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Comparative toxicokinetics, absolute oral bioavailability and biotransformation of zearalenone in different poultry species Mathias Devreese, Gunther Antonissen, N. Broekaert, Siegrid De Baere , Lynn Vanhaecke, Patrick De Backer, and Siska Croubels J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01608 • Publication Date (Web): 07 May 2015 Downloaded from http://pubs.acs.org on May 10, 2015

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Journal of Agricultural and Food Chemistry

Comparative toxicokinetics, absolute oral bioavailability and biotransformation of zearalenone in different poultry species Mathias Devreese*1, Gunther Antonissen1,2, Nathan Broekaert1, Siegrid De Baere1, Lynn Vanhaecke³, Patrick De Backer1, Siska Croubels1 1

Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke

² Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke ³ Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke E-mail addresses: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected] *Corresponding Author: E-mail address: [email protected], Tel: + 32 9 264 73 24, Fax: +32 9 264 74 97

1 ACS Paragon Plus Environment


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Abstract – After oral (PO) and intravenous (IV) administration of zearalenone (ZEN) to broiler

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chickens, laying hens and turkey poults, the mycotoxin was rapidly absorbed (Tmax = 0.32 –

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0.97 h) in all three species, however, the absolute oral bioavailability was low (F% = 6.87 –

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10.28%). Next, also a rapid elimination of the mycotoxin in all poultry species was observed

5

(T1/2el = 0.29 – 0.46 h). Both α- and β-zearalenone (ZEL) were formed equally after IV

6

administration in all species studied, whereas an increased biotransformation to β-ZEL was

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demonstrated after PO administration indicating pre-systemic biotransformation mainly in

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broiler chickens and laying hens. In comparison to the latter, turkey poults demonstrated a

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more extensive biotransformation of ZEN to α-ZEL after PO administration which could, in

10

combination with the observed higher volume of distribution of ZEN, indicate a higher

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sensitivity of this species to the effects of ZEN in comparison to other poultry species.

12

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Keywords – poultry; laying hen; turkey; broiler chicken; zearalenone; toxicokinetics;

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biotransformation


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Introduction

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Mycotoxins are secondary metabolites produced by different fungal species contaminating

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several food and feed commodities. Over 400 mycotoxins have been identified, although

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only a few of them are known to have distinct toxic effects. The most prevalent mycotoxin

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producing fungi in moderate climates are Fusarium species. Zearalenone (ZEN) is one of the

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most frequently occurring Fusarium mycotoxins. In a recent study by Streit 1, 87% of the 83

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investigated feed and feed raw material samples were contaminated with ZEN, with a

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median contamination level of 14 µg/kg and a maximum of 5.3 mg/kg. ZEN can be listed as a

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non-steroidal estrogen or myco-estrogen 2. It resembles 17β-estradiol, the principal

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hormone produced by the ovary, which allows ZEN to bind to estrogen receptors in target

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cells 3. Once the estrogen receptor is bound, it undergoes a conformational change allowing

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the receptor to interact with chromatin and to modulate transcription of target genes 4. Not

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all compounds have the same affinity for estrogen receptors. It has been shown that some

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phase I metabolites of ZEN can express lower or even higher affinities to estrogen receptors

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than the parent compound. In general, ZEN is metabolized by 3α- and 3β-hydroxysteroid

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dehydrogenase (HSD) into α- and β-zearalenol (ZEL), respectively. β-ZEL has a 2.5 times

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lower affinity for the estrogen receptor, whereas α-ZEL has a 92 times higher binding affinity

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compared to ZEN. The biotransformation to β-ZEL can therefore be regarded as an

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inactivation pathway, whereas the biotransformation to α-ZEL can be seen as a bio-

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activation5. The rate and extent of α- or β-ZEL production, and consequently the

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susceptibility, is species dependent. Pigs are regarded as the most sensitive species, as

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suggested by in vitro data demonstrating that pig liver microsomes dominantly convert ZEN

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into α-ZEL 5. This has been confirmed in vivo where α-ZEL was the only phase I metabolite

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detected in plasma, urine and faeces of pigs intravenously (IV) dosed with ZEN 6. Next, in a 3 ACS Paragon Plus Environment


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study by Zöllner

the α-ZEL/β-ZEL ratios were 2.5/1 and 3/1 in pig liver and urine,

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respectively, after oral intake of ZEN. In vitro results suggest that laying hens and cattle

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metabolize ZEN to a large extent into β-ZEL, which confirms that they are less prone to the

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effects of ZEN

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detoxification pathway. The extent of conjugation has also been found to have major inter-

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species variation. Migdalof

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the total ZEN recovered in humans, whereas in dogs glucuronidation only accounts for 1% of

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the excreted ZEN metabolites.

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To date, no in vivo data are available in poultry regarding the phase I and phase II

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biotransformation of ZEN after oral exposure, which is mandatory to confirm the in vitro

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findings and the species dependent sensitivity. Also, limited information on in vivo

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toxicokinetics and absolute oral bioavailability is available. Osselaere

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elimination of ZEN after IV administration of 0.3 mg ZEN/kg body weight (BW) to broiler

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chickens (T1/2el = 0.53 h), whereas after oral (PO) administration of the same dose, no ZEN or

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phase I metabolites could be detected in plasma (limit of quantification: 1-5 ng/mL). The

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rapid elimination and the assumed low oral bioavailability of the mycotoxin supports the

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limited toxicity of ZEN in this animal species. Furthermore, Dailey11 administered 10 mg

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[14C]ZEN/kg BW to laying hens and demonstrated that 94% of the administered dose was

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eliminated via the excreta within 72 h, with one-third as unchanged [14C]ZEN and the other

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part as unidentified metabolites. Feeding mature laying hens a diet contaminated with ZEN

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up to 800 mg/kg did not affect their reproductive performance 12, 13. In contrast, feeding 100

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mg ZEN/kg feed to mature female turkeys, reduced the egg production by 20%

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feeding 800 mg ZEN/kg feed to male turkeys for 2 weeks induced strutting behavior and an

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increased size and coloration of caruncles and dewlaps, which was not present in birds fed

5, 8

. Next, also phase II biotransformation (conjugation) is regarded as a

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reported that glucuronidated metabolites account for 99% of

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reported a rapid

14

. Next,


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the uncontaminated diet

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turkey poults is most probably attributed to differences in toxicokinetics and/or

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biotransformation processes.

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Therefore, the aim of the present study was to unravel the toxicokinetic behavior, absolute

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oral bioavailability and biotransformation of ZEN after PO as well as IV administration in the

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economically most important poultry species, namely broiler chickens, laying hens and

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turkey poults.

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Materials and Methods

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Chemicals, products and reagents

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The analytical standard of ZEN, used for both plasma analysis and the animal experiment

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was obtained from Fermentek (Jerusalem, Israel). The analytical standards of α-ZEL, β-ZEL, α-

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zearalanol (α-ZAL), β-zearalanol (β-ZAL) and zearalanone (ZAN) used for the analytical

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experiments were obtained from Sigma-Aldrich (Bornem, Belgium). The internal standard

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(IS),

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Austria). All analytical standards were stored at ≤ -15 °C. Working solutions of ZEN, the

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described metabolites, and the IS were prepared by appropriate dilution of the stock

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solutions with acetonitrile (ACN) and stored at 2-8 °C. Water, methanol (MeOH), ACN and

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glacial acetic acid used for the plasma analysis were of LC-MS grade and obtained from

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Biosolve (Valkenswaard, The Netherlands). Water and dimethylsulfoxide (DMSO) used for

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the animal experiment were of analytical grade and obtained from Filterservice (Eupen,

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Belgium). Millex®-GV PVDF filter units (0.22 µm) were obtained from Merck-Millipore

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(Overijse, Belgium).

13

. This difference in sensitivity between broiler chickens and

C18-ZEN, used for the analytical experiments was obtained from Romer Labs (Tulln,



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Animals and experimental procedure

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Six turkey Hybrid Converter poults (3 ♂ / 3 ♀, 0.95 ± 0.06 kg BW), broiler chickens (Ross 308,

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3 ♂ / 3 ♀, 1.05 ± 0.05 kg BW) and laying hens (Brown Leghorn, 6 ♀, 1.04 ± 0.05 kg BW) were

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obtained from a commercial breeder. During the experiment, animals were housed in group

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and a 16 h light / 8 h dark light cycle was applied. Feed and water were given ad libitum

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throughout the one week acclimatization period. Subsequently, twelve hours before the

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start of the experiment, the animals were deprived of feed. After this period, three birds per

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species were administered ZEN (3 mg/kg BW) by oral gavage (PO) whereas the other 3

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birds/species were injected the same dose of ZEN in the wing vein (IV). The PO and IV bolus

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solution was prepared by dissolving the ZEN standard in DMSO (10 mg/mL) and further

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diluted with water (PO) or physiological saline (IV) up to a volume of 1 mL. Following

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administration of the mycotoxin, 0.5 mL of blood was drawn from the leg vein at various

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time points: 0 (just before administration) and 2, 5, 10, 20, 30, 40, 50, 60, 120, 180 and 240

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min post-administration. Samples were centrifuged (2851 x g, 10 min, 4 °C) and aliquots of

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plasma (150 µL) were stored at ≤ -15 °C until analysis. After a two-day wash-out period, the

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protocol was repeated in a two-way cross-over design. The birds that received an IV injection

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of the mycotoxin then received a PO bolus and vice versa. The dosing, blood collection and

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sample storage was performed in the same way as the first administration. The animal

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experiment was approved by the Ethical Committee of the Faculty of Veterinary Medicine

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and Bioscience Engineering of Ghent University (EC 2014/118).

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LC-MS/MS analysis

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Sample treatment and quantification of ZEN, and its phase I metabolites (α-ZEL, β-ZEL, α-

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ZAL, β-ZAL and ZAN, see Supplementary Figure 1), in poultry plasma was performed as 6 ACS Paragon Plus Environment

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previously described by De Baere et al. (2012) with minor modifications. Briefly, 50 µL of IS

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working solution (50 ng/mL) were added to 150 µL of plasma, followed by the addition of

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ACN up to 1 mL. Next, the sample was vortex mixed (15 sec) and centrifuged (8517 x g, 10

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min, 4 °C). The supernatant was evaporated to dryness using nitrogen (N2, 45 ± 5 °C). The dry

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residue was reconstituted in 200 µL of water/MeOH (85/15, v/v), vortex mixed (15 sec),

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filtered through a Millex® GV-PVDF syringe filter and transferred into a conical autosampler

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vial. An aliquot (5 µL) was injected onto the LC-MS/MS instrument.

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Chromatographic separation was achieved on a Hypersil® Gold column (50 mm x 2.1 mm i.d.,

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dp: 1.9 µm) in combination with a guard column of the same type (10 mm x 2.1 mm i.d., dp:

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3 µm), both from ThermoFisher Scientific (Breda, The Netherlands). Mobile phase A

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consisted of 0.1% acetic acid in water whereas mobile phase B was ACN. Following gradient

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elution program was run: 0-0.5 min (70% A, 30% B), 0.5-5.0 min (linear gradient to 70% B),

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5.0-6.4 min (30% A, 70% B), 6.4-6.5 min (linear gradient to 30% B), 6.5-8.5 min (70% A, 30%

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B). Flow rate was set at 300 µL/min and the temperature of the column oven and

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autosampler tray were set at 45 and 5 °C, respectively.

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Detection was performed on a Waters Xevo® TQ-S triple quadrupole mass spectrometer by

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means of electrospray ionization (ESI) in the negative ionization mode. The instrument was

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tuned by direct infusion of a 10 ng/mL working solution of ZEN, the metabolites and the IS.

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The following parameters were retained for optimal detection: capillary voltage: 2.5 kV,

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cone voltage: 15 V, source temperature: 150 °C, desolvation temperature: 600 °C, cone gas

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flow: 150 L/h, desolvation gas flow: 1000 L/h. Acquisition was performed in the selected

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reaction monitoring (SRM) mode. Following transitions (m/z) were used as quantifier and

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qualifier ion, respectively, for ZEN: 317.1 > 175.0 and 317.1 >131.0, for α-ZEL: 319.0 > 275.0



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and 319.0 > 159.9, for β-ZEL: 319.0 > 159.9 and 319.0 > 275.0, for ZAN: 319.0 > 205.0 and

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319.0 > 275.0, for α-ZAL: 321.1 > 277.1 and 321.1 > 303.0, for β-ZAL: 321.1 > 277.1 and 321.1

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> 303.0 and for the IS: 335.2 > 168.9 and 335.2 > 185.0. The method was validated for the

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compounds listed above according to a validation protocol described by De Baere

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on European

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following parameters: linearity (correlation coefficient and goodness-of-fit), within- and

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between-run accuracy and precision, carry-over, limit of quantification (LOQ) and limit of

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detection (LOD). The LOQ was set at 0.5 ng/mL for ZEN, 0.2 ng/mL for α-ZEL and α-ZAL and

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0.1 ng/mL for β-ZEL, β-ZAL and ZAN.

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UHPLC-HRMS analysis

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The extracted samples were also analyzed by UHPLC coupled to HRMS analysis for

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identification and semi-quantification of the glucuronide conjugates of ZEN and of its phase I

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metabolites as previously described by De Baere 19. This was performed based on the peak

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area ratios of the metabolites, versus the IS. The UHPLC system consisted of an Accela® type

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1250 High Speed LC and autosampler both from ThermoFisher Scientific. The same HPLC

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column and mobile phases were used as described above. The UHPLC was interfaced to an

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Exactive Orbitrap® HR mass spectrometer, equipped with a heated ESI probe operating in the

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negative ionization mode (ThermoFisher Scientific). Accurate masses were based on the

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predicted masses (m/z; [M-H]-) described by De Baere 19 and Stevenson 20: ZEN-glucuronide

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(ZEN-GlcA, C24H30O11): 493.17153; ZAN-GlcA, α-ZEL-GlcA and β-ZEL-GlcA (C24H32O11, no

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chromatographic separation or distinction between these compounds based on accurate

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masses possible): 495.18718; α-ZAL-GlcA, β-ZAL-GlcA (C24H34O11, no chromatographic

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separation or distinction between these compounds based on accurate masses possible):

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and international

18

16

based

guidelines. The validation protocol encompassed


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497.20283. Confirmation of the GlcA peaks observed in the extracted ion chromatogram

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(EIC) was performed by comparing the theoretically calculated 13C/12C isotope ratios for the

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[M-H]- ion with the corresponding isotope ratios detected in the analyzed samples, as

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described by De Baere 19.

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Toxicokinetic analysis

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Non compartmental toxicokinetic analysis of ZEN, α- and β-ZEL was performed with

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WinNonlin 6.3 (Pharsight, St-Louis, MI, USA). For ZEN, following toxicokinetic parameters

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were calculated for IV and PO administration: maximal plasma concentration for PO (Cmax),

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plasma concentration at time 0 for IV (C0), time to maximal plasma concentration (Tmax), area

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under the plasma concentration-time curve from time 0 to 3 h (AUC0-t), area under the

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plasma concentration-time curve from time 0 to infinite (AUC0-inf), elimination rate constant

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(kel), elimination half-life (T1/2el), total body clearance (Cl), volume of distribution (Vd) and

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mean residence time (MRT).

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The absolute oral bioavailability (F, expressed as %, F%) for each individual bird was

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calculated according to the formula:

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F% = AUC0-inf PO / AUC0-inf IV x 100

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The Cl and Vd after PO administration was calculated by multiplying the data generated by

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the toxicokinetic software (i.e. Vd/F and Cl/F) by the F from each individual bird.

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The mean absorption time (MAT) was calculated according to the formula:

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MAT = MRTPO – MRTIV The absorption rate constant (ka) and absorption half-life (T1/2a) were derived from the MAT:



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ka = MAT-1

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T1/2a = 0.693 / ka

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For α- and β-ZEL, the Cmax, Tmax, AUC0-t (from time 0 to the last time point above the LOQ, i.e.

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2 or 3 h) and AUC0-inf were determined after both IV and PO administration of ZEN. Next,

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ZEL/ZEN ratios were calculated according to the formulas:

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α-ZEL/ZEN = (AUC0-inf α-ZEL) / (AUC0-inf ZEN)

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β-ZEL/ZEN = (AUC0-inf β-ZEL) / (AUC0-inf ZEN)

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ZEL/ZEN = (AUC0-inf α-ZEL + AUC0-inf β-ZEL) / (AUC0-inf ZEN)

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Finally, the α-ZEL/β-ZEL ratio was calculated according to the formula: α-ZEL/β-ZEL = (AUC0-inf α-ZEL) / (AUC0-inf β-ZEL)

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Statistical analysis

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All toxicokinetic parameters from each compound (ZEN, α- and β-ZEL) and each

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administration route were compared between animal species using one-way analysis of

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variance (ANOVA) (SPSS 21, IBM, USA). The level of significance was set at 0.05.

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Results and Discussion

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The goal of this study was to unravel the toxicokinetic behavior, absolute oral bioavailability

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and biotransformation of ZEN in different poultry species in order to assess potential species

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dependent sensitivity to this mycotoxin. During the animal experiment, no clinical symptoms

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were observed after PO or IV administration of ZEN to the birds. Furthermore, no sex

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differences in toxicokinetic parameters were observed for broiler chickens and turkey poults.


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In figure 1A, B and C, the plasma concentration-time profiles of ZEN, α- and β-ZEL after IV

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and PO administration of ZEN to broiler chickens, laying hens and turkey poults are

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presented, respectively. Only trace amounts of α- and β-ZAL and ZAN were detected but

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none above the LOQ of 0.2, 0.1 and 0.1 ng/mL, respectively. As can be seen in these figures

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and Table 1, ZEN is rapidly absorbed in all studied poultry species, namely broiler chickens

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(Tmax = 0.35 h, MAT = 0.37 h), laying hens (Tmax = 0.32 h, MAT = 0.35 h) and turkey poults

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(Tmax = 0.97 h, MAT = 0.44 h). However, the F% is low in all studied poultry species, 8.34%,

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10.28% and 6.87%, in broiler chickens, laying hens and turkey poults, respectively. This might

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indicate either a low extent of absorption and/or extensive pre-systemic biotransformation,

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and attributes to the generally accepted high tolerance of poultry to ZEN since F is a

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measurement for systemic exposure. Next to rapid absorption, the mycotoxin is rapidly

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eliminated in all species as well with T1/2el between 0.34 – 0.36 h and 0.29 – 0.46 h after PO

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and IV administration, respectively. Osselaere

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administration of 0.3 mg ZEN/kg BW to broiler chickens. Furthermore, a high Vd is observed

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after PO and IV administration in all animal species (Vd = 3.21 – 10.65 L/kg). For broiler

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chickens, an even higher Vd has been reported previously by Osselaere

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L/kg, however this value was reported with a high uncertainty (15.15 L/kg). Of interest here

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is that turkey poults have a significant higher Vd (9.03 – 10.65 L/kg) compared to broiler

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chickens (3.21 – 4.16 L/kg) and laying hens (6.18 – 6.24 L/kg) which supports the hypothesis

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of a higher sensitivity to ZEN of this species. Furthermore, the higher Vd explains why the C0

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is significantly lower in turkey poults (C0 = 700.5 ng/mL) compared to broiler chickens (C0 =

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2607.0 ng/mL) and laying hens (C0 = 2789.2 ng/mL) although they were administered the

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same dose of ZEN (3 mg/kg BW). Also pigs display a high Vd of ZEN, comparable to turkey

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poults, after IV administration, namely 10.84 L/kg, leading to a high tissue distribution of the

10

reported a similar T1/2el of 0.53 h after IV

10

, namely 22.26



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mycotoxin 6. Besides a higher Vd in turkey poults, also a significantly higher Cl in turkeys (Cl =

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16.57 – 19.66 L/h/kg) compared to broiler chickens (Cl = 9.06 – 11.1 L/h/kg) and laying hens

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(Cl = 9.39 – 11.38 L/h/kg) was noticed. Since both Vd and Cl are higher in turkey poults, this

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leads to a comparable T1/2el between the latter (T1/2el = 0.35 – 0.38 h) and broiler chickens

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(T1/2el = 0.29 – 0.34 h) and laying hens (T1/2el = 0.36 – 0.46 h). Next, due to the higher Cl in

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turkey poults and comparable F, the AUC0-inf is significantly lower in this species after both

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administration routes (AUC0-inf PO = 10.21 h.ng/mL, AUC0-inf IV = 183.12 h.ng/mL) compared to

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broiler chickens (AUC0-inf PO = 23.59 h.ng/mL, AUC0-inf IV = 292.10 h.ng/mL) and laying hens

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(AUC0-inf PO = 30.04 h.ng/mL, AUC0-inf IV = 324.93 h.ng/mL). Similarly, also the AUC0-t was

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significantly lower.

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As described above, species dependent biotransformation of ZEN has a distinct impact on

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sensitivity to this mycotoxin, and the in vitro estrogenic potency of its phase I metabolites is

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as follows: α-ZAL > α-ZEL > β-ZAL > ZEN > β-ZEL 21. Phase I biotransformation of ZEN consists

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of reduction of the ketone group of ZEN to its corresponding alcohol by 3α-/3β-HSD, thereby

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forming α/β-ZEL, and aromatic hydrogenation of α/β-ZEL to α/β-ZAL by cytochrome P450

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(CYP450) enzymes. Finally, α/β-ZAL can be oxidized by 3α-/3β-HSD to ZAN. The

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biotransformation of ZEL to ZAL and ZAN has been reported for pigs and humans, however,

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the extent is negligible 21. Also in the present study only trace amounts of α/β-ZAL and ZAN

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were detected demonstrating that formation of α- and β-ZEL is also the predominant phase I

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biotransformation in poultry. In all three poultry species both α- and β-ZEL could be detected

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although the extent differed between species and routes of administration (Figure 1 and

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Table 2). Turkey poults have a more efficient biotransformation of ZEN to α- and β-ZEL

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compared to broiler chickens and laying hens, with higher α-ZEL/ZEN and β-ZEL/ZEN ratios

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after both PO and IV administration (although not significant for β-ZEL/ZEN after PO), 12 ACS Paragon Plus Environment

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whereas these ratios are comparable between broiler chickens and laying hens (Table 2).

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Consequently, also the ZEL/ZEN ratio in turkey poults is significantly higher after PO

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(ZEL/ZENPO = 5.226) as well as IV administration (ZEL/ZENIV = 0.152) compared to broiler

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chickens (ZEL/ZENPO = 1.149, ZEL/ZENIV = 0.032) and laying hens (ZEL/ZENPO = 2.086,

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ZEL/ZENIV = 0.042). Furthermore, the biotransformation of ZEN to α- and β-ZEL occurs to a

248

greater extent after PO than after IV administration in all animal species. This difference in

249

ZEL formation between both administration routes indicates pre-systemic biotransformation

250

of ZEN in the gut and/or the liver. After IV administration, the α-ZEL/β-ZEL ratio is not

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statistically different between bird species and is around 1 (α-ZEL/β-ZELIV = 1.073 to 1.629).

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In contrast, after PO administration the β-ZEL isomer is predominantly formed in broiler

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chickens (α-ZEL/β-ZELPO = 0.153) and laying hens (α-ZEL/β-ZELPO = 0.027), but this is

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significantly different in turkey poults (α-ZEL/β-ZELPO = 0.749). Again, this indicates pre-

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systemic formation of mainly β-ZEL, especially in broiler chickens and laying hens. These data

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confirm the in vitro findings in laying hens, namely a predominant β-ZEL formation 5. Since

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the α-ZEL/ZEN ratio after PO and IV ZEN administration is significantly higher in turkey poults

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compared to both other bird species, this supports the hypothesis of increased sensitivity of

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turkey poults to the estrogenic effects of the mycotoxin.

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Phase II biotransformation of ZEN and its phase I metabolites comprises glucuronidation

261

which is catalyzed by uridine 5’-diphosphate glucuronosyltransferase (UGT)

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analytical standards of glucuronidated ZEN (ZEN-GlcA) nor glucuronidated α- or β-ZEL (ZEL-

263

GlcA) are commercially available, no accurate quantitative analysis could be performed.

264

However, a semi-quantitative using HR-MS was done (Figure 2). After IV administration of

265

ZEN, ZEN-GlcA is the predominant phase II metabolite in all three bird species. Although the

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ZEL/ZEN ratio is higher in turkey poults, namely 0.152, compared to broiler chickens and

22

. Since no



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laying hens, 0.032 and 0.042 respectively, no ZEL-GlcA could be detected in this species after

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IV administration, which is in contrast to the other species. After oral ZEN administration, a

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comparable amount of ZEN-GlcA and ZEL-GlcA was detected in broiler chickens and laying

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hens, whereas in turkey poults only ZEL-GlcA was detected, attributed to the high ZEL/ZEN

271

ratio in this species after PO administration (ZEL/ZEN = 5.226). This confirms the phase I

272

biotransformation data, namely a pre-systemic ZEL formation and consequent predominant

273

ZEL glucuronidation. Differences in phase II biotransformation might also account for

274

differences in sensitivity. For the mycotoxin DON, for instance, limited sulfation was

275

described for turkeys in contrast to broiler chickens, which might explain the increased

276

sensitivity of turkeys to deoxynivalenol

277

turkeys are more sensitive compared to other poultry and animal species based on

278

differences in biotransformation of AFB1. In turkeys, the phase I bio-activation of AFB1 to

279

AFB1-8,9-epoxide, is highly efficient and the epoxide metabolite is held responsible for the

280

hepatotoxic and carcinogenic effects of AFB1 24. Furthermore, turkeys have a glutathione-S-

281

transferase deficiency (phase II biotransformation) which adds to the sensitivity of this

282

species 25.

283

In conclusion, this paper describes – for the first time – the toxicokinetic behavior, absolute

284

oral bioavailability and comparative phase I and II biotransformation of ZEN in broiler

285

chickens, laying hens and turkey poults. ZEN is rapidly absorbed in all studied poultry

286

species, but the absolute oral bioavailability of the mycotoxin is low. Furthermore, it is

287

rapidly eliminated as well after both PO and IV administration. Both α- and β-ZEL were

288

formed equally after IV administration in all bird species, whereas an increased

289

biotransformation to β-ZEL was demonstrated after oral administration indicating pre-

290

systemic biotransformation processes, primarily in broiler chickens and laying hens. Finally,

23

. Also for the mycotoxin aflatoxin B1 (AFB1),


Page 15 of 32


291

the presented data suggest that turkey poults might be more sensitive to the effects of ZEN

292

based on the higher Vd of ZEN and the more extensive pre-systemic biotransformation of

293

ZEN to α-ZEL in comparison to the two other poultry species.



Page 16 of 32

294

Supporting Information Available

295

Chemical structure and phase I biotransformation pathways of zearalenone (adapted from

296

Mukherjee21). This material is available free of charge via the Internet at http://pubs.acs.org.

297

References

298

1.

299

mycotoxin screening reveals the occurrence of 139 different secondary metabolites in feed

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P. T.; van der Burg, P.; Gustafsson, J. A., Interaction of estrogenic chemicals and

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phytoestrogens with estrogen receptor beta. Endocrinology 1998, 139, 4252-4263.

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hepatic biotransformation of zearalenone. Vet J 2006, 172, 96-102.

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deoxynivalenol (DON) in the pig. Archives of Animal Nutrition-Archiv Fur Tierernahrung 2004,

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Lindner, W., Concentration levels of zearalenone and its metabolites in urine, muscle tissue,

Streit, E.; Schwab, C.; Sulyok, M.; Naehrer, K.; Krska, R.; Schatzmayr, G., Multi-

Tiemann, U.; Danicke, S., In vivo and in vitro effects of the mycotoxins zearalenone

Greenman, D. L.; Mehta, R. G.; Wittliff, J. L., Nuclear interaction of Fusarium

Kuiper, G. G. J. M.; Lemmen, J. G.; Carlsson, B.; Corton, J. C.; Safe, S. H.; van der Saag,

Malekinejad, H.; Maas-Bakker, R.; Fink-Gremmels, J., Species differences in the

Dänicke, S.; Valenta, H.; Doll, S., On the toxicokinetics and the metabolism of

Zollner, P.; Jodlbauer, J.; Kleinova, M.; Kahlbacher, H.; Kuhn, T.; Hochsteiner, W.;


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deoxynivalenol, T-2 toxin and zearalenone in broiler chickens. Food Chem Toxicol 2013, 51,

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of Dietary Zearalenone on Reproduction of Chickens. Poultry Sci 1981, 60, 1165-1174.

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Concentrations of Dietary Zearalenone by Young Male Turkey Poults. Poultry Sci 1986, 65,

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Zinedine, A.; Soriano, J. M.; Molto, J. C.; Manes, J., Review on the toxicity, occurrence,

Migdalof, B. H.; Dugger, H. A.; Heider, J. G.; Coombs, R. A.; Terry, M. K.,

Osselaere, A.; Devreese, M.; Goossens, J.; Vandenbroucke, V.; De Baere, S.; De

Dailey, R. E.; Reese, R. E.; Brouwer, E. A., Metabolism of [Zearalenone-C-14 in Laying

Allen, N. K.; Aakhusallen, S.; Mirocha, C. J., Effect of Zearalenone on Reproduction of

Allen, N. K.; Mirocha, C. J.; Aakhusallen, S.; Bitgood, J. J.; Weaver, G.; Bates, F., Effect

Allen, N. K.; Peguri, A.; Mirocha, C. J.; Newman, J. A., Effects of Fusarium Cultures, T-

Olsen, M.; Mirocha, C. J.; Abbas, H. K.; Johansson, B., Metabolism of High-



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De Baere, S.; Goossens, J.; Osselaere, A.; Devreese, M.; Vandenbroucke, V.; De

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and deepoxy-deoxynivalenol in animal body fluids using LC-MS/MS detection. Journal of

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Volume 8: Notice to applicants and guideline on the establishment of maximum residue limits

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(MRLs) for residues of veterinary medicinal products in foodstuffs of animal origin. 2005 weblink:

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Kinetics of Veterinary Drugs in Food-Producing Animals: Validation of Analytical Methods Used in

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Residue Depletion Studies. 2015 weblink:

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Quantitative determination of zearalenone and its major metabolites in animal plasma using

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Miles, C. O., Preparative enzymatic synthesis of glucuronides of zearalenone and five of its

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metabolites. J Agr Food Chem 2008, 56, 4032-4038.

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V.; Zarbl, H.; Georgopoulos, P. G., Physiologically-Based Toxicokinetic Modeling of

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Zearalenone and Its Metabolites: Application to the Jersey Girl Study. Plos One 2014, 9.

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VICH GL49(R). Guidance for Industry Studies to Evaluate the Metabolism and Residue

De Baere, S.; Osselaere, A.; Devreese, M.; Van Haecke, L.; De Backer, P.; Croubels, S.,

Stevenson, D. E.; Hansen, R. P.; Loader, J. I.; Jensen, D. J.; Cooney, J. M.; Wilkins, A. L.;

Mukherjee, D.; Royce, S. G.; Alexander, J. A.; Buckley, B.; Isukapalli, S. S.; Bandera, E.


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Pfeiffer, E.; Hildebrand, A.; Mikula, H.; Metzler, M., Glucuronidation of zearalenone,

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zeranol and four metabolites in vitro: Formation of glucuronides by various microsomes and

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human UDP-glucuronosyltransferase isoforms. Mol Nutr Food Res 2010, 54, 1468-1476.

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De Backer, P.; Croubels, S., Toxicokinetic study and oral bioavailability of deoxynivalenol in

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turkey poults, and comparative biotransformation between broilers and turkeys World

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Mycotoxin J 2015, in press.

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the relative roles of cytochromes P450 1A5 and 3A37. Toxicol Appl Pharmacol 2011, 254,

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349-54.

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Devreese, M.; Antonissen, G.; Broekaert, N.; De Mil, T.; De Baere, S.; Vanhaecke, L.;

Rawal, S.; Coulombe, R. A., Jr., Metabolism of aflatoxin B1 in turkey liver microsomes:

Chen, C-H. Activation and Detoxification Enzymes: Functions and Implications. 2012,



Page 20 of 32

375

Figure Captions

376

Figure 1. Plasma concentration-time profile of zearalenone (ZEN), α-zearalenol (α-ZEL) and

377

β-zearalenol (β-ZEL) after intravenous (IV) and oral (PO) administration of 3 mg ZEN/kg body

378

weight to broiler chickens (A), laying hens (B) and turkey poults (C) (n=6). Values are

379

presented as mean + SD.

380

Figure 2. Ratio of the peak area detected by ultra-high performance liquid chromatography

381

coupled to high resolution-mass spectrometry of zearalenone (ZEN) or its metabolites,

382

namely α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), ZEN-glucuronide (ZEN-GlcA) and ZEL-

383

glucuronide (ZEL-GlcA), and the internal standard (13C18-ZEN) plotted against the time after

384

intravenous (IV) or oral (PO) administration (p.a.) of 3 mg ZEN/kg body weight to broiler

385

chickens (A), laying hens (B) or turkey poults (C) (n=6). Values are presented as mean + SD.


Page 21 of 32




Page 22 of 32

Table 1. Main Toxicokinetic Parameters Of Zearalenone After Oral (PO) And Intravenous (IV) Administration (3 mg/kg BW) To Broiler Chickens, Laying Hens And Turkey Poults (n=6). Values Are Presented As Mean ± SD. Broiler chickens Laying hens Turkey poults PO

IV

PO

IV

PO

IV

39.17 ± 18.67 a,b

2607.0 ± 506.03 a

54.12 ± 22.90 a

2789.2 ± 189.78 a

8.33 ± 2.21 b

700.5 ± 301.18 b

Tmax (h) e

0.35 ± 0.16 a

/

0.32 ± 0.08 a

/

0.97 ± 0.36 b

/

AUC0-t (h.ng/mL) f

21.87 ± 12.12 a,b

290.9 ± 72.78 a

29.31 ± 6.78 a

322.8 ± 37.27 a

8.53 ± 3.07 b

180.0 ± 21.12 b

AUC0-inf (h.ng/mL) g

23.59 ± 15.62 a

292.1 ± 72.50 a

30.04 ± 6.45 a

324.9 ± 35.94 a

10.21 ± 5.57 b

183.1 ± 18.92 b

ka (h-1) h

3.36 ± 1.52 a

/

3.50 ± 1.41 a

/

3.68 ± 2.28 a

/

T1/2a (h) i

0.26 ± 0.10 a

/

0.24 ± 0.09 a

/

0.30 ± 0.19 a

/

MRT (h) j

0.53 ± 0.13 a

0.16 ± 0.02 a

0.53 ± 0.07 a

0.22 ± 0.04 a,b

0.68 ± 0.19 a

0.33 ± 0.09 b

MAT (h) k

0.37 ± 0.14 a

/

0.35 ± 0.13 a

/

0.44 ± 0.27 a

/

Cl (L/h/kg) l

9.06 ± 4.61 a

11.1 ± 2.91 a

11.38 ± 3.03 a

9.39 ± 1.08 a

19.66 ± 3.87 b

16.57 ± 1.64 b

kel (h-1) m

2.14 ± 0.35 a

2.86 ± 1.06 a

1.98 ± 0.37 a

1.72 ± 0.07 a

2.04 ± 0.57 a

1.83 ± 0.10 a

T1/2el (h) n

0.34 ± 0.07 a

0.29 ± 0.11 a

0.36 ± 0.07 a

0.46 ± 0.12 a

0.35 ± 0.10 a

0.38 ± 0.02 a

Cmax (PO) c or C0 (IV) d (ng/mL)

22

ACS Paragon Plus Environment

Page 23 of 32


Vd (L/kg) o

3.21 ± 2.13 a

4.16 ± 0.75 a

6.18 ± 2.52 a

6.24 ± 0.92 a

10.65 ± 1.05 b

9.03 ± 1.43 b

F (%) p

8.34 ± 5.96 a

100 a

10.28 ± 2.53 a

100 a

6.87 ± 3.54 a

100 a

A different superscript (a or b) denotes a significant difference between animal species for each administration route at p < 0.05 Cmax: maximal plasma concentration, d C0: plasma concentration at time 0, e Tmax: time to maximal plasma concentration, f AUC0-t: area under the plasma concentration-time curve from time 0 to 3 h; g AUC0-inf: area under the plasma concentration-time curve from time 0 to infinite, h ka: absorption rate constant, i T1/2a: absorption half-life, j MRT: mean residence time, k MAT: mean absorption time, l Cl: total body clearance, m kel: elimination rate constant, n T1/2el: elimination half-life, o Vd: volume of distribution, p F: absolute oral bioavailability c

23



Page 24 of 32

Table 2. Main Toxicokinetic Parameters Of α- And β-zearalenol (α-/β-ZEL) After Oral (PO) And Intravenous (IV) Administration Of Zearalenone (3 mg/kg BW) To Broiler Chickens, Laying Hens And Turkey Poults (n=6). Values Are Presented As Mean ± SD. Broiler chickens Laying hens Turkey poults PO

IV

PO

IV

PO

IV

Cmax (ng/mL) c

3.99 ± 2.53 a

30.31 ± 10.23 a

3.20 ± 1.99 a

37.91 ± 10.07 a

6.45 ± 0.33 a

27.32 ± 2.21 a

Tmax (h) d

0.63 ± 0.11 a

0.03 ± 0.00 a

0.27 ± 0.08 a

0.03 ± 0.00 a

0.58 ± 0.08 a

0.09 ± 0.03 b

AUC0-t (h.ng/mL) e

2.44 ± 0.80 a

5.68 ± 1.61 a

1.57 ± 0.87 a

6.81 ± 1.80 a

11.60 ± 0.14 b

17.71 ± 2.35 b

AUC0-inf (h.ng/mL) f

2.79 ± 1.37 a

5.93 ± 1.68 a

1.63 ± 0.89 a

6.99 ± 1.75 a

15.58 ± 8.39 b

20.04 ± 5.12 b

Cmax (ng/mL) c

25.25 ± 13.96 a

13.30 ± 6.48 a

96.16 ± 38.95 b

38.92 ± 16.14 b

28.52 ± 15.36 a

15.14 ± 1.61 a

Tmax (h) d

0.61 ± 0.11 a

0.07 ± 0.19 a

0.42 ± 0.08 a,b

0.03 ± 0.00 a

0.37 ± 0.14 b

0.15 ± 0.03 b

AUC0-t (h.ng/mL) e

24.55 ± 15.09 a

3.50 ± 1.51 a

58.48 ± 14.60 a

6.64 ± 2.81 b

29.66 ± 13.43 a

9.83 ± 1.43 b

AUC0-inf (h.ng/mL) f

27.36 ± 17.51 a

4.09 ± 1.87 a

58.84 ± 14.54 a

6.90 ± 2.71 a

38.83 ± 20.04 a

15.36 ± 3.42 b

α-ZEL/ZEN g

0.138 ± 0.073 a

0.020 ± 0.001 a

0.057 ± 0.026 a

0.021 ± 0.004 a

1.015 ± 0.369 b

0.109 ± 0.033 b

β-ZEL/ZEN h

1.607 ± 1.159 a

0.018 ± 0.011 a

2.029 ± 0.262 a

0.021 ± 0.006 a

4.888 ± 3.124 a

0.089 ± 0.028 b

α-ZEL

β-ZEL

24


Page 25 of 32


ZEL/ZEN i

1.149 ± 0.525 a

0.032 ± 0.009 a

2.086 ± 0.271 a

0.042 ± 0.009 a

5.226 ± 2.898 b

0.152 ± 0.040 b

α-ZEL/β-ZEL j

0.153 ± 0.049 a

1.629 ± 0.566 a

0.027 ± 0.013 a

1.073 ± 0.151 a

0.749 ± 0.292 b

1.076 ± 0.243 a

A different superscript (a or b) denotes a significant difference between animal species for each administration route at p < 0.05 c Cmax: maximal plasma concentration, d Tmax: time to maximal plasma concentration, e AUC0-t: area under the plasma concentration-time curve from time 0 to 2 or 3 h, f AUC0-inf: area under the plasma concentration-time curve from time 0 to infinite, g α-ZEL/ZEN = (AUC0-inf α-ZEL) / (AUC0-inf ZEN), h β-ZEL/ZEN = (AUC0-inf β-ZEL) / (AUC0-inf ZEN), i ZEL/ZEN = (AUC0-inf α-ZEL + AUC0-inf β-ZEL) / (AUC0-inf ZEN), j α-ZEL/β-ZEL = (AUC0-inf α-ZEL ) / (AUC0-inf β-ZEL)

25



Page 26 of 32

Figure 1 (A)

Plasma concentration (ng/mL)

IV 10000.0 1000.0 100.0 ZEN 10.0

α-ZEL β-ZEL

1.0 0.1 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time p.a. (h)


PO 100.00

10.00

1.00

ZEN α-ZEL

0.10

β-ZEL

0.01 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time p.a. (h)


Page 27 of 32


(B)


IV 10000.0 1000.0 100.0 ZEN 10.0

α-ZEL β-ZEL

1.0 0.1 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time p.a. (h)


PO 1000.00 100.00 10.00 ZEN 1.00

α-ZEL β-ZEL

0.10 0.01 0.0

0.5

1.0

1.5

2.0

Time p.a. (h)



Page 28 of 32

(C)


IV 1000

100 ZEN α-ZEL

10

β-ZEL 1 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time p.a. (h)


PO 100.00

10.00

1.00

ZEN α-ZEL

0.10

β-ZEL

0.01 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time p.a. (h)


Page 29 of 32


Figure 2 (A)

IV 1000.000

Area ratios

100.000 10.000

ZEN

1.000

α-ZEL

0.100

β-ZEL ZEN-GlcA

0.010

ZEL-GlcA 0.001 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time p.a. (h)

PO 10.000

Area ratios

1.000 ZEN 0.100

α-ZEL β-ZEL

0.010

ZEN-GlcA ZEL-GlcA

0.001 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time p.a. (h)



Page 30 of 32

(B)

IV 1000.000

Area ratios

100.000 10.000

ZEN

1.000

α-ZEL

0.100

β-ZEL ZEN-GlcA

0.010

ZEL-GlcA 0.001 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time p.a. (h)

PO 10.000

Area ratios

1.000 ZEN 0.100

α-ZEL β-ZEL

0.010

ZEN-GlcA ZEL-GlcA

0.001 0.0

0.5

1.0

1.5

2.0

Time p.a. (h)


Page 31 of 32


(C)

IV Area ratios

100.00 10.00 ZEN

1.00

α-ZEL β-ZEL

0.10

ZEN-GlcA 0.01 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time p.a. (h)

PO Area ratios

10.000 1.000 ZEN

0.100

α-ZEL β-ZEL

0.010

ZEL-GlcA 0.001 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Time p.a. (h)



Page 32 of 32

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