueous phase was collected and 8 mL of hexane were added for removing the lipids. Part of the aqueous phase was passed through a FUMONIPREP column. Extracts were evaporated to dryness and stored at −20 °C. The method was linear (Fisher test, p
< 0.01 and r2 ≥ 0.99) for a concentration range of 0.25 to 25 ng/g for FB1, FB2 and FB3, the lowest concentrations validated corresponded to the LOQ [29
The method used to measure the total forms of ZEN and its metabolites in liver was previously described [30
]. Briefly, five g of liver were homogenized in 5 mL of acetate buffer in presence of 1000 U of H-2 β-glucuronidase and placed overnight in a shaking bath at 37 °C. The mixture was placed in a conical tube containing of 12.5 ng of [13
]-ZEN and thirty ml of acetonitrile/hexane (2:1) were added. Samples were placed 15min on a stir table, then centrifuged. The upper phase was removed and the lower phase was centrifuged again to collect the supernatant that was evaporated to dryness. The dry residue was suspended in 2.5 mL methanol; then, 22.5 mL of PBS was added and the solution was passed through the Easi-Extract Zearalenone Column. Extracts were evaporated to dryness and stored at −20 °C. The method was linear (Fisher test, p
< 0.01 and r2 ≥ 0.99) for a concentration range of 0.25 to 4 ng/g for α-ZOL and β-ZOL and 1 to 4 ng/g for ZEN; the lowest concentrations validated corresponded to the LOQ [30