Deoxynivalenol (DON) is a widespread trichothecene mycotoxin that commonly contaminates cereal crops and has various toxic effects in animals and humans. DON primarily targets the gastrointestinal tract, the first barrier against ingested food contaminants. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based phosphoproteomic approach was employed to elucidate the molecular mechanisms underlying DON-mediated intestinal toxicity in porcine epithelial cells (IPEC-J2) exposed to 20 μM DON for 60 min. There were 4153 unique phosphopeptides, representing 389 phosphorylation sites, detected in 1821 phosphoproteins. We found that 289 phosphopeptides corresponding to 255 phosphoproteins were differentially phosphorylated in response to DON. Comprehensive Gene Ontology (GO) analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that, in addition to previously well-characterized mitogen-activated protein kinase (MAPK) signaling, DON exposure altered phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase/signal transducer, and activator of transcription (JAK/STAT) pathways. These pathways are involved in a wide range of biological processes, including apoptosis, the intestinal barrier, intestinal inflammation, and the intestinal absorption of glucose. DON-induced changes are likely to contribute to the intestinal dysfunction. Overall, identification of relevant signaling pathways yielded new insights into the molecular mechanisms underlying DON-induced intestinal toxicity, and might help in the development of improved mechanism-based risk assessments in animals and humans.

Fluorene-9-bisphenol (BHPF), which has been used as a substitute for bisphenol A (BPA) in consumer goods and industrial products, can be detected in environmental media and human urine. BHPF has been reported to have endocrine-disrupting effects, whereas deleterious effects on steroidogenesis in H295R cells and underlying mechanisms are still unclear. Here, we investigated effects of BHPF on steroidogenesis using human adrenocortical carcinoma cells (H295R). Cytotoxicity was initially assessed and half-maximal inhibitory concentration (IC₅₀) was determined based on proliferation of cells. Responses of four steroid hormones, aldosterone, cortisol, testosterone and 17β-estradiol (E₂), and ten critical genes, StAR, HMGR, CYP11A1, CYP11B1, CYP11B1, HSD3B2, CYP21, CYP17, 17β-HSD, and CYP19, involved in steroidogenesis after exposure to non-cytotoxic concentrations of BHPF were determined in the presence or absence of 100 μM dbcAMP. Adenylate cyclase (AC) activity, intracellular concentrations of cAMP, PKA activity and amounts of steroidogenic factor-1 (SF-1) gene and expressions of proteins were determined to elucidate underlying mechanisms of effects on steroidogenesis. BHPF was cytotoxic to H295R cells in a dose- and time-dependent manner. Effects on production of hormones results demonstrated that exposure to greater concentrations of BHPF inhibited productions of aldosterone, cortisol, testosterone and E₂ by down-regulation of steroidogenic genes. Inhibition of AC activity, intercellular cAMP content and PKA activity after exposure to BHPF implied that the AC/cAMP/PKA signaling pathway was involved in BHPF-induced suppression of steroidogenesis in H295R cells. Additionally, BHPF inhibited steroidogenesis and expressions of steroidogenic genes via decreasing expression of SF-1 protein, both in basal and dbcAMP-induced treatment. These results contributed to understanding molecular mechanisms of BHPF-induced effects on steroidogenesis and advancing the comprehensive risk assessment of BPs.

A novel determination method using flow-through cartridge purification and ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was developed for the identification and quantification of 30 mycotoxins (aflatoxin B1, B2, G1, G2, sterigmatocysin, T-2 toxin, diacetoxyscirpenol, roquefortine C, methylergonovine, ergocornine, lysergol, enniatin A, enniatin A1, enniatin B, enniatin B1, beauvericin, deoxynivalenol, 3-acetylDeoxynivalenol, 15-acetylDeoxynivalenol, patulin, verruculogen, neosolaniol, gliotoxin, HT-2 toxin, wortmannin, zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, and β-zearalanol) in feedstuffs. Mycotoxins were extracted from sample by 0.1% formic acid aqueous solution/acetonitrile (16/84 v/v), then purified by a Cleanert® MC clean-up column. As a result, the pigments and other matrices were efficient removed from the sample extract solution. The purified eluate was collected, then evaporated and redissolved by 0.1% formic acid aqueous solution/acetonitrile (70:30 v/v) for UPLC-MS/MS detection. A BEH Shield RP18 chromatographic column was employed for separation of 30 mycotoxins in a total of 14 min. Multiple-reaction monitoring (MRM) mode was applied for qualitative and quantitative analysis, and matrix calibration curves obtained with the external-standard method was used for quantitation of target analytes. Under optimized conditions, the linearity range was from 2 to 1000 ng/g, and the limit of quantification of the developed method was from 2 to 50 ng/g. The recoveries of 30 mycotoxins spiked in urine samples were from 72.0% to 118.5%, and the relative standard deviation was below 20%. The method was also well approved by certified reference sample, and applied on the real feedstuff samples testing successfully.