Zearalenone-16-O-glucoside: A New Masked ... - ACS Publications

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Zearalenone-16‑O‑glucoside: A New Masked Mycotoxin Maria Paula Kovalsky Paris,†,△ Wolfgang Schweiger,†,▽ Christian Hametner,⊗ Romana Stückler,† Gary J. Muehlbauer,§ Elisabeth Varga,‡ Rudolf Krska,‡ Franz Berthiller,‡ and Gerhard Adam*,† †

Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Konrad Lorenz Straße 24, A-3430 Tulln, Austria ⊗ Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163, A-1060 Vienna, Austria § Department of Plant Biology, Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108 United States ‡ Center for Analytical Chemistry and Christian Doppler Laboratory for Mycotoxin Metabolism, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna, Konrad Lorenz Straße 20, A-3430 Tulln, Austria S Supporting Information *

ABSTRACT: This paper reports the identification of a barley UDP-glucosyltransferase, HvUGT14077, which is able to convert the estrogenic Fusarium mycotoxin zearalenone into a near-equimolar mixture of the known masked mycotoxin zearalenone-14O-β-glucoside and a new glucose conjugate, zearalenone-16-O-β-glucoside. Biocatalytical production using engineered yeast expressing the HvUGT14077 gene allowed structural elucidation of this compound. The purified zearalenone-16-O-β-glucoside was used as an analytical calibrant in zearalenone metabolization experiments. This study confirmed the formation of this new masked mycotoxin in barley seedlings as well as in wheat and Brachypodium distachyon cell suspension cultures. In barley roots, up to 18-fold higher levels of zearalenone-16-O-β-glucoside compared to the known zearalenone-14-O-β-glucoside were found. Incubation of zearalenone-16-O-β-glucoside with human fecal slurry showed that this conjugate can also be hydrolyzed rapidly by intestinal bacteria, converting the glucoside back to the parental mycotoxin. Consequently, it should be considered as an additional masked form of zearalenone with potential relevance for food safety. KEYWORDS: Fusarium, zearalenone, xenoestrogen, UDP-glucosyltransferase, heterologous expression, yeast, barley, wheat, Brachypodium

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INTRODUCTION Zearalenone (structure 1 in Figure 1) is a secondary metabolite produced by several Fusarium species during infection of cereal host plants in the field and also during suboptimal grain storage.1,2 Zearalenone has low acute toxicity for animals and humans, but poses a health risk due to its strong estrogenic activity,3 as it can bind to both human estrogen receptors alpha and beta and acts as an endocrine disruptor.4 The European Food Safety Authority Panel on Contaminants in the Food Chain established a tolerable daily intake for zearalenone of 0.25 μg/kg body weight.5 To protect consumers, maximum tolerated zearalenone levels for various food commodities have been enacted in the European Union.6 The maximum limit for zearalenone in wheat is rarely reached under dry conditions, yet it has been observed that delayed harvesting due to cool and wet conditions can cause significant zearalenone contamination of small grain cereals.7 For example, in the exceptional year 2008 it was estimated that in the United Kingdom 28.6% of wheat samples exceeded the European limit for unprocessed cereals of 100 μg/kg. High zearalenone levels have also been observed in countries where warm and humid conditions prevail.8 Infected plants are capable of metabolizing fungal toxins, predominantly by formation of glucose conjugates.9 Because such conjugates typically escape routine detection methods, but are potentially hydrolyzed in the digestive tract of consumers of © 2014 American Chemical Society

contaminated grain, they are receiving increased attention as “masked mycotoxins”. Recently, a review on the state of the field of masked mycotoxins highlighted that only limited information about the occurrence, bioavailability, and toxicological relevance of such compounds is available.9 When radiolabeled zearalenone was used to treat maize suspension culture cells, about 12% of the input was recovered as a proposed β-glucoside (zearalenone-4-O-glucoside).10 Meanwhile, due to a widely accepted5 proposed change in the numbering system of atoms in the zearalenone structure (Figure 1) compound 2 is named zearalenone-14-O-β-glucoside in the newer literature.11 The glucoside was found in naturally zearalenone-contaminated Bavarian wheat samples with concentration levels of about 10% of total zearalenone on a molar basis.12 The first UDP-glucosyltransferase (UGT) gene encoding an enzyme capable of converting zearalenone into zearalenone-14O-β-glucoside was identified in 200613 from the model plant Arabidopsis thaliana (UGT73C6, AT2G36790). Yeast cells expressing this gene proved very useful as a tool to produce this glucoside13 and the corresponding α- and β-zearalenol-14-O-βReceived: Revised: Accepted: Published: 1181

December 14, 2013 January 3, 2014 January 4, 2014 January 4, 2014 dx.doi.org/10.1021/jf405627d | J. Agric. Food Chem. 2014, 62, 1181−1189

Journal of Agricultural and Food Chemistry

Article

Figure 1. Chemical structures of zearalenone metabolites and numbering of atoms in zearalenone: zearalenone (1); zearalenone-14-O-β-glucoside (2); zearalenone-16-O-β-glucoside (3); β-zearalenol (4); α-zearalenol (5).

glucosides.14 Analysis of the metabolization of zearalenone by Arabidopsis seedlings revealed that they very rapidly convert zearalenone to zearalenone-14-O-β-glucoside.15 However, the accumulation is transient, and mass spectrometric analysis revealed that further derivatives were formed, such as zearalenone-malonyl-glucoside, zearalenone-dihexoside, and a zearalenone-pentosylhexoside. Also, the glucosides of the phase I metabolites (β-zearalenol (4) and α-zearalenol (5)) were observed.15 The natural occurrence of a zearalenol-glucoside in maize was recently reported.16 Zearalenone-14-O-β-glucoside does not interact with the human estrogen receptor in vitro,13 but when fed to pigs it is rapidly converted back to zearalenone.17 Thus, on a molar basis the estrogenicity of zearalenone-14-O-β-glucoside may be nearly equal to that of zearalenone in mammals (and humans), but no solid data are available. Zearalenone co-occurs with other Fusarium toxins such as deoxynivalenol, but is typically formed rather late during the infection.1,7 Previously, we reported several UDP-glucosyltransferases of barley, which were transcriptionally induced specifically by a deoxynivalenol-producing strain of Fusarium graminearum18 and by deoxynivalenol application,19 and characterized these genes by heterologous expression in yeast.20 Because most of these genes did not confer increased resistance to deoxynivalenol, we tested for activity with zearalenone.

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zearalenone stock solution (5 mg/mL) was prepared in acetone. Zearalenone and zearalenone-14-O-β-glucoside were purified at the IFA-Tulln. Other chemicals, unless stated otherwise, were obtained from Sigma-Aldrich (Vienna, Austria). Cultures were incubated at 30 °C and 180 rpm, and samples were collected for HPLC-MS/MS analysis after 4, 8, 18, and 24 h. HPLC-MS/MS Analysis. Zearalenone and its glucosylated derivatives were analyzed as described previously,15 with slight modifications. Briefly, an 1100 HPLC system (Agilent Technologies, Waldbronn, Germany) was coupled to a QTrap LC-MS/MS system (AB Sciex, Foster City, CA, USA). Ionization was performed with an electrospray interface in negative ionization mode. Deviating from the original method, the column used was a 150 mm × 4.6 mm i.d., 5 μm, Zorbax Eclipse C-8 (Agilent Technologies) for separation of the analytes by applying the same solvent gradient system (5 mM aqueous ammonium acetate/methanol). For zearalenone, the deprotonated precursor (m/z 317.1, declustering potential (DP) −51 V) was fragmented to m/z 130.9 (collision energy (CE) −38 V). The same selected reaction monitoring transition (m/z 479.1 → m/z 317.1, DP 31 V, CE 22 eV) was used for the detection and quantification of both zearalenone-14-O-β-glucoside and zearalenone-16-O-β-glucoside. To verify the apparent recovery of the method using complex fermentation media (with fecal bacteria), blanks were spiked at three concentration levels in triplicates. LC-HR-MS spectra of the purified zearalenone-16-O-β-glucoside were acquired on a 6550 iFunnel QTOF coupled to a 1290 Infinity UHPLC system (Agilent Technologies). One microliter of a 1 mg/L solution of zearalenone-16-O-β-glucoside in MeOH/H2O (30:70, v/v) was injected on a 150 mm × 2.1 mm i.d., 1.8 μm, Zorbax SB-C18 rapid-resolution high-definition column (Agilent Technologies). A linear water/methanol gradient (both with 0.1% HCOOH) was applied. Full scan spectra over a range of m/z 50−1000 were acquired after positive and negative electrospray ionization. MassHunter B.05.01 was used for data acquisition, whereas MassHunter Qualitative Analysis B.06.00 was used for data evaluation. Biocatalytical Production and Purification of Zearalenone16-O-β-glucoside. Yeast strain YZGA515 expressing HvUGT14077 was cultivated in a 20 L single-wall glass autoclavable bioreactor (Applikon, Schiedeam, Netherlands) at 30 °C and 100 rpm. An overnight culture was added to 10 L of selective medium buffered with 50 mM sodium phosphate, resulting in an OD600 of 0.05. The cells were incubated overnight at 30 °C and 100 rpm, and the pH was kept

MATERIALS AND METHODS

Incubation of Toxin-Sensitive Yeast Expressing HvUGT14077 with Zearalenone. Three independent transformants of the toxin-sensitive Saccharomyces cerevisiae strain YZGA515 expressin g t he UDP-gluco syltransferase HvUGT14077 (GU170356.1),20 or containing the empty vector, were propagated in liquid selective medium lacking leucine (SC-LEU) to an OD600 of 0.7 at 30 °C and 180 rpm. Fifty milliliters of the yeast suspensions was harvested by centrifugation, and the pellets were resuspended in 5 mL of SC-LEU, which contained 5 mg/L (=15.7 μM) of zearalenone and was buffered with 50 mM sodium phosphate to pH 7.0. The 1182

dx.doi.org/10.1021/jf405627d | J. Agric. Food Chem. 2014, 62, 1181−1189


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O-β-glucoside solution in the respective medium was added to 500 μL of fecal slurry, and samples were flushed with nitrogen for 10 s. Tubes were incubated at 37 °C, and the reaction was stopped after 0.5 and 2 h of incubation by adding 1 mL of ethanol. As a negative control, 500 μL of water was added to 500 μL of fecal slurry, and as a positive control 500 μL of water was added to the incubation medium containing 2 mg/L of zearalenone-16-O-β-glucoside. Supernatants were sterilized using Ultrafree-CL sterile filters with a pore size of 0.2 μm (Millipore, Schwalbach, Germany) prior to analysis by HPLC-MS/ MS.

constant during the fermentation at 7.0 by addition of 10 N NaOH. The zearalenone stock was added to reach 5 mg/L. After 4, 8, and 22 h, additional zearalenone was administered corresponding to further steps of 5 mg/L. After 24 h of incubation, the yeast supernatant was collected in 500 mL portions by centrifugation and 800 mL of methanol was added to the 8 L of supernatant. Zearalenone-14-O-βglucoside and zearalenone-16-O-β-glucoside were purified from the supernatant as previously described.14 Structure Characterization by NMR. NMR spectra were measured in CD3OD solution on an Avance DRX-400 FT-NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany), operating at 400.13 MHz for 1H and at 100.62 MHz for 13C, at 300 K using a 5 mm inverse broadband Z-gradient probe head. Data were recorded and evaluated using TOPSPIN 1.3 (Bruker Biospin). All pulse programs were taken from the Bruker software library. Chemical shifts (established on the basis of the residual solvent resonances) are given in parts per million (ppm) relative to tetramethylsilane and coupling constants are in hertz (Hz). Incubation of Barley, Wheat, and Brachypodium distachyon with Zearalenone. Barley seeds of the varieties ‘Morex’ (six-rowed spring malting, University of Minnesota) and ‘Signora’ (a two-rowed spring barley brewing variety dominant in Austria) were surfacesterilized by incubation with a solution containing 5% (v/v) sodium hypochlorite (14% active chlorine) and 0.1% (v/v) Triton X-100 for 7 min. The sterilization solution was removed completely, and seeds were incubated with 70% (v/v) ethanol for 1 min and washed three times with sterile distilled water. The seeds were placed on Murashige and Skoog basal medium containing 0.5% (w/v) sucrose and 2% (w/v) agar in magenta plant culture boxes with a cycle of 15 h of light at 20 °C and 9 h of darkness at 10 °C for 1 week. Seedlings were transferred to liquid macronutrient salt medium (1650 mg/L ammonium nitrate, 1900 mg/L potassium nitrate, 370 mg/L ammonium sulfate, 50 mM phosphoric acid, buffered to pH 7.0 with 10 N NaOH) without sugar containing 5 mg/L zearalenone and 1 mg/L deoxynivalenol. The 5 mg/mL zearalenone stock was prepared in acetone, and the 1 mg/mL deoxynivalenol stock was prepared in water. Plants were incubated at 21 °C with constant light at 100 rpm. Metabolites were extracted from roots and leaves separately after 24 h of incubation by adding 1 mL of ethanol to 200 mg of plant material and wet grinding of the tissue with a Retsch Mixer Mill (Retsch, Haan, Germany) for 1 min at high speed. The Triticum aestivum suspension culture (derived from wheat cultivar Heines Koga II, DSMZ PC-998) was obtained from the Plant Cell Culture Collection of the Leibniz Institute, DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and weekly subcultured in Gamborg B5 medium (Duchefa Biochemie, Netherlands) in constant light at 21 °C and 100 rpm. The B. distachyon cell culture21 derived from line Bd21 was kindly provided by Dr. Carl Ng (University College, Dublin, Ireland) and cultivated in SCM medium containing 4.4 g/L Linsmaier and Skoog medium basal salt mix including vitamins (Duchefa Biochemie, The Netherlands), 3% (w/v) maltose (Roth, Germany), 7.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.75 mg/L kinetin with pH set to 5.8 with 1 M KOH. Bd21 cells were propagated at 21 °C in darkness and also subcultured weekly by combining 50 mL of culture with 50 mL of fresh medium. About 200 mg of each cell culture was harvested by centrifugation and resuspended in 1 mL of the respective medium containing 2 mg/L of zearalenone and 1 mg/L deoxynivalenol. Samples were incubated at 21 °C and shaken for 4, 18, and 24 h. The cells were centrifuged, and the metabolites were extracted from the cell pellet as described for barley roots and leaves. All supernatants were analyzed by HPLC-MS/MS. Incubation of Intestinal Bacteria with Zearalenone-16-O-βglucoside. The in vitro fecal hydrolysis assay was conducted using samples collected from six healthy donors (four women and two men, ages 28−38 years) who had signed informed consent forms. Two incubation media were tested: (a) distilled water and (b) the complex fermentation medium as previously described by Dall’Erta et al.22 For the fecal slurry, 4 mL of water was added to 1 g of fecal material and homogenized. Five hundred microliters of a 2 mg/L zearalenone-16-

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RESULTS HvUGT14077 Expression in Yeast Interferes with Estrogen Receptor Activation. In a previous study, we reported the functional analysis of four barley UDPglucosyltransferase genes,20 which were highly induced by the application of the Fusarium mycotoxin deoxynivalenol.19 Although recombinant HvUGT14077 protein was clearly detected in yeast, no deoxynivalenol resistance was observed. To test for activity with zearalenone, we transformed the expression vectors into the previously described yeast estrogen reporter strain YZCP90813 and observed diminished activation. Production of a New Glucoside by HvUGT14077 Expression in Yeast Transformants Treated with Zearalenone. To determine the mechanism in which HvUGT14077 metabolizes zearalenone and hence interferes with estrogen receptor activation, we incubated YZGA515 expressing HvUGT14077 and the empty vector control with 5 mg/L (15.7 μM) zearalenone. Analysis of the supernatant by LC-MS/MS revealed the production of zearalenone-14-O-βglucoside13 and a second compound with the same mass, however, with a different retention time (Figure 2). As shown

Figure 2. Chromatogram of metabolites produced by zearalenonetreated yeast strain YZGA515 expressing HvUGT14077. Each mass transition is displayed as a separate line. Zearalenone (1) has a retention time of 9.9 min; α-zearalenol (5), 9.8 min; β-zearalenol (4), 9.5 min; and zearalenone-14-O-β-glucoside (2), 8.5 min. The new compound, zearalenone-16-O-β-glucoside (3), with a retention time of 7.5 min, has the same mass-to-charge-ratio as compound 2.

later, the hypothesis that this new metabolite might be zearalenone-16-O-β-glucoside (3) was confirmed. In a smallscale experiment (50 mL shake culture), the transformed yeast strain was able to metabolize the administered zearalenone rapidly, most of it within 4 h (Figure 3). At time point 0 only about 50% of the input was detected in the supernatant. After 1183



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The 1H and 13C NMR data for zearalenone-16-O-β-glucoside are shown in Figure 4 and summarized in Table 1. For structure elucidation, HH-COSY, HC-HSQC, and HC-HMBC spectra were recorded. Using these two-dimensional methods, the structure of zearalenone-16-O-β-glucoside was confirmed, and complete signal assignments were established. In particular, the carbon signals of the benzene ring were assigned on the basis of direct and long-range correlations of the aromatic (H13, H15) and olefinic (H11, H12) protons. Then, the attachment position of the glucose moiety at the zearalenone skeleton could be determined by long-range coupling of the glucosyl-H1 to the C16 carbon. Formation of Zearalenone-16-O-β-glucoside in Zearalenone-Treated Barley. To test whether zearalenone-16-Oβ-glucoside is also formed in planta, we treated barley seedlings with an initial root length of about 1.5 cm with 5 mg/L zearalenone in liquid cultures. The average values of two biological replicates each containing pools of three seedlings are shown in Figure 5. In the control medium without added zearalenone, none was detected, whereas in the spiked control medium with a theoretical input of 78.5 nmol only 61.9 nmol was determined after 24 h. Zearalenone was not detected in the supernatants or in the roots of ‘Morex’ and ‘Signora’ seedlings incubated in medium lacking zearalenone. Approximately 1−7% of the added zearalenone was still present in the supernatants of both varieties, and only a small portion of the input (0.5−1%) was recovered as zearalenone-14-O-β-glucoside and zearalenone-16-O-β-glucoside in the supernatant. Although a significant amount of the added zearalenone was recovered in the root extracts, 66 and 87% of the input were converted to zearalenone-16-O-β-glucoside by ‘Morex’ and ‘Signora’ seedlings, respectively. The cotyledons were upright above the zearalenone-containing medium, without direct contact. They contained only minimal amounts of zearalenone and zearalenone metabolites. Only 3−5% of the administered zearalenone was metabolized to the previously described masked mycotoxin zearalenone-14-O-β-glucoside in barley; about 16−18-fold more zearalenone-16-O-β-glucoside than zearalenone-14-O-β-glucoside was detected in the barley root samples. Zearalenone Glycosylation by Wheat and Brachypodium Suspension Cultures. Due to a higher sensitivity of suspension cultures, only 2 mg/L of zearalenone was added to the media instead of the 5 mg/L used in the barley seedling test. Panels a and b of Figure 6 show the results of a time course of treatment of the Brachypodium suspension culture with zearalenone as an average of three biological replicates. The input of 6.2 μmol of zearalenone was metabolized by the approximately 200 mg of wet weight of cells, and within 18 h only 21% remained in the medium. A significant portion of zearalenone (50% after 4 h and 14% after 24 h) was found to be present in the extracted cell pellet. About 46% of the input was converted to zearalenone-16-O-β-glucoside within 4 h, which further increased to 76% after 18 h. A very different situation was observed with a wheat suspension culture (Figure 6c,d). Again, zearalenone added to the medium rapidly became undetectable, but compared to Brachypodium the intracellular concentrations of zearalenone, zearalenone-14-O-β-glucoside, and zearalenone-16-O-β-glucoside remained low. After 24 h, only 13% of the input was converted to zearalenone-14-O-β-glucoside and zearalenone16-O-β-glucoside, respectively, and detected in the cell extracts. Compared to Brachypodium, a larger portion of zearalenone-14-

Figure 3. Time course of metabolization of zearalenone in a smallscale experiment.

24 h, a nearly complete conversion of the zearalenone input, to a mixture of both glucosides with roughly 1:1 ratio, was observed (about 44% zearalenone-16-O-β-glucoside, Figure 3). Biocatalytic Production and Purification of the New Glucoside. To obtain enough of the new zearalenoneglucoside for structure elucidation, the strain YZGA515 expressing HvUGT14077 was cultivated in 10 L of medium at pH 7.0 in a 20 L bioreactor. Due to its limited solubility in aqueous medium and its toxicity for the yeast strain at higher concentrations, 5 mg/L zearalenone was added consecutively at the beginning and again after 4, 8, and 22 h of incubation. The pH levels remained stable between 6.7 and 7.0, and the yeast showed normal growth starting at an OD600 of 5.3 and reaching 10.7 after 24 h. In the fermentation supernatant with a zearalenone input of 200 mg after 24 h, 39 mg of zearalenone14-O-β-glucoside and 38 mg of zearalenone-16-O-β-glucoside were obtained. As a first purification step, 500 mL portions of the fermentation supernatant containing 10% (v/v) methanol were loaded to equilibrated C18 solid phase extraction columns. None of the zearalenone metabolites were detected in the flow-through. Washing the column with 10 mL of 30% methanol resulted in some losses (1.9 mg of zearalenone-14-Oβ-glucoside and 9.5 mg of zearalenone-16-O-β-glucoside). The glucosides were eluted from the columns with 60% (v/v) methanol, which resulted in a recovery of 34.4 mg of zearalenone-14-O-β-glucoside and 27.8 mg of zearalenone-16O-β-glucoside. A second purification step was conducted using preparative HPLC. The retention time for zearalenone-14-O-βglucoside was 5.0 and that for zearalenone-16-O-β-glucoside was 3.7 min. After evaporation of the solvent, totals of 24.3 mg of zearalenone-14-O-β-glucoside and 21.4 mg of zearalenone16-O-β-glucoside were obtained. Structural Characterization by HR-MS and NMR. HRMS verified the isolated substance to be a C 24 H 32 O 10 compound, consistent with the hypothesis of a zearalenoneglucoside. In positive ion mode the [M + H]+ ion showed an m/z value of 481.2064 (Δm = −0.81 ppm), whereas the [M + Na]+ ion was measured at m/z 503.1887 (Δm = −0.23 ppm). After negative electrospray ionization, the [M − H]− ion showed an m/z value of 479.1929 (Δm = 1.37 ppm) and the [M + HCOO]− ion, m/z 525.1969 (Δm = −1.62 ppm). MS/MS spectra showed the cleavage of the glucose moiety. Further fragments from zearalenone could not be used to differentiate between zearalenone-16-O-β-glucoside and zearalenone-14-O-β-glucoside. 1184



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Figure 4. 1H and 13C(-APT) NMR spectra of zearalenone-16-O-β-glucoside.

O-β-glucoside also appeared in the supernatant of wheat cells, but practically no zearalenone-16-O-β-glucoside was detected. Zearalenone, zearalenols, and zearalenone-glucosides add up to only about 40%; a significant portion of the administered zearalenone is unaccounted for. A search for masses of other possible zearalenone metabolites revealed that a large portion of the input might be present in the cells as zearalenonemalonyl-glucoside, for which a reference substance is not yet available. The crude assumption that zearalenone-malonylglucoside shows the same response during ionization in the mass spectrometer leads to the estimate that potentially 30% of the added zearalenone may be converted to zearalenonemalonyl-glucoside in the wheat suspension culture. Hydrolysis of Zearalenone-16-O-β-glucoside by Intestinal Bacteria. Dall’Erta et al.22 showed a high conversion of zearalenone-14-O-β-glucoside by intestinal bacteria incu-

bated in a complex medium containing starch, peptone, tryptone, yeast extract, pectin, mucin, casein, arabinogalactan, etc., in grams per liter amounts. In pilot experiments using 1 mg/L zearalenone-16-O-β-glucoside also an almost complete conversion to zearalenone was observed by intestinal bacteria incubated in water (data not shown). To test the differences between incubating fecal samples in the complex medium of Dall’Erta22 and water, a concentration of 1 mg/L zearalenone16-O-β-glucoside was selected. The fecal samples are a complex analytical matrix causing high variability. The recovery of zearalenone in the feces resuspended in the Dall’Erta fermentation mix was only 35%, with large variability (22% relative standard deviation), whereas the water-resuspended samples were rather unproblematic (on average 81% recovery with 11% relative standard deviation). Overall, due to the high relative standard deviation, the results are to be regarded as 1185



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Table 1. 1H and 13C NMR Shifts of Zearalenone-16-O-β-glucoside position 1 3 3-CH3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1′ 2′, 3′, 4′, 5′ 6′

1

H (ppm)

13

mult

C (ppm)

5.27 1.35 1.75, 1.59 1.77, 1.58 2.48, 2.28

m d, 6.3 Hz m m m

2.67, 2.26 2.00, 1.62 2.30, 2.04 6.00 6.33 6.65

m m m ddd, 15.5, 9.7, 4.4 Hz d, 15.5 Hz d, 1.7 Hz

6.61

d, 1.7 Hz

4.87 3.37−3.41 3.88 3.69

d m dd, 12.0, 1.5 Hz dd, 12.0, 4.8 Hz

170.0 73.1 20.3 36.1 22.3 44.8 214.3 38.7 22.8 32.6 134.4 130.1 107.5 161.2 104.0 157.1 117.4 138.5 102.9 78.4 (2), 75.0, 71.4 62.7

Figure 5. Distribution of zearalenone and its metabolites 24 h after incubation with 15.7 μM zearalenone with axenic cultures of seedlings of the barley cultivars ‘Morex’ and ‘Signora’. Shown are zearalenone and detected metabolites remaining in the medium (supernatant, abbreviated supern.) and extracted from the roots.

hypothesis will be tested with the new metabolite at hand in future in vitro interaction studies. Yeast expressing the HvUGT14077 gene allowed straightforward production of zearalenone-16-O-β-glucoside. The use of an ABC transporterdeficient strain increases the uptake of zearalenone23 but has the disadvantage of increasing sensitivity. Concentrations higher than 5 mg/L are toxic for the strain YZGA515. The conditions used for fermentation can still be optimized to increase the yield of glucosides, for example, by more sophisticated zearalenone administration regimes. The cleanup from large volumes of rather dilute fermentation broth is clearly a viable alternative to chemical synthesis. So far, we are only aware of one publication describing the chemical synthesis of zearalenone-16-O-β-glucoside, as a byproduct of synthesis of zearalenone-14-O-β-glucoside.24 Presumably, only small amounts were obtained, which were insufficient for structural

semiquantitative. In both cases very rapid hydrolysis of zearalenone-16-O-β-glucoside was observed and nearly all of the input was recovered as zearalenone within 30 min of incubation, regardless of the suspension medium (Table 2). No remaining zearalenone-16-O-β-glucoside was found in the spiked fecal slurry of any of the volunteers after 2 h.

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DISCUSSION

We elucidated that the UDP-glucosyltransferase HvUGT14077 can produce a mixture of the known masked mycotoxin zearalenone-14-O-β-glucoside (2) and the new zearalenone-16O-β-glucoside (3) in roughly equimolar amounts. The diminished response to zearalenone in the engineered yeast strain YZCP908 expressing this gene is a first indication that zearalenone-16-O-β-glucoside, like zearalenone-14-O-β-glucoside,13 no longer interacts with the estrogen receptor. This 1186



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Figure 6. Time course of zearalenone metabolization by suspension cultures Brachypodium distachyon Bd21 (a, b) and wheat (PC-998) (c, d). Initial concentration was 6.2 μM zearalenone.

Table 2. Hydrolysis of Zearalenone-16-O-glucoside (3) to Zearalenone (1) by Intestinal Bacteriaa t = 0.5 h medium

t=2h

sample

3 (nmol)

1 (nmol)

conversion (%)

3 (nmol)

1 (nmol)

conversion (%)

H2O

A B C D E F

0.02 0.00 0.11 0.00 0.00 0.00

2.34 2.21 1.90 1.78 1.86 1.62

113 106 92 86 90 78

0.00 0.00 0.00 0.00 0.00 0.00

2.29 1.85 2.38 1.87 2.01 2.51

110 89 115 90 97 121

Dall’Erta mix

A B C D E F

0.34 0.00 0.40 0.00 0.00 0.09

2.26 2.14 1.57 1.69 2.01 2.26

109 103 75 81 97 109

0.00 0.00 0.00 0.00 0.01 0.05

2.03 2.17 1.96 2.26 2.39 2.57

98 105 94 109 115 124

a

The input of 2.08 nmol of 3 was used as 100% to calculate conversion. Samples A−F represent the six feces donors. Values shown have a relative standard deviation of 11% for samples in water and 22% for the Dall’Erta fermentation mix,22 respectively.

characterization. However, the authors did test three commercially available zearalenone immuno-affinity columns with the compound “tentatively identified as zearalenone-16-Oβ-glucoside” and reported recoveries below 1%.23 It is therefore highly likely that zearalenone-16-O-β-glucoside escapes widely used antibody-based detection methods, one of the criteria for a “masked” mycotoxin. The yeast biocatalytical production and cleanup described in this work are straightforward, and with this approach it should be possible to produce the worldwide need of an analytical reference substance. It would also be

possible to produce larger amounts for future toxicological studies. Using the yeast-produced reference substance, we investigated zearalenone metabolization in two barley cultivars (‘Morex’ and ‘Signora’) and found that both preferentially convert zearalenone into the new glucoside (16−18 times more zearalenone-16-O-β-glucoside than zearalenone-14-O-β-glucoside was observed in roots). Because HvUGT14077 produces a near 1:1 mixture, the preferential accumulation of zearalenone16-O-β-glucoside in barley suggests that other UGTs exist in 1187



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therefore available for absorption. Zearalenone-16-O-β-glucoside fulfills the criteria of a masked mycotoxin. Studies on the natural occurrence and toxicological relevance are therefore warranted.

the barley genome that can nearly exclusively generate zearalenone-16-O-β-glucoside. UDP-glycosyltransferases, most of which are expected to be glucosyltransferases, are encoded in plant genomes by an extremely large family. The model plants A. thaliana and B. distachyon25,26 contain approximately 120 and 178 predicted UGTs, respectively.27,28 An alternative hypothesis is that the zearalenone-glucosides transiently observed are the result of a dynamic equilibrium, as the glucosides are rapidly metabolized into further compounds such as malonyl-gucoside and diglucoside and other unknown forms. Potentially, the speed of downstream reactions could be largely different for the two zearalenone-glucosides and different in various plant species. In zearalenone-treated Arabidopsis, zearalenone-14-Oβ-glucoside was found inside the plant transiently in high levels, but after 24 h, intracellular zearalenone-14-O-β-glucoside was decreased and much of the input was converted to unknown compounds.15 Zill et al.29 showed that in maize suspension cells treated with radiolabeled zearalenone 50% of the input was found in the nonextractable “insoluble residue”. In zearalenone-treated transgenic yeast, nearly all of the zearalenone-glucosides that are produced are efficiently transported across the plasma membrane by unknown transporters and released into the medium. In contrast, in the plant cell experiments only a small percentage of the zearalenoneglucosides was found in the medium; most of it is presumably stored in vacuoles. The soluble derivative is a “masked” mycotoxin if it is hydrolyzed by intestinal hydrolytic enzymes of the intestinal flora. In principle, two different types of hydrolytic enzymes could be relevant for reactivation of masked mycotoxins. Some bacteria contain either periplasmatic β-glucosidases (e.g., Sphingomonas30) or presumably cytosolic glycoside hydrolase family 1 members (e.g., the lactic acid bacterium Oenococcus31). Such enzymes can be easily expressed in Escherichia coli and are not sensitive to aerobic conditions or require cell integrity. The complex nutrient-rich incubation buffer used by Dall’Erta et al.22 to generate the fecal slurry should be unnecessary, or in the worst case some ingredients may even be inhibitory (e.g., glucose released from starch due to end product inhibition). Alternatively bacteria have phosphoenolpyruvate-dependent phosphotransferase systems, where uptake of β-glucosides is coupled with phosphorylation of the incoming glucose. After translocation, intracellular 6-phospho-β-glucosidases32 cleave the β-glycosidic bond, releasing 6-P-β-glucose, which is utilized by the bacteria. Avoiding oxidative damage of cells by maintaining strictly anaerobic conditions during resuspension and the incubation22 may be relevant in this case to allow efficient uptake. Yet phosphoenolpyruvate-dependent phosphotransferase systems for β-glucosides are generally transcriptionally repressed by the presence of preferred nutrients (as present in the Dall’Erta mix), but this process is expected to become evident only after longer incubation. We have not found evidence that resuspending fecal samples in water instead of the complex mixture described by Dall’Erta reduces the ability of fecal bacteria to hydrolyze zearalenone-glucosides. An advantage of resuspending the samples in water is that matrix effects are lower and the recovery rates much higher. In pilot experiments, we furthermore found that fecal samples stored for 24 h under aerobic conditions at 4 °C have essentially unchanged ability to hydrolyze zearalenone-14-O-β-glucoside (data not shown). Maintaining strict anaerobic conditions does not seem to be critical. In conclusion, one can assume that ingested zearalenone-16-O-β-glucoside is cleaved in the intestines and

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ASSOCIATED CONTENT

* Supporting Information S

Supplementary Figures S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(G.A.) E-mail: [email protected]. Phone: +43 147654 6380. Fax: +43 147654 1186. Present Addresses △

(M.P.K.P.) BIOMIN GmbH, Industriestraße 21, A-3130 Herzogenburg, Austria. ▽ (W.S.) Institute for Biotechnology in Plant Production, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna, Konrad Lorenz Straße 20, A-3430 Tulln, Austria. Funding

This work was funded by Austrian Science Fund (FWF) special research project F37 (F3706, F3708) and by funding from the U.S. Wheat and Barley Scab Initiative (U.S. Department of Agriculture, Agricultural Research Service) and the Minnesota Small Grains Initiative to G.J.M. Furthermore, we acknowledge the Federal Ministry of Economy, Family and Youth, the National Foundation for Research, Technology and Development, BIOMIN Holding GmbH. and Nestec Ltd. for funding the Christian Doppler Laboratory for Mycotoxin Metabolism. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Andreas Hartig and Fabian Istl (University of Vienna) for providing access to the fermentor and Carl Ng (University College, Dublin, Ireland) for generously providing the Brachypodium suspension culture. Franziska Löschenberger (Saatzucht Donau, Probstdorf, Austria) kindly provided barley seeds. We thank Johannes Leitner and Christoph Schüller for critically reading the manuscript.

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ABBREVIATIONS USED UGT, UDP-glucosyltransferase; TDI, tolerable daily intake; SC-LEU-HIS, synthetic complete lacking leucine and histidine; SCM, suspension culture medium; OD600, optical density at 600 nm

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REFERENCES

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