Metabolic Profile of Zearalenone in Liver ... - ACS Publications

Article Cite This: J. Agric. Food Chem. 2017, 65, 11292−11303

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Metabolic Profile of Zearalenone in Liver Microsomes from Different Species and Its in Vivo Metabolism in Rats and Chickens Using Ultra High-Pressure Liquid Chromatography-Quadrupole/Time-of-Flight Mass Spectrometry Shupeng Yang,†,‡,§,∥ Huiyan Zhang,‡,§,∥ Feifei Sun,§ Karl De Ruyck,‡ Jinzhen Zhang,† Yue Jin,† Yanshen Li,⊥ Zhanhui Wang,§ Suxia Zhang,§ Sarah De Saeger,‡ Jinhui Zhou,*,† Yi Li,*,† and Marthe De Boevre‡ †

Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Key Laboratory of Bee Products for Quality and Safety Control, Laboratory of Risk Assessment for Quality and Safety of Bee Products, Bee Product Quality Supervision and Testing Center, Ministry of Agriculture, Beijing 100093, People’s Republic of China ‡ Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium § China Agricultural University, College of Veterinary Medicine, Beijing Laboratory for Food Quality and Safety, Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing 100193, People’s Republic of China ⊥ College of Life Science, Yantai University, Yantai, Shandong 264005, P. R. China S Supporting Information *

ABSTRACT: To explore differences of zearalenone (ZEN) metabolism between various species, phase I and II metabolism by liver microsomes of animals and human were investigated using ultra high-pressure liquid chromatography-quadrupole/time-offlight mass spectrometry (UHPLC-Q/TOF MS). A total of 24 metabolites were identified, among which 12 were reported for the first time. Reduction, hydroxylation, and glucuronidation were the major metabolic pathways of ZEN, and significant differences in various species were also observed. Reduction was the main reaction in swine and human, whereas hydroxylation was predominant in rats, chickens, goats, and cows in in vitro systems. Furthemore, in vivo metabolism of ZEN in rats and chickens was investigated, and 23 and 6 metabolites were identified in each species, respectively. Reduction, hydroxylation, and glucuronidation were the major metabolic pathways in rats, while reduction and sulfation predominated in chickens. These results further enrich the biotransformation profile of ZEN, providing a helpful reference for assessing the risks to animals and humans. KEYWORDS: zearalenone, metabolism, biotransformation, phase I and phase II

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respectively.3,11 Additionally, some national governments set legal regulations/guidance for ZEN in specific foods and feeds, as in China and Japan.12 The toxicity of ZEN is highly associated with its metabolism in animals and humans.7,13 Drug metabolisms can significantly affect the pharmacokinetics and bioavailability of ZEN in the body, thereby affecting its exposure in the body. Toxicokinetics of ZEN in animals demonstrate that the mycotoxin is rapidly and extensively absorbed from the gastrointestinal tract (80− 85% in pigs).14 However, the oral bioavailability of ZEN is quite low: less than 10% in poultry and rats.15−18 The poor metabolic stability and extensive metabolism of ZEN could account for the phenomenon.7,19 In order to clarify the metabolic profile of ZEN, numerous in vitro and in vivo metabolism studies of ZEN have been carried out in animals and human.7,13 It has been demonstrated that liver, gastro-

INTRODUCTION Fusarium mycotoxins are secondary metabolites produced by colonies of various f ungal species on grains before harvesting but also postharvest under poor storage conditions.1 Up until now, more than 300 Fusarium mycotoxins have been identified, in which zearalenone (ZEN) is one with the most concern due to its frequent occurrence in cereals worldwide and high estrogenic activity. ZEN is mainly produced by F. graminearum, F. culmorum, F. equiseti, and F. verticillioides.2−4 Maize is the most prominent cereal with high incidence and concentrations of ZEN. However, other cereal crops, such as wheat, rice, oats, and barley are also susceptible to contamination with ZEN.5 Besides significant estrogenic effects, ZEN also exhibits immunotoxicity, genotoxicity, and reproductive toxicity.3,6,7 Furthermore, ZEN could stimulate growth of human breast cancer cells, indicating it has potential carcinogenicity for human.8−10 Owing to its high toxicity and exposure in contaminated food and feed, tolerable daily intakes (TDI) for ZEN have been established by the European Food Safety Authority (EFSA) and the Joint Committee FAO/WHO, at 0.25 μg kg−1 (b.w.) and 0.50 μg kg−1 (b.w.) per day, © 2017 American Chemical Society

Received: Revised: Accepted: Published: 11292

October 8, 2017 December 1, 2017 December 5, 2017 December 5, 2017 DOI: 10.1021/acs.jafc.7b04663 J. Agric. Food Chem. 2017, 65, 11292−11303

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Journal of Agricultural and Food Chemistry

human livers, suggesting the complex metabolism of ZEN.31−33 The hydroxylated sites existed not only on the aromatic ring but on the aliphatic positions as well. Hydroxylation was mainly catalyzed by rats (CYP2C6, CYP2C11, and CYP2C13) and human (CYP2C8, CYP3A4, and CYP3A5) enzymes.34 The analogues of ZEN, cis-ZEN and α-ZAL, could also be hydroxylated.35 The C-8, C-13, and C-15 position were the major metabolic reactive sites of ZEN in rats and human. However, the estrogen-like effects of these hydroxylated products, 8-OH-ZEN, 13-OH-ZEN, and 15-OH-ZEN, were less than that of ZEN, indicating that oxidation is a detoxification pathway for ZEN.20,31,32 In addition, the hydroxylation could also occur at the C-5, 9, and 10 positions of the aliphatic ring but not at the C-6 position.36 Even though significant differences have been observed in the metabolic profile of ZEN among animal species, a detailed clarification was not available. The published literature mainly focused on its reduction products, α-ZEL and β-ZEL, whereas information on the other two major detoxification ways (glucuronidation and oxidation) was limited.7,13,22 Many hydroxylated metabolites of ZEN produced by rat and human liver microsomes have been identified, but still no research was done in liver microsomes of other species to clarify the interspecies differences.13,36 Moreover, limited information is available for whether the hydroxylated metabolites of ZEN could be formed in vivo.36 In recent years, liquid chromatography coupled with high-resolution mass spectrometry (LC− HRMS), along with advanced data postprocessing techniques, have become common technologies in the field of metabolic studies and may now also effectively characterize the metabolic profile of ZEN.37−40 To clearly elucidate the various metabolic profiles of ZEN in different species, phase I and phase II in vitro metabolism of ZEN by rat, chicken, pig, sheep, cow, and human liver microsomes and its in vivo metabolism in rats and chickens were investigated using LC−HRMS. The results further enrich the published characterizations of ZEN and provide useful references for a better understanding of its biotransformation and risk assessment.

intestinal mucosal cells, and microbes are able to biotransform ZEN, with the liver playing a particularly vital role.2,7 Biotransformation of ZEN principally occurs via four major pathways, namely, reduction, oxidation, and conjugation with glucuronic acid and sulfate.7 α-Zearalenol (α-ZEL) and βzearalenol (β-ZEL) were the metabolites of ZEN discovered first, their formation being catalyzed by 3α- and 3βhydroxysteroid dehydrogenases (HSDs), respectively.7 Both of these reduction metabolites have different estrogenic activities, in which α-ZEL is almost 500-fold stronger in comparison to ZEN, while β-ZEL is 16 times lower than ZEN.20,21 Therefore, the pathway to produce α-ZEL is considered a toxic pathway whereas to form β-ZEL a detoxifying pathway.7,13 The rate and extent of α- or β-ZEL production significantly influences ZEN toxicity.7,13 The rate of α-ZEL and β-ZEL varies in different species: ZEN is predominantly converted into α-ZEL in rats and pigs, while for poultry, cows and sheep, β-ZEL is the dominant hepatic metabolite.22 In general, the yield of α-ZEL is five-folds higher than β-ZEL in pigs after oral dosage with ZEN.3,7 This is one of the most important reasons that pigs are the most sensitive animal species to ZEN.3,7,19,23,24 Besides hepatic activity, reduction could also occur among the intestinal mucosa and microbes, from which β-ZEL and α-ZEL are the predominant metabolites.7,13 There is also a minor reductive metabolic pathway of ZEN in animals that occurs at the C-11 double bond, and produces zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol(β-ZAL).4,7 Early studies have reported these three metabolites, but they were not commonly observed in the plasma, urine or feces of animals in later researches, indicating that reduction at C-11, and C-12 is only a minor metabolic pathway of ZEN.15,25 In addition, ZEN and its reduced metabolites could be efficiently conjugated with glucuronic acid (GlcA) and sulfate, which is catalyzed by uridine diphosphateglucuronosyltransferases (UGT) and sulfotransferase (SULT), respectively.26,27 In general, conjugation with GlcA is regarded as a detoxification reaction, since the glucuronide conjugates lack the biological activity with regard to oestrogenicity.20,28 Although the sulfate conjugates of ZEN show some oestrogenicity, the estrogenic activity of ZEN-14S is very low in comparison to ZEN.20 For ZEN, there are two hydroxyl groups at C-14 and C-16 which are conjugated with GlcA, while for α- or β-ZEL, three sites at C-7, C-14 or C-16 are prone to glucuronidation.27 In addition, it was found that glucuronidation preferentially occurs at the C-14 hyroxyl group, and a relatively limited amount of C-16-glucuronides was observed in animals and human.3,7,27 On the basis of in vitro and in vivo studies, glucuronidation is the dominant metabolic pathway of ZEN in mammals, especially for pigs and human, since almost all metabolites are present in conjugated forms in the plasma and urine after oral dosages of ZEN.7,13,23,29 ZEN conjugates significantly affected ZEN pharmacokinetics and excretion patterns in swine, and the conjugates produced by liver were excreted into the intestine through the bile. These conjugates are hydrolyzed into free forms by intestinal microbes and then reabsorbed by the intestine, resulting in two consecutive peaks in plasma concentration of ZEN after oral administration in swine: a typical enterohepatic circulation.14 In general, it may be maintained that reduction and conjugation are the two major metabolic pathways for ZEN.7,30 However, Pfeiffer et al. proposed that ZEN could be metabolized into many hydroxylated products by rat and

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MATERIALS AND METHODS

Reagents and Chemicals. The standards of ZEN, α-ZEL, and βZEL were purchased in powder form from Fermentek Ltd. (Jerusalem, Israel). β-Nicotinamide adenine dinucleotide phosphates (NADPH) was acquired from Roche Chemical Co. (Beijing, China), while 5′diphosphoglucuronic acid (UDPGA) was purchased from Sigma Chemical Co. (Beijing, China). Pooled rat and human liver microsomes were purchased from the RILD Research Institute for Liver Diseases (Shanghai, China) and stored at −80 °C until use. Acetonitrile, methanol, and formic acid were purchased from Fisher Chemical Co. (New Jersey), all of LC−MS grade. Water was purified using a Milli-Q system (Millipore, MA). All other chemicals and reagents were of the highest analytical grade available. Animals. Six male and six female Wistar rats (weight, 0.18−0.20 kg) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Six male and six female Avian chickens (weight, 1.0−1.2 kg) were purchased from Beijing Huadu Co. Ltd. (Beijing, China). All animals were housed in an animal room with standardized temperature (25−28 °C), humidity (50−60%), and a 12 h light/dark cycle. The experiments started after 1 week of acclimatization allowing the animals to adapt to the environment. During the whole experiment, the animals were fed with commercial standard diets, and given water ad libitum. Additionally, the animal trial had been approved by the Animal Care Center of the China Agricultural University (2016-SYXK-0042). All care and handling of 11293

DOI: 10.1021/acs.jafc.7b04663 J. Agric. Food Chem. 2017, 65, 11292−11303

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Journal of Agricultural and Food Chemistry animals was performed with approval of the Institutional Authority for Laboratory Animal Care. Microsomal Incubations of ZEN. Chicken, swine, goat and cow liver microsomes adopted in the present study were prepared in house as described by Yang et al., which were stored at −80 °C prior to the incubation assays.37−39 For the phase I metabolism trail, animals and human liver microsomes were separately incubated with ZEN under the same conditions. The incubation reactions were initiated by adding liver microsome proteins (0.5 mg mL−1) to a solution of ZEN (15.81 μM) and NADPH (1 mM) in Tris-HCl buffer (500 mM, pH 7.4). The total volume of the incubation was 500 μL. The control incubations were performed in the absence of ZEN or NADPH. After incubation for 2 h at 37 °C on a mechanical shaker, the reaction was terminated by adding 500 μL of ice-cold acetonitrile. After thorough mixing by shaking and centrifugation at 12 000g for 15 min, the clear supernatant was analyzed by UPLC-Q/TOF MS. All experiments were conducted in triplicate. In parallel, phase I metabolism experiments on α-ZEL and β-ZEL with liver microsomes from various species were also conducted. For phase II metabolism experiments, the incubation systems were performed as described above, except that the NADPH was replaced by UDPGA (3.8 mM). For these, sample preparation was also carried out as per the phase I trials. Oral Gavage of ZEN to Rats and Chickens. After 1 week of acclimatization, all animals were deprived of feed for 12 h before the start of the experiment, but water was provided ad libitum. Subsequently, ZEN was administered to rats and chickens at a single dose of 5.0 mg kg−1 b.w. by oral gavage. The standard of ZEN was dissolved in ethanol/water (20/80, v/v). The same volume of ethanol/ water (20/80, v/v) without any mycotoxin was orally administered to control group chickens. Following administration of the mycotoxin, urine and fecal samples were collected according to the following time frame: 0−24 h, 24−48 h, and 48−72 h. All samples were frozen at −20 °C until analysis. Rats’ urine samples were centrifuged at 12 000 g at 4 °C for 15 min, then, 2 mL clear supernatant was purified using Oasis HLB cartridge (3 mL, 60 mg). First, the cartridges were preconditioned with 3 mL of methanol, followed by 3 mL of water. Subsequently, sample extracts were loaded onto the HLB cartridge and washed by 3 mL of methanol/water (5/95, v/v). After drying, analytes were eluted from the cartridge by 3 mL of methanol, then the elution was evaporated under a gentle stream of nitrogen and reconstituted with 1 mL of acetonitrile/water (15/85, v/v). As for the fecal samples of rats and chickens, they were separately homogenized before weighing. To 2 g of fecal samples, 10 mL of ethyl acetate was added and the mixture was shaken for 5 min. Then, the samples were centrifuged at 12 000g at 4 °C for 15 min. The organic layer was separated and evaporated to dryness under a stream of nitrogen at 40 °C. Afterward, the residue was redissolved in 0.5 mL of acetonitrile/ water (15/85, v/v), followed by a dilution to 4.5 mL with water. The obtained solution was also purified using an HLB cartridge for purification as mentioned above. Instrumental Conditions. For the identification of ZEN metabolites, an Acquity UPLC system (Waters Co., Milford, MA) coupled to a hybrid Q/TOF-MS SYNAPT HDMS (Waters, Manchester, U.K.) was used. The raw data were acquired and processed by Masslynx, MetaboLynxXS software (version 4.1), and MassFragment software. The MS was performed in the negative ionization mode (ESI−). The capillary and cone voltage were 3.0 kV and 35 V, respectively. The desolvation gas (N2) flow rate was 600 L h−1 at 300 °C, and the source temperature was 100 °C. Leucine enkephalin was used as an external reference at m/z 554.26145 in ESI−. The survey scan mass range was acquired from 200 to 800 Da (centroid) during acquisition. The collision energy was ramped from 10 to 40 eV to the [M − H]− of all potential ZEN metabolites. A gradient elution consisting of mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) was adopted for separating ZEN metabolites, which was pumped at a flow rate of 0.3 mL min−1. For phase I metabolism in vitro and in vivo, the separation was performed on an Acquity HSS T3 column (100 mm × 2.1 mm, 1.7 μm). The gradient elution program was performed as follows: 0−1.0 min, 25% B; 1.0−11.0 min, 25% B to 50% B; 11.0−12.0

min, 50% B to 70% B; 12.0−13.0 min, 100% B; and 13.0−15.0 min, 25% B. In addition, the Acquity BEH RP18 column (50 mm × 2.1 mm, 1.7 μm) was used for phase II metabolism of ZEN in vitro, and the gradient elution program was performed as follows: 0−2.0 min, 15% B; 2.0−12.0 min, 15% B to 50% B; 12.0−13.0 min, 50% B to 100% B; 13.0−14.0 min, 100% B; and 14.0−15.0 min, 15% B. The injection volume was 10 μL.

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RESULTS AND DISCUSSION Fragmentation Pattern of ZEN. During electrospray ionization, ZEN is prone to form the deprotonated ion m/z 317.1393 ([M − H]−, C18H21O5−) with a high response value, since there are two phenolic hydroxyl groups in its chemical structure. The lactone group of ZEN (m/z 317.1393) is susceptible for fragmentation under collision induced dissociation (CID) to form fragment ions m/z 299.1280 and m/z 273.1497 by the loss of H2O (18.0106 Da, error = 0.7 mDa) or CO2 (43.9898 Da, error = −0.2 mDa), respectively. The αbond on C-7 of the fragment ion m/z 273.1497 is further dissociated to form the product ion, m/z 203.0708, by losing C5H10 (70.0783 Da, error = 0.6 mDa). The presence of an octatomic ring was proposed in the structure of m/z 203.0708. Because of its instability, the octatomic ring was likely to form product ions of m/z 175.0392 and m/z 187.0393 under CID by the loss of 28 and 16 Da, respectively. A loss of 28 Da may be due to the loss of CO or C2H4. On the basis of the error of accurate mass (36.7 mDa error for CO; 0.3 mDa error for C2H4) provided by high-resolution mass spectrometry, we concluded that 28 Da was due to the loss of C2H4, which could verify our assumption mentioned above to some extent. Similarly, the neutral loss of 16 Da was identified as CH4 (error = 0.2 mDa) not O (error = 36.6 mDa). As shown in Figure 1, we observed the product ion m/z 175.0392 was the base peak, which also verified the aforementioned proposal. In addition, on the basis of the product ion m/z 175.0392, the product ions m/z 131.0489, m/z 147.0443, and m/z 160.0158 were formed by the loss of CO2 (43.9898 Da, error = 0.5 mDa), CO (27.9949 Da, error = 0.1 mDa), and CH3 (15.0235 Da, error = 0.1 mDa), individually. The detailed fragmentation process along with the chemical structure of each product ion is described in Figure 1. Structural Characterization of ZEN Metabolites Observed in Vitro. After ZEN was incubated with the liver microsomes of rats, chicken, swine, goats, cows, and humans for 2 h, ice-cold acetonitrile was added into the incubation medium to precipitate proteins. The sample was determined by UHPLC-Q/TOF MS followed by high-speed centrifugation at 4 °C. The acquired data was further analyzed by the postacquisition data mining software MetabolynxXS. As a result, a total of 20 metabolites were identified. The representative EICs chromatogram (with mass window of 0.05 Da) and MS/ MS spectra of ZEN phase I metabolites are shown in Figure 2 and Figure S1. Detailed information about the accurate mass, retention time, and typical fragment ions of each metabolite is listed in Table 1. As for the chemical structures of individual metabolites, they are described as follows. Hydrogenation Metabolites MZ-1 and MZ-2. In comparison with the standards of α-ZEL and β-ZEL, MZ-1 and MZ-2 have the same MS/MS spectra and retention times. Therefore, MZ-1 was confirmed to be β-ZEL, while MZ-2 was α-ZEL. Oxidation Metabolites MZ-3 to MZ-20. MZ-3 to MZ-20 shared the same accurate mass values at m/z 333.1338 ([M − 11294


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dissociation of the lactone ring would be significantly influenced if it was on C-2, because an internal hydrogen bonding would delay the observed retention time. Thus, observing from the MS/MS spectra retention time of MZ-3 and MZ-4, these were temporarily identified as 3-OH-ZEN and 2OH-ZEN, individually. Although the retention time of MZ-15 (8.12 min) and MZ20 (10.16 min) differed, they possessed similar MS/MS spectra, indicating that they could be potential epimers. According to their MS/MS spectra, we could observe an apparent H2O loss, and the fragment ion (m/z 191) was 16 Da higher than m/z 175 (ZEN). Therefore, the hydroxylation position of MZ-15 and MZ-20 were deduced to be on C-9 to C-12, inclusive. Consequently, taken together with the fact that a double bond may be epoxidized to form a pair of isomer, MZ-15 and MZ-20 were temporarily identified as 11, 12-endo/exoepoxidized-ZEN. As for the six pairs of epimers (MZ-6 and MZ-9, MZ-7 and MZ-13, MZ-12 and MZ-16), they all had the fragment ions of m/z 187, m/z 175, and m/z 131 in their MS/MS spectra, which also appeared in the MS/MS spectrum of ZEN, indicating that the hydroxylation position would be on C-4, C-5, or C-6. If the hydroxylation occurred on C-6, the internal hydroxyl bond would significantly decrease its polarity, consequently delaying the retention time. Thus, taking the retention time and MS/MS spectra into consideration, the hydroxylation positions of MZ12 and MZ-16 would be on C-6. In the 6 epimers, there were fragment ions m/z 187, 175, and 131 in the MS/MS spectra of metabolites MZ-6 and MZ-9, MZ-7 and MZ-13, and MZ-12 and MZ-16, respectively. This is the same as the fragment ions in ZEN, suggesting that the hydroxylation should be at position C-4, C-5, or C-6. Considering the molecular structure of ZEN, C-6 or C-8 hydroxylation may easily form intramolecular hydrogen bonds with the adjacent carbonyl group on C-7, thus forming a pair of epimeric structures. The polarity of the molecule is weakened, and the retention time is delayed. Compared with the other four major metabolites, the retention time of MZ-12 and M-16 is the longest, and it is speculated that these two may possess C-6 hydroxylation. Hildebrand et al. reported that ZEN could be extensively hydroxylated by rat liver microsomes, and approximately 10 hydroxylated metabolites were identified.36 By aligning the abundance of the major fragment ions in reported MS/MS spectra with those in the present study, MZ-7 and MZ-13 were identified as hydroxylated products at the C-5 position and MZ-6 and MZ-9, MZ-7 and MZ-13, MZ-12 and MZ-16, MZ-8 and MZ-17, MZ-5 and MZ-10, MZ-11 and MZ-14 were considered as 4-OH-ZEN, 5OH-ZEN, 6-OH-ZEN, 8-OH-ZEN, 10-OH-ZEN, and 9-OHZEN, respectively.36 Glucuronidation Metabolites MZ-21, MZ-22, MZ-23, and MZ-24. UDPGA (3.8 mM) was added into the in vitro microsomal incubation test that mimics glucuronidation in vivo. ZEN was incubated with liver microsomes of livestock and humans. Then the samples were acquired by UHPLC-Q/TOF MS and analyzed in MetabolynxXS software. As a result, a total of four phase II metabolites were discovered. EICs and MS/MS spectrum of the four metabolites are shown in Figures S1 and S2. Additionally, the accurate mass, retention time and typically observed fragments are listed in Table 1. The accurate masses of MZ-21 and MZ-22 were m/z 493.1679 ([M − H]−, C24H29O11−), which is 176 Da higher than the parent molecule, ZEN, indicating that these two metabolites would be the conjugates of glucuronic acid with ZEN. In the MS/MS spectra, we observed the typical neutral

Figure 1. Accurate MS/MS spectrum of ZEN and its proposed fragmentation pathways under the condition of CID in ESI− mode.

H]−, C18H23O6−), which was 16 Da higher than that of ZEN, however, their retention times varied from 3.70 to 10.16 min. The gained 16 Da indicated that these metabolites could potentially be monohydroxylated or oxidized metabolites. Because of the similar chemical polarities of some metabolites (MZ-3 and MZ-4; MZ-6, MZ-7, and MZ-8; MZ-9, MZ-10, and MZ-11; MZ-16 and MZ-17; MZ-18 and MZ-19), they could not be completely separated. As for MZ-18 and MZ-19, their retention time was 8.75 and 8.90 min, respectively, but they had the same product ions, m/z 305, 289, 191, 176, and 148. From the product ions, we could apparently know that m/z 191 and m/z 176 were 16 Da higher than the corresponding ions of ZEN, m/z 175 and m/z 160, respectively, suggesting that hydroxylation would be present in the aromatic or lactone ring. In addition, the MS/MS spectra showed that MZ-18 and MZ19 is not susceptible to losing H2O (18 Da). On the basis of the polarity of MZ-18 and MZ-19, along with published literature supporting the hydroxylation of ZEN on its aromatic ring, we could conclude that MZ-18 and MZ-19 were representative of 13-OH-ZEN and 15-OH-ZEN, respectively.32,36 Except for MZ-15, for metabolites MZ-3 to MZ-17, a significant loss of H2O (18 Da) was observed in their MS/MS spectra. This indicated that the hydroxylation should be located in the lactone ring of ZEN, except at the position of the double bond located between C-11 and C-12. Given the chemical structure of ZEN and MS/MS spectra of MZ-5 and MZ-10, MZ-6 and MZ-9, MZ-7 and MZ-13, MZ-8 and MZ-17, MZ-11 and MZ-14, MZ-12 and MZ-16, we were easily led to infer that the hydroxylation positions should be on the number C-4, C-5, C-6, C-8, C-9, and C-10 carbon atoms. Thus, the hydroxylation position of MZ-3 and MZ-4 should be on C-2 and C-3. We hypothesize that the hydroxylation was on C-3. The 11295


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Figure 2. Representative extracted ion chromatograms (EICs, the extraction window is 50 mDa) of ZEN phase I metabolites (left, reduction metabolites M1 and M2; right, oxidation metabolites M3−M20) detected in rat, chicken, swine, goat, cow, and human liver microsomes.

conjugated in C-16.27 Hence, MZ-21 and MZ-22 were identified to be ZEN-16-GlcA and ZEN-14-GlcA, respectively. MZ-23 and MZ-24 shared the same observed ion peak m/z of 669.2031 ([M − H]−, C30H37O17−), which is 352 Da larger than the original ZEN, suggesting that they were conjugates of ZEN with a double GlcA. In the MS/MS spectrum of MZ-23, two typical neutral losses of 176 Da were observed whereas

loss of GlcA. Given the two phenolic hydroxyl groups of ZEN, considered along with the retention times of these two metabolites (5.54 and 6.95 min), we could initially surmise that if the conjugation position is at the C-16, the retention time would be shortened, relative to C-14 conjugation, due to the destroyed intramolecular hydrogen bonds when GlcA is 11296


11297

319.1546 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 333.1338 493.1710 493.1710 669.2031 669.2031 509.1659 495.1866 495.1866 397.0957 399.1114

C18H23O5− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C18H21O6− C24H29O11− C24H29O11− C30H37O17− C30H37O17− C24H29O12− C24H31O11− C24H31O11− C18H21SO8− C18H23SO8−

β-ZEL/α-ZEL 3-OH-ZEN 2-OH-ZEN 10-OH-ZEN 4-OH-ZEN

5-OH-ZEN

8-OH-ZEN

4-OH-ZEN

10-OH-ZEN 9-OH-ZEN 6-OH-ZEN

5-OH-ZEN

9-OH-ZEN epoxy-ZEN 6-OH-ZEN

8-OH-ZEN

13-OH-ZEN 15-OH-ZEN epoxy-ZEN ZEN-16-GlcA ZEN-14-GlcA ZEN-14,16-di-GlcA ZEN-14-2GlcA OH-ZEN-GlcA β/α-ZEL-16-GlcA β/α-ZEL-14-GlcA ZEN-14-SO3H β/α-ZEL-14-SO3H

MZ-1/2b MZ-3 MZ-4 MZ-5 MZ-6b

MZ-7

MZ-8

MZ-9b

MZ-10 MZ-11b MZ-12b

MZ-13b

MZ-14 MZ-15 MZ-16b

MZ-17

MZ-18 MZ-19b MZ-20 MZ-21b,c MZ-22b,c MZ-23b,c MZ-24c MZ-25−MZ-31b MZ-32/MZ-34b MZ-33/MZ-35b MZ-36b MZ-37/MZ-38b

−0.9 0.4 0.8 −0.5 −0.9 −1.2 −1.5 −1.3−1.8 0.6/-1.1 −1.1/0.9 −0.6 −1.4

−1.4

0.5 −1.3 1.1

0.8

0.4 1.3 −1.2

−0.5

−1.2

−0.8

1.4/0.9 −1.1 0.7 −0.3 0.9

1.3

error (ppm)

8.75 8.90 10.16 5.55 6.95 3.96 7.43 2.05−2.94 4.57/4.24 6.26/5.23 7.32 5.78/6.32

8.39

7.87 8.15 8.29

7.53

7.06 7.14 7.32

6.93

6.75

6.69

7.91/9.50 3.70 6.02 6.25 6.60

11.92

retention time (min) rat

√ √ √ √ √ ND √

√

√ ND ND

√

√ ND √

√

√

√

√ √ √ √ √

√

chicken

√ √ √ √ √ √ ND

√

√ ND √

√

√ ND √

√

√

ND

√ √ √ √ √

√

swine

√ √ √ √ √ √ √

√

√ ND √

√

ND √ √

√

√

ND

√ √ √ √ √

√

goat

√ √ √ √ √ ND √

√

√ √ √

√

√ √ √

√

√

ND

√ √ √ √ √

√

cow

observed in excreta of chickens after oral dose ZEN

√ √ √ √ √ √ ND

√

√ √ √

√

√ ND √

√

√

ND

√ √ √ √ √

√

human

observed in urine and feces of rats after oral dose ZEN

√ √ ND √ √ ND ND

√

√ ND ND

√

√ √ √

√

ND

√

√ √ √ √ √

√

liver microsomes major fragments 299, 289, 273, 261, 203, 187, 175d, 160, 147, 131 301, 291, 275, 257, 213, 188, 174, 160d, 130 305d, 287, 277, 191, 175, 163, 135 305d, 287, 261, 243, 205, 174, 160 315, 289, 271, 219, 191, 175, 163, 161d, 133 315, 289, 271, 227, 203, 187, 175d, 161, 149, 131 315, 297, 271, 245d, 227, 217, 201, 189, 187, 175, 149,131 315, 305, 289, 271, 247, 203, 201, 189, 187d, 175, 159 315, 289, 271, 227, 203, 187, 175d, 161, 149, 131 315, 289, 271, 219, 191, 175, 163, 161d, 133 315, 271, 247, 203d, 175, 161, 160, 131 315, 289, 271, 247, 245, 216, 187, 175d, 161, 149, 131 315, 297, 271, 245d, 227, 217, 201, 189, 187, 175, 149,131 315, 271, 247, 203d, 175, 161, 160, 131 305d, 289, 261, 220, 191, 176 315, 289, 271, 247, 245, 216, 187, 175d, 161, 149, 131 315, 305, 289, 271, 247, 203, 201, 189, 187d, 175, 159 305, 289, 265, 191d, 190, 176, 148, 146 315, 305, 289, 201, 191, 190, 176d, 148 305, 289, 191, 175, 160, 147d, 119 449, 359, 317d, 273, 175, 113 475, 317d, 273, 175, 113 651, 493d, 449, 317, 273, 175, 113 631, 607, 351d, 317, 193 333d, 303, 289, 187 175, 113 451, 433, 361, 319d, 275, 175, 113 477, 361, 319d, 275, 175, 113 317d, 273, 203, 175, 149 381, 355, 319d, 301, 251

a The [M − H]− (m/z) values were calculated from the proposed structural formulas. The error (ppm) is the difference between the calculated and observed m/z values. bThese metabolites were observed in vivo of rats and chicken. cThese metabolites observed in vitro were separated using the Acquity BEH RP18 column (50 mm × 2.1 mm, 1.7 μm), and thus, their retention times were very different from others metabolites. dThe base peak in the MS/MS spectra; √, detected; and ND, not detected.

317.1389

C18H21O5−

[M − H] (m/z)

composition

ZEN

metabolites

ZEN

no.

−

Table 1. Summary of ZEN Metabolites Detected in The Liver Microsomes from Various Animals and Humans and in Vivo of Rats and Chickensa

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Figure 3. Proposed metabolic pathway of ZEN in the liver microsomes of five animal species and human. Reduction, hydroxylation, and glucuronidation were the main metabolic pathway of ZEN in vitro systems.

fragment m/z 351 was observed for MZ-24, suggesting that two GlcA were bound together for MZ-24. The two hydroxyl groups of ZEN at C-14 and C-16 were considered most likely to bind GlcA. However, considering the yield of ZEN-14-GlcA (MZ-22) was significantly larger than that of ZEN-16-GlcA (MZ-21) in liver microsomes system, we assume that MZ-24 should be the product of GlcA binding to ZEN at C-14, and thus MZ-24 was regarded as ZEN-14-(GlcA)2. Characteristic fragments in the MS/MS spectrum of MZ-23 at m/z 493 and 317 were also observed, indicating that the two GlcA would be at two different hydroxylated sites of the ZEN melecule. Hence, MZ-23 was identified as ZEN-14,16-di-GlcA. Metabolite Profile of ZEN Observed in Vivo of Rats and Chickens after Oral Dose. On the basis of the in vitro metabolic results, we conducted an in vivo metabolism of ZEN in rats and chickens. ZEN was administrated to these two animals at a dosage rate of 5.0 mg kg−1 b.w. Then, urine, plasma and fecal samples were collected, extracted, and cleaned up for analysis by UHPLC-Q/TOF MS. As a result, 23 and 8 metabolites were detected in samples from rats and chicken,

respectively. The EICs and MS/MS spectra are listed in Figure S3 and Figure S1. Also, the analysis of each metabolite was described as follows. Phase I Metabolites of ZEN Detected in Vivo of Rats and Chickens. A total of 9 and 2 phase I metabolites were observed in vivo for rats and chickens, respectively. This was also observed in vitro: α-ZEL and β-ZEL as well as seven hydroxylated metabolites MZ-6 and MZ-9 (5-OH-ZEN), MZ11 (9-OH-ZEN), MZ-12 and MZ-16 (6-OH-ZEN), MZ-13 (5OH-ZEN), and MZ-19 (15-OH-ZEN). Structural analysis for these metabolites has been fully described above. Phase II Metabolites of ZEN Detected in Vivo of Rats and Chickens. Compared with the phase II metabolism in vitro, three GlcA binding products (MZ-21, MZ-22, and MZ23) were also detected in rats. Furthermore, another 11 conjugates were identified in rats. Hydroxylated and GlcA Binding Products MZ-25 to MZ-31. Metabolites MZ-25 to MZ-31 had the same accurate mass of m/z 509, which was 176 Da higher than OH-ZEN, indicating that they were likely to be the GlcA binding 11298


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Figure 4. Relative content of phase I metabolites of ZEN after incubation with liver microsomes from rat, chicken, swine, goat, cow, and human at 37 °C for 120 min. The error bars represent standard deviations of the mean (n = 3).

was the sulfate conjugate. There are two phenolic hydroxyl groups for ZEN to react with sulfate, but the phenolic hydroxyl group at C-14 was easier to conjugate with sulfate than C-16, due to the influence from lactone groups. Thus, MZ-36 was temporarily identified as ZEN-14-SO3H. MZ-37 and MZ-38 possessed the same accurate mass, m/z 399, which was 80 Da higher than that of α-/β-ZEL, indicating that they represented their respective sulfate-bound forms. In addition, the characteristic neutral loss, 80 Da, as well as the typical fragment ions, m/ z 399 and m/z 319, verified that they were sulfate-binding metabolites. Given the likelihood of reaction at C-14 and the polarity, MZ-37 and MZ-38 were identified as β-ZEL-14-SO3H and α-ZEL-14-SO3H, respectively. Metabolic Pathways of ZEN in Animals and Human Liver Microsomes. On the basis of the metabolite analysis described above, the major metabolic pathways of ZEN by animals and human liver microsomes are regarded as reduction, hydroxylation, and glucuronidation. Phase I and II metabolic pathways of ZEN in vitro are illustrated in Figure 3. The reduction of ZEN was mainly generated at the carbonyl of C-7, producing α-ZEL and β-ZEL. The hydroxylation can occur mainly at C-13 and C-15 as well as at C-2, C-3, C-4, C-5, C-6, C-8, C-9, and C-10 of the aliphatic ring. The binding sites with GlcA were at C-14 and C-16. In addition, significant metabolic differences in species and yields were observed. To compare metabolic differences, each metabolite in the EICs observed from phase I and II systems, was integrated and plotted into Figure 4 and Figure 5, respectively. Figure 4 illustrates that αZEL, 8-OH-ZEN, 9-OH-ZEN, 13-OH-ZEN, and 15-OH-ZEN are the dominant metabolites of ZEN and have a high yield in different species and humans. Moreover, in swine and human, the main reaction of ZEN was reduction, followed by hydroxylation, whereas in rats, chickens, goats and cows, the main reaction was hydroxylation, followed by reduction. In comparison with β-ZEL, α-ZEL has been demonstrated as the dominant metabolite and had a larger relative yield in human and animals, especially in rats, chickens, swine, and human. Many studies have explored the interspecies metabolic differences of ZEN. In rats, swine, goats, and cows, the yield of α-ZEL was much higher than that of β-ZEL, which was consistent with the present study.3,7,13,22,29 However, in chickens, the yield of α-ZEL was often less or equal to that of β-ZEL, which was quite different from the observations made

conjugates with OH-ZEN. The retention times of these seven metabolites were 1.26 min, 2.10 min, 2.26 min, 2.41 min, 2.65 min, 2.80 min, and 2.95 min, respectively, suggesting that they were isomers. In addition, the MS/MS spectra of these seven metabolites were all similar and characteristically simple, with only signals of m/z 509 and m/z 333 observed. However, because of low absolute response, it was difficult to acquire reliable MS/MS spectra for m/z 333. Thus, it was difficult to identify the hydroxylated position along with the conjugative positions. Reductive GlcA Conjugates MZ-32 and MZ-35. Metabolites MZ-32, MZ-33, MZ-34, and MZ-35 shared the same accurate mass, observed as m/z 495.1866 ([M − H]−, C24H31O11−), which was 176 Da higher than α-ZEL or β-ZEL. However, their retention time varied, implying that they are GlcA conjugates of α-ZEL or β-ZEL. As for their MS/MS spectra, they were almost the same with the neutral loss of 176 Da, which further demonstrated they were GlcA conjugates. However, it was still unable to identify their chemical structures. To differentiate the conjugated positions, phase II metabolism of α-ZEL and β-ZEL was investigated. Consequently, MZ-33 and MZ-35 were only observed in the metabolic system incubating α-ZEL, while MZ-32 and MZ-34 were only observed after metabolism of β-ZEL. According to the results of phase II metabolism of α-ZEL, we inferred that MZ-33 and MZ-35 were GlcA binding compounds. α-ZEL has three hydroxyl groups (C-7, C-14, and C-16). Moreover, the MS/MS spectrum of a GlcA conjugate at C-7 differed from those of C-14 and C-16. Thus, MZ-35 and MZ-33 are representative of GlcA binding to C-14 or C-16. If GlcA binds with the hydroxyl group at C-16, the intramolecular hydrogen bonds would be destroyed, and the final conformation would exhibit enhanced polarity. Therefore, MZ-33 and MZ-35 were identified as α-ZEL-16-GlcA, and α-ZEL-14-GlcA, respectively. Similarly, MZ-32 and MZ-34 were regarded as β-ZEL-16-GlcA and β-ZEL-14-GlcA, respectively. Sulfate Binding Compounds MZ-36, MZ-37, and MZ38. In addition to reactions forming GlcA conjugates, ZEN is also able to bind with sulfate. Three sulfation metabolites were detected in chickens’ feces. The mass of MZ-36 was m/z 397, which was 80 Da higher than that of ZEN, suggesting that MZ36 can be a sulfation compound. In addition, typical fragment ions, m/z 397 and m/z 317, further demonstrated that MZ-36 11299


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the yield of ZEN-16-GlcA was apparently higher, implying interspecies differences for glucuronidation pathways. MZ-23, detected in swine, goats, and cows, was considered to represent the combination of GlcA binding metabolites at C-14 and C-16. The amount of MZ-23 was almost equal to that of ZEN-16GlcA, suggesting that the GlcA binding reaction is prone to occur in swine. Unlike MZ-23, MZ-24, only detected in ruminant animals such as goats and cows, was generated by binding GlcA at C-14, followed by another GlcA binding on the basis of the characteristic binding motif. Little information was available regard to the double GlcA binding reaction.43 However, this phenomenon further enriched the GlcA binding reaction, so the unique GlcA binding reaction in ruminants was advanced. Correlation of ZEN in Vitro and in Vivo Metabolism in Rats and Chickens. Results from the in vitro metabolism indicated that hydroxylation was a major metabolic pathway for ZEN, which needed to be demonstrated in vivo. Therfore, in the present study, metabolism of ZEN in living rats and chickens was conducted. The results showed that reduction, hydroxylation, and glucuronidation were major metabolic pathways in rats, but reduction and sulfation were principal pathways in chickens. The in vivo metabolic pathways of ZEN are shown in Figure 6. Metabolites in urine and feces accumulated during 24−48 h after administration, while limited metabolites were detected in samples collected during the 0−12 h, 12−24 h, and 48−72 h periods. This observed phenomenon might be explained by the enterohepatic circulation. α-ZEL was the dominant product of ZEN in rats, which was consistent with the in vitro metabolic results. However, in chickens, the yield of α-ZEL and β-ZEL was similar, which was significantly different from that of in vitro results. In addition to reduction reaction, the hydroxylation sites of ZEN in rats were regarded as the C-4, C-5, C-6, C-9, and C-15 positions. For the hydroxylated metabolites of ZEN, the in vitro and in vivo results varied. 8-OH-ZEN, 13-OH-ZEN, and 15-OH-ZEN were the dominant metabolites of ZEN from rat liver microsomes but in living rats are different: 4-OH-ZEN and 5-OH-ZEN. The hydroxylation in liver, intestinal mucosa, and microbial flora of rats are likely the cause for these differences. ZEN could be hydroxylated in chicken liver microsomes, but no hydroxylated metabolite was identified in vivo. A total of 14 GlcA binding products were detected and identified in the urine of rats. Results indicated that glucuronidation occurred in the reduced and hydroxylated metabolites of ZEN. This phenomenon was not observed in chickens. Except for the reported ZEN-14GlcA, ZEN-16-GlcA, α-ZEL-14-GlcA, α-ZEL-16-GlcA, β-ZEL14-GlcA, and β-ZEL-16-GlcA, glucuronidation further occurred on these hydroxylated metabolites. ZEN-14,16-di-GlcA, observed in vitro, was also identified in the urine of rats. The glucuronidation of ZEN along with its reduction products (at C-14) was demonstrated in the published data. In conclusion, the phase I and II metabolism of ZEN in rat, chicken, swine, goat, cow, and human liver microsomes were investigated and compared using UHPLC-Q/TOF MS. As a result, reduction, hydroxylation, and glucuronidation were the major metabolic pathways of ZEN, and significant differences in various species were also observed. However, α-ZEL, 8-OHZEN, 13-OH-ZEN, 15-OH-ZEN, and ZEN-14-GlcA are the dominant metabolites in in vitro systems. In addition, ZEN, αZEL, and β-ZEL can be mutually transformed by liver microsomes. Furthermore, in vivo metabolism of ZEN showed that reduction, hydroxylation, and glucuronidation were the

Figure 5. Comparative the formation of glucuronidation metabolites of ZEN after incubation with liver microsomes from rat, chicken, swine, goat, cow, and human at 37 °C for 120 min. The error bars represent standard deviations of the mean (n = 3).

in this study.15 The reduction reaction of ZEN could be mediated by not only enzymes in liver but also by the intestinal mucosa and microbial flora. Unlike reduction in the liver, βZEL is the dominant metabolite in intestinal microsomes.3 In the present study, we found that hydrogenation and dehydrogenation of ZEN in the liver was a reversible process, namely, ZEN, α-ZEL and β-ZEL could be mutually transformed (Figure S4 and S5). In liver microsomes, α-ZEL or βZEL can be directly oxidized into ZEN in the absence of NADPH. Earlier literature has reported that ZEN, α-ZEL, and β-ZEL could be mutually transformed in erythrocytes but neglected the similar phenomenon in liver.41 Hildebrand et al. and Pfeiffer et al. reported that the hydroxylation sites of ZEN in rats and human liver microsomes were at C-8, C-9, C-10, C-13, and C-15.32,33,36,42 Apart from these sites, we found new hydroxylated sites at C-2, C-3, C-4, C-5, C-11, and C-12. In addition, the results demonstrated that hydroxylation of ZEN occurred not only in rats and human but also in chickens, swine, goats, and cows, with significant metabolic differences. Figure 4 shows that 13-OH-ZEN, 15OH-ZEN, and 8-OH-ZEN were the more abundant species, which is in accord with the literature.32,36,42 Other hydroxylated metabolites were observed at trace levels, which might account for the limited metabolites by Hildebrand et al. and Pfeiffer et al.32,36,42 4-OH-ZEN, 5-OH-ZEN, and 9-OH-ZEN had larger yield in chickens, which was different from other species. Compared with its metabolism in swine and human, ZEN seemed more prone to hydroxylation by chicken metabolism. The estrogen-like effect of α-ZEL was 92 times higher than that of ZEN, but that effect was markedly reduced for β-ZEL, 8-OHZEN, 13-OH-ZEN, 15-OH-ZEN, and ZEN-GlcA conjugates.20,28 ZEN was likely to be hydroxylated in rats and chickens but not in swine where α-ZEL was generated. The lower extent of hydroxylation of ZEN could partially acount for higher sensitivity to ZEN in swine. Glucuronidation is regarded as a vital detoxification pathway. In addition, two di-GlcA binding metabolites were identified in swine, goats, and cows. In Figure 5, the yield of ZEN-14GlcA was far more than that of ZEN-16-GlcA, indicating the C-14 hydroxyl group was the principal site for binding GlcA, which is in line with the literature.13,19,27 However, in goats and cows, 11300


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Figure 6. Proposed metabolic pathways of ZEN in rats and chickens after oral dose.

major metabolic pathways in rats, while reduction and sulfation predominate in chickens.

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Zhanhui Wang: 0000-0002-0167-9559 Sarah De Saeger: 0000-0002-2160-7253 Yi Li: 0000-0002-2752-7406 Marthe De Boevre: 0000-0002-6151-5126

ASSOCIATED CONTENT

S Supporting Information *

Author Contributions

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04663. Accurate MS/MS spectra of ZEN metabolites; EICs of ZEN phase II metabolites observed in in vitro system; EICs of ZEN metabolites detected in vivo of rats and chickens; and EICs of α-ZEL and β-ZEL phase I metabolites observed in in vitro systems (PDF)

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∥

S.Y. and H.Z. contributed equally to this work.

Funding

This study was supported by the National Natural Science Foundation of China (Grant No. 31702296). Yanshen Li was supported by the National Natural Science Foundation of China (Grant No. 31402246). Notes

AUTHOR INFORMATION

The authors declare no competing financial interest.

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Corresponding Authors

*Phone: +86-10-6259-4943. Fax: +86-10-6259-6429. E-mail: [email protected]. *E-mail: [email protected].

ACKNOWLEDGMENTS

We are grateful to Marthe De Boevre and Karl De Ruyck for providing many professional suggestions and polishing the language of the present study.

ORCID

Shupeng Yang: 0000-0001-7879-403X 11301


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Zearalenone in Different Poultry Species. J. Agric. Food Chem. 2015, 63, 5092−5098. (16) Winkler, J.; Kersten, S.; Meyer, U.; Engelhardt, U.; Daenicke, S. Residues of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites in plasma of dairy cows fed Fusarium contaminated maize and their relationships to performance parameters. Food Chem. Toxicol. 2014, 65, 196−204. (17) Shin, B. S.; Hong, S. H.; Bulitta, J. B.; Hwang, S. W.; Kim, H. J.; Lee, J. B.; Yang, S. D.; Kim, J. E.; Yoon, H. S.; Kim, D. J.; Yoo, S. D. Disposition, Oral Bioavailability, and Tissue Distribution of Zearalenone in Rats at Various Dose Levels. J. Toxicol. Environ. Health, Part A 2009, 72, 1406−1411. (18) Osselaere, A.; Devreese, M.; Goossens, J.; Vandenbroucke, V.; De Baere, S.; De Backer, P.; Croubels, S. Toxicokinetic study and absolute oral bioavailability of deoxynivalenol, T-2 toxin and zearalenone in broiler chickens. Food Chem. Toxicol. 2013, 51, 350− 355. (19) Binder, S. B.; Schwartz-Zimmermann, H. E.; Varga, E.; Bichl, G.; Michlmayr, H.; Adam, G.; Berthiller, F. Metabolism of Zearalenone and Its Major Modified Forms in Pigs. Toxins 2017, 9, 56. (20) Drzymala, S. S.; Binder, J.; Brodehl, A.; Penkert, M.; Rosowski, M.; Garbe, L. A.; Koch, M. Estrogenicity of novel phase I and phase II metabolites of zearalenone and cis-zearalenone. Toxicon 2015, 105, 10−12. (21) Molina-Molina, J. M.; Real, M.; Jimenez-Diaz, I.; Belhassen, H.; Hedhili, A.; Torne, P.; Fernandez, M. F.; Olea, N. Assessment of estrogenic and anti-androgenic activities of the mycotoxin zearalenone and its metabolites using in vitro receptor-specific bioassays. Food Chem. Toxicol. 2014, 74, 233−239. (22) Malekinejad, H.; Maas-Bakker, R.; Fink-Gremmels, J. Species differences in the hepatic biotransformation of zearalenone. Vet. J. 2006, 172, 96−102. (23) Brezina, U.; Rempe, I.; Kersten, S.; Valenta, H.; Humpf, H. U.; Danicke, S. Determination of zearalenone, deoxynivalenol and metabolites in bile of piglets fed diets with graded levels of Fusarium toxin contaminated maize. World Mycotoxin J. 2016, 9, 179−193. (24) Gambacorta, L.; Solfrizzo, M.; Visconti, A.; Powers, S.; Cossalter, A. M.; Pinton, P.; Oswald, I. P. Validation study on urinary biomarkers of exposure for aflatoxin B-1, ochratoxin A, fumonisin B-1, deoxynivalenol and zearalenone in piglets. World Mycotoxin J. 2013, 6, 299−308. (25) Songsermsakul, P.; Bohm, J.; Aurich, C.; Zentek, J.; RazzaziFazeli, E. The levels of zearalenone and its metabolites in plasma, urine and faeces of horses fed with naturally, Fusarium toxin-contaminated oats. J. Anim. Physiol. Anim. Nutr. 2013, 97, 155−161. (26) Pfeiffer, E.; Kommer, A.; Dempe, J. S.; Hildebrand, A. A.; Metzler, M. Absorption and metabolism of the mycotoxin zearalenone and the growth promotor zeranol in Caco-2 cells in vitro. Mol. Nutr. Food Res. 2011, 55, 560−567. (27) Pfeiffer, E.; Hildebrand, A.; Mikula, H.; Metzler, M. Glucuronidation of zearalenone, zeranol and four metabolites in vitro: Formation of glucuronides by various microsomes and human UDP-glucuronosyltransferase isoforms. Mol. Nutr. Food Res. 2010, 54, 1468−1476. (28) Frizzell, C.; Uhlig, S.; Miles, C. O.; Verhaegen, S.; Elliott, C. T.; Eriksen, G. S.; Sorlie, M.; Ropstad, E.; Connolly, L. Biotransformation of zearalenone and zearalenols to their major glucuronide metabolites reduces estrogenic activity. Toxicol. In Vitro 2015, 29, 575−581. (29) Ueberschar, K. H.; Brezina, U.; Danicke, S. Zearalenone (ZEN) and ZEN metabolites in feed, urine and bile of sows: Analysis, determination of the metabolic profile and evaluation of the binding forms. Landbauforschung 2016, 66, 21−28. (30) Metzler, M.; Pfeiffer, E.; Hildebrand, A. A. Zearalenone and its metabolites as endocrine disrupting chemicals. World Mycotoxin J. 2010, 3, 385−401. (31) Pfeiffer, E.; Hildebrand, A. A.; Becker, C.; Schnattinger, C.; Baumann, S.; Rapp, A.; Goesmann, H.; Syldatk, C.; Metzler, M. Identification of an aliphatic epoxide and the corresponding

ABBREVIATIONS USED ZEN, zearalenone; α-ZEL, α-zearalenol; β-ZEL, β-zearalenol; ZAN, zearalanone; α-ZAL, α-zearalanol; β-ZAL, β-zearalanol; TDI, tolerable daily intake; EFSA, European Food Safety Authority; HSDs, hydroxysteroid dehydrogenases; GlcA, glucuronic acid; UGT, uridine diphosphate-glucuronosyltransferases; SULT, sulfotransferase; UDPGA, 5′-diphosphoglucuronic acid; NADPH, β-nicotinamide adenine dinucleotide phosphate; CID, collision induced dissociation; EIC, extracted ion chromatogram; OH, hydroxyl; LC−HRMS, liquid chromatography combined with high-resolution mass spectrometry.

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Metabolic Profile of Zearalenone in Liver ... - ACS Publications

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