TAK1 inhibition mitigates intracerebral hemorrhage-induced brain injury through reduction of oxidative stress and neuronal pyroptosis via the NRF2 signaling pathway.

Introduction: Intracerebral hemorrhage (ICH) often triggers oxidative stress through reactive oxygen species (ROS). Transforming growth factor-ß-activated kinase 1 (TAK1) plays a pivotal role in regulating oxidative stress and inflammation across various diseases. 5Z-7-Oxozeaenol (OZ), a specific inhibitor of TAK1, has exhibited therapeutic effects in various conditions. However, the impact of OZ following ICH and its underlying molecular mechanisms remain elusive. This study aimed to explore the possible role of OZ in ICH and its underlying mechanisms by inhibiting oxidative stress-mediated pyroptosis. Methods: Adult male Sprague-Dawley rats were subjected to an ICH model, followed by treatment with OZ. Neurobehavioral function, blood-brain barrier integrity, neuronal pyroptosis, and oxidative stress markers were assessed using various techniques including behavioral tests, immunofluorescence staining, western blotting, transmission electron microscopy, and biochemical assays. Results: Our study revealed that OZ administration significantly inhibited phosphorylated TAK1 expression post-ICH. Furthermore, TAK1 blockade by OZ attenuated blood-brain barrier (BBB) disruption, neuroinflammation, and oxidative damage while enhancing neurobehavioral function. Mechanistically, OZ administration markedly reduced ROS production and oxidative stress by facilitating nuclear factor-erythroid 2-related factor 2 (NRF2) nuclear translocation. This was accompanied by a subsequent suppression of the NOD-like receptor protein 3 (NLRP3) activation-mediated inflammatory cascade and neuronal pyroptosis. Discussion: Our findings highlight that OZ alleviates brain injury and oxidative stress-mediated pyroptosis via the NRF2 pathway. Inhibition of TAK1 emerges as a promising approach for managing ICH.

Disruption of TIGAR-TAK1 alleviates immunopathology in a murine model of sepsis.

Macrophage-orchestrated inflammation contributes to multiple diseases including sepsis. However, the underlying mechanisms remain to be defined clearly. Here, we show that macrophage TP53-induced glycolysis and apoptosis regulator (TIGAR) is up-regulated in murine sepsis models. When myeloid Tigar is ablated, sepsis induced by either lipopolysaccharide treatment or cecal ligation puncture in male mice is attenuated via inflammation inhibition. Mechanistic characterizations indicate that TIGAR directly binds to transforming growth factor ß-activated kinase (TAK1) and promotes tumor necrosis factor receptor-associated factor 6-mediated ubiquitination and auto-phosphorylation of TAK1, in which residues 152-161 of TIGAR constitute crucial motif independent of its phosphatase activity. Interference with the binding of TIGAR to TAK1 by 5Z-7-oxozeaenol exhibits therapeutic effects in male murine model of sepsis. These findings demonstrate a non-canonical function of macrophage TIGAR in promoting inflammation, and confer a potential therapeutic target for sepsis by disruption of TIGAR-TAK1 interaction.

Oxomollugin, an oxidized substance in mollugin, inhibited LPS-induced NF-κB activation via the suppressive effects on essential activation factors of TLR4 signaling.

Oxomollugin is a degraded product of mollugin and was found to be an active compound that inhibits LPS-induced NF-κB activation. In this study, we investigated the inhibitory activity of oxomollugin, focusing on TLR4 signaling pathway, resulting in NF-κB activation. Oxomollugin inhibited the LPS-induced association of essential factors for initial activation of TLR4 signaling, MyD88, IRAK4 and TRAF6. Furthermore, oxomollugin showed suppressive effects on LPS-induced modification of IRAK1, IRAK2 and TRAF6, LPS-induced association of TRAF6-TAK1/TAB2, and followed by IKKα/ß phosphorylation, which critical in signal transduction leading to LPS-induced NF-κB activation. The consistent results suggested that oxomollugin inhibits LPS-induced NF-κB activation via the suppression against signal transduction in TLR4 signaling pathway.The activities of oxomollugin reported in this study provides a deeper understanding on biological activity of mollugin derivatives as anti-inflammatory compounds.

5Z-7-Oxozaenol attenuates cuprizone-induced demyelination in mice through microglia polarization regulation.

INTRODUCTION: Demyelination is a key factor in axonal degeneration and neural loss, leading to disability in multiple sclerosis (MS) patients. Transforming growth factor beta activated kinase 1 (TAK1) is a critical molecule involved in immune and inflammatory signaling pathways. Knockout of microglia TAK1 can inhibit autoimmune inflammation of the brain and spinal cord and improve the outcome of MS. However, it is unclear whether inhibiting TAK1 can alleviate demyelination. METHODS: Eight-week-old male c57bl/6j mice were randomly divided into five groups: (a) the control group, (b) the group treated with cuprizone (CPZ) only, (c) the group treated with 5Z-7-Oxozaenol (OZ) only, and (d) the group treated with both cuprizone and 15 µg/30 µg OZ. Demyelination in the mice of this study was induced by administration of CPZ (ig) at a daily dose of 400 mg/kg for consecutive 5 weeks. OZ was intraperitoneally administered at mentioned doses twice a week, starting from week 3 after beginning cuprizone treatment. Histology, rotarod test, grasping test, pole test, Western blot, RT-PCR, and ELISA were used to evaluate corpus callosum demyelination, behavioral impairment, oligodendrocyte differentiation, TAK1 signaling pathway expression, microglia, and related cytokines. RESULTS: Our results demonstrated that OZ protected against myelin loss and behavior impairment caused by CPZ. Additionally, OZ rescued the loss of oligodendrocytes in CPZ-induced mice. OZ inhibited the activation of JNK, p65, and p38 pathways, transformed M1 polarized microglia into M2 phenotype, and increased brain-derived neurotrophic factor (BDNF) expression to attenuate demyelination in CPZ-treated mice. Furthermore, OZ reduced the expression of proinflammatory cytokines and increases anti-inflammatory cytokines in CPZ-treated mice. CONCLUSION: These findings suggest that inhibiting TAK1 may be an effective approach for treating demyelinating diseases.

bFGF stimulated plasminogen activation factors, but inhibited alkaline phosphatase and SPARC in stem cells from apical Papilla: Involvement of MEK/ERK, TAK1 and p38 signaling.

INTRODUCTION: Basic fibroblast growth factor (bFGF) plays a critical role in odontoblast differentiation and dentin matrix deposition, thereby aiding pulpo-dentin repair and regeneration. OBJECTIVES: The purpose of this study was to clarify the effects of bFGF on plasminogen activation factors, TIMP-1), ALP; and SPARC (osteonectin) expression/production of stem cells from apical papilla (SCAP) in vitro; and the involvement of MEK/ERK, p38, Akt, and TAK1 signaling. METHODS: SCAP were exposed to bFGF with/without pretreatment and co-incubation with various signal transduction inhibitors (U0126, SB203580, LY294002, and 5Z-7-oxozeaenol). The expression of FGF receptors (FGFRs), PAI-1, uPA, p-ERK, p-TAK1, and p-p38 was analyzed via immunofluorescent staining. The gene expression and protein secretion of SCAP were determined via real-time PCR and ELISA. ALP activity was evaluated via ALP staining. RESULTS: SCAP expressed FGFR1, 2, 3, and 4. bFGF stimulated the PAI-1, uPA, uPAR, and TIMP-1 mRNA expression (p < 0.05). bFGF induced PAI-1, uPA, and soluble uPAR production (p < 0.05) but suppressed the ALP activity and SPARC production (p < 0.05) of SCAP. bFGF stimulated ERK, TAK1, and p38 phosphorylation of SCAP. U0126 (a MEK/ERK inhibitor) and 5Z-7-oxozeaenol (a TAK1 inhibitor) attenuated the bFGF-induced PAI-1, uPA, uPAR, and TIMP-1 expression and production of SCAP, but SB203580 (a p38 inhibitor) did not. LY294002, SB203580, and 5Z-7oxozeaenol could not reverse the inhibition of ALP activity caused by bFGF. Interestingly, U0126 and 5Z-7-oxozeaenol prevented the bFGF-induced decline of SPARC production (p < 0.05). CONCLUSION: bFGF may regulate fibrinolysis and matrix turnover via modulation of PAI-1, uPA, uPAR, and TIMP-1, but bFGF inhibited the differentiation (ALP, SPARC) of SCAP. These events are mainly regulated by MEK/ERK, p38, and TAK1. Combined use of bFGF and SCAP may facilitate pulpal/root repair and regeneration via regulation of the plasminogen activation system, migration, matrix turnover, and differentiation of SCAP.

Zearalenone alters the excitability of rat neuronal networks after acute in vitro exposure.

Zearalenone (ZEA) is a mycotoxin produced by Fusarium species, detectable in various cereals and processed food products worldwide. ZEA displays a significant estrogenic activity, thus its main health risk is the interference with sexual maturation and reproduction processes. However, in addition to being key hormonal regulators of reproductive function, estrogenic compounds have a widespread role in brain, as neurotrophic and neuroprotective factors, and they may influence the activity of several brain areas not directly linked to reproduction, as well. Therefore, in the present study, acute effects of ZEA were studied on certain neuronal functions in rats. Experiments were performed on rat brain slices or live rats. Slices were incubated in ZEA-containing (10-100 µM) solution for 30 min. Electrically evoked and spontaneous field potentials were studied in the neocortex and in the hippocampus. At higher concentrations, ZEA incubation of the slices altered excitability and the pattern of epileptiform activity in neocortex and inhibited the development of LTP in hippocampus. For the verification of these in vitro results, in vivo electrophysiological and immunohistochemical investigations were also performed. ZEA was administered systemically (5 mg/kg, i.p.) to male rats and somatosensory evoked potentials and neuronal activation studied by c-fos expression were analyzed. No neuronal activation could be demonstrated in the hippocampus within 2 h of the injection. In the somatosensory cortex, ZEA did not change in vivo evoked potential parameters, but the activation of a small neuronal population could be demonstrated with the c-fos technique in this brain area. This result could be associated with the ZEA-induced alteration of epileptiform activity observed in vitro. Altogether, the toxin altered the excitability and plasticity of neuronal networks after direct treatment in slices, but the effects were less prominent on the given brain areas after systemic treatment in vivo. A probable explanation for the partial lack of in vivo effects may be that after a single injection, ZEA did not cross the blood-brain barrier at sufficient rate to allow the build-up of comparable concentrations in the investigated brain areas. However, in case of compromised blood-brain barrier functions or long-term repeated exposure, alterations in cortical and hippocampal functions cannot be ruled out.

Concentration of Zearalenone, Alpha-Zearalenol and Beta-Zearalenol in the Myocardium and the Results of Isometric Analyses of the Coronary Artery in Prepubertal Gilts.

The carry-over of zearalenone (ZEN) to the myocardium and its effects on coronary vascular reactivity in vivo have not been addressed in the literature to date. Therefore, the objective of this study was to verify the hypothesis that low ZEN doses (MABEL, NOAEL and LOAEL) administered per os to prepubertal gilts for 21 days affect the accumulation of ZEN, α-ZEL and ß-ZEL in the myocardium and the reactivity of the porcine coronary arteries to vasoconstrictors: acetylcholine, potassium chloride and vasodilator sodium nitroprusside. The contractile response to acetylcholine in the presence of a cyclooxygenase (COX) inhibitor, indomethacin and / or an endothelial nitric oxide synthase (e-NOS) inhibitor, L-NAME was also studied. The results of this study indicate that the carry-over of ZEN and its metabolites to the myocardium is a highly individualized process that occurs even at very low mycotoxin concentrations. The concentrations of the accumulated ZEN metabolites are inversely proportional to each other due to biotransformation processes. The levels of vasoconstrictors, acetylcholine and potassium chloride, were examined in the left anterior descending branch of the porcine coronary artery after oral administration of ZEN. The LOAEL dose clearly decreased vasoconstriction in response to both potassium chloride and acetylcholine (P < 0.05 for all values) and increased vasodilation in the presence of sodium nitroprusside (P = 0.021). The NOAEL dose significantly increased vasoconstriction caused by acetylcholine (P < 0.04), whereas the MABEL dose did not cause significant changes in the vascular response. Unlike higher doses of ZEN, 5 µg/kg had no negative influence on the vascular system.

Low dose of zearalenone elevated colon cancer cell growth through G protein-coupled estrogenic receptor.

Colon cancer is one of the leading causes of cancer death worldwide. It is widely believed that environmental factors contribute to colon cancer development. Zearalenone (ZEA) is non-steroidal estrogenic mycotoxin that is widely found in the human diet and animal feeds. Most cancer studies of ZEA focused on estrogen sensitive cancers, while few focused on other types, such as colon cancer; despite the gastrointestinal tract being the first barrier exposed to food contaminants. This study investigated the stimulatory effects of ZEA on colon cancer cell lines and their underlying molecular mechanisms. ZEA promoted anchorage independent cell growth and cell cycle progression through promoting G1-to-S phase transition. Proliferative marker, cyclin D1 and Ki67 were found to be upregulated upon ZEA treatment. G protein-coupled estrogenic receptor 1 (GPER) protein expression was promoted upon ZEA treatment suggesting the involvement of GPER. The growth promoting effect mediated through GPER were suppressed by its antagonist G15. ZEA were found to promote the downstream parallel pathway, MAPK signaling pathway and Hippo pathway effector YAP1. Altogether, our observations suggest a novel mechanism by which ZEA could promote cancer growth and provide a new perspective on the carcinogenicity of ZEA.

The Effect of Different Doses of Zearalenone in Feed on the Bioavailability of Zearalenone and Alpha-Zearalenol, and the Concentrations of Estradiol and Testosterone in the Peripheral Blood of Pre-Pubertal Gilts.

The objective of this study was to determine the effect of long-term (48 days), per os administration of specific zearalenone (ZEN) doses (20 and 40 µg ZEN/kg BW in experimental groups EI and EII, which were equivalent to 200% and 400% of the upper range limit of the no-observed-adverse-effect-level (NOAEL), respectively) on the bioavailability of ZEN and the rate of changes in estradiol and testosterone concentrations in the peripheral blood of pre-pubertal gilts. ZEN and α-ZEL levels were similar until day 28. After day 28, α-ZEL concentrations increased significantly in group EI, whereas a significant rise in ZEN levels was noted in group EII. The presence of estradiol in peripheral blood plasma was not observed until day 20 of the experiment. Spontaneous secretion of estradiol was minimal, and it was determined at very low levels of up to 10 pg/mL in EI and EII groups. Testosterone concentrations ranged from 4 to 9 ng/mL in all groups. A decrease in the concentrations of both analyzed hormones was reported in the last stage of the experiment. The results of the experiment indicate that: (i) The bioavailability of ZEN in peripheral blood has low diagnostic value, (ii) exposure to low doses of ZEN induces minor changes in the concentrations of the analyzed hormones, which could lead to situational supraphysiological hormone levels and changes in endogenous hormonal balance.

Effects of zearalenone-induced oxidative stress and Keap1-Nrf2 signaling pathway-related gene expression in the ileum and mesenteric lymph nodes of post-weaning gilts.

Zearalenone (ZEA) contamination of feed affects animal husbandry and the human health. Currently, the molecular mechanism underlying small intestine-related diseases caused by ZEA-induced oxidative stress is not well understood. In this study, we aimed to identify the mechanisms involved in ZEA (0.5-1.5 mg/kg)-induced oxidative stress in the ileum and mesenteric lymph nodes (MLNs) and the role of the Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in post-weaning gilts. Forty post-weaning gilts (Landrace × Yorkshire × Duroc) with an average body weight of 14.01 ± 0.86 kg were randomly allocated to four groups and fed a corn-soybean meal basal diet supplemented with < 0.1, 0.5, 1.0, or 1.5 mg/kg ZEA. The results showed that the activity of total superoxide dismutase and glutathione peroxidase decreased (p < 0.05) linearly and quadratically and that the content of malondialdehyde increased (p < 0.05) quadratically in the ileum and MLNs with increasing ZEA in the diet. Immunohistochemical analysis showed that the expression of Nrf2 and glutathione peroxidase 1 (Gpx1) immunoreactive proteins in the ileum and MLNs were significantly enhanced with increasing ZEA. The relative mRNA and protein expression of Nrf2, Gpx1, quinone oxidoreductase 1 (Nqo1), hemeoxygenase 1 (Ho1), modifier subunit of glutamate-cysteine ligase (Gclm), and catalytic subunit of glutamate-cysteine ligase (Gclc) increased (p < 0.05) linearly and quadratically, and the relative mRNA and protein expression of Keap1 decreased (p < 0.05) linearly and quadratically in the ileum with increasing ZEA concentrations in the diet. Further, the relative mRNA and protein expression of Nrf2 and Gpx1 increased (p < 0.05) linearly and quadratically, and the relative mRNA and protein expression of Nqo1, Ho1, and Gclm decreased (p < 0.05) quadratically in the MLNs as ZEA concentrations increased in the diet. Our results provide valuable genetic information on ZEA-induced oxidative stress in the ileum and MLNs of post-weaning gilts and have elucidated the key regulatory genes involved in the Keap1-Nrf2 signaling pathway. Results indicated that the Keap1-Nrf2 signaling pathway might be a key target to further prevent and treat ZEA-induced injury to the ileum in post-weaning gilts.

Metabolic Profile, Bioavailability and Toxicokinetics of Zearalenone-14-Glucoside in Rats after Oral and Intravenous Administration by Liquid Chromatography High-Resolution Mass Spectrometry and Tandem Mass Spectrometry.

Zearalenone-14-glucoside (ZEN-14G), a key modified mycotoxin, has attracted a great deal of attention due to the possible conversion to its free form of zearalenone (ZEN) exerting toxicity. In this study, the toxicokinetics of ZEN-14G were investigated in rats after oral and intravenous administration. The plasma concentrations of ZEN-14G and its major five metabolites were quantified using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The data were analyzed via non-compartmental analysis using software WinNonlin 6.3. The results indicated that ZEN-14G was rapidly hydrolyzed into ZEN in vivo. In addition, the major parameters of ZEN-14G following intravenous administration were: area under the plasma concentration-time curve (AUC), 1.80 h·ng/mL; the apparent volume of distribution (VZ), 7.25 L/kg; and total body clearance (CL), 5.02 mL/h/kg, respectively. After oral administration, the typical parameters were: AUC, 0.16 h·ng/mL; VZ, 6.24 mL/kg; and CL, 4.50 mL/h/kg, respectively. The absolute oral bioavailability of ZEN-14G in rats was about 9%, since low levels of ZEN-14G were detected in plasma, which might be attributed to its extensive metabolism. Therefore, liquid chromatography high-resolution mass spectrometry (LC-HRMS) was adopted to clarify the metabolic profile of ZEN-14G in rats' plasma. As a result, eight metabolites were identified in which ZEN-14-glucuronic acid (ZEN-14GlcA) had a large yield from the first time-point and continued accumulating after oral administration, indicating that ZEN-14-glucuronic acid could serve a potential biomarker of ZEN-14G. The obtained outcomes would prompt the accurate safety evaluation of ZEN-14G.

Fumonisin B1, B2 and B3 in Muscle and Liver of Broiler Chickens and Turkey Poults Fed with Diets Containing Fusariotoxins at the EU Maximum Tolerable Level.

Although provisional maximum tolerable daily intake and recommended guidelines have been established for fumonisins (FB) in food, few data are available concerning levels of FB in edible animal tissues. Such data are of particular interest in avian species that can tolerate relatively high levels of fumonisins in their feed. Also, even if multiple contamination of animal feed by toxins produced by Fusarium is very frequent, little is known about the consequences of multiple contamination for FB levels in tissues. The aim of this study was to analyze the concentrations of FB in the muscle and liver of chickens and turkeys fed with FB alone and with FB combined with deoxynivalenol (DON), and with zearalenone (ZEN). Experimental diets were formulated by incorporating ground cultured toxigenic Fusarium strains in corn-soybean based feeds. Control diets were free of mycotoxins, FB diets contained 20 mg FB1+FB2/kg, and FBDONZEN diets contained 20, 5, and 0.5 mg/kg of FB1+FB2, DON, and ZEN, respectively. Animals were reared in individual cages with free access to water and feed. The feed was distributed to male Ross chickens from the 1st to the 35th day of age and to male Grade Maker turkeys from the 55th to the 70th day of age. On the last day of the study, the birds were starved for eight hours, killed, and autopsied for tissues sampling. No sign of toxicity was observed. A UHPLC-MS/MS method with isotopic dilution and immunoaffinity clean-up of samples has been developed for analysis of FB in muscle (n = 8 per diet) and liver (n = 8 per diet). Only traces of FB that were below the LOQ of 0.25 µg/kg were found in most of the samples of animals fed with the control diets. Mean concentrations of FB1, FB2, and FB3 in muscle were 17.5, 3.39, and 1.26 µg/kg, respectively, in chickens, and 5.77, 1.52, and 0.54 µg/kg in turkeys, respectively. In the liver, the respective FB1, FB2, and FB3 concentrations were 44.7, 2.61, and 0.79 µg/kg in chickens, and 41.47, 4.23, and 1.41 µg/kg, in turkeys. Cumulated level of FB1+FB2+FB3 in the highly contaminated samples were above 60 and 100 µg/kg in muscle and liver, respectively. The concentrations of FB in the tissues of animals fed the FBDONZEN diet did not greatly differ from the concentrations measured in animals fed the diet containing only FB.

The effect of subchronic oral exposure to zearalenone on hematologic and biochemical analytes, and the blood redox status of adult rabbit bucks.

BACKGROUND: Zearalenone (ZEN) is a mycoestrogen with a ubiquitous presence in animal feeds, which also has hematotoxic, hepatotoxic, nephrotoxic, and immunotoxic properties. However, there is a paucity of literature that discusses the effects of ZEN on rabbits. OBJECTIVES: The aim of this study was to evaluate the effect of a prolonged, low-level (50 µg ZEN/kg body weight) exposure on the clinicopathologic and redox status analytes of rabbit bucks. METHODS: Ten adult bucks were included in the study. Each underwent a 7-week control period, followed by a 7-week exposure period. Water or ZEN solutions were daily administered orally (0.5 mL) during the control and exposure periods, respectively. Blood samples were collected weekly for Complete Blood Counts, serum biochemical analyte and reactive oxygen metabolite (ROM) measurements. Data were analyzed using a mixed model, and the level of significance was set at a P of <0.05. RESULTS: During the ZEN exposure period, significant increases were noted in the red blood cell distribution width (RDW) and mean platelet volumes (MPVs), as well as in the white blood cell, monocyte, and eosinophil counts. Significant increases were observed in aspartate aminotransferase and total bilirubin, whereas urea, creatinine, glucose, total calcium, sodium, and potassium concentrations were significantly decreased. The ROM concentrations did not differ significantly between the control and ZEN exposure periods. CONCLUSIONS: Under the present experimental conditions, ZEN affected some of the clinicopathologic analytes of adult rabbit bucks; these changes were mostly indicative of mild hepatocellular damage and dysfunction, inflammatory and/or allergic responses, and renal tubular damage. A ZEN dose of 50 µg/kg body weight did not seem to affect the blood redox status of bucks, as evaluated by the ROM concentrations.

Feeding study for the mycotoxin zearalenone in yellow mealworm (Tenebrio molitor) larvae-investigation of biological impact and metabolic conversion.

Edible insects as additional food and/or feed source may represent one important component to solve the problem of food security for a growing human population. Especially for covering the rising demand for protein of animal origin, seven insect species currently allowed as feed constituents in the European Union are gaining more interest. However, before considering insects such as yellow mealworm larvae (Tenebrio molitor) as suitable for, e.g. human consumption, the possible presence and accumulation of contaminants must be elucidated. The present work investigates the effects of the mycotoxin zearalenone (ZEN) and its metabolites on insect larvae. Seven different diets were prepared: toxin-free control, spiked and artificially contaminated (both containing approx.500 µg/kg and approx. 2000 µg/kg of ZEN) as well as two naturally contaminated diets (600 µg/kg and 900 µg/kg ZEN). The diets were used in a multiple-week feeding trial using T. molitor larvae as model insects. The amount of ZEN and its metabolites in the feed, larvae and the residue were measured by HPLC-MS/MS. A significantly enhanced individual larval weight was found for the insects fed on the naturally contaminated diets compared to the other feeding groups after 8 weeks of exposure. No ZEN or ZEN metabolites were detected in the T. molitor larvae after harvest. However, ZEN, α- and ß-stereoisomers of zearalenol were found in the residue samples indicating an intense metabolism of ZEN in the larvae. No further ZEN metabolites could be detected in any sample. Thus, ZEN is not retained to any significant amount in T. molitor larvae.

Feed contamination with zearalenone promotes growth but affects the immune system of rainbow trout.

To investigate the effects of feed contamination with zearalenone (ZEN) at the current European Commission (EC) guidance value (2 mg⋅kg-1 feed) on the growth and health of rainbow trout, we performed a long-term feeding trial under aquaculture conditions. It started with the external feeding of the fish larvae, and continued for 96 weeks, at which point the fish had reached market size. To assess the growth of fish and their feeding efficiency throughout this period, the fish were regularly weighed and measured, and their feed consumption was monitored. Additionally, to investigate potential health effects, after 72 weeks of the exposure to ZEN, the fishes' blood was analyzed for major hematological and biochemical indices, and their head kidney, spleen, and liver were examined for morphological, histopathological, cytological, and molecular changes. Finally, to gain insight into the metabolism and distribution of ZEN in fish, the content of free and glucuronidated forms of ZEN and its major metabolites was measured in the intestine, liver, and muscles of the exposed fish. The feed-borne exposure of rainbow trout to ZEN at a dose of 2 mg⋅kg-1 feed resulted in higher feeding efficiency and growth rate, most probably due to the anabolic properties of the ZEN metabolite. Importantly for the consumers of fish, despite absorption and metabolism of ZEN in the digestive system of the fish that had been exposed for 72 weeks, the residuals of ZEN were not transferred to the fishes' muscles, which rules out a potential risk to human health related to the consumption of fish meat. However, the increased growth of fish fed with the contaminated feed may come at some cost, as the exposure to ZEN was associated with modulation of key components of the adaptive and innate immune systems. Moreover, the trunk kidney of ZEN-fed fish showed massive inflammation that was likely caused by pathogen infection. These findings raise concerns about fish health under the current recommended EC guidance values.

Toxicogenomic responses to zearalenone in Caenorhabditis elegans reveal possible molecular mechanisms of reproductive toxicity.

In this study, the possible molecular mechanisms of zearalenone (ZEA)-induced reproductive and developmental toxic effects in Caenorhabditis elegans (C. elegans) were investigated. Differential gene expression profiles were identified, and 171, 245, and 3149 genes were down- or up-regulated (>2.0 fold) in 10, 20, and 40 µg/ml ZEA treated groups, respectively, as compared to untreated controls. Pathway specific mapping showed that the major differentially expressed genes were collagen synthetic pathways regulating genes, col-121 and dpy-17. Real-time PCR reconfirmation of key genes, related to cuticle collagen synthetic pathway, found dramatic changes in the expression of the genes dpy-31, sqt-3, col-121, and dpy-17 following exposure to ZEA (40 µg/ml), which indicated the significance of these genes in ZEA-induced toxicity. Cuticle collagen plays many key roles in the development and reproduction of C. elegans. The hypersensitive responses in transgenic and mutant worms also confirmed the roles of these genes in lethality and reproductive response to ZEA exposure, which indicates that ZEA blocked the normal collagen processing and cuticle formation. Taken together, our results demonstrate that disruption of the collagen biosynthetic pathway might be a key mechanism in ZEA-induced reproductive and developmental toxic effects in C. elegans.

First study on trichothecene and zearalenone exposure of the Romanian population through wheat-based products consumption.

In this study, a dietary exposure assessment of mycotoxins was conducted for the Romanian population using the contamination data of a various categories of wheat-based products for direct human consumption. Wheat-based foods (n = 181) commercialized in Romania, including flour, bread, biscuits, breakfast cereals and pasta, were evaluated by GC-QqQ-MS/MS for the occurrence of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3AcDON), 15-acetyldeoxynivalenol (15AcDON), fusarenon-X, nivalenol, HT-2 and T-2 toxins, diacetoxyscirpenol, neosolaniol and zearalenone (ZEA). DON and 15AcDON were detected in 63 and 5% of all the analyzed samples, whereas 13-AcDON, HT-2, T-2, NIV and ZEA were not detected. Exposure of Romanian adult population was assessed, the EDIs for the sum of DON+3AcDON+15AcDON were 669 ng kg-1 bw day-1 at low-bound estimation, and 690 ng kg-1 bw day-1 at upper-bound estimation, being lower than the TDI set (1000 ng kg-1 bw day-1).

Effect of subchronic oral exposure to zearalenone on the reproductive system of rabbit bucks.

OBJECTIVE To determine the effect of subchronic oral exposure to zearalenone (ZEA) at a daily dose of 50 µg of ZEA/kg of body weight (an environmentally relevant concentration) on the reproductive system of rabbit bucks. ANIMALS 8 healthy sexually mature New Zealand White rabbits. PROCEDURES During the experimental period (March to June), each rabbit underwent a 7-week control protocol and then a 7-week treatment protocol. Water (0.5 mL) or ZEA solution (50 µg/kg [0.5 mL]) was administered orally once daily during the control and treatment period, respectively; ejaculates were collected weekly. Studied end points included semen quality variables (spermatozoa kinetics, morphology, viability, and DNA fragmentation), serum testosterone concentration, and results of histologic examination of the testes and epididymides following euthanasia at the end of the experimental period. RESULTS Treatment with ZEA solution resulted in significant increases in spermatozoa beat-cross frequency, in the percentages of spermatozoa with head and midpiece abnormalities, and in the percentages of DNA-fragmented spermatozoa, compared with effects of the control treatment. Serum testosterone concentration, other spermatozoa velocity variables, and percentages of progressive and total motility, rapidly or slowly moving spermatozoa, and live spermatozoa did not differ significantly between the 2 periods. Histologic examination revealed no patterns of abnormal findings in the testes and epididymides. CONCLUSIONS AND CLINICAL RELEVANCE Oral treatment with ZEA solution at an enviromentally relevant concentration caused minor interference with rabbit bucks' sperm quality. Although mostly considered mild, the sperm quality changes warrant further investigation in terms of fertilizing capacity impairment.

Growth performance, selected blood biochemical parameters and body weights of pre-pubertal gilts fed diets supplemented with different doses of zearalenone (ZEN).

The aim of this study was to determine whether exposure to low doses of zearalenone (ZEN) induces changes in the serum biochemical profile and body weights (BW). Pre-pubertal gilts (with BW of up to 14.5 kg) were administered ZEN in daily doses of 5 µg/kg BW (group 1, n = 15), 10 µg/kg BW (group 2, n = 15), 15 µg/kg BW (group 3, n = 15) or placebo (control group C, n = 15) throughout the experiment. Blood was sampled for analysis on 10 dates (at five-day intervals). Minor but statistically significant differences in the analysed serum biochemical parameters (ALT, AST, ALP, total cholesterol, total bilirubin, glucose, total protein, iron, BUN and urea) were observed in the studied groups. The biochemical parameters of the analysed gilts indicate that the maintenance of homeostasis and biotransformation of ZEN require considerable energy expenditure. Beginning on the fourth analytical date, BW gains were consistently higher in the experimental groups than in group C. The observed decrease in glucose and total protein levels can probably be attributed to higher BW gains and the ongoing ZEN biotransformation processes in the enterocytes and the liver.

Molecular and biochemical evidence on the role of zearalenone in rat polycystic ovary.

Globally, food and animal feed contamination with mycotoxins is one of the most important challenges affecting human health. Zearalenone is a non-steroidal mycotoxin with estrogen-like activity that has been reported to induce reproductive dysfunctions including polycystic ovary in women. The aim of this study was to assess the possible impact of prolonged low dose zearalenone (0.1 mg/kg b.w.) exposure to increase the risk of developing polycystic ovary in rats. We found that zearalenone increases the plasma insulin, glucose, testosterone, progesterone and luteinizing hormone levels, while the plasma estradiol level was reduced. Zearalenone also incited tumor necrosis factor-α and the secreted frizzled-related protein-4 expressions. Histological examination showed atresia of follicles in the treated group. It is concluded that zearalenone intoxication intensely manipulates the plasma hormonal factors and the level of gene expressions related to the polycystic ovary in rats, thus increases the risk of its progression.