Response of intestinal HT-29 cells to the trichothecene mycotoxin deoxynivalenol and its sulfated conjugates

Abstract The sulfated forms of the Fusarium toxin deoxynivalenol (DON), deoxynivalenol-3-sulfate (DON-3-Sulf) and deoxynivalenol-15-sulfate (DON-15-Sulf) were recently described, however little is known about their mechanism of action in mammalian cells. DON-3-Sulf and DON-15-Sulf were taken up by HT-29 colon carcinoma cells, although to a lesser extent compared to DON. All three compounds were found to enhance the intracellular ROS level in the dichlorofluorescein assay (≥ 1μM), even though substantial differences were observed in their cytotoxic potential. In silico modelling highlighted that DON-sulfates do not share the classical mechanism of action of DON, being unable to fit into the ribosomal pocket and trigger the classical ribotoxic stress response. However, DON-3-Sulf and DON-15-Sulf sustained a distinctive proliferative stimulus in HT-29 and activated autophagy. The mechanisms of action of DON-3-Sulf and DON-15-Sulf suggest a potential interplay between the onset of ribosomal inhibition and autophagy activation as an alternative and/or complementary mode of action for DON and its sulfated analogues

Deoxynivalenol induces structural alterations in epidermoid carcinoma cells A431 and impairs the response to biomechanical stimulation

Morphology together with the capability to respond to surrounding stimuli are key elements governing the spatial interaction of living cells with the environment. In this respect, biomechanical stimulation can trigger significant physiological cascades that can potentially modulate toxicity. Deoxynivalenol (DON, vomitoxin) is one of the most prevalent mycotoxins produced by Fusarium spp. and it was used to explore the delicate interaction between biomechanical stimulation and cytotoxicity in A431 cells. In fact, in addition of being a food contaminant, DON is a relevant toxin for several organ systems. The combination between biomechanical stimulation and the mycotoxin revealed how DON can impair crucial functions affecting cellular morphology, tubulin and lysosomes at concentrations even below those known to be cytotoxic in routine toxicity studies. Sub-toxic concentrations of DON (0.1\u20131 \u3bcM) impaired the capability of A431 cells to respond to a biomechanical stimulation that normally sustains trophic effects in these cells. Moreover, the effects of DON (0.1\u201310 \u3bcM) were partially modulated by the application of uniaxial stretching (0.5 Hz, 24 h, 15% deformation). Ultimately, proteomic analysis revealed the potential of DON to alter several proteins necessary for cell adhesion and cytoskeletal modulation suggesting a molecular link between biomechanics and the cytotoxic potential of the mycotoxin

Pro-Inflammatory Effects of NX-3 Toxin Are Comparable to Deoxynivalenol and not Modulated by the Co-Occurring Pro-Oxidant Aurofusarin

The type A trichothecene NX-3, produced by certain Fusarium graminearum strains, is similar to the mycotoxin deoxynivalenol (DON), with the exception that it lacks the carbonyl moiety at the C-8 position. NX-3 inhibits protein biosynthesis and induces cytotoxicity to a similar extent as DON, but so far, immunomodulatory effects have not been assessed. In the present study, we investigated the impact of NX-3 on the activity of the nuclear factor kappa B (NF-κB) signaling pathway in direct comparison to DON. Under pro-inflammatory conditions (IL-1β treatment), the impact on cytokine mRNA levels of NF-κB downstream genes was studied in human colon cell lines, comparing noncancer (HCEC-1CT) and cancer cells (HT-29). In addition, potential combinatory effects with the co-occurring Fusarium secondary metabolite aurofusarin (AURO), a dimeric naphthoquinone known to induce oxidative stress, were investigated. NX-3 and DON (1 μM, 20 h) significantly activated a NF-κB regulated reporter gene to a similar extent. Both trichothecenes also enhanced transcript levels of the known NF-κB-dependent pro-inflammatory cytokines IL-8, IL-6, TNF-α and IL-1β. Comparing the colon cancer HT-29 and noncancer HCEC-1CT cells, significant differences in cytokine signaling were identified. In contrast, AURO did not affect NF-κB pathway activity and respective cytokine expression levels at the tested concentration. Despite its pro-oxidant potency, the combination with AURO did not significantly affect the immunomodulatory effects of the tested trichothecenes. Taken together, the present study reveals comparable potency of DON and NX-3 with respect to immunomodulatory and pro-inflammatory potential. Consequently, not only DON but also NX-3 should be considered as factors contributing to intestinal inflammatory processes

The Influence of Processing Parameters on the Mitigation of Deoxynivalenol during Industrial Baking

Deoxynivalenol (DON), a frequent contaminant of flour, can be partially degraded by baking. It is not clear: (i) How the choice of processing parameter (i.e., ingredients, leavening, and baking conditions) affects DON degradation and thus (ii) how much DON can be degraded during the large-scale industrial production of bakery products. Crackers, biscuits, and bread were produced from naturally contaminated flour using different processing conditions. DON degradation during baking was quantified with the most accurate analytical methodology available for this Fusarium toxin, which is based on liquid chromatography tandem mass spectrometry. Depending on the processing conditions, 021%, 416%, and 25% DON were degraded during the production of crackers, biscuits, and bread, respectively. A higher NaHCO3 concentration, baking time, and baking temperature caused higher DON degradation. NH4HCO3, yeast, vinegar, and sucrose concentration as well as leavening time did not enhance DON degradation. In vitro cell viability assays confirmed that the major degradation product isoDON is considerably less toxic than DON. This proves for the first time that large-scale industrial baking results in partial detoxification of DON, which can be enhanced by process management.(VLID)365712

Stable Isotope-Assisted Metabolomics for Deciphering Xenobiotic Metabolism in Mammalian Cell Culture

We present a workflow based on stable isotope-assisted metabolomics and the bioinformatics tool MetExtract II for deciphering xenobiotic metabolites produced by human cells. Its potential was demonstrated by the investigation of the metabolism of deoxynivalenol (DON), an abundant food contaminatn, in a liver cracinoma cell line (HeoG2) and a model for colon carcinoma (HT29). Detected known metabolites included DON-3-sulfate, DON-10-sulfonate, and DON-10-glutathione as well as DON-cysteine. Conjugation with amino acids and antibiotics was confirmed for the first time. The approach allows the untargeted elucidation of human xenobiotic products in tissue culture.<br /

Supplementary Table 2 from Discovery of a Novel Potent EGFR Inhibitor Against EGFR Activating Mutations and On-Target Resistance in NSCLC

KINOMEscan selectivity profiling of BI-4732</p

Supplementary Figure S1 from Discovery of a Novel Potent EGFR Inhibitor Against EGFR Activating Mutations and On-Target Resistance in NSCLC

Tumor growth curve and body weight for each individual mouse in YU-1097 xenograft model</p

Supplementary Table 1 from Discovery of a Novel Potent EGFR Inhibitor Against EGFR Activating Mutations and On-Target Resistance in NSCLC

The EGFR mutation status of patient-derived models using whole exome sequencing analysis</p

Supplementary Figure S3 from Discovery of a Novel Potent EGFR Inhibitor Against EGFR Activating Mutations and On-Target Resistance in NSCLC

In vitro activity of BI-4732 in PDC cells harboring EGFR-activating mutations and T790M mutation</p