Impact of Fusarium-Derived Mycoestrogens on Female Reproduction: A Systematic Review.

Contamination of the world's food supply and animal feed with mycotoxins is a growing concern as global temperatures rise and promote the growth of fungus. Zearalenone (ZEN), an estrogenic mycotoxin produced by Fusarium fungi, is a common contaminant of cereal grains and has also been detected at lower levels in meat, milk, and spices. ZEN's synthetic derivative, zeranol, is used as a growth promoter in United States (US) and Canadian beef production. Experimental research suggests that ZEN and zeranol disrupt the endocrine and reproductive systems, leading to infertility, polycystic ovarian syndrome-like phenotypes, pregnancy loss, and low birth weight. With widespread human dietary exposure and growing experimental evidence of endocrine-disrupting properties, a comprehensive review of the impact of ZEN, zeranol, and their metabolites on the female reproductive system is warranted. The objective of this systematic review was to summarize the in vitro, in vivo, and epidemiological literature and evaluate the potential impact of ZEN, zeranol, and their metabolites (commonly referred to as mycoestrogens) on female reproductive outcomes. We conducted a systematic review (PROSPERO registration CRD42020166469) of the literature (2000-2020) following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The data sources were primary literature published in English obtained from searching PubMed, Web of Science, and Scopus. The ToxR tool was applied to assess risk of bias. In vitro and in vivo studies (n = 104) were identified and, overall, evidence consistently supported adverse effects of mycoestrogens on physiological processes, organs, and tissues associated with female reproduction. In non-pregnant animals, mycoestrogens alter follicular profiles in the ovary, disrupt estrus cycling, and increase myometrium thickness. Furthermore, during pregnancy, mycoestrogen exposure contributes to placental hemorrhage, stillbirth, and impaired fetal growth. No epidemiological studies fitting the inclusion criteria were identified.

Study of enzymatic activity in human neuroblastoma cells SH-SY5Y exposed to zearalenone's derivates and beauvericin.

Beauvericin (BEA), α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL), are produced by several Fusarium species that contaminate cereal grains. These mycotoxins can cause cytotoxicity and neurotoxicity in various cell lines and they are also capable of produce oxidative stress at molecular level. However, mammalian cells are equipped with a protective endogenous antioxidant system formed by no-enzymatic antioxidant and enzymatic protective systems such as glutathione peroxidase (GPx), glutathione S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD). The aim of this study was evaluating the effects of α-ZEL, ß-ZEL and BEA, on enzymatic GPx, GST, CAT and SOD activity in human neuroblastoma cells using the SH-SY5Y cell line, over 24 h and 48 h with different treatments at the following concentration range: from 1.56 to 12.5 µM for α-ZEL and ß-ZEL, from 0.39 to 2.5 µM for BEA, from 1.87 to 25 µM for binary combinations and from 3.43 to 27.5 µM for tertiary combination. SH-SY5Y cells exposed to α-ZEL, ß-ZEL and BEA revealed an overall increase in the activity of i) GPx, after 24 h of exposure up to 24-fold in individual treatments and 15-fold in binary combination; ii) GST after 24 h of exposure up to 10-fold (only in combination forms), and iii) SOD up to 3.5- and 5-fold in individual and combined treatment, respectively after 48 h of exposure. On the other hand, CAT activity decreased significantly in all treatments up to 92% after 24 h except for ß-ZEL + BEA, which revealed the opposite.

The mycoestrogen zeranol at high dosage antagonizes transient receptor potential channel activities in 3T3 L1 cells.

Recent findings have revealed that exposure to environmental contaminants may result in obesity and pose a health threat to the general public. As the activity of transient receptor potential channels (TRPs) plays a permissive role in adipogenesis, the interactions between TRPs and some food pollutants, i.e. bisphenol A, di (2-ethylhexyl) phthalate, zearalenone, and zeranol at 10 µM were investigated in the present study. TRP-V1,-V3, -C4 and -C6 are reported to be differentially expressed in the adipocyte differentiation, and immunoblotting was performed to quantify changes in these TRPs affected by the pollutants. Our result indicated that the mycoestrogen zeranol or α-zearalanol suppressed the expression of the V1 and C6 isoforms. Subsequently, confocal microscopy was used to measure the calcium inflow repressed by zeranol from 0.1 µM to 10 µM. Oil Red O staining was used to determine the differentiation of 3T3 L1 preadipocytes. Zeranol could suppress the expression of TRP-V1 and -C6 protein and inhibit the associated flow of calcium into the cytosol of 3T3 L1 cells. Its IC50 value for inhibiting calcium inflow stimulated by 40 µM capsaicin or 10 µM GSK1702934A was estimated to be around 6 µM. Reduced TRP-V1 or -C6 activity might result in promoting adipogenesis. In conclusion, this study demonstrated that zeranol could potentiate fat cell differentiation through antagonizing TRP-V1 and -C6 activities.

Coffee Silverskin and Spent Coffee Suitable as Neuroprotectors against Cell Death by Beauvericin and α-Zearalenol: Evaluating Strategies of Treatment.

Coffee silverskin and spent coffee have been evaluated in a neuroblastoma cell line (SH-SY5Y cells) against beauvericin (BEA) and α-zearalenol (α-ZEL)-induced cytotoxicity with different strategies of treatment. First, the direct treatment of mycotoxins and coffee by-products extracts in SH-SY5Y cells was assayed. IC50 values for α-ZEL were 20.8 and 14.0 µM for 48 h and 72 h, respectively and, for BEA only at 72 h, it was 2.5 µM. Afterwards, the pre-treatment with spent coffee obtained by boiling water increased cell viability for α-ZEL at 24 h and 48 h from 10% to 16% and from 25% to 30%, respectively; while with silverskin coffee, a decrease was observed. Opposite effects were observed for BEA where an increase for silverskin coffee was observed at 24 h and 48 h, from 14% to 23% and from 25% to 44%, respectively; however, a decrease below 50% was observed for spent coffee. Finally, the simultaneous treatment strategy for the highest concentration assayed in SH-SY5Y cells provided higher cytoprotection for α-ZEL (from 44% to 56% for 24 h and 48 h, respectively) than BEA (30% for 24 h and 48 h). Considering the high viability of coffee silverskin extracts for SH-SY5Y cells, there is a forthcoming promising use of these unexploited residues in the near future against mycotoxins effects.

Oxidative stress, glutathione, and gene expression as key indicators in SH-SY5Y cells exposed to zearalenone metabolites and beauvericin.

The co-presence of mycotoxins from fungi of the genus Fusarium is a common fact in raw food and food products, as trace levels of them or their metabolites can be detected, unless safety practices during manufacturing are carried out. Zearalenone (ZEA), its metabolites α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL) and, beauvericin (BEA) are co/present in cereals, fruits or their products which is a mixture that consumer are exposed and never evaluated in neuronal cells. In this study the role of oxidative stress and intracellular defense systems was assessed by evaluating reactive oxygen species (ROS) generation and glutathione (GSH) ratio activity in a human neuroblastoma cell line, SH-SY5Y cells, treated individually and combined with α-ZEL, ß-ZEL and BEA. It was further examined the expression of genes involved in cell apoptosis (CASP3, BAX, BCL2) and receptors of (endogenous or exogenous) estrogens (ERß and GPER1), by RT-PCR in those same conditions. These results demonstrated elevated ROS levels in combinations where α-ZEL was involved (2.8- to 8-fold compared to control); however, no significant difference in ROS levels were detected when single mycotoxin was tested. Also, the results revealed a significant increase in GSH/GSSG ratio at all concentrations after 24 h. Expression levels of CASP3 and BAX were up regulated by α-ZEL while CASP3 and BCL2 were down regulated by ß-ZEL, revealing how ZEA´s metabolites can induce the expression of cell apoptosis genes. However, BEA down-regulated the expression of BCL2. Moreover, ß-ZEL + BEA was the only combination treatment which was able to down regulate the levels of cell apoptosis gene expression. Relying to our findings, α-ZEL, ß-ZEL and BEA, induce injury in SH-SY5Y cells elevating oxidative stress levels, disturbing the antioxidant activity role of glutathione system and finally, causing disorder in the expressions and activities of the related apoptotic cell death genes.

Zearalenone and Metabolites in Livers of Turkey Poults and Broiler Chickens Fed with Diets Containing Fusariotoxins.

Zearalenone (ZEN) and metabolites were measured in livers of turkeys and broilers fed a control diet free of mycotoxins, a diet that contained 0.5 mg/kg ZEN (ZEN diet), and a diet that contained 0.5, 5, and 20 mg/kg of ZEN, fumonisins, and deoxynivalenol, respectively (ZENDONFB diet). The feed was individually distributed to male Grade Maker turkeys from the 55th to the 70th day of age and to male Ross chickens from the 1st to the 35th day of age, without any signs of toxicity. Together, the free and conjugated forms of ZEN, α- and ß-zearalenols (ZOLs), zearalanone (ZAN), and α- and ß-zearalanols (ZALs) were measured by UHPLC-MS/MS with [13C18]-ZEN as an internal standard and immunoaffinity clean-up of samples. ZAN and ZALs were not detected. ZEN and ZOLs were mainly found in their conjugated forms. α-ZOL was the most abundant and was found at a mean concentration of 2.23 and 1.56 ng/g in turkeys and chickens, respectively. Consuming the ZENDONFB diet significantly increased the level of total metabolites in the livers of chickens. Furthermore, this increase was more pronounced for the free forms of α-ZOL than for the conjugated forms. An investigation of the presence of ZEN and metabolites in muscle with the methods validated for the liver failed to reveal any traces of these contaminants in this tissue. These results suggest that concomitant dietary exposure to deoxynivalenol (DON) and fumonisins (FB) may alter the metabolism and persistence of ZEN and its metabolites in the liver.

The livestock growth-promoter zeranol facilitates GLUT4 translocation in 3T3 L1 adipocytes.

Zeranol is an approved but controversial growth-promoting agent for livestock in North America. It is a mycotoxin metabolite secreted by the Fusarium family fungi. The regulatory bodies in this region have established the acceptable daily intake and exposure below the level would not significantly increase the health risk for humans. However, their European counterparts have yet to establish an acceptable level and do not permit the use of this agent in farm animals. Given the growth-promoting ability of zeranol, its effect on energy metabolism was investigated in the current study. Our results indicated that zeranol could induce glucose transporter type 4 (GLUT4) expression in 3T3 L1 cells at 10 µM and initiate the translocation of the glucose transporter to the membrane as assayed by confocal microscopy. The translocation was likely triggered by the increase of GLUT4 and p-Akt. The insulin signal transduction pathway of glucose translocation was analyzed by Western blot analysis. Since no increase in the phosphorylated insulin receptor substrate in zeranol-treated cells was evidenced, the increased p-Akt and GLUT4 amount should be the mechanism dictating the GLUT4 translocation. In summary, this study showed that zeranol could perturb glucose metabolism in differentiated 3T3 L1 adipocytes. Determining the growth-promoting mechanism is crucial to uncover an accepted alternative to the general public.

Individual and Combined Effect of Zearalenone Derivates and Beauvericin Mycotoxins on SH-SY5Y Cells.

Beauvericin (BEA) and zearalenone derivatives, α-zearalenol (α-ZEL), and ß-zearalenol (ß-ZEL), are produced by several Fusarium species. Considering the impact of various mycotoxins on human's health, this study determined and evaluated the cytotoxic effect of individual, binary, and tertiary mycotoxin treatments consisting of α-ZEL, ß-ZEL, and BEA at different concentrations over 24, 48, and 72 h on SH-SY5Y neuronal cells, by using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide). Subsequently, the isobologram method was applied to elucidate if the mixtures produced synergism, antagonism, or additive effects. Ultimately, we determined the amount of mycotoxin recovered from the media after treatment using liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight mass spectrometry (LC-ESI-qTOF-MS). The IC50 values detected at all assayed times ranged from 95 to 0.2 µM for the individual treatments. The result indicated that ß-ZEL was the most cytotoxic mycotoxin when tested individually. The major effect detected for all combinations assayed was synergism. Among the combinations assayed, α-ZEL + ß-ZEL + BEA and α-ZEL + BEA presented the highest cytotoxic potential with respect to the IC value. At all assayed times, BEA was the mycotoxin recovered at the highest concentration in individual form, and ß-ZEL + BEA was the combination recovered at the highest concentration.

Toxicokinetics of α-zearalenol and its masked form in rats and the comparative biotransformation in liver microsomes from different livestock and humans.

Alpha-zearalenol (α-ZEL) and its masked form α-zearalenol-14 glucoside (α-ZEL-14G) have much higher oestrogenic activity than zearalenone. Owing to very limited toxicokinetic and metabolic data, no reference points could be established for risk assessment. To circumvent it, the toxicokinetic, metabolic profiles, and phenotyping of α-ZEL and α-ZEL-14G were comprehensively investigated in this study. As a result, the plasma concentrations of α-ZEL and α-ZEL-14G were all below LOQ after oral administration, while after iv injection, both could be significantly bio-transformed into various metabolites. A complete hydrolysis of α-ZEL-14G contributed to α-ZEL overall toxicity. Additionally, 31 phase I and 10 phase II metabolites of α-ZEL, and 9 phase I and 5 phase II metabolites were identified for α-ZEL-14G. For α-ZEL, hydroxylation, dehydrogenation, and glucuronidation were the major metabolic pathways, while for α-ZEL-14G, it was deglycosylation, reduction, hydroxylation, and glucuronidation. Significant metabolic differences were observed for α-ZEL and α-ZEL-14G in the liver microsomes of rats, chickens, swine, goats, cows and humans. Phenotyping studies indicated that α-ZEL and α-ZEL-14G were mediated by CYP 3A4, 2C8, and 1A2. Moreover, the deglycosylation of α-ZEL-14G was critically mediated by CES-I and CES-II. The acquired data would provide fundamental perspectives for risk evaluation of mycotoxins and their modified forms.

Evaluation of the epigenetic alterations and gene expression levels of HepG2 cells exposed to zearalenone and α-zearalenol.

Zearalenone, produced by various Fusarium species, is a non-steroidal estrogenic mycotoxin that contaminates cereals, resulting in adverse effects on human health. We investigated the effects of zearalenone and its metabolite alpha zearalenol on epigenetic modifications and its relationship with metabolic pathways in human hepatocellular carcinoma cells following 24 h of exposure. Zearalenone and alpha zearalenol at the concentrations of 1, 10 and 50 µM significantly increased global levels of DNA methylation and global histone modifications (H3K27me3, H3K9me3, H3K9ac). Expression levels of the chromatin modifying enzymes EHMT2, ESCO1, HAT1, KAT2B, PRMT6 and SETD8 were upregulated by 50 µM of zearalenone exposure using PCR arrays, consistent with the results of global histone modifications. Zearalenone and alpha zearalenol also changed expression levels of the AhR, LXRα, PPARα, PPARÉ£, L-fabp, LDLR, Glut2, Akt1 and HK2 genes, which are related to nuclear receptors and metabolic pathways. PPARÉ£, a key regulator of lipid metabolism, was selected from among these genes for further analysis. The PPARÉ£ promoter reduced methylation significantly following zearalenone exposure. Taken together, the epigenetic mechanisms of DNA methylation and histone modifications may be key mechanisms in zearalenone toxicity. Furthermore, effects of zearalenone in metabolic pathways could be mediated by epigenetic modifications.

Manganese protects wheat from the mycotoxin zearalenone and its derivatives.

Searching for factors that reduce zearalenone (ZEN) toxicity is an important challenge in wheat production, considering that this crop is a basic dietary ingredient. ZEN, absorbed by cells, is metabolized into α-zearalenol and α-zearalanol, and this study focused on the function of manganese ions as potential protectants against the mycotoxins. Stress effects were invoked by an application of 30 µM ZEN and its derivatives. Manganese ions were applied at 100 µM, not stress-inducing concentration. Importance of the biomembrane structures in the absorption of the mycotoxins was demonstrated in in vitro wheat calli and on model membranes. ZEN showed the greatest and α-zearalanol the smallest stressogenic effect manifested as a decrease in the calli growth. This was confirmed by variable increase in antioxidant enzyme activity. Mn ions added to the toxin mixture diminished stressogenic properties of the toxins. Variable decrease in total lipid content and the percentage of phospholipid fraction detected in calli cells exposed to ZEN and its metabolites indicated significance of the membrane structure. An analysis of physicochemical parameters of model membranes build from phosphatidylcholine, a basic lipid in native membranes, and its mixture with the tested toxins made by Langmuir technique and verified by Brewster angle microscopy, confirmed variable contribution of ZEN and its derivatives to the modification of membrane properties. The order of toxicity was as follows: ZEN ≥ α-zearalenol > α-zearalanol. Manganese ions present in the hydrophilic phase interacted with polar lipid groups and reduced the extent of membrane modification caused by the mycotoxins.

Melatonin alleviates ß-zearalenol and HT-2 toxin-induced apoptosis and oxidative stress in bovine ovarian granulosa cells.

ß-zearalenol (ß-zol) and HT-2 are mycotoxins which cause apoptosis and oxidative stress in mammalian reproductive cells. Melatonin is an endogenous antioxidant involved in apoptosis and oxidative stress-related activities. This study investigated the effects of ß-zol and HT-2 on bovine ovarian granulosa cells (BGCs), and how melatonin may counteract these effects. ß-zol and HT-2 inhibited cell proliferation in a dose-dependent manner, and induced apoptosis of BGCs. They also yielded upregulation of the apoptosis-related genes Bax/Bcl-2 and Caspase3 and phosphorylation of p38MAPK. Increases in intracellular ROS were observed along with higher levels of mRNA anti-oxidation markers SOD1, SOD2, and CAT. SOD1, SOD2, malonaldehyde (MDA), and glutathione peroxidase (GSH-px) activities increased, as did the levels of SOD1 and SOD2 proteins. All of these effects were reduced or entirely attenuated in BGCs pre-treated with melatonin. Our results demonstrate that melatonin has protective effects against mycotoxin-induced apoptosis and oxidative stress in BGCs.

Cytotoxic and inflammatory effects of individual and combined exposure of HepG2 cells to zearalenone and its metabolites.

Zearalenone (ZEA), a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain. ZEA derivatives (α-zearalenol (α-ZOL), ß-zearalenol (ß-ZOL)) can also be produced by Fusarium spp. in corn stems infected by fungi in the field. Also, following oral exposure, zearalenone is metabolized in various tissues, particularly in the liver, the major metabolites being α-ZOL and ß-ZOL. The co-exposure of cells to mixture of a combination of mycotoxins may cause an increase of toxicity produced by these mycotoxins. In this in vitro study, we investigated the combined effects of ZEA, α-ZOL, ß-ZOL in binary mixtures on the viability and inflammatory response of human liver cancer cell line (HepG2). Cell viability was assessed after 72 h using a neutral red assay. Effect of the toxins and their binary combinations on the expression of genes involved in inflammation (IL-1ß, TNF-α, and IL-8) were assessed through qPCR. Our viability data showed that irrespective of the toxin combinations, the toxins have synergistic effect. ZEA + α-ZOL and ZEA + ß-ZOL mixtures have induced a slight to high antagonistic response on inflammatory cytokines at low concentrations that have turned into strong synergism for high concentrations. α-ZOL + ß-ZOL showed antagonistic effects on inflammation for IL-1ß and TNF-α, but act synergic for IL-8 at high toxin concentrations. This study clearly shows that co-contamination of food and feed with ZEA metabolites should be taken into consideration, as the co-exposure to mycotoxins might result in stronger adverse effect than resulted from the exposure to individual toxin.

Insights into In Vivo Absolute Oral Bioavailability, Biotransformation, and Toxicokinetics of Zearalenone, α-Zearalenol, ß-Zearalenol, Zearalenone-14-glucoside, and Zearalenone-14-sulfate in Pigs.

The aim of this study was to determine the toxicokinetic characteristics of ZEN and its modified forms, α-zearalenol (α-ZEL), ß-zearalenol (ß-ZEL), zearalenone-14-glucoside (ZEN14G), and zearalenone-14-sulfate (ZEN14S), including presystemic and systemic hydrolysis in pigs. Crossover pig trials were performed by means of intravenous and oral administration of ZEN and its modified forms. Systemic plasma concentrations of the administered toxins and their metabolites were quantified and further processed via tailor-made compartmental toxicokinetic models. Furthermore, portal plasma was analyzed to unravel the site of hydrolysis, and urine samples were analyzed to determine urinary excretion. Results demonstrate complete presystemic hydrolysis of ZEN14G and ZEN14S to ZEN and high oral bioavailability for all administered compounds, with further extensive first-pass glucuronidation. Conclusively, the modified-ZEN forms α-ZEL, ß-ZEL, ZEN14G, and ZEN14S contribute to overall ZEN systemic toxicity in pigs and should be taken into account for risk assessment.

Synergistic estrogenic effects of Fusarium and Alternaria mycotoxins in vitro.

Mycotoxins are toxic secondary metabolites formed by various fungal species that are found as natural contaminants in food. This very heterogeneous group of compounds triggers multiple toxic mechanisms, including endocrine disruptive potential. Current risk assessment of mycotoxins, as for most chemical substances, is based on the effects of single compounds. However, concern on a potential enhancement of risks by interactions of single substances in naturally occurring mixtures has greatly increased recently. In this study, the combinatory effects of three mycoestrogens were investigated in detail. This includes the endocrine disruptors zearalenone (ZEN) and α-zearalenol (α-ZEL) produced by Fusarium fungi and alternariol (AOH), a cytotoxic and estrogenic mycotoxin formed by Alternaria species. For evaluation of effects, estrogen-dependent activation of alkaline phosphatase (AlP) and cell proliferation were tested in the adenocarcinoma cell line Ishikawa. The estrogenic potential varied among the single substances. Half maximum effect concentrations (EC50) for AlP activation were evaluated for α-ZEL, ZEN and AOH as 37 pM, 562 pM and 995 nM, respectively. All three mycotoxins were found to act as partial agonists. The majority of binary combinations, even at very low concentrations in the case of α-ZEL, showed strong synergism in the AlP assay. These potentiating phenomena of mycotoxin mixtures highlight the urgent need to incorporate combinatory effects into future risk assessment, especially when endocrine disruptors are involved. To the best of our knowledge, this study presents the first investigation on synergistic effects of mycoestrogens.

SIRT1 protects cardiac cells against apoptosis induced by zearalenone or its metabolites α- and ß-zearalenol through an autophagy-dependent pathway.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. The major ZEN metabolites are α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL). In the present study, we investigated the underlying mechanism of the toxicity induced by ZEN, α-ZOL and ß-ZOL in cardiac cells (H9c2). We show that treatment with ZEN or its metabolites induces the activation of the mitochondrial pathway of apoptosis as characterized by an increase in ROS generation, a loss of mitochondrial transmembrane potential (ΔΨm) and an activation of caspases. Besides, we demonstrate that these mycotoxins promote the activation of autophagy before the onset of apoptosis. Indeed, we observed that a short-time (6h) treatment with ZEN, α-ZOL or ß-ZOL, increased the level of Beclin-1 and LC3-II and induced the accumulation of the CytoID® autophagy detection probe. Moreover, the inhibition of autophagy by Chloroquine significantly increased cell death induced by ZEN, α-ZOL or ß-ZOL, suggesting that the activation of autophagy serves as a cardioprotective mechanism against these mycotoxins. In addition, we found that the inhibition (EX527) or the knockdown of SIRT1 (siRNA) significantly increased apoptosis induced by ZEN or its derivatives, whereas SIRT1 activation with RSV greatly prevents the cytotoxic effects of these mycotoxins. By contrast, when autophagy was inhibited by CQ, the activation of SIRT1 by RSV had no protection against the cardiotoxicity of ZEN or its metabolites, suggesting that SIRT1 protects cardiac cells by an autophagy-dependent pathway.

Toxicological effects of fumonisin B1 alone and in combination with other fusariotoxins on bovine granulosa cells.

There is now overwhelming evidence of global contamination of commodities with Fusarium mycotoxins. Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in corn in combination with deoxynivalenol (DON), α-zearalenol (α-ZEA) and ß-zearalenol (ß-ZEA). The aim of this study was to determine if FB1, alone and combined with DON or α-ZEA or ß-ZEA, can affect cell proliferation and steroid production of bovine granulosa cells (GC). A species-specific model with bovine granulosa cells (GC) was used to study the potential endocrine disruptor effects of FB1 alone and in co-exposure. In the presence of ß-ZEA (30 ng/mL), FB1 at 30 ng/mL showed a stimulatory effect on GC numbers. Insulin-like growth factor-1 (IGF1)-stimulated cell proliferation was decreased after exposure to ß-ZEA alone at 5.0 µg/mL and FB1 with α-ZEA and ß-ZEA at the same concentration. Regarding steroid production, FB1 at 30 ng/mL and 100 ng/mL amplified the inhibitory effect of ß-ZEA (30 ng/mL) on estradiol (E2) production, while FB1 alone increased (P < 0.05) IGF1-induced E2 production. α-ZEA alone decreased (P < 0.05) E2 production, whereas ß-ZEA alone and in combination with FB1 decreased (P < 0.05) E2 production. These studies indicate for the first time that the Fusarium mycotoxin FB1 along with other mycotoxins can affect GC proliferation and steroid production, which ultimately could influence reproductive function in cattle.

Crocin protects human embryonic kidney cells (HEK293) from α- and ß-Zearalenol-induced ER stress and apoptosis.

α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL) are the major metabolites of Zearalenone (ZEN) and are known to induce many toxic effects. In the present study, we investigated the involvement of endoplasmic reticulum (ER) stress in α- and ß-ZOL-mediated toxicity in human kidney cells (HEK293) and evaluated the effect of a common dietary compound Crocin (CRO), from saffron. We show that α- and ß-ZOL treatment induces ER stress as evidenced by the upregulation of the 78 kDa glucose-regulated protein (GRP78) and the Growth arrest and DNA damage-inducible protein (GADD34). Activation of the ER stress response is associated with activation of the mitochondrial pathway of apoptosis. This apoptotic process is characterized by an increase in ROS generation and lipid peroxidation, a loss of mitochondrial transmembrane potential (ΔΨm) and activation of caspases. We also demonstrate that the antioxidant properties of CRO help to prevent ER stress and reduce α- and ß-ZOL-induced apoptosis in HEK293 cells. Our results suggest that saffron consumption might be helpful to prevent α- and ß-ZOL-induced ER stress and toxicity.

Effects of Dietary Exposure to Zearalenone (ZEN) on Carp (Cyprinus carpio L.).

The mycotoxin zearalenone (ZEN) is frequently contaminating animal feeds including feed used in aquaculture. In the present study, the effects of dietary exposure to ZEN on carp (Cyprinus carpio L.) were investigated. ZEN at three different concentrations (low dose: 332 µg kg(-1), medium dose: 621 µg kg(-1) and high dose: 797 µg kg(-1) final feed, respectively) was administered to juvenile carp for four weeks. Additional groups received the mycotoxin for the same time period but were fed with the uncontaminated diet for two more weeks to examine the reversibility of the ZEN effects. No effects on growth were observed during the feeding trial, but effects on haematological parameters occurred. In addition, an influence on white blood cell counts was noted whereby granulocytes and monocytes were affected in fish treated with the medium and high dose ZEN diet. In muscle samples, marginal ZEN and α-zearalenol (α-ZEL) concentrations were detected. Furthermore, the genotoxic potential of ZEN was confirmed by analysing formation of micronuclei in erythrocytes. In contrast to previous reports on other fish species, estrogenic effects measured as vitellogenin concentrations in serum samples were not increased by dietary exposure to ZEN. This is probably due to the fact that ZEN is rapidly metabolized in carp.

Food contaminant zearalenone and its metabolites affect cytokine synthesis and intestinal epithelial integrity of porcine cells.

The intestinal epithelium is the first barrier against food contaminants. Zearalenone (ZEN) is an estrogenic mycotoxin that was identified as a common contaminant of cereal grains and food and feedstuffs. In the present study, we have investigated the in vitro effects of ZEN and some of its metabolites (α-ZOL, ß-ZOL) in concentrations of 10-100 µM on a swine epithelial cell line: Intestinal porcine epithelial cells (IPEC-1). We demonstrated that both ZEN metabolites were more toxic for IPEC cells as resulted from the XTT test, while for doses lower than 10 µM, only ß-ZOL showed a more pronounced cytotoxicity versus epithelial cells as resulted from neutral red assay. ZEN has no effect on TER values, while α-ZOL significantly decreased the TER values, starting with day 4 of treatment. ß-ZOL had a dual effect, firstly it induced a significant increase of TER, and then, starting on day 6, it induced a dramatic decrease of TER values as compared with on day 0. Concerning the cytokine synthesis, our results showed that ZEN has a tendency to increase the synthesis of IL-8 and IL-10. By contrast, α- and ß-ZOL decreased the expression of both IL-8 and IL-10, in a dose dependent manner. In conclusion, our results showed that ZEN and its metabolites differently affected porcine intestinal cell viability, transepithelial resistance and cytokine synthesis with important implication for gut health.