Cereulide and Deoxynivalenol Increase LC3 Protein Levels in HepG2 Liver Cells.

Food contaminants of bacterial or fungal origin frequently contaminate staple foods to various extents. Among others, the bacterial toxin cereulide (CER) and the mycotoxin deoxynivalenol (DON) co-occur in a mixed diet and are absorbed by the human body. Both toxins exert dis-tinctive mitotoxic potential. As damaged mitochondria are removed via autophagy, mitochondrial and lysosomal toxicity were assessed by applying low doses of single and combined toxins (CER 0.1-50 ng/mL; DON 0.01-5 µg/mL) to HepG2 liver cells. In addition to cytotoxicity assays, RT-qPCR was performed to investigate genes involved in lysosomal biogenesis and autophagy. CER and DON caused significant cytotoxicity on HepG2 cells after 5 and 24 h over a broad concentration range. CER, alone and in combination with DON, increased the transcription of the autophagy related genes coding for the microtubule associated protein 1A/1B light chain 3 (LC3) and sequestome 1 (SQSTM1) as well as LC3 protein expression which was determined using immunocytochemistry. DON increased LC3 protein expression without induction of gene transcription, hence it seems plausible that CER and DON act on different pathways. The results support the hypothesis that CER induces autophagy via the LC3 pathway and damaged mitochondria are therefore eliminated.

Assessing Mixture Effects of Cereulide and Deoxynivalenol on Intestinal Barrier Integrity and Uptake in Differentiated Human Caco-2 Cells.

The human intestine is regularly exposed to ingested food contaminants, such as fungal and bacterial toxins, which have been described to co-occur in a mixed diet. Thus, it is of utmost importance to understand possible interactions between contaminants of different origin. Hence, we investigated the single and combined effects of one of the most abundant mycotoxins, deoxynivalenol (DON; 0.1 to 10 µg/mL), and the bacterial toxin cereulide (CER; 1 to 100 ng/mL) on differentiated human Caco-2 (C2BBe1) cells cultured in a transwell system. We tested the capacity of the two toxins to alter the intestinal integrity and further investigated the uptake of both compounds and the formation of selected DON metabolites. CER alone (10 and 100 ng/mL) and in combination with DON (10 ng/mL CER with 1 µg/mL DON) was found to alter the barrier function by increasing the transepithelial electrical resistance and the expression of the tight junction protein claudin-4. For the first time, DON-3-sulfate was identified as a metabolite of human intestinal cells in vitro. Moreover, co-incubation of CER and DON led to an altered ratio between DON and DON-3-sulfate. Hence, we conclude that co-exposure to CER and DON may alter the intestinal barrier function and biotransformation of intestinal cells.

Combinatory effects of cereulide and deoxynivalenol on in vitro cell viability and inflammation of human Caco-2 cells.

Deoxynivalenol (DON), one of the most abundant mycotoxins in cereal products, was recently detected with other mycotoxins and the emetic bacterial toxin cereulide (CER) in maize porridge. Within a cereal-based diet, co-exposure to these toxins is likely, hence raising the question of combinatory toxicological effects. While the toxicological evaluation of DON has quite progressed, consequences of chronic, low-dose CER exposure are still insufficiently explored. Information about the combinatory toxicological effects of these toxins is lacking. In the present study, we investigated how CER (0.1-100 ng/mL) and DON (0.01-10 µg/mL) alone and in a constant ratio of 1:100 (CER:DON) affect the cytotoxicity and immune response of differentiated human intestinal Caco-2 cells. While DON alone reduced cell viability only in the highest concentration (10 µg/mL), CER caused severe cytotoxicity upon prolonged incubation (starting from 10 ng/mL after 24 h and 48 h, 2.5 ng/mL and higher after 72 h). After 72 h, synergistic effects were observed at 2.5 ng/mL CER and 0.25 µg/mL DON. Different endpoints of inflammation were investigated in interleukin-1ß-stimulated Caco-2 cells. Notably, DON-induced interleukin-8 transcription and secretion were diminished by the presence of 10 and 25 ng/mL CER after short-term (5 h) incubation, indicating immunosuppressive properties. We hypothesise that habitual consumption of cereal-based foods co-contaminated with CER and DON may cause synergistic cytotoxic effects and an altered immune response in the human intestine. Therefore, further research concerning effects of co-occurring bacterial toxins and mycotoxins on the impairment of intestinal barrier integrity, intestinal inflammation and the promotion of malnutrition is needed.

The Hop Polyphenols Xanthohumol and 8-Prenyl-Naringenin Antagonize the Estrogenic Effects of Fusarium Mycotoxins in Human Endometrial Cancer Cells.

The Fusarium toxin zearalenone (ZEN) and its reductive metabolite α-zearalenol (α-ZEL) are well-documented endocrine disruptors that are frequently found to contaminate cereal products, including beer. But also hop is known to represent a source for endocrine active compounds, containing amongst others xanthohumol (XAN), which might be converted to the potent phytoestrogen 8-prenylnaringenin (8-PN). In the present study, we investigated the interaction of these xenoestrogens in mixtures which might occur in beer. Estrogenicity was measured as induction of alkaline phosphatase (AlP) expression in estrogen-sensitive Ishikawa cells. In binary combinations, XAN was found to act as a potent antagonist of mycotoxin-induced estrogenicity, significantly suppressing the AlP-inducing impact of both ZEN and α-ZEL at nanomolar concentrations. Also 8-PN antagonized the estrogenic stimulus of the two fungal metabolites, although less pronounced. These effects also manifested in combinations of three or four test compounds, and at the level of cell proliferation, that was assessed via an E-screen-like approach in Ishikawa cells. Of note, co-exposure to the investigated myco- and phyto-estrogens did not result in additive or overadditive/synergistic estrogenic effects in the applied test system. Being aware that the actual study is still limited to the in vitro situation, our results even suggest that prenylated chalkones from hops might protect against Fusarium toxin-induced endocrine disruptive activities at concentrations that can be reached by moderate beer consumption.

Delphinidin protects colon carcinoma cells against the genotoxic effects of the mycotoxin altertoxin II.

Alternaria spp. are ubiquitous molds that are able to produce toxic secondary metabolites which may contaminate food globally. One of those is the mycotoxin altertoxin II (ATX-II), a genotoxic and mutagenic compound. In recent years, different flavonoids that may co-occur with mycotoxins in food were demonstrated to temper toxic effects of molds, mostly through their anti-oxidant properties. Thus, in this study, we assessed the influence of the berry anthocyanidin delphinidin on the toxicity of ATX-II in HT-29 colon carcinoma cells. We performed coupled SRB/WST-1 cytotoxicity assays which revealed only weak antagonistic interactions, and single-cell gel electrophoresis ("comet") assays, where we observed a potent protective effect of delphinidin on the DNA-damaging properties of ATX-II. Furthermore, we investigated the mechanism for this interaction. In the DCF assay delphinidin was found to reduce intracellular oxidative stress levels, which might contribute partly to the latter protection. However, LC-MS experiments showed that co-incubation of the mycotoxin with either delphinidin or its potential degradation product phloroglucinol aldehyde significantly decreased ATX-II concentrations in aqueous solutions, indicating that a direct chemical reaction of ATX-II with these components is likely responsible for the observed loss of toxicity. Our results indicate that delphinidin - and possibly other anthocyanins as well - might play a role in the protection of the gut from Alternaria-induced genotoxicity.

The secondary Fusarium metabolite aurofusarin induces oxidative stress, cytotoxicity and genotoxicity in human colon cells.

Aurofusarin (AURO), a dimeric naphthoquinone, is produced by Fusarium fungi. Although frequently found in food and feed, toxicological studies are limited. Hence, the in vitro toxicity of AURO was investigated in the colon adenocarcinoma cell line HT29 and the non-tumorigenic colon cells HCEC-1CT. Cytotoxic effects were found at concentrations ≥1 µM by evaluating mitochondrial activity (WST-1) and cellular proliferation (sulforhodamine B assay). 10 µM of AURO induced a decrease of cells in the S-phase, measured by flow cytometry. Confocal microscopy revealed AURO-mediated increase of intracellular p53 protein. In accordance, DNA-damage was seen in the comet assay (≥1 µM) together with enhanced levels of formamidopyrimidine-DNA-glycosylase (fpg)-sensitive sites, indicative for oxidative stress. An increase of intracellular reactive oxygen species was observed in the dichlorofluorescein (DCF) assay (≥5 µM). The GSSG/GSH ratio was elevated, but no impact on redox-sensitive Nrf2-dependent genes (Nrf2, γ-GCL, NQO1) was found at the gene expression level. However, induction of cytochrome P450 monooxygenase (CYP) 1A1 was measured at the gene expression and protein level. In conclusion, these in vitro data suggest that, when co-occurring, AURO might be considered as a potential contributor to the overall toxicity of respective Fusarium mycotoxin mixtures.

Genistein and delphinidin antagonize the genotoxic effects of the mycotoxin alternariol in human colon carcinoma cells.

SCOPE: Although associated with anti-oxidative properties, genistein has been reported to induce DNA strand breaks, whereby oxidative stress and topoisomerase poisoning are considered as potential mechanisms. In contrast, delphinidin, a catalytic topoisomerase inhibitor, is known to suppress the DNA-damaging properties of several topoisomerase poisons. Recently, alternariol, a mycotoxin produced by Alternaria spp., was found not only to induce oxidative stress but also to act as a topoisomerase poison. As both, polyphenols and mycotoxins, might occur in our nutrition simultaneously, the question was addressed whether potential combinatory effects on DNA integrity have to be considered. METHODS AND RESULTS: We determined combinatory effects of either genistein or delphinidin with alternariol in HT-29 cells. Cytotoxicity was assessed by WST-1 and SRB assays, whereby only weak interactions were observed. The comet assay revealed significant antagonistic interactions of both polyphenols with the genotoxicity of AOH. The underlying mechanism comprises the suppression of alternariol-mediated stabilization of DNA/topoisomerase-II-intermediates, as observed in the ICE assay. Furthermore, DEL but not GEN was found to suppress AOH-mediated oxidative stress. CONCLUSION: Our data indicate that a respective polyphenol-rich diet might aid to protect against genotoxic damages caused by AOH, whereby bioactive concentrations of DEL are predominantly expected locally in the intestines.