Deoxynivalenol don

Human prostate adenocarcinoma cell lines LNCaP, PC3, and 22Rv1 were purchased from the European Collection of Authenticated Cell Cultures (ECACC) (Sigma Aldrich, Saint Louis, MO, USA) and DU-145 from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI (LNCaP, PC3, 22Rv1) or Dulbecco’s modified Eagle’s medium (DMEM) (DU-145) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES) together with 50 U/mL penicillin and 50 μg/mL streptomycin (Thermo Fisher Scientific Inc, Waltham, MA, USA), in a humidified atmosphere of 5% CO₂ at 37 °C. To avoid any possible involvement of serum components, the cells were treated without antibiotics and serum.
Stock solution of deoxynivalenol (DON) (Sigma-Aldrich, Saint Louis, MO, USA) was prepared in ethanol. The cells were treated with ethanol at the highest concentrations used in the study and no statistically significant decrease in viability was observed. The final concentrations of DON were achieved by adding culture medium. Cells were treated with DON for 24 h. Non-treated cells were used as controls.

For the cytotoxicity test, toxins were purchased from Sigma (St Quentin Fallavier, France): deoxynivalenol (DON) (purity > 98%), tryptophol (TRPT) (purity > 97%), apicidin (API) (purity > 98%) and emodin (EMO) (purity > 90%). Enniatins (ENNs) (A1, B, B1, purity > 99%), cyclo-(L-Pro-L-Tyr) (Cyclo) (purity > 98%), and brevianamide-F (BRV-F) (purity > 95%) were purchased from BioAustralis (Smithfield, Australia), beauvericin (BEA) (purity > 95%) and aurofusarin (AFN), (purity > 97%) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). All mycotoxins were dissolved in dimethyl sulfoxide (DMSO) (Sigma) to prepare stock solutions stored at −20 °C. Working dilutions were freshly prepared in cell culture medium for each experiment.
To convert the concentration in the pig feed into the concentration to which intestinal cells might be exposed, we assumed, as already done in previous studies [53 (link)], that mycotoxins were ingested in one meal, diluted in 1 L of gastrointestinal fluid and were entirely bioaccessible. Next, the ratio of DON to emerging mycotoxins was calculated based on three plausible scenarios according to the concentration of DON and emerging mycotoxins in the feed (Supplementary Table S1).
Several 3-fold dilutions of each individual toxin and mixtures at different ratios were performed to account for the concentrations present in feed.

Deoxynivalenol (DON) were purchased from Sigma-Aldrich (Shanghai, China). PCV2 strains were kindly donated by the Laboratory of Infectious Disease, Department of Prevention Veterinary Medicine, Nanjing Agricultural University. It was isolated and sequenced from the kidneys of piglets, naturally infected with multiple system failure syndrome of weaned piglets, and stored at −80 °C.

To determine the effects of DON on PCa steroidogenesis, cells were treated with 0, 1 or 5 µM DON or DHEA 0–100 nM over 2 or 3 days. Non-treated cells were used as a control. Deoxynivalenol (DON) (Sigma-Aldrich, Saint Louis, MI, USA) and dehydroepiandrosterone (DHEA) (Avanti Polar Lipids, Inc., Alabaster, AL, USA) stock solutions were prepared in ethanol. Stocks were dissolved in experimental medium before use.
The doses and experimental scheme were based on our previous study [8 (link)]. All experiments were carried out with three different pools of cells. Because DHEA can serve as a precursor for the more potent androgens T and DHT [44 (link)], as well as estrogens, its effects on prostate health are of interest.

Deoxynivalenol (DON) was purchased from Sigma (St. Quentin Fallavier, France), and fusarenon-X (FX) was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). The toxins were dissolved to 60 mM in dimethylsulfoxide (DMSO) and stored at −20 °C before dilution in complete culture media.

Deoxynivalenol (DON) (D-0156) was obtained from Sigma-Aldrich (St. Louis, MO, USA). It was dissolved in water for use in the cell exposure studies. Dimethyl sulfoxide (DMSO) and forskolin were from Sigma-Aldrich. Alamar blue, propidium iodide, RNAse A, and DRAQ5 were purchased from ThermoFisher Scientific (Waltham, MA, USA). Dulbecco’s Modified Eagle Medium (DMEM), phosphate buffered saline (PBS), trypsin-versene (EDTA) mixture, penicillin-streptomycin mixture, and foetal bovine serum EU standard (FBS) were from Lonza (Verviers, Belgium). The CellTox Green cytotoxicity assay and cAMP-Glo assay were bought from Promega (Madison, WI, USA). BD BioCoat Cellware poly-L-lysine 12 mm coverslips and the TNF-α ELISA were obtained from BD Biosciences (Bedford, MA, USA). The IL-1β ELISA was from R &D Systems (Minneapolis, MN, USA).