zearalenol and taleranol

Topics: Animals; Antibodies, Monoclonal; Equipment Design; Food Analysis; Food Contamination; Gold Colloid; Immunoassay; Milk; Sensitivity and Specificity; Zearalenone; Zeranol

Topics: Animal Feed; Animals; Antibodies, Monoclonal; Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Female; Food Contamination; Haptens; Inhibitory Concentration 50; Mice, Inbred BALB C; Reproducibility of Results; Sensitivity and Specificity; Swine; Tandem Mass Spectrometry; Zea mays; Zearalenone; Zeranol

Zearalenone (ZEN) and its metabolites are found in many food products and are known to induce many toxic effects. The major ZEN metabolites are α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). The mechanisms by which they mediate their cytotoxic effects are not well known and seem to differ depending on the type of cells. We investigated the possible underlying mechanism in α-ZOL and β-ZOL-induced toxicity in HCT116 cells. We showed that cell treatment with α-ZOL/β-ZOL generated endoplasmic reticulum (ER) stress and activated the Unfolded Protein Response (UPR) as evidenced by XBP1 mRNA splicing and up-regulation of GADD34, GRP78, ATF4 and CHOP. Apoptosis was triggered by ZEN metabolites-induced ER stress, and executed through a mitochondria-dependent pathway, characterized by a loss of mitochondrial transmembrane potential (ΔΨm), a downstream generation of O2•(-) and caspase 3 activation. Cellular deficiency of the pro-apoptotic proteins Bax and Bak protected cells against α/β-ZOL-induced toxicity. However, treatment with α-ZOL or β-ZOL combined with Quercetin (QUER), a common dietary flavonoid with well-known antioxidant activity, significantly reduced damage induced by α and β-ZOL in all tested markers. We concluded that QUER protects against the cellular toxicity of α and β-ZOL.×.

Topics: Activating Transcription Factor 4; Analysis of Variance; Antioxidants; Apoptosis; Cell Line, Tumor; Dose-Response Relationship, Drug; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Flow Cytometry; Gene Expression Regulation, Neoplastic; HCT116 Cells; HSP70 Heat-Shock Proteins; Humans; Mitochondrial Proteins; Protein Disulfide-Isomerases; Protein Phosphatase 1; Quercetin; Reactive Oxygen Species; Transcription Factor CHOP; X-Box Binding Protein 1; Zearalenone; Zeranol

In this work, a simple and environmental friendly methodology has been developed for the analysis of a group of six mycotoxins with estrogenic activity produced by Fusarium species (i.e. zearalanone, zearalenone, α-zearalanol, β-zearalanol, α-zearalenol, and β-zearalenol), using microdispersive SPE the symbol micro should de before dSPE with multiwalled carbon nanotubes as sorbent. Separation, determination, and quantification were achieved by HPLC coupled to ion trap MS with an ESI interface. Parameters affecting the extraction efficiency of µ-dSPE such as pH of the sample, amount of multiwalled carbon nanotubes, and type and volume of elution solvent, were studied and optimized. The methodology was validated for mineral, pond, and wastewater as well as for powdered infant milk using 17β-estradiol-2,4,16,16,17-d5 (17β-E2 -D5 ) as internal standard, obtaining recoveries ranging from 85 to 120% for the three types of water samples and from 77 to 115% for powdered infant milk. RSD values were lower than 10%. The LOQs achieved were in the range 0.05-2.90 μg/L for water samples and 2.02-31.9 μg/L for powdered infant milk samples.

Topics: Chromatography, High Pressure Liquid; Infant Formula; Limit of Detection; Mass Spectrometry; Mineral Waters; Mycotoxins; Nanotubes, Carbon; Ponds; Solid Phase Extraction; Wastewater; Zearalenone; Zeranol

In this work, the suitability of a methodology based on dispersive liquid-liquid microextraction (DLLME) has been evaluated for the extraction of four endoestrogens (estriol, 17α-estradiol, 17β-estradiol, and estrone), an exoestrogen (17α-etynylestradiol), and a mycotoxin (zearalenone), together with some of their major metabolites (2-methoxyestradiol, α-zearalanol, β-zearalanol, α-zearalenol, and β-zearalenol) from different types of milk (whole and skimmed cow milk and semiskimmed goat milk) and whole natural yogurt. The methodology includes a previous protein precipitation with acidified ACN and a defatting step with n-hexane. Separation of the analytes, determination, and quantification were developed by MEKC coupled to ESI-MS using a BGE containing an aqueous solution of ammonium perfluorooctanoate as MS friendly surfactant. Calibration, precision, and accuracy studies of the described DLLME-MEKC-MS/MS method were evaluated obtaining a good linearity and LODs in the low micrograms per liter range.

Topics: Animals; Chromatography, Micellar Electrokinetic Capillary; Estrogens; Estrone; Food Analysis; Food Contamination; Limit of Detection; Liquid Phase Microextraction; Mass Spectrometry; Milk; Reproducibility of Results; Yogurt; Zearalenone; Zeranol

A novel, simple, rapid and eco-friendly method based on dispersive liquid-liquid microextraction using a bromosolvent was developed to determine six estrogenic mycotoxins (zearalenone, zearalanone, α-zearalanol, β-zearalanol, α-zearalenol and β-zearalenol) in water samples by liquid chromatography-electrospray ionization tandem mass spectrometry in the negative mode (LC-ESI-MS/MS). The optimal conditions for this method include the use of 100 μL bromocyclohexane as an extraction solvent (using a non-dispersion solvent), 10 mL of aqueous sample (adjusted to pH 4), a vortex extraction time of 2 min, centrifugation for 10 min at 3500 rpm and no ionic strength adjustment. The calibration function was linear and was verified by applying the Mandel fitting test with a 95% confidence level. No matrix effect was observed. According to the relative standard deviations (RSDs), the precision was better than 13% for the repeatability and intermediate precision. The average recoveries of the spiked compounds ranged from 81 to 118%. The method limits of detection (LOD) and quantification (LOQ) considering a 125-fold pre-concentration step were 4-20 and 8-40 ng L(-1), respectively. Next, the method was applied to the analysis of the environmental aqueous samples, demonstrating the presence of β-zearalanol and zearalanone in the river water samples.

Topics: Chromatography, Liquid; Estrogens, Non-Steroidal; Fresh Water; Limit of Detection; Liquid Phase Microextraction; Mycotoxins; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Zearalenone; Zeranol

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, β-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 β-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. β-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 β-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.

Topics: Animals; Cell Line; Cell Survival; Epithelial Cells; Estrogens, Non-Steroidal; Food Contamination; Interleukin-10; Interleukin-8; Intestinal Mucosa; Swine; Zearalenone; Zeranol

A novel highly sensitive chemiluminescence immunoassay (CLIA) was developed to detect zearalenone in food samples by using both biotinylated zearalenone conjugates and gold (Au) nanoparticles labeled with streptavidin-horseradish peroxidase for signal amplification. Biotinylated zearalenone-ovalbumin conjugates and Au nanoparticles labeled with streptavidin-horseradish peroxidase were synthesized separately. The concentrations of immunoreagents and the reaction times of these immunoreagents were optimized to improve the performances of analytical methods. For the CLIA based on biotinylated zearalenone conjugates and Au nanoparticles labeled with streptavidin-horseradish peroxidase, the limit of detection was 0.008 ng/mL and the IC50 was 0.11 ng/mL. The linear working range was 0.02-0.51 ng/mL. The cross-reactivities with the zearalenone analogues (α-zearalanol, zearalanone, α-zearalenol, β-zearalanol, and β-zearalenol) were 32, 17, 12, 0.3, and 0.1%, respectively. The recovery rates in spiked food samples were 97-117%, and the intraday and interday relative standard deviations were both <10%. Parallel analysis of natural food samples showed a good correlation between this novel CLIA and liquid chromatography-tandem mass spectrometry. This method provides a rapid, accurate, and highly sensitive method to determine levels of zearalenone in food samples.

Topics: Chromatography, Liquid; Food Analysis; Food Contamination; Gold; Horseradish Peroxidase; Immunoassay; Luminescence; Metal Nanoparticles; Streptavidin; Tandem Mass Spectrometry; Zearalenone; Zeranol

α- and β-zearalenol (α-ZOL and β-ZOL, respectively) are metabolites of the mycotoxin zearalenone (ZEN). All three individual mycotoxins have shown to be biological active i.e. being estrogenic and able to stimulate cellular proliferation albeit at different strengths. In this work, cytosol protein expression was determined by using stable-isotope labelling by amino acids in cell culture (SILAC) upon exposure of α-ZOL and β-ZOL to the steroidogenesis cell model H295R. A total of 14 and 5 individual proteins were found to be significantly regulated by α-ZOL and β-ZOL, respectively. Interestingly, there were no common protein regulations by the metabolites or the parent mycotoxin ZEN. Furthermore, the regulated proteins were assigned to networks and groups of actions that also differed from one another suggesting that the three individual mycotoxins may have unique biological activities.

Topics: Cell Line; Cytosol; Gene Expression Regulation; Humans; Protein Interaction Maps; Proteins; Steroids; Zearalenone; Zeranol

Zearalenone (ZEN) and α-zearalanol (α-ZAL, zeranol) were studied in differentiated Caco-2 cells and in the Caco-2 Millicell® system in vitro to simulate their in vivo intestinal absorption and metabolism in humans.. In addition to metabolic reduction/oxidation, extensive conjugation with glucuronic acid and sulfate of the parent compounds and their phase I metabolites was observed. The positional isomers of the glucuronides and sulfates were unambiguously identified: Sulfonation occurred specifically at the 14-hydroxyl group, whereas glucuronidation was less specific and, in addition to the preferred 14-hydroxyl group, involved the 16- and 7-hydroxyl groups. Using the Caco-2 Millicell® system, an efficient transfer of the glucuronides and sulfates of ZEN and α-ZAL and their phase I metabolites into both the basolateral and the apical compartment was observed after apical administration. The apparent permeability coefficients (P(app) values) of ZEN, α-ZAL and the ZEN metabolite α-zearalenol were determined, using an initial apical concentration of 20 μM and a permeation time of 1 h.. According to the P(app) values, the three compounds are expected to be extensively and rapidly absorbed from the intestinal lumen in vivo and reach the portal blood both as aglycones and as glucuronide and sulfate conjugates in humans.

Topics: Biotransformation; Caco-2 Cells; Cell Membrane Permeability; Cell Polarity; Chromatography, High Pressure Liquid; Enterocytes; Estrogens, Non-Steroidal; Glucuronides; Growth Substances; Humans; Hydroxysteroid Dehydrogenases; Intestinal Absorption; Isomerism; Oxidation-Reduction; Sulfates; Tandem Mass Spectrometry; Zearalenone; Zeranol

Glucuronidation constitutes an important pathway in the phase II metabolism of the mycotoxin zearalenone (ZEN) and the growth promotor α-zearalanol (α-ZAL, zeranol), but the enzymology of their formation is yet unknown. In the present study, ZEN, α-ZAL and four of their major phase I metabolites were glucuronidated in vitro using hepatic microsomes from steer, pig, rat and human, intestinal microsomes from humans, and eleven recombinant human UDP-glucuronosyltransferases (UGTs). After assigning chemical structures to the various glucuronides by using previously published information, the enzymatic activities of the various microsomes and UGT isoforms were determined together with the patterns of glucuronides generated. All six compounds were good substrates for all microsomes studied. With very few exceptions, glucuronidation occurred preferentially at the sterically unhindered phenolic 14-hydroxyl group. UGT1A1, 1A3 and 1A8 had the highest activities and gave rise to the phenolic glucuronide, whereas glucuronidation of the aliphatic hydroxyl group was mostly mediated by UGT2B7 with low activity. Based on these in vitro data, ZEN, α-ZAL and their metabolites must be expected to be readily glucuronidated both in the liver and intestine as well as in other extrahepatic organs of humans and various animal species.

Topics: Animals; Cattle; Estrogens, Non-Steroidal; Female; Glucuronides; Glucuronosyltransferase; Humans; Intestine, Small; Isoenzymes; Male; Microsomes; Microsomes, Liver; Rats; Rats, Wistar; Recombinant Proteins; Swine; Zearalenone; Zeranol

A simple analytical scheme for the screening and quantification of zearalenone and its metabolites, alpha-zearalenol and beta-zearalenol, is reported. Extracts from maize flour samples were collected by supercritical fluid extraction and afterwards, they were analyzed by CE with amperometric detection. This scheme allowed a rapid and reliable identification of contaminated flour samples according to the reference value established for zearalenone by directive 2005/38/EC (200 microg/kg). The sample screening method was carried out by CZE using 25 mM borate separation buffer at pH 9.2 and 25.0 kV as separation voltage, monitoring the amperometric signal at +700 mV with a carbon paste electrode. In this way, total amount of mycotoxins was determined and samples were processed in 4 min with a detection limit of 12 microg/L, enough to discriminate between positive (more than 200 microg/L total mycotoxins) and negative samples (less than 200 microg/L total mycotoxins). Positive samples were then subjected to CZE separation and quantification of each analyte was done with 50 mM borate running buffer modified with 30% methanol at pH 9.7 and 17.5 kV as separation voltage. Under these conditions, separation was achieved in 15 min with detection limits from 20 to 35 microg/L for each analyte.

Topics: Electrophoresis, Capillary; Flour; Mycotoxins; Zea mays; Zearalenone; Zeranol

The enzymes 3alpha- and 3beta-hydroxysteroid dehydrogenase (3alpha- and 3beta-HSD) play a pivotal role in synthesis of various steroid hormones including oestradiol and testosterone. The structure of the mycotoxin zearalenone resembles many characteristics of steroids and binds to oestrogen receptors as an agonist. Consequently, it is suggested that zearalenone is also a substrate for 3alpha-HSD and 3beta-HSD. 3alpha-HSD and 3beta-HSD isoforms are expressed in the liver and kidney but also in many steroidogenic tissues. It was the aim of the present study to demonstrate the presence of these enzymes in granulosa cells, which were obtained from bovine and porcine ovaries, and to investigate whether zearalenone is a substrate for these enzymes. The results show a species-specific expression pattern in the granulosa cells of both species. Moreover, it was demonstrated that zearalenone when added to the culture medium, is converted into alpha-zearalenol and beta-zearalenol. Corresponding to the apparent expression profile, in porcine granulosa cells predominantly alpha-zearalenol was formed, whereas bovine granulosa cells preferentially converted zearalenone into beta-zearalenol. This is the first report demonstrating the extrahepatic biotransformation of zearalenone in target tissues.

Topics: Animals; Cattle; Cells, Cultured; Female; Granulosa Cells; Hydroxysteroid Dehydrogenases; Swine; Zearalenone; Zeranol

A method is described for the quantitative determination of the veterinary drug zeranol, its epimer taleranol and the mycotoxins zearalenone, alpha-zearalenol and beta-zearalenol in bovine urine. The method is based on liquid chromatography coupled to negative-ion electrospray mass spectrometry-mass spectrometry of urine extracts prepared by solid-phase extraction with C(18) columns. Two transition ions at m/z 277 and 91 are monitored for zeranol and taleranol along with the transition ion at m/z 281 for their respective deuterated (d(4)) internal standards. Similarly, two transitions are monitored for each of the three mycotoxins along with a transition ion for each of their corresponding internal standards. The method has been validated according to the new European Union criteria for analysis of veterinary drug residues, and is suitable for monitoring urine samples taken under National Surveillance Schemes. The method has been validated at 1, 1.5 and 2 ng ml(-1) for zeranol and taleranol and at 5, 10 and 15 ng ml(-1) for each of the three mycotoxins. Correlation between the described method and a routine method, based on gas chromatography-mass spectrometry, was assessed using a range of naturally incurred samples.

Topics: Animals; Cattle; Chromatography, Liquid; Fusarium; Gas Chromatography-Mass Spectrometry; Mycotoxins; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Zearalenone; Zeranol