Biotransformation of zearalenone to non-estrogenic compounds with two novel recombinant lactonases from Gliocladium.

BACKGROUND: The mycotoxin zearalenone (ZEA) produced by toxigenic fungi is widely present in cereals and its downstream products. The danger of ZEA linked to various human health issues has attracted increasing attention. Thus, powerful ZEA-degrading or detoxifying strategies are urgently needed. Biology-based detoxification methods are specific, efficient, and environmentally friendly and do not lead to negative effects during cereal decontamination. Among these, ZEA detoxification using degrading enzymes was documented to be a promising strategy in broad research. Here, two efficient ZEA-degrading lactonases from the genus Gliocladium, ZHDR52 and ZHDP83, were identified for the first time. This work studied the degradation capacity and properties of ZEA using purified recombinant ZHDR52 and ZHDP83. RESULTS: According to the ZEA degradation study, transformed Escherichia coli BL21(DE3) PLySs cells harboring the zhdr52 or zhdp83 gene could transform 20 µg/mL ZEA within 2 h and degrade > 90% of ZEA toxic derivatives, α/ß-zearalanol and α/ß-zearalenol, within 6 h. Biochemical analysis demonstrated that the optimal pH was 9.0 for ZHDR52 and ZHDP83, and the optimum temperature was 45 °C. The purified recombinant ZHDR52 and ZHDP83 retained > 90% activity over a wide range of pH values and temperatures (pH 7.0-10.0 and 35-50 °C). In addition, the specific activities of purified ZHDR52 and ZHDP83 against ZEA were 196.11 and 229.64 U/mg, respectively. The results of these two novel lactonases suggested that, compared with ZHD101, these two novel lactonases transformed ZEA into different products. The slight position variations in E126 and H242 in ZDHR52/ZEA and ZHDP83/ZEA obtained via structural modelling may explain the difference in degradation products. Moreover, the MCF-7 cell proliferation assay indicated that the products of ZEA degradation using ZHDR52 and ZHDP83 did not exhibit estrogenic activity. CONCLUSIONS: ZHDR52 and ZHDP83 are alkali ZEA-degrading enzymes that can efficiently and irreversibly degrade ZEA into non-estrogenic products, indicating that they are potential candidates for commercial application. This study identified two excellent lactonases for industrial ZEA detoxification.

Enhancing the activity of zearalenone lactone hydrolase toward the more toxic α-zearalanol via a single-point mutation.

Zearalenone (ZEN) and its derivatives are estrogenic mycotoxins known to pose significant health threats to humans and animals. Especially, the derivative α-zearalanol (α-ZAL) is over 10 times more toxic than ZEN. Simultaneous degradation of ZEN and its derivatives, especially α-ZAL, using ZEN lactone hydrolases (ZHDs) is a promising solution to eliminate their potential hazards to food safety. However, most available ZHDs exhibit limited activity toward the more toxic α-ZAL compared to ZEN. Here, we identified a broad-substrate spectrum ZHD, named ZHDAY3, from Exophiala aquamarina CBS 119918, which could not only efficiently degrade ZEN but also exhibited 73% relative activity toward α-ZAL. Through rational design, we obtained the ZHDAY3(N153H) mutant, which exhibited the highest specific activity (253.3 ± 4.3 U/mg) reported so far for degrading α-ZAL. Molecular docking, structural comparative analysis, and kinetic analysis collectively suggested that the shorter distance between the side chain of the catalytic residue His242 and the lactone bond of α-ZAL and the increased binding affinity to the substrate were mainly responsible for the improved catalytic activity of ZHDAY3(N153H) mutant. This mechanism was further validated through additional molecular docking of 18 mutants and experimental verification of six mutants.IMPORTANCEThe mycotoxins zearalenone (ZEN) and its derivatives pose a significant threat to food safety. Here, we present a highly promising ZEN lactone hydrolase (ZHD), ZHDAY3, which is capable of efficiently degrading both ZEN and the more toxic derivative α-ZAL. Next, the ZHDAY3(N153H) mutant obtained by single-point mutation exhibited the highest specific activity for degrading α-ZAL reported thus far. We further elucidated the molecular mechanisms underlying the enhanced hydrolytic activity of ZHDAY3(N153H) toward α-ZAL. These findings represent the first investigation on the molecular mechanism of ZHDs against α-ZAL and are expected to provide a significant reference for further rational engineering of ZHDs, which will ultimately contribute to addressing the health risks and food safety issues posed by ZEN-like mycotoxins.

Interaction of mycotoxins zearalenone, α-zearalenol, and ß-zearalenol with cytochrome P450 (CYP1A2, 2C9, 2C19, 2D6, and 3A4) enzymes and organic anion transporting polypeptides (OATP1A2, OATP1B1, OATP1B3, and OATP2B1).

Zearalenone (ZEN) is a mycoestrogen produced by Fusarium fungi. ZEN is a frequent contaminant in cereal-based products, representing significant health threat. The major reduced metabolites of ZEN are α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL). Since the toxicokinetic interactions of ZEN/ZELs with cytochrome P450 enzymes (CYPs) and organic anion transporting polypeptides (OATPs) have been barely characterized, we examined these interactions applying in vitro models. ZEN and ZELs were relatively strong inhibitors of CYP3A4 and moderate inhibitors of CYP1A2 and CYP2C9. Both CYP1A2 and CYP3A4 decreased ZEN and ß-ZEL concentrations in depletion assays, while only CYP1A2 reduced α-ZEL levels. OATPs tested were strongly or moderately inhibited by ZEN and ZELs; however, these mycotoxins did not show higher cytotoxicity in OATP-overexpressing cells. Our results help the deeper understanding of the toxicokinetic/pharmacokinetic interactions of ZEN, α-ZEL, and ß-ZEL.

Toxicokinetics of Zearalenone following Oral Administration in Female Dezhou Donkeys.

Zearalenone (ZEN) is a mycotoxin produced by various Fusarium strains, that is present in food and feed raw materials worldwide, causing toxicity effects in animals and humans. This research aimed to explore the toxicokinetics of ZEN on female Dezhou donkeys following a single oral exposure dosage of 2 mg/kg BW (body weight). The sample collection of donkeys plasma was carried out at 0, 5, 10, 15, 20, 30, 45, 60, 90 min, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 6 h, 9 h, 12 h, 24 h, 48 h, 72 h, 96 h and 120 h via intravenous catheter, and fecal and urinary samples were severally collected at 0 h and every 6 h until 120 h. The concentrations of ZEN, α-zearalenol (α-ZOL), ß-zearalenol (ß-ZOL), α-zearalanol (α-ZAL), ß-zearalanol (ß-ZAL), zearalanone (ZAN) in plasma, urine, and feces were detected by UPLC-MS/MS. Only ZEN was detected in plasma, and the maximum was 15.34 ± 5.12 µg/L occurred at 0.48 h after gavage. The total plasma clearance (Cl) of ZEN was 95.20 ± 8.01 L·kg·BW-1·h-1. In addition, the volume of distribution (Vd) was up to 216.17 ± 58.71 L/kg. The percentage of total ZEN (ZEN plus the main metabolites) excretion in feces and urine was 2.49% and 2.10%, respectively. In summary, ZEN was fast absorbed and relatively slowly excreted in female donkeys during 120 h after a single gavage, indicating a trend of wider tissue distribution and longer tissue persistence.

A mechanistic toxicology study to grasp the mechanics of zearalenone estrogenicity: Spotlighting aromatase and the effects of its genetic variability.

Zearalenone (ZEN) is a mycoestrogen produced by Fusarium fungi contaminating cereals and in grain-based products threatening human and animal health due to its endocrine disrupting effects. Germane to the mechanisms of action, ZEN may activate the estrogen receptors and inhibit the estrogens-producing enzyme aromatase (CYP19A1). Both show single nucleotide variants (SNVs) among humans associated with a diverse susceptibility of being activated or inhibited. These variations might modify the endocrine disrupting action of ZEN, requiring dedicated studies to improve its toxicological understanding. This work focused on human aromatase investigating via 3D molecular modelling whether some of the SNVs reported so far (n = 434) may affect the inhibitory potential of ZEN. It has been also calculated the inhibition capability of α-zearalenol, the most prominent and estrogenically potent phase I metabolite of ZEN, toward those aromatase variants with an expected diverse sensitivity of being inhibited by ZEN. The study: i) described SNVs likely associated with a different susceptibility to ZEN and α-zearalenol inhibition - like T310S that is likely more susceptible to inhibition, or D309G and S478F that are possibly inactive variants; ii) proofed the possible existence of inter-individual susceptibility to ZEN; iii) prioritized aromatase variants for future investigations toward a better comprehension of ZEN xenoestrogenicity at an individual level.

Estrogen receptor α interaction of zearalenone and its phase I metabolite α-zearalenol in combination with soy isoflavones in hERα-HeLa-9903 cells.

Risk assessment primarily relies on toxicological data of individual substances, with limited information on combined effects. Recent in vitro experiments using Ishikawa cells, an endometrial carcinoma cell line expressing both estrogen receptor isoforms, demonstrated interactive effects of phyto- and mycoestrogens. The mycoestrogens, zearalenone (ZEN), and α-zearalenol (α-ZEL) exhibited significantly enhanced estrogenic responses in the presence of isoflavones (ISF), depending on substance ratios and concentrations. This study investigated the impact of phyto- and mycoestrogen combinations on estrogenic response following OECD guideline 455, utilizing hERα-HeLa-9903 cells. Test substances included mycoestrogens (ZEN and α-ZEL) and isoflavones (genistein (GEN), daidzein (DAI), and S-equol (EQ), a gut microbial metabolite of DAI). Mycoestrogens were tested in the range of 0.001 to 100 nM, while isoflavones were used at concentrations 1000 times higher based on relevant occurrence ratios. Results showed that ZEN and α-ZEL induced ERα-dependent luciferase expression in concentrations above 1 nM and 0.01 nM, respectively. However, ISF caused a superinduction of the luciferase signal above 1 µM. A superinduction is characterized by an unusually strong or heightened increase in the activity of the luciferase enzyme. This signal is not affected by the estrogen receptor antagonist 4-hydroxytamoxifen (4-OH-TAM), which was additionally used to verify whether the increase of signal is a true reflection of receptor activation. This superinduction was observed in all combinations of ZEN and α-ZEL with ISFs. Contrary to the luciferase activity findings, RT-qPCR experiments and a stability approach revealed lower real ERα activation by ISFs than measured in the ONE-Glo™ luciferase test system. In conclusion, the OECD protocol 455 appears unsuitable for testing ISFs due to their superinduction of luciferase and interactions with the test system, resulting in experimental artifacts. Further studies are necessary to explore structure-activity relationships within polyphenols and clarify the test system's applicability.

A sensitive monoclonal antibody-based ELISA integrated with immunoaffinity column extraction for the detection of zearalenone in food and feed samples.

Zearalenone (ZEN) is one of the most toxic mycotoxins widely found in agricultural products. In this study, a sensitive enzyme-linked immunosorbent assay (ELISA) integrated with immunoaffinity column extraction for the detection of ZEN in food and feed samples was developed. A ZEN derivative containing a carboxylic group was first synthesized and then linked to bovine serum albumin (BSA). The formed ZEN-BSA conjugate was used as the immunogen for the production of the monoclonal antibody (mAb) against ZEN. The hybridoma clones (1G5) capable of secreting antibodies against ZEN were successfully selected. Based on this mAb, the IC50 and LOD of the ELISA for ZEN were 0.37 ng mL-1 and 0.04 ng mL-1, respectively, which were 1.6-308.1 times lower than those in the published ELISAs, indicating the high sensitivity of our assay. There was no cross-reactivity of the mAb with other four mycotoxins (patulin, AFB1, DON, and OTA). Due to the high similarity in molecular structures among ZEN and its homologs (α-zearalanol, ß-zearalanol, zearalanone, α-zearalenol, ß-zearalenol), the CR values of the mAb with the homologs were within 3.59%-105.71%. Taking advantage of plenty of mAb, the immunoaffinity column was prepared by immobilizing the mAb on Sepharose-4B gel and filling it into an SPE column. ZEN spiked samples (corn, wheat, feed) were extracted using an immunoaffinity column and measured by ELISA and HPLC-FLD simultaneously. The recoveries of the ELISA for ZEN in the spiked samples were 92.46-105.48% with RSDs of 4.87-10.11%. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation y = 1.0589x + 1.43815 (R2 = 0.998, n = 6) was obtained. To verify the applicability, the proposed ELISA was also applied to some real samples randomly collected from a local market. It was proven that the newly produced mAb-based ELISA was a feasible and sensitive method for the detection of ZEN in food and feed samples.

Identification and Modification of Enzymatic Substrate Specificity through Residue Alteration in the Cap Domain: A Thermostable Zearalenone Lactonase.

Zearalenone (ZEN) and its derivatives are prevalent contaminants in cereal crops. This study investigated a novel thermostable ZEN lactonase (ZENM) from Monosporascus sp. GIB2. ZENM demonstrated its highest activity at 60 °C, maintaining over 90% relative activity from 50 to 60 °C. Notably, efficient hydrolysis of ZEN and its two derivatives was achieved using ZENM, with specific activities of 333 U/mg for ZEN, 316 U/mg for α-zearalenol (α-ZOL), and 300 U/mg for α-zearalanol (α-ZAL). The activity of ZENM toward α-ZOL is noteworthy as most ZEN lactonases rarely achieve such a high degradation rate of α-ZOL. Based on the sequence-structure analysis, five residues (L123, G163, E171, S199, and S202) conserved in other ZEN lactonases were substituted in ZENM. Of interest was the G163S mutant in the cap domain that displayed enhanced activity toward α-ZOL compared to the wild-type enzyme. Notably, the mutant G163S exhibited higher catalytic activity toward α-ZOL (kcat/Km 0.223 min-1 µM-1) than ZEN (kcat/Km 0.191 min-1 µM-1), preferring α-ZOL as its optimum substrate. In conclusion, a thermostable ZEN lactonase has been reported, and the alteration of residue G163 in the cap domain has been shown to modify the substrate specificity of ZEN lactonase.

A comparison of four liquid chromatography-mass spectrometry platforms for the analysis of zeranols in urine.

Targeted biomonitoring studies quantifying the concentration of zeranols in biological matrices have focused on liquid chromatography interfaced to mass spectrometry (LC-MS). The MS platform for measurement, quadrupole, time-of-flight (ToF), ion trap, etc., is often chosen based on either sensitivity or selectivity. An instrument performance comparison of the benefits and limitations using matrix-matched standards containing 6 zeranols on 4 MS instruments, 2 low-resolution (linear ion traps), and 2 high-resolution (Orbitrap and ToF) was undertaken to identify the best measurement platform for multiple biomonitoring projects characterizing the endocrine disruptive properties of zeranols. Analytical figures of merit were calculated for each analyte to compare instrument performance across platforms. The calibration curves had correlation coefficients r = 0.989 ± 0.012 for all analytes and LODs and LOQs were ranked for sensitivity: Orbitrap > LTQ > LTQXL > G1 (V mode) > G1 (W mode). The Orbitrap had the smallest measured variation (lowest %CV), while the G1 had the highest. Instrumental selectivity was calculated using full width at half maximum (FWHM) and as expected, the low-resolution instruments had the broadest spectrometric peaks, concealing coeluting peaks under the same mass window as the analyte. Multiple peaks from concomitant ions, unresolved at low resolution (within a unit mass window), were present but did not match the exact mass predicted for the analyte. For example, the high-resolution platforms were able to differentiate between a concomitant peak at 319.1915 from the analyte at 319.1551, included in low-resolution quantitative analyses demonstrating the need to consider coeluting interfering ions in biomonitoring studies. Finally, a validated method using the Orbitrap was applied to human urine samples from a pilot cohort study.

Occurrence of resorcyclic acid lactones in porcine urine: discrimination between illegal use and contamination.

Zeranol (α-zearalanol, α-ZAL), is a resorcyclic acid lactone (RAL). Its administration to farm animals to improve meat production has been prohibited in the European Union due to the potential risk to human health. However, it has been demonstrated that α-ZAL may be present in livestock animals due to Fusarium fungi that produce fusarium acid lactones contamination in feed. The fungi produce a small amount of zearalenone (ZEN), which is metabolized to zeranol. The potential endogenous origin of α-ZAL makes it difficult to correlate positive samples to a potential illicit treatment with α-ZAL. We present two experimental studies that investigated the origin of natural and synthetic RALs in porcine urine. Urine samples from pigs that were either fed with ZEN-contaminated feed or administered α-ZAL by injection were analyzed by liquid chromatography coupled to tandem mass spectrometry, with the method validated according to Commission Implementing Regulation (EU) 2021/808. The data show that although the concentration of α-ZAL in the ZEN feed-contaminated samples is significantly lower than in the illicit administration samples, α-ZAL can occur in porcine urine via natural metabolism. Additionally, the feasibility of using the ratio of forbidden/fusarium RALs in porcine urine as a reliable biomarker for illicit treatment with α-ZAL administration was evaluated for the first time. This study demonstrated that the obtained ratio in the contaminated ZEN feed study was close to 1, while in the illegally administered α-ZAL samples the ratio is always higher than 1 (up to 135). Therefore, this study proves that the ratio criteria (already used when a forbidden RAL is detected in bovine urine) may also be used for porcine urine.

Study of Competitive Displacement of Curcumin on α-zearalenol Binding to Human Serum Albumin Complex Using Fluorescence Spectroscopy.

α-zearalenol (α-ZOL) is a mycotoxin with a strong estrogen effect that affects the synthesis and secretion of sex hormones and is transported to target organs through human serum albumin (HSA). Additionally, it has been reported that curcumin can also bind to HSA with high affinity at the same binding site as α-ZOL. Additionally, several studies reported that reducing the bound fraction of α-ZOL contributes to speeding up the elimination rate of α-ZOL to reduce its hazard to organs. Therefore, to explore the influence of a nutrition intervention with curcumin on α-ZOL effects, the competitive displacement of α-ZOL from HSA by curcumin was investigated using spectroscopic techniques, ultrafiltration techniques and HPLC methods. Results show that curcumin and α-ZOL share the same binding site (subdomain IIA) on HSA, and curcumin binds to HSA with a binding constant of 1.12 × 105 M-1, which is higher than that of α-ZOL (3.98 × 104 M-1). Ultrafiltration studies demonstrated that curcumin could displace α-ZOL from HSA to reduce α-ZOL's binding fraction. Synchronous fluorescence spectroscopy revealed that curcumin could reduce the hydrophobicity of the microenvironment of an HSA-α-ZOL complex. This study is of great significance for applying curcumin and other highly active foodborne components to interfere with the toxicokinetics of α-ZOL and reduce its risk of its exposure.

Hydrophilic Covalent Organic Frameworks Coated Steel Sheet As a Mass Spectrometric Ionization Source for the Direct Determination of Zearalenone and Its Derivatives.

Zearalenone has attracted worldwide attention due to its toxic properties and threat to public health. A rapid determination method for zearalenone and its derivatives by hydrophilic covalent organic frameworks coated steel sheet (HCOFCS) combined with ambient mass spectrometry (AMS) was developed. The HCOFCS behaved as both a tip for solid-phase microextraction and a solid substrate for electrospray ionization mass spectrometry (ESI-MS). To evaluate the HCOFCS-ESI-MS method, five zearalenone and its derivatives in milk samples were determined, including zearalenone (ZEA), α-zearalenol (α-ZEL), ß-zearalenol (ß-ZEL), α-zearalanol (α-ZAL), and ß-zearalanol (ß-ZAL). After the extraction procedure, the HCOFCS was directly added with a high voltage for ESI-MS, and the analysis could be completed within 1 min. The developed method showed good linearity in the range 0.1-100 µg/L with a coefficient of determination (R2) > 0.9991. The limits of detection (LODs) and limits of quantitation (LOQs) ranged from 0.05 to 0.1 and 0.2 to 0.3 µg/L, respectively. The results demonstrated that the HCOFCS combined with ESI-MS can be used for the rapid and sensitive determination of trace ZEA and its derivatives in milk samples with satisfactory recoveries from 80.58% to 109.98% and reproducibility with relative standard deviations (RSDs) no more than 11.18%. Furthermore, HCOFCS showed good reusability, which could reuse at least 10 extraction cycles with satisfactory adsorption performance.

Estrogenic in vitro evaluation of zearalenone and its phase I and II metabolites in combination with soy isoflavones.

Humans and animals are exposed to multiple substances in their food and feed that might have a negative health impact. Among these substances, the Fusarium mycoestrogen zearalenone (ZEN) and its metabolites α-zearalenol (α-ZEL) and α-zearalanol (α-ZAL) are known to possess endocrine disruptive properties. In a mixed diet or especially animal feed, these potential contaminants might be ingested together with naturally occurring phytoestrogens such as soy isoflavones. So far, risk assessment of potential endocrine disruptors is usually based on adverse effects of single compounds whereas studies investigating combinatorial effects are scarce. In the present study, we investigated the estrogenic potential of mycoestrogens and the isoflavones genistein (GEN), daidzein (DAI) and glycitein (GLY) as well as equol (EQ), the gut microbial metabolite of DAI, in vitro alone or in combination, using the alkaline phosphatase (ALP) assay in Ishikawa cells. In the case of mycoestrogens, the tested concentration range included 0.001 to 10 nM with multiplication steps of 10 in between, while for the isoflavones 1000 times higher concentrations were investigated. For the individual substances the following order of estrogenicity was obtained: α-ZEL > α-ZAL > ZEN > GEN > EQ > DAI > GLY. Most combinations of isoflavones with mycoestrogens enhanced the estrogenic response in the investigated concentrations. Especially lower concentrations of ZEN, α-ZEL and α-ZAL (0.001-0.01 nM) in combination with low concentrations of GEN, DAI and EQ (0.001-0.1 µM) strongly increased the estrogenic response compared to the single substances.

Effects of Pubertal Exposure to Butyl Benzyl Phthalate, Perfluorooctanoic Acid, and Zeranol on Mammary Gland Development and Tumorigenesis in Rats.

Endocrine-disrupting chemicals (EDCs)-including butyl benzyl phthalate (BBP), perfluorooctanoic acid (PFOA), and zeranol (α-ZAL, referred to as ZAL hereafter)-can interfere with the endocrine system and produce adverse effects. It remains unclear whether pubertal exposure to low doses of BBP, PFOA, and ZAL has an impact on breast development and tumorigenesis. We exposed female Sprague Dawley rats to BBP, PFOA, or ZAL through gavage for 21 days, starting on day 21, and analyzed their endocrine organs, serum hormones, mammary glands, and transcriptomic profiles of the mammary glands at days 50 and 100. We also conducted a tumorigenesis study for rats treated with PFOA and ZAL using a 7,12-dimethylbenz[a]anthracene (DMBA) model. Our results demonstrated that pubertal exposure to BBP, PFOA, and ZAL affected endocrine organs and serum hormones, and induced phenotypic and transcriptomic changes. The exposure to PFOA + ZAL induced the most phenotypic and transcriptomic changes in the mammary gland. PFOA + ZAL downregulated the expression of genes related to development at day 50, whereas it upregulated genes associated with tumorigenesis at day 100. PFOA + ZAL exposure also decreased rat mammary tumor latency, reduced the overall survival of rats after DMBA challenge, and affected the histopathology of mammary tumors. Therefore, our study suggests that exposure to low doses of EDCs during the pubertal period could induce changes in the endocrine system and mammary gland development in rats. The inhibition of mammary gland development by PFOA + ZAL might increase the risk of developing mammary tumors through activation of signaling pathways associated with tumorigenesis.

Raman spectral analysis for rapid determination of zearalenone and alpha-zearalanol.

Mycotoxins, including zearalenone, are important natural products produced by fungi that occasionally contaminate agricultural commodities and pose serious health risks to consumers of food and feed. Zearalenone and its metabolite, α-zearalanol, are of significant concern due to their estrogenic and anabolic steroid activity. Several governments have regulatory standards and advisory guidelines for zearalenone and α-zearalanol. Raman and ultraviolet spectroscopy were employed with density functional theory methods to evaluate spectroscopic properties to distinguish between zearalenone and α-zearalanol systematically. Raman bands were assigned based on vibrational frequency calculations. A portable Raman spectroscopy instrument (785 nm laser) distinguished between zearalenone and α-zearalanol in a label-free manner. Many vibrational bands of zearalenone and α-zearalanol are similar, including high-intensity peaks at 1315 cm-1 and 1650 cm-1. However, the intensities in the Raman spectra at 1465 cm-1, 1495 cm-1, and 1620 cm-1 enabled the identification of zearalenone. The Raman peak at 1450 cm-1 is associated with α-zearalanol. These vibrational bands serve as spectral indicators to differentiate between the structurally similar zearalenone and α-zearalanol.

Testing the extraction of 12 mycotoxins from aqueous solutions by insoluble beta-cyclodextrin bead polymer.

Mycotoxins are toxic metabolites of filamentous fungi; they are common contaminants in numerous foods and beverages. Cyclodextrins are ring-shaped oligosaccharides, which can form host-guest type complexes with certain mycotoxins. Insoluble beta-cyclodextrin bead polymer (BBP) extracted successfully some mycotoxins (e.g., alternariol and zearalenone) from aqueous solutions, including beverages. Therefore, in this study, we aimed to examine the ability of BBP to remove other 12 mycotoxins (including aflatoxin B1, aflatoxin M1, citrinin, dihydrocitrinone, cyclopiazonic acid, deoxynivalenol, ochratoxin A, patulin, sterigmatocystin, zearalanone, α-zearalanol, and ß-zearalanol) from different buffers (pH 3.0, 5.0, and 7.0). Our results showed that BBP can effectively extract citrinin, dihydrocitrinone, sterigmatocystin, zearalanone, α-zearalanol, and ß-zearalanol at each pH tested. However, for the removal of ochratoxin A, BBP was far the most effective at pH 3.0. Based on these observations, BBP may be a suitable mycotoxin binder to extract certain mycotoxins from aqueous solutions for decontamination and/or for analytical purposes.

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.

Case-Control Study of Nodding Syndrome in Acholiland: Urinary Multi-Mycotoxin Screening.

This case-control study adds to the growing body of knowledge on the medical, nutritional, and environmental factors associated with Nodding Syndrome (NS), a seizure disorder of children and adolescents in northern Uganda. Past research described a significant association between NS and prior history of measles infection, dependence on emergency food and, at head nodding onset, subsistence on moldy maize, which has the potential to harbor mycotoxins. We used LC-MS/MS to screen for current mycotoxin loads by evaluating nine analytes in urine samples from age-and-gender matched NS cases (n = 50) and Community Controls (CC, n = 50). The presence of the three mycotoxins identified in the screening was not significantly different between the two groups, so samples were combined to generate an overall view of exposure in this community during the study. Compared against subsequently run standards, α-zearalenol (43 ± 103 µg/L in 15 samples > limit of quantitation (LOQ); 0 (0/359) µg/L), T-2 toxin (39 ± 81 µg/L in 72 samples > LOQ; 0 (0/425) µg/L) and aflatoxin M1 (4 ± 10 µg/L in 15 samples > LOQ; 0 (0/45) µg/L) were detected and calculated as the average concentration ± SD; median (min/max). Ninety-five percent of the samples had at least one urinary mycotoxin; 87% were positive for two of the three compounds detected. While mycotoxin loads at NS onset years ago are and will remain unknown, this study showed that children with and without NS currently harbor foodborne mycotoxins, including those associated with maize.

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.