Article pubs.acs.org/JAFC
Comparative Oral Bioavailability, Toxicokinetics, and Biotransformation of Enniatin B1 and Enniatin B in Broiler Chickens Sophie Fraeyman,† Mathias Devreese,† Gunther Antonissen,†,‡ Siegrid De Baere,† Michael Rychlik,§ and Siska Croubels*,† †
Department of Pharmacology, Toxicology and Biochemistry and ‡Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium § Chair of Analytical Food Chemistry, Technische Universität München, Alte Akademie 10, 85354 Freising, Germany S Supporting Information *
ABSTRACT: A toxicokinetic study of the Fusarium mycotoxins enniatin B1 (ENN B1) and enniatin B (ENN B) was performed in broiler chickens. Each animal received ENN B1 or B orally via an intracrop bolus and intravenously at a dose of 0.2 mg/kg body weight. Both enniatins were poorly absorbed after oral administration, with absolute oral bioavailabilities of 0.05 and 0.11 for ENNs B1 and B, respectively. Both enniatins were readily distributed to the tissues, with mean volumes of distribution of 25.09 and 33.91 L/kg for ENNs B1 and B, respectively. The mean total body clearance was rather high, namely, 6.63 and 7.10 L/ h/kg for ENNs B1 and B, respectively. Finally, an UHPLC-HRMS targeted approach was used to investigate the phase I and II biotransformations of both mycotoxins. Oxygenation was the major phase I biotransformation pathway for both ENNs B1 and B. Neither glucuronide nor sulfate phase II metabolites were detected. KEYWORDS: mycotoxin, enniatin B, enniatin B1, broiler chickens, oral bioavailability, toxicokinetic analysis, biotransformation, phase I and phase II metabolites, HRMS
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respectively.2 For poultry feed, 78 and 67% of the poultry feed samples analyzed by de Lourdes Mendes de Souza et al.3 were contaminated with ENNs B and B1, respectively, with maximum levels up to 4.6 μg/kg ENN B and 12 μg/kg ENN B1. ENNs have ionophoric properties and can form complexes with cations. In vitro studies have demonstrated antimicrobial, insecticidal, phytotoxic, and cytotoxic actions of ENNs.1 ENN B showed a higher cytotoxic effect (IC50 = 6.3 μM) in cells of the human colorectal adenocarcinoma cell line Caco-2, compared to the other Fusarium mycotoxins such as nivalenol (IC50 = 6.9 μM), deoxynivalenol (IC50 = 13.0 μM), and zearalenone (IC50 = 49.5 μM).4 However, in vivo data concerning health effects of ENNs in humans and animals are scarce. In vivo studies evaluating chronic toxicity, reproduction and developmental toxicity, neurotoxicity, genotoxicity, and carcinogenicity are lacking. The European Food Safety Authority (EFSA) recognized that the data were insufficient to establish a tolerable daily intake (TDI) or an acute reference dose (ARfD). Due to the lack of toxicity data, risk assessment was not possible for humans and most livestock animal species. For broilers and laying hens, however, noobserved-adverse-effect levels (NOAELs) of 244 and 763 μg/kg body weight (bw)/day and NOAELs of 216 and 674 μg/kg bw/day were established for ENN B1 and ENN B, respectively.5
INTRODUCTION
Enniatins (ENNs) are secondary metabolites produced by various Fusarium fungi, including F. avenaceum, F. poae, and F. tricinctum. These mycotoxins are highly prevalent food and feed contaminants and are considered “emerging” mycotoxins.1 The ENNs most frequently contaminating feed samples are ENNs A, 1, A1, 2, B, 3, and B1, 4 (Figure 1). Streit et al.2 investigated 83 feed and feed raw material samples, of which 72, 79, 76, and 76% were contaminated with ENNs A, A1, B, and B1, with maximum levels of 1745, 2216, 780, and 2690 μg/kg,
Received: June 28, 2016 Revised: August 31, 2016 Accepted: September 6, 2016
Figure 1. Chemical structure of enniatins A, A1, B, and B1. © XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.6b02913 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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design, in which each animal received a single dose orally (po) via an intracrop bolus and an intravenous injection (iv) in the wing vein (vena basilica). A 2-day wash-out period was respected between the two routes of administration. Blood was collected by direct venipuncture of the leg vein (vena metatarsalis plantaris superf icialis) just before and at different time points (5, 10, 20, 30 min and 1, 2, 4, 6, 8, 10, 24 h) after administration (pa). The blood samples were centrifuged (2095g, 10 min, 4 °C), and aliquots of plasma (25 μL for the samples at 5, 10, 20, 30, 60, and 120 min pa iv and 250 μL for the other time points) were stored at ≤ −15 °C until analysis. The animal experiments were approved by the Ethical Committee of the Faculty of Veterinary Medicine and Bioscience Engineering of Ghent University (EC 2014/09). Plasma Sample Preparation. To the plasma aliquots (25 or 250 μL) was added 12.5 μL of the IS (2 ng/mL). After a vortex-mixing step, 750 μL of acetonitrile was added followed by vortex-mixing and centrifugation (8517g, 10 min, 4 °C). The supernatant was evaporated under a gentle nitrogen (N2) stream (60 ± 5 °C). The dry residue was dissolved in 250 μL of acetonitrile/water (80:20, v/v). After a vortexmixing step, the sample was filtered through a Millex-GN nylon filter and transferred to an autosampler vial. A 5 μL aliquot was injected onto the LC-MS/MS instrument. Liquid Chromatography−Tandem Mass Spectrometry (LCMS/MS). ENN B and ENN B1 plasma concentrations were quantitated using UHPLC-MS/MS. The UHPLC-MS/MS system consisted of an Acquity H-Class UPLC coupled to a Xevo TQ-S mass spectrometer (Waters, Zellik, Belgium). The column used was a 50 mm × 2.1 mm i.d., 1.7 μm, C18 Acquity UPLC BEH column, with a 5 mm × 2.1 mm i.d. VanGuard BEH C18 guard column of the same material (Waters). Mobile phase A consisted of 0.1% glacial acetic acid in water, whereas mobile phase B consisted of acetonitrile. The following gradient elution program was run: 0.0−0.5 min (50% B), 0.5−6.0 min (linear gradient to 85% B), 6.0−8.0 min (85% B), 8.0− 8.1 min (linear gradient to 50% B), 8.1−10.0 min (50% B). The flow rate was set at 300 μL/min. The column oven and tray temperature were set at 45 and 7 °C, respectively. The LC column effluent was interfaced to a Xevo TQ-S mass spectrometer (Waters) equipped with an electrospray ionization (ESI) probe operating in the positive ionization mode. Instrument parameters were optimized by syringe infusion of a 10 ng/mL working solution of ENN B, ENN B1, and the IS. The following MS/MS parameters were used: capillary voltage, 3.60 kV; cone voltage, 30.00 V; source offset, 60.00 V; source temperature, 150 °C; desolvation temperature, 550 °C; collision gas flow, 0.15 mL/min; nebulizer gas flow, 7.00 bar. Dwell time was set at 0.025 s. Acquisition was performed in the multiple reaction monitoring (MRM) mode. Product ions with the highest signal intensity were used as quantitation ion. The following transitions (m/z) were monitored for qualification and quantitation, respectively: for ENN B, 640.40 → 214.20 and 640.40 → 196.20; for ENN B1, 654.40 → 214.10 and 654.40 → 196.20; and for IS, 643.30 → 215.30 and 643.30 → 197.10. Cone voltage was set at 80.0 V for both ENNs and the IS. The collision energy was set at 20.0, 25.0, and 20.0 eV for ENN B, ENN B1, and the IS, respectively. Ultraperformance Liquid Chromatography−High-Resolution Mass Spectrometry (UPLC-HRMS). An Acquity I-Class UPLC coupled to a Synapt G2-Si HDMS instrument (Waters) was used to identify phase I and II metabolites of ENN B and ENN B1 in broiler chicken plasma samples. The chromatographic conditions were the same as described above. HRMS instrument parameters were optimized by syringe infusion of a 10 ng/mL working solution of ENN B, ENN B1, and the IS. The following HRMS parameters were used: capillary voltage, 3.00 kV; sampling cone voltage, 40.00 V; source offset, 60.00 V; source temperature, 115 °C; desolvation temperature, 200 °C; cone gas flow, 50 L/h; desolvation gas flow, 650 L/h; nebulizer gas flow, 6.00 bar; lock spray capillary voltage, 2.00 kV. HRMS acquisition was performed from 0 to 7 min in the positive ESI resolution mode using the MSE continuum scan function. Time-offlight (TOF) MS settings were as follows: low mass, 50 Da; high mass, 1200 Da; scan time, 0.1 s; data format, continuum. The lock mass solution consisted of leucine encephalin (200 pg/uL). The lock spray
Knowledge of the absorption, distribution, metabolism, and excretion (ADME) processes of ENNs is essential to assess the health risk in affected animals and also to estimate the possible carry-over of these mycotoxins into animal-derived tissues and products. Devreese et al.6 demonstrated a high absolute oral bioavailability of ENN B1 in pigs (F = 0.91), resulting in a high systemic exposure. On the other hand, the oral absorption of ENN A and ENN A1 in pigs was low.7 However, data on the toxicokinetic characteristics of ENNs in general in poultry is largely lacking. Nevertheless, in vitro and in vivo studies demonstrated different biotransformation pathways of ENN B. After incubation of ENN B (0.66 μM) with rat, dog, and human liver microsomes, 12 metabolites could be identified (M1−12). Five metabolites were monohydroxylated (M1−M5) and two were N-demethylated (M6, M7) species, whereas metabolites M8−M12 were the result of multiple oxidations.8 After incubation of chicken liver microsomes with ENN B (0.66 μM), only eight metabolites were identified, namely, five hydroxylated (M1−M4 and M13) and three carboxylated metabolites (M9, M11, and M12). After an ENN B contaminated diet had been fed to broilers (12.7 mg/kg) and laying hens (11.2 mg/kg), M11 and M13 were the dominant metabolites in liver and serum samples. In eggs, only M13 and M4 were detected.9 The objective of the present study was to investigate the toxicokinetic parameters, absolute oral bioavailability, and phase I and II metabolites of ENN B and ENN B1 in broiler chickens in order to extend the knowledge of the ADME processes of these mycotoxins in this species. First, a liquid chromatography−tandem mass spectrometry (LC-MS/MS) method to quantitate ENNs B and B1 in broiler chicken plasma was developed and validated. Second, a toxicokinetic study of both ENNs was performed in broiler chickens. Finally, an ultrahighperformance liquid chromatography−high-resolution mass spectrometry (UHPLC-HRMS) method was developed, and plasma samples were analyzed to identify possible phase I and phase II metabolites.
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MATERIALS AND METHODS
Chemicals, Products, and Reagents. Analytical standards of ENNs B and B1 were purchased from Fermentek (Fermentek, Jerusalem, Israel) and Sigma-Aldrich (Sigma-Aldrich, Diegem, Belgium), respectively. The internal standard (IS) [15N3]-ENN B was synthesized according to the method of Hu and Rychlik.10 ENN B, ENN B1, and the IS were stored at ≤ −15 °C. Glacial acetic acid, water, methanol, and acetonitrile were of LC-MS grade (Biosolve, Valkenswaard, The Netherlands). Absolute ethanol was of analytical grade (VWR, Leuven, Belgium). Millex-GN nylon filters (0.20 μm) were purchased from Merck-Millipore (Overijse, Belgium). Stock and Working Solutions. Stock solutions of ENN B1 (100 μg/mL), ENN B (100 μg/mL), and the IS (10 μg/mL) for analytical experiments were prepared in methanol and stored at −20 °C. Combined working solutions of ENN B1 and ENN B (ranging from 0.250 to 50 ng/mL) were prepared by appropriate dilution of the stock solutions in acetonitrile. These working solutions were used for the preparation of matrix-matched calibration standards and quality control samples and were freshly prepared for each analytical batch. For the animal experiments, ENN B1 and ENN B were dissolved in ethanol (2 mg/mL). Animal Experiments. Twelve 3-week-old broiler chickens (Ross 308) of mixed gender and a mean bw of 960 ± 142 g were randomly divided into two equal groups of six animals. Each group was composed of an equal number of male and female broilers. The animals received a bolus of ENN B1 (group 1) or ENN B (group 2) at a dose of 0.2 mg/kg bw. The study was set up in a two-way crossover B
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from time 0 to infinity (AUC0−inf, ng·h/mL), maximum concentration after oral administration (Cmax, ng/mL), plasma concentration at time of iv injection (C0, ng/mL), time to maximal plasma concentration (Tmax, h), elimination rate constant (kel, h−1), elimination half-life (T1/2el, h), total body clearance (Cl, L/h/kg), and volume of distribution (Vd, L/kg), with Cl and Vd scaled for the absolute oral bioavailability (F). F was calculated as the ratio of the AUC0−inf after oral administration to the AUC0−inf after intravenous injection. Statistical Analysis. One-way analysis of variance (ANOVA) was performed on all toxicokinetic parameters for the different routes of administration and for both ENNs, using SPSS Statistics 23. The level of significance was set at 0.05.
was acquired during HRMS acquisition, but no correction was applied. The lock spray settings were as follows: scan time, 0.1 s; interval, 30 s; scans to average, 3; mass window, 0.5 Da. Data processing and lock mass correction (m/z 556.276575) was performed using Unify 1.7 software (Waters). No standards of the metabolites were commercially available; however, the search for possible phase I metabolites was based on those reported previously,8 using a targeted approach. Therefore, the chemical formulas and the theoretical accurate masses of possible phase I metabolites were added to the Metabolite Identification−MSe processing method, together with some nontargeted transformations (2 × oxidation, glucuronide, and sulfate conjugation). Method Validation. The developed LC-MS/MS method was inhouse validated on the basis of the protocol described by De Baere et al.,11 using spiked blank plasma samples obtained from healthy, untreated broiler chickens. Linearity, accuracy, precision, limit of quantitation (LOQ), limit of detection (LOD), carry-over, matrix effects, and extraction recovery were determined in compliance with the recommendations and guidelines defined by the European Community and with criteria described in the literature.11−16 Calibration Curves. Matrix-matched calibration curves were prepared in 250 μL (low range, 0.025−5 ng/mL) or 25 μL (high range, 0.25−75 ng/mL) blank broiler chicken plasma. The correlation coefficients (r) and goodness-of-fit coefficients (gof) were calculated, and limits were set at ≥0.99 and ≤20%, respectively.11,14,15 Accuracy and Precision. Within-run accuracy and precision (repeatability) were determined by analyzing six blank samples that were spiked at 0.05, 0.5, and 1 ng/mL (250 μL) or 0.5, 5, and 50 ng/ mL (25 μL) in the same run. The between-run accuracy and precision (reproducibility) were determined by analyzing two blank samples spiked at 0.05, 0.1, and 1 ng/mL (250 μL) or 0.5, 5, and 50 ng/mL (25 μL) on three different days (n = 6). The acceptance criteria for accuracy were as follows: −50 to +20%, −30 to +10%, and −20 to +10% for concentration levels ≤1 ng/mL, between 1 and 10 ng/mL, and ≥10 ng/mL, respectively. The precision was evaluated by the determination of the relative standard deviation (RSD), which had to be below the RSDmax value. For the within-day precision, RSDmax is fixed at 30, 25, and 15% for concentrations 0.9998 and the gof coefficients were 5.62 and 6.57% for ENN B1 and ENN B, respectively. There was no carry-over, because no peak higher than the LOD was detected in the elution zone of ENNs B and B1 or the IS after injection of the reconstitution solvent. The results of the evaluation of the accuracy and precision fell within the acceptance criteria. RE was 76.2% (ENN B) and 72.8% (ENN B1), SSE was 93.4% (ENN B) and 98.1% (ENN B1), and RA was 71.1% (ENN B) and 71.4% (ENN B1). This is the first study describing the disposition of ENN B1 and ENN B in broiler chickens. The plasma concentration− time curves are depicted in Figure 2, whereas Table 1
Figure 2. Plasma concentration−time curve after intravenous (iv) and oral (po) administration of 0.2 mg/kg bw enniatin B1 or enniatin B to broiler chickens (n = 6). Values are presented as the mean + or − SD.
summarizes the main toxicokinetic parameters. After iv administration of ENNs B and B1, the toxicokinetic parameters did not significantly differ between the toxins. For both ENN B1 and ENN B, the mean AUC0−t and the mean AUC0−inf were significantly higher after iv administration compared to po administration (p < 0.001). This resulted in a mean F of 0.05 and 0.11 for ENNs B1 and B, respectively. The low absolute oral bioavailability of both ENNs in broiler chickens is in strong contrast with the high oral bioavailability of ENN B1 in pigs (mean F = 0.91),6 demonstrating the importance of speciesspecific studies. This is in accordance with other Fusarium toxins. For example, the oral bioavailability of deoxynivalenol after a single oral bolus is much higher in pigs (F = 0.54) compared to broiler chickens (F = 0.19).17,18 The low fraction C
DOI: 10.1021/acs.jafc.6b02913 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Table 1. Main Toxicokinetic Parameters of ENN B1 and ENN B after Intravenous (iv) and Oral (po) Administration (Dose = 0.2 mg/kg bw) to Broiler Chickens (n = 6)a ENN B1 toxicokinetic parameter
iv
AUC 0−t (ng·h/mL) AUC 0−inf (ng·h/mL) F Cmax (ng/mL) C0 (ng/mL) Tmax (h) kel (h−1) T1/2el (h) Cl (L/h/kg) Vd (L/kg)
29.71 ± 3.37 a 30.57 ± 3.53 a
ENN B po 1.48 1.56 0.05 1.37
± ± ± ±
iv
1.03 1.04 0.04 0.68
po
28.37 ± 6.12 a 29.17 ± 6.29 a
b b a a
60.07 ± 8.15 a
2.87 3.21 0.11 0.96
± ± ± ±
1.59 1.76 0.06 0.75
b b a a
50.35 ± 18.79 a 0.63 ± 0.34 a 0.52 ± 0.19 b 1.49 ± 0.61 b 6.64 ± 0.81 a 14.36 ± 6.16 b
0.28 ± 0.07 a 2.63 ± 0.70 abc 6.63 ± 0.81 a 25.09 ± 6.84 ab
0.29 ± 0.15 a 0.25 ± 0.03 a 2.87 ± 0.38 ac 7.18 ± 1.34 a 29.39 ± 5.49 a
0.22 ± 0.04 a 3.32 ± 0.65 c 7.10 ± 1.32 a 33.91 ± 8.70 a
a
AUC0−t, area under the plasma concentration−time curve from time 0 to last concentration > LOQ; AUC0−inf, area under the plasma concentration−time curve from time 0 to infinite; F, absolute oral bioavailability (AUC0−inf po/AUC0−inf iv); Cmax, maximal plasma concentration; C0, plasma concentration at time 0; Tmax, time to maximal plasma concentration; kel, elimination rate constant; T1/2el, elimination half-life; Cl, total body clearance corrected for F; Vd, volume of distribution corrected for F. Values sharing a lower case letter within a row for the same parameter do not differ from one another at p < 0.05. Values are presented as the mean ± SD.
Table 2. Molecular Formula, Retention Time, Theoretical Neutral Mass, and Mean Mass Error of ENN B, ENN B1, and their Metabolites compound ENN ENN ENN ENN ENN ENN ENN ENN ENN ENN
B B1 B_M1a B_M1b B1_M1a B1_M1b B_M2a B_M2b B1_M2 B1_M3
compound detected previously8
M1−M5 M1−M5
M8−M12 M8−M12
metabolite
formula
retention time (min)
theoretical neutral mass (Da)
parent parent mono-oxygenated mono-oxygenated mono-oxygenated mono-oxygenated multiple oxidations multiple oxidations multiple oxidations dioxygenated
C33H57N3O9 C34H59N3O9 C33H57N3O10 C33H57N3O10 C34H59N3O10 C34H59N3O10 C33H55N3O11 C33H55N3O11 C34H57N3O11 C34H59N3O11
4.9 5.6 2.7 3.2 3.0 3.4 2.6 2.7 1.9 2.3
639.4095 653.4251 655.4044 655.4044 669.4200 669.4200 669.3837 669.3837 683.3993 685.4149
mean mass error ± SD (ppm) 0.7842 1.2124 −0.1587 0.0595 −1.5217 −2.3048 0.0015 0.2074 −2.1543 −2.1547
± ± ± ± ± ± ± ± ± ±
2.1386 2.8052 2.4192 2.5347 1.8782 1.8812 2.5685 2.4294 2.5783 3.2900
liver samples.20 Jestoi et al.21 demonstrated the high prevalence of beauvericin and ENNs in Finnish egg samples, namely, 79 and 56% of eggs collected in 2004 and 2005, respectively. However, concentrations in whole egg samples were low, with maximum levels of 0.7 μg/kg ENN B and 1.12 μg/kg ENN B1. The prevalence (366/367 samples contaminated with ENNs and/or beauvericin) and concentrations were higher in egg yolk samples, with maximum concentrations of 1.3, 1.3, 7.5, 3.8, and 7.0 μg/kg for beauvericin and ENNs A, A1, B, and B1, respectively. These findings suggest bioaccumulation of beauvericin and ENNs in egg yolk.21 Carry-over of ENN B and ENN B1 from feed to broiler tissues was determined after feeding of a multimycotoxin contaminated diet for 14 days, including 12.716 mg/kg ENN B and 4.057 mg/kg ENN B1. The carry-over rates from feed to liver were 0.16 and 0.12%, whereas carry-over rates in skin were 0.39 and 0.37% for ENNs B and B1, respectively. ENNs were slowly eliminated from liver and skin after withdrawal of the contaminated feed.22 However, tissue concentrations were usually low.20,22 This could be attributed to the low oral bioavailability of ENNs as demonstrated in the present study. The plasma samples obtained during the toxicokinetic study were analyzed to identify the major phase I and II metabolites of ENN B and ENN B1. The knowledge of metabolites of ENNs is limited, and thus targeted HRMS was preferred to obtain as much information as possible. Table 2 summarizes the molecular formulas, retention times, theoretical masses, and
of the dose that was absorbed reached its maximum concentration rather rapidly with mean Tmax values of 0.63 h (ENN B1) and 0.29 h (ENN B) in broiler chickens. Similarly, the mean Tmax was 0.24 h for ENN B1 in pigs.6 In broiler chickens, the mean T1/2el after iv administration was 2.63 and 3.32 h for ENN B1 and ENN B, respectively. Both Cl and Vd were high, with mean Cl values of 6.6 and 7.1 L/h/kg and mean Vd values of 25.09 and 33.91 L/kg after iv administration for ENN B1 and ENN B, respectively. The mean T1/2el, mean Cl, and mean Vd did not significantly differ between both routes of administration. Notwithstanding the rather high mean Cl, the mean T1/2el was rather long for both ENN B1 and ENN B in broiler chickens. This could be attributed to the high mean Vd of both ENN B1 and ENN B. The Vd of ENN B1 in pigs after iv administration was lower, with volumes of distribution in the central (Vc) and peripheral compartments (Vp) of 0.57 and 0.69 L/kg, respectively.6 Compared to broiler chickens, the mean Cl was lower in pigs (Cliv = 2.00 L/h/kg), whereas T1/2el was 1.13 h in pigs.6 The high mean Vd in broiler chickens suggests that binding to plasma proteins is limited and/or that both ENNs easily distribute into the tissues.19 The latter could be explained by the lipophilic nature of these compounds. Residue studies have confirmed their presence in the liver, meat, and skin of broiler chickens and in eggs of laying hens.20−22 Traces of ionophoric coccidiostats, beauvericin, ENN A, ENN A1, ENN B, and ENN B1 were found in a limited number of Finnish poultry meat and D
DOI: 10.1021/acs.jafc.6b02913 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry mean mass errors of the metabolites of ENNs B and B1. Several phase I metabolites of ENN B1 and ENN B were detected after single administration and, as expected, the metabolic pathways of both ENNs were similar. Peaks were observed at mean m/z values of 678.3936 (ENN B_M1a), 678.3937 (ENN B_M1b), 692.4091 (ENN B1_M1a), and 692.4073 (ENN B1_M1b), which correspond to two Na+ adducts of the theoretical accurate masses of the mono-oxygenated metabolites of ENN B (m/z 655.4044) and ENN B1 (m/z 669.4200), respectively. Peaks were also observed at mean m/z values of 692.3729 (ENN B_M2a), 692.3733 (ENN B_M2b), and 706.3868 (ENN B1_M2). These peaks correspond to the Na+ adducts of the oxygenated and dehydrogenated metabolites of ENN B (theoretical accurate mass = 669.3837) and B1 (theoretical accurate mass = 683.3993), respectively. ENN B1 was also dioxygenated, with an observed m/z value of 708.4032 (ENN B1_M3), which corresponds to the Na+ adduct of the theoretical accurate mass of 685.4149. These results are in accordance with the results of Ivanova et al.,9 who detected five hydroxylated and three carboxylated metabolites after incubation of chicken liver microsomes with ENN B. The latter study also confirmed these findings in vivo. After feeding of an ENN B contaminated diet to broiler chickens and laying hens, both hydroxylated and carboxylated metabolites of ENN B were found in liver and serum. In eggs, only hydroxylated metabolites were found. Neither ENN B nor its metabolites were found in liver samples 3 days after withdrawal of the contaminated feed. However, both ENN B and a hydroxylated metabolite were detected in eggs collected 3 days after withdrawal of the contaminated diet.9 In the present study, no sulfated or glucuronidated phase II metabolites of ENN B or ENN B1 were detected. Figure 3 shows the mean chromatographic peak area of the parent compounds and detected metabolites at the different time points after iv administration. The mean peak areas of the parent compounds were higher than the mean peak areas of the individual metabolites. In the plasma samples collected after po administration, the peak areas of the phase I metabolites were usually low or were not detected, which could be attributed to the low concentrations of the parent components. On the basis of the information obtained in the present work, a more thorough identification of the metabolites could be performed in future studies, using, for example, MS/MS. Feed is frequently contaminated with ENNs, and contamination levels can be as high as a few hundreds of micrograms per kilogram of feed.2 However, both ENN B1 and ENN B are poorly absorbed in broiler chickens after oral administration, resulting in a low systemic exposure to these mycotoxins. Consequently, systemic adverse effects due to the consumption of contaminated feed are probably limited. This was acknowledged by the EFSA opinion stating that “The chronic exposure for poultry indicated that adverse health effects from beauvericin and enniatins were unlikely”.5 Nevertheless, ENN concentrations in the gastrointestinal tract can be high, and in vitro studies demonstrated the cytotoxicity of ENNs in different cell types.1,4,5 ENN B was more cytotoxic compared to nivalenol, deoxynivalenol, and zearalenone in Caco-2 cells.4 Besides, the impact of ENNs on the gastrointestinal microbiota should be investigated. Therefore, further research is imperative to examine the local adverse health effects of ENNs in the intestine of broiler chickens.
Figure 3. Absolute chromatographic peak area−time curve of enniatin B (A) or enniatin B1 (B) and metabolites at different time points after intravenous administration of 0.2 mg/kg bw ENN B or ENN B1 to broiler chickens (n = 6). Values are presented as the mean + SD.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b02913. Table S1: Results of the within- and between-run precision and accuracy evaluation for the analysis of ENN B1 and ENN B in broiler chicken plasma (PDF)
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AUTHOR INFORMATION
Corresponding Author
*(S.C.) Phone: +32 9 264 73 47. Fax: +32 9 264 74 97. E-mail:
[email protected]. Funding
The Synapt High Definition Mass Spectrometer (Synapt G2-Si HDMS, Waters) was funded by the Hercules project (no. AUGE 13/13). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Joren De Smet, Marianne Lauwers, Elke Gasthuys, Wim Schelstraete, and Thomas De Mil for their aid during the animal experiments and Anneleen Watteyn for her assistance in the laboratory experiments. E
DOI: 10.1021/acs.jafc.6b02913 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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(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) Toutain, P. L.; Bousquet-Mélou, A. Volumes of distribution. J. Vet. Pharmacol. Ther. 2004, 27, 441−453. (20) Jestoi, M.; Rokka, M.; Peltonen, K. An integrated sample preparation to determine coccidiostats and emerging Fusariummycotoxins in various poultry tissues with LC-MS/MS. Mol. Nutr. Food Res. 2007, 51, 625−637. (21) Jestoi, M.; Rokka, M.; Järvenpäa,̈ E.; Peltonen, K. Determination of Fusarium mycotoxins beauvericin and enniatins (A, A1, B, B1) in eggs of laying hens using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Food Chem. 2009, 115, 1120−1127. (22) Callebaut, F.; Tangni, E. K.; Debongnie, P.; Stals, E.; Huybrechts, B.; Waegeneers, N.; Delezie, E.; Van Pamel, E.; Daeseleire, E. Carry-over of mycotoxins to animal products: case study poultry. Scientifec Report 2011/2012 CODA-CERVA (Centrum voor Onderzoek in Diergeneeskunde en Agrochemie-Centre d’Étude et de Recherches Vétérinaires et Agrochemiques), 2011/2012; pp 141−144, http://coda-cerva.be/images/pdf/CODA_CERVA_ Scientific_Report_2011_2012.pdf (accessed Aug 11, 2016).
REFERENCES
(1) Jestoi, M. Emerging Fusarium-mycotoxins fusaproliferin, beauvericin, enniatins, and moniliformin: a review. Crit. Rev. Food Sci. Nutr. 2008, 48, 21−49. (2) Streit, E.; Schwab, C.; Sulyok, M.; Naehrer, K.; Krska, R.; Schatzmayr, G. Multi-mycotoxin screening reveals the occurrence of 139 different secondary metabolites in feed and feed ingredients. Toxins 2013, 5, 504−523. (3) de Souza, M. L. M.; Sulyok, M.; Freitas-Silva, O.; Costa, S. S.; Brabet, C.; Machinski Junior, M.; Sekiyama, B. L.; Vargas, E. A.; Krska, R.; Schuhmacher, R. Cooccurrence of mycotoxins in maize and poultry feeds from Brazil by liquid chromatography/tandem mass spectrometry. Sci. World J. 2013, 2013, 1−9. (4) Vejdovszky, K.; Warth, B.; Sulyok, M.; Marko, D. Non-synergistic cytotoxic effects of Fusarium and Alternaria toxin combinations in Caco-2 cells. Toxicol. Lett. 2016, 241, 1−8. (5) European Food Safety Authority. Scientific Opinion on the risks to human and animal health related to the presence of beauvericin and enniatins in food and feed. EFSA J. 2014 12 (8), 380210.2903/ j.efsa.2014.3802. (6) Devreese, M.; Broekaert, N.; De Mil, T.; Fraeyman, S.; De Backer, P.; Croubels, S. Pilot toxicokinetic study and absolute oral bioavailability of the Fusarium mycotoxin enniatin B1 in pigs. Food Chem. Toxicol. 2014, 63, 161−165. (7) Devreese, M.; De Baere, S.; De Backer, P.; Croubels, S. Quantitative determination of the Fusarium mycotoxins beauvericin, enniatin A, A1, B and B1 in pig plasma using high performance liquid chromatography-tandem mass spectrometry. Talanta 2013, 106, 212− 219. (8) Ivanova, L.; Faeste, C. K.; Uhlig, S. In vitro phase I metabolism of the depsipeptide enniatin B. Anal. Bioanal. Chem. 2011, 400, 2889− 2901. (9) Ivanova, L.; Faeste, C. K.; Delezie, E.; Van Pamel, E.; Daeseleire, E.; Callebaut, A.; Uhlig, S. Presence of enniatin B and its hepatic metabolites in plasma and liver samples from broilers and eggs from laying hens. World Mycotoxin J. 2014, 7, 167−175. (10) Hu, L.; Rychlik, M. Biosynthesis of 15N3-labeled enniatins and beauvericin and their application to stable isotope dilution assays. J. Agric. Food Chem. 2012, 60, 7129−7136. (11) De Baere, S.; Goossens, J.; Osselaere, A.; Devreese, M.; Vandenbroucke, V.; De Backer, P.; Croubels, S. Quantitative determination of T-2 toxin, HT-2 toxin, deoxynivalenol and deepoxy-deoxynivalenol in animal body fluids using LC-MS/MS detection. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2011, 879, 2403−2415. (12) Commission Decision. 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performances of analytical methods and the interpretation of results. Off. J. Eur. Communities 2002, L221, 8−36. (13) Guidance for Industry. Studies to Evaluate the Metabolism and Residue Kinetics of Veterinary Drugs in Food Producing Animals: Validation of Analytical Methods Used in Residue Depletion Studies. VICH GL 49 (R), http://www.fda.gov/downloads/AnimalVeterinary/ Gu i d anc e Co m pl i anc e Enf o r c em e n t / G u i da n c ef or In du s t r y / UCM207942.pdf (accessed Aug 5, 2016). (14) Knecht, J.; Stork, G. Prozentuales und logarithmisches Verfahren zur Berechnung von Eichkurven. Fresenius’ Z. Anal. Chem. 1974, 270, 97−99. (15) Hill, H. M.; Causey, A. G.; Lessard, D.; Selinger, K.; Herman, J. Choice and optimization of calibration functions. In Bioanalytical Approaches for Drugs, including Anti-asthmatics and Metabolites; Royal Society of Chemistry: London, UK, 1992; Vol. 20, pp 111−118. (16) Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal. Chem. 2003, 75, 3019−3030. (17) Goyarts, T.; Dänicke, S. Bioavailability of the Fusarium toxin deoxynivalenol (DON) from naturally contaminated wheat for the pig. Toxicol. Lett. 2006, 163, 171−182. F
DOI: 10.1021/acs.jafc.6b02913 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
