- Email: [email protected]
Pilot toxicokinetic study and absolute oral bioavailability of the Fusarium mycotoxin enniatin B1 in pigs
Food and Chemical Toxicology 63 (2014) 161–165
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Brief communication
Pilot toxicokinetic study and absolute oral bioavailability of the Fusarium mycotoxin enniatin B1 in pigs Mathias Devreese ⇑, Nathan Broekaert, Thomas De Mil, Sophie Fraeyman, Patrick De Backer, Siska Croubels Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
a r t i c l e
i n f o
Article history: Received 1 October 2013 Accepted 5 November 2013 Available online 13 November 2013 Keywords: Toxicokinetics Absolute oral bioavailability Mycotoxin Enniatin B1 Pig
a b s t r a c t The aim of present study was to reveal the toxicokinetic properties and absolute oral bioavailability of enniatin B1 in pigs. Five pigs were administered this Fusarium mycotoxin per os and intravenously in a two-way cross-over design. The toxicokinetic profile fitted a two-compartmental model. Enniatin B1 is rapidly absorbed after oral administration (T1/2a = 0.15 h, Tmax = 0.24 h) and rapidly distributed and eliminated as well (T1/2ela = 0.15 h; T1/2elb = 1.57 h). The absolute oral bioavailability is high (90.9%), indicating a clear systemic exposure. After intravenous administration, the mycotoxin is distributed and eliminated rapidly (T1/2ela = 0.15 h; T1/2elb = 1.13 h), in accordance with oral administration. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Fusarium fungi frequently infest crops in temperate regions such as Western Europe and North America. They can produce a wide range of mycotoxins, including several extensively studied compounds such as trichothecenes, zearalenone and fumonisins. They are also capable of producing other less well-known mycotoxins like enniatins (ENNs) (enniatin (ENN) A, A1, B and B1) and beauvericin (BEA). ENNs are cyclic hexadepsipeptides consisting of alternating D-a-hydroxyisovaleric acids and L-methyl-amino acids (Zhukhlistova et al., 1999) (Fig. 1). Over the last decade, ENNs were found to be common contaminants of grains, maize and other feedstuffs (Jestoi, 2008; Devreese et al., 2013). Recently, Streit et al. (2013) analyzed 83 feed and feed raw material samples mainly from European origin for contamination with less well-known mycotoxins. The results indicated that ENNs were amongst the most prevalent contaminants. In 92% of the samples, ENN B1 was detected, the median contamination level was 14 lg/kg, whereas the maximum concentration was 2690 lg/kg. The primary toxic action of ENNs results from its ionophoric characteristics. Several in vitro toxicity studies elucidated their antibacterial, antifungal, antihelmintic, insecticidal and herbicidal potency. Also their cytotoxic effect on a variety of cell types was demonstrated previously (reviewed by Jestoi, 2008). However, the biological activity of ENNs has only been tested in few in vivo studies and there are no cases of mycotoxicosis in humans and animals reported. In mice, acute tox⇑ Corresponding author. Tel.: +32 9 264 73 24. E-mail address: [email protected] (M. Devreese). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.11.005
icity and death occurred only after application of 10–40 mg/kg BW per day intraperitoneally over 6 days (McKee et al., 1997), whereas oral dosing of 0.5–1 mg/kg BW over 6 days to mice and single doses of up to 50 mg/kg BW in rats, did not produce toxic effects (Gäumann et al., 1950; Bosch et al., 1989). To date, no in vivo toxicity data of ENNs are available for pigs. For a better understanding of the in vivo toxic effects and correlating in vitro toxicity data to in vivo, elucidating the toxicokinetic characteristics of ENNs is crucial. The goal of the present study was to gain more information regarding the absorption, distribution, metabolisation and excretion (ADME) of ENN B1 in pigs. 2. Materials and methods 2.1. Chemicals, products and reagents The analytical standard of ENN B1 (molecular weight: 653.85, purity P 95%, stored at 6 15 °C), used for both plasma analysis and the animal experiment was obtained from Sigma–Aldrich (Bornem, Belgium). The internal standard (IS) maduramicin (MAD) was a kind gift from Alpharma (Wilrijk, Belgium) and was stored at 2–8 °C. Water, methanol and acetonitrile (ACN) used for the plasma analysis were of LC–MS grade and obtained from Biosolve (Valkenswaard, The Netherlands). Glacial acetic acid for analytical experiments and water and ethanol for the animal experiment were of analytical grade and obtained from VWR (Leuven, Belgium). 2.2. Animals and experimental procedure Five piglets (BW = 21.7 ± 1.2 kg BW) were housed together and acclimatized for one week. Twelve hours before the start of the experiment, the animals were famished. After this period, all animals were administered ENN B1 (0.05 mg/kg BW), either by oral gavage (pig 1–3, PO) or by intravenous (IV) injection in the ear vein
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Fig. 1. Chemical structure (insert) and plasma concentration–time profile of enniatin B1 after oral (PO) and intravenous (IV) administration (0.05 mg/kg BW) to pigs (n = 5). Results are expressed as mean + SD.
(pig 4–5). This dose resembles a feed contamination level of 1 mg/kg, since the feed intake of a 20 kg weighing pig is about 1 kg/day. This theoretical feed contamination level was chosen arbitrarily as it is included in the wide range of ENN B1 contamination levels that have been detected in feed (±10 lg/kg to P5 mg/kg) (Jestoi, 2008; Streit et al., 2013). The oral bolus solution was prepared instantly before administration by dissolving the ENN B1 standard in ethanol (1 mg/mL) and further diluted with 50 mL of water. This solution was given by gavage using an intragastric tube. The tube was rinsed sufficiently with water to assure complete delivery in the stomach. For IV injection, a stock solution 10 mg ENN B1/mL ethanol was prepared and diluted with physiological saline to obtain a final concentration of 0.25 mg/mL. Following the oral and intravenous administration, blood was drawn at various time points: 0 (just before administration) and 5, 10, 20, 30, 40, 60, 90 and 120 min post-administration. Samples were centrifugated (2851g, 10 min, 4 °C). Aliquots of plasma (250 lL) were stored at 615 °C until analysis. After a threeday wash-out period, pig 1–3 received an intravenous injection of the mycotoxin and pig 4–5 an oral bolus (two-way cross-over design). The dosing, blood collection and sample storage was performed in the same way as the first administration. This animal experiment was approved by the Ethical Committee of Ghent University (case number 2013/27).
accuracy was 4.7%, 3.5% and 2.9%, respectively. The between-run precision (reproducibility) and accuracy were determined by analyzing quality control samples (1, 10 and 100 ng/mL) together with each analytical batch of samples, run on three different days. The results for reproducibility were 7.0%, 7.0% and 7.4%, respectively, and for between-run accuracy 6.3%, 1.9% and 5.9%, respectively. All the results for within- and between-run accuracy and precision fell within the acceptability ranges specified by Devreese et al. (2013). The limit of quantification (LOQ) was the lowest concentration of the analyt for which the method was validated with an accuracy and precision that fell within the recommended ranges. The LOQ was also established as the lowest point of the calibration curve. The LOQ was determined by analyzing six samples spiked at 0.2 ng/mL, on the same day, and fell within the acceptability ranges for accuracy and within- and between-run precision, 50% to +20% and 30.2 and 45.3%, respectively. The limit of detection (LOD) was defined as the lowest concentration of ENN B1 that could be recognized by the detector with a signal-to-noise (S/N) ratio of P3. The LOD was 0.08 ng/mL. For the recovery experiments, two types of matrix-matched calibration curves were prepared, namely by spiking the blank calibrator samples before (spiked) and after extraction (spiked extract). One calibration curve was prepared using standard solutions. The slopes of the resulting linear, 1/x weighted, calibration curves with spiked and spiked extracts samples were compared with the related slopes of the calibration curves with spiked extracts and standard solution. The recovery of the extraction step (RE) was 88.31% and the signal suppression/enhancement (SSE) 92.26%. 2.4. Toxicokinetic analysis Toxicokinetic analysis was performed with WinNonlin 6.3. (Pharsight, St-Louis, MI, USA). The most important toxicokinetic parameters were calculated for IV and PO administration (Table 1): maximal plasma concentration (Cmax), plasma concentration at time 0 (C0), time to maximal plasma concentration (Tmax), area under the plasma concentration–time curve from time 0 to infinite (AUC0-inf), absorption rate constant (ka), absorption half-life (T1/2a), distribution rate constant (kela), distribution half-life (T1/2ela), elimination rate constant (kelb), elimination half-life (T1/2elb), clearance (Cl), volume of distribution in the central (Vc) and peripheral (Vp) compartment. The absolute oral bioavailability (OBB) was calculated according to the formula:
OBB ¼ AUC0inf PO =AUC0inf IV
2.5. Statistical analysis All toxicokinetic parameters were compared between both routes of administration with a Student’s t-test (SPSS 19, IBM, USA). The level of significance was set at 0.05 and indicated with ⁄.
3. Results and discussion 2.3. LC–MS/MS analysis Sample treatment and ENN B1 quantification in plasma was performed as previously described by Devreese et al. (2013). Briefly, to 250 lL of plasma were added 12.5 lL of the IS working solution and 750 lL of ACN, followed by a vortex mixing (15 s) and centrifugation step (8517g, 10 min, 4 °C). Next, the supernatant was transferred to another tube and evaporated using a gentle nitrogen (N2) stream (45 ± 5 °C). The dry residue was reconstituted in 200 lL of ACN/water (80/20, v/ v). After vortex mixing (15 s), the sample was transferred into an autosampler vial. An aliquot (5 lL) was injected onto the LC–MS/MS instrument. Chromatographic separation was achieved on a Hypersil Gold column (50 mm 2.1 mm i.d., dp: 1.9 lm) in combination with a guard column of the same type (10 mm 2.1 mm i.d., dp: 3 lm), both from ThermoFisher Scientific (Breda, The Netherlands). Mobile phase A consisted of 0.1% glacial acetic acid in water whereas mobile phase B was ACN. Following gradient elution program was run: 0–0.5 min (70% A, 30% B), 0.5–2.5 min (linear gradient to 20% A), 2.5–8.5 min (20% A, 80% B), 8.5–10.0 min (linear gradient to 70% A), 10.0–12.0 min (70% A, 30% B). Flow rate was set at 300 lL/min. The LC column effluent was interfaced to a TSQÒ Quantum Ultra triple quadrupole mass spectrometer, equipped with a heated electrospray ionization (h-ESI) probe operating in the positive ionization mode (all from ThermoFisher Scientific). Acquisition was performed in the selected reaction monitoring (SRM) mode. Following transitions (m/z) were monitored for qualification and quantification, respectively, for ENN B1: 654.46 > 214.07 and 654.46 > 196.08 and for MAD: 934.49 > 393.14 and 629.23 (Fig. 2). The method was validated by a set of parameters previously described by De Baere et al. (2011). Linearity was evaluated by preparing matrix-matched calibration curves (range 0.2–200 ng/mL). The correlation coefficient (r) and goodnessof-fit coefficient (g) were 0.9992 and 11.31%, respectively. Within-run precision (repeatability) and accuracy were determined by analyzing six blank samples spiked at 1, 10 and 100 ng/mL in the same run. The repeatability, expressed as relative standard deviation (RSD) was 3.4%, 5.7% and 4.2%, respectively. Within-run
Graphically, the plasma concentration–time profiles of ENN B1 (Fig. 1) fitted a two-compartmental model. This two-compartmental Table 1 Main toxicokinetic parameters of enniatin B1 after oral (PO) and intravenous (IV) administration (0.05 mg/kg BW) to pigs (n = 5).
Cmax (ng/mL) C0 (ng/mL) Tmax (h) AUC0-inf (h ng/mL) ka (h1) T1/2a (h) Cl (L/h/kg) kel a (h1) kel b (h1) T1/2el a (h) T1/2el b (h) Vc (L/kg) Vp (L/kg) OBB (%)
PO
IV
29.69 ± 5.904 – 0.24 ± 0.063 25.28 ± 8.050 4.66 ± 0.239 0.15 ± 0.008 2.00 ± 0.637 4.60 ± 0.386 0.57 ± 0.272 0.15 ± 0.013 1.57 ± 0.746 0.60 ± 0.170 0.86 ± 0.395 90.9 ± 12.85
– 89.51 ± 22.693 – 27.80 ± 6.670 – – 1.91 ± 0.457 5.03 ± 1.557 0.92 ± 0.524 0.15 ± 0.047 1.13 ± 0.645 0.57 ± 0.172 0.69 ± 0.373 –
Cmax: maximal plasma concentration, C0: plasma concentration at time 0, Tmax: time to maximal plasma concentration, AUC0-inf: area under the plasma concentration– time curve from time 0 to infinite, ka: absorption rate constant, T1/2a: absorption half-life, Cl: clearance, kel a: distribution rate constant, kel b: elimination rate constant, T1/2el a: distribution half-life, T1/2el b: elimination half-life, Vc: volume of distribution in the central compartment, Vp: volume of distribution in the peripheral compartment, OBB: absolute oral bioavailability.
M. Devreese et al. / Food and Chemical Toxicology 63 (2014) 161–165
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Fig. 2. LC–MS/MS chromatogram showing the SRM traces of enniatin B1 (ENN B1) and the internal standard maduramicin (MAD) for the analysis of a pig plasma sample that was taken 5 min after oral (a) and intravenous (b) administration of 0.05 mg ENN B1/kg BW, and a blank control sample (c).
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M. Devreese et al. / Food and Chemical Toxicology 63 (2014) 161–165
Fig. 2 (continued)
fitting was confirmed by toxicokinetic analysis, with a correlation coefficient of 0.97 (±0.058). The results revealed that ENN B1 is rapidly absorbed after oral intake (ka = 4.66 h1, Tmax = 0.24 h) and the OBB is remarkably high (90.9%). Furthermore, the mycotoxin is rapidly distributed and eliminated as well (T1/2ela,PO = 0.15 h; T1/2elb,PO = 1.57 h). After IV administration, ENN B1 was distributed and eliminated rapidly (T1/2ela,IV = 0.15 h; T1/2elb,IV = 1.13 h), in accordance with oral administration. A good level of agreement was also found for plasma clearance (ClPO = 2.00 L/h/kg; ClIV = 1.91 L/h/kg). No significant differences in the toxicokinetic parameters were found between PO and IV administration. Due to the discrepancy of significant in vitro but low in vivo acute toxicity of ENNs, several authors attributed this to a low bioavailability and/or rapid elimination of these mycotoxins after oral uptake (Faeste et al., 2011; Ivanova et al., 2011). In a recent study, the intestinal biovailability of ENNs was assessed in vitro with Caco-2 cells (Meca et al., 2012). For ENN B1, the apparent bioavailability was 55–66%. The present in vivo trial demonstrated an even higher OBB of ENN B1 in pigs of 91%. On the other hand, rapid metabolisation and elimination might explain the low acute in vivo toxicity of these compounds, which was confirmed by the present study. The metabolisation pathways of ENNs in vivo remain unclear, only in vitro data for ENN B is available. After incubating ENN B with human liver microsomes, the toxin undergoes extensive metabolisation, mainly by CYP3A4 (Faeste et al., 2011), resulting in oxidation and N-demethylation products (Ivanova et al., 2011). Furthermore, it was suggested that CYP3A and CYP1A might also be relevant in rats and dogs (Faeste et al., 2011). Bearing this in vivo and previous in vitro studies in mind, future research should focus on elucidating the phase I and II metabolisation pathways of ENNs and the toxicity of these metabolites. Next,
toxicokinetic studies of ENN B1, and other ENNs, should be performed in other animal species to compare mycotoxin and species dependent differences in toxicity and sensitivity. Conflict of Interest None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgments The authors would like to thank the ‘Agency for Innovation by Science and Technology’ (IWT, Brussels, Belgium) for its financial support (SB Grant 2010 No. 101301). References Bosch, U., Mirocha, C.J., Abbas, H.K., Dimenna, M., 1989. Toxicity and toxin production by Fusarium isolates from New Zealand. Mycopathologia 108, 73– 79. De Baere, S., Goossens, J., Osselaere, A., Devreese, M., Vandenbroucke, V., De Backer, P., Croubels, S., 2011. Quantitative determination of T-2 toxin, HT-2 toxin, deoxynivalenol and de-epoxy-deoxynivalenol in animal body fluids using LC– MS/MS detection. J. Chromatogr. B 879, 2403–2415. Devreese, M., De Baere, S., De Backer, P., Croubels, S., 2013. 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 106, 212–219. Faeste, C.K., Ivanova, L., Uhlig, S., 2011. In vitro metabolism of the mycotoxin enniatin B in different species and cytochrome P450 enzyme phenotyping by chemical inhibitors. Drug Metab. Dispos. 39, 1768–1776. Gäumann, E., Naef-Roth, S., Ettlinger, L., 1950. Zur gewinnung von enniatinen aus dem myzel verschiedener Fusarien. J. Phytopathol. 16, 289–299. Ivanova, L., Faeste, C.K., Uhlig, S., 2011. In vitro phase I metabolism of the depsipeptide enniatin B. Anal. Bioanal. Chem. 400, 2889–2901.
M. Devreese et al. / Food and Chemical Toxicology 63 (2014) 161–165 Jestoi, M., 2008. Emerging Fusarium mycotoxins fusaproliferin, beauvericin, enniatins, and moniliforme – a review. CRC Rev. Food Sci. 48, 21–49. McKee, T.C., Bokesch, H.R., McCormick, J.L., Rashid, M.A., Spielvogel, D., Gustafson, K.R., Alavanja, M.M., Cardellina, J.H., Boyd, M.R., 1997. Isolation and characterization of newanti-HIV and cytotoxic leads from plants, marine and microbial origins. J. Nat. Prod. 60, 431–438. Meca, G., Manes, J., Font, G., Ruiz, M.J., 2012. Study of the potential toxicity of commercial crispy breads by evaluation of bioaccessibility and bioavailability of minor Fusarium mycotoxins. Food Chem. Toxicol. 50, 288–294.
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Streit, E., Schwab, C., Sulyok, M., Naehrer, K., Krska, R., Schatzmayr, R., 2013. Multimycotoxin screening reveals the occurence of 139 different secondary metabolites in feed and feed ingredients. Toxins 5, 504–523. Zhukhlistova, N.E., Tishchenko, G.N., Tolstykh, I.V., Zenkova, V.A., 1999. Molecular and crystal structure of the antibiotic enniatin B, a secondary microbial metabolite. Crystallogr. Rep. 44, 8–12.
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Brief communication
Pilot toxicokinetic study and absolute oral bioavailability of the Fusarium mycotoxin enniatin B1 in pigs Mathias Devreese ⇑, Nathan Broekaert, Thomas De Mil, Sophie Fraeyman, Patrick De Backer, Siska Croubels Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
a r t i c l e
i n f o
Article history: Received 1 October 2013 Accepted 5 November 2013 Available online 13 November 2013 Keywords: Toxicokinetics Absolute oral bioavailability Mycotoxin Enniatin B1 Pig
a b s t r a c t The aim of present study was to reveal the toxicokinetic properties and absolute oral bioavailability of enniatin B1 in pigs. Five pigs were administered this Fusarium mycotoxin per os and intravenously in a two-way cross-over design. The toxicokinetic profile fitted a two-compartmental model. Enniatin B1 is rapidly absorbed after oral administration (T1/2a = 0.15 h, Tmax = 0.24 h) and rapidly distributed and eliminated as well (T1/2ela = 0.15 h; T1/2elb = 1.57 h). The absolute oral bioavailability is high (90.9%), indicating a clear systemic exposure. After intravenous administration, the mycotoxin is distributed and eliminated rapidly (T1/2ela = 0.15 h; T1/2elb = 1.13 h), in accordance with oral administration. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Fusarium fungi frequently infest crops in temperate regions such as Western Europe and North America. They can produce a wide range of mycotoxins, including several extensively studied compounds such as trichothecenes, zearalenone and fumonisins. They are also capable of producing other less well-known mycotoxins like enniatins (ENNs) (enniatin (ENN) A, A1, B and B1) and beauvericin (BEA). ENNs are cyclic hexadepsipeptides consisting of alternating D-a-hydroxyisovaleric acids and L-methyl-amino acids (Zhukhlistova et al., 1999) (Fig. 1). Over the last decade, ENNs were found to be common contaminants of grains, maize and other feedstuffs (Jestoi, 2008; Devreese et al., 2013). Recently, Streit et al. (2013) analyzed 83 feed and feed raw material samples mainly from European origin for contamination with less well-known mycotoxins. The results indicated that ENNs were amongst the most prevalent contaminants. In 92% of the samples, ENN B1 was detected, the median contamination level was 14 lg/kg, whereas the maximum concentration was 2690 lg/kg. The primary toxic action of ENNs results from its ionophoric characteristics. Several in vitro toxicity studies elucidated their antibacterial, antifungal, antihelmintic, insecticidal and herbicidal potency. Also their cytotoxic effect on a variety of cell types was demonstrated previously (reviewed by Jestoi, 2008). However, the biological activity of ENNs has only been tested in few in vivo studies and there are no cases of mycotoxicosis in humans and animals reported. In mice, acute tox⇑ Corresponding author. Tel.: +32 9 264 73 24. E-mail address: [email protected] (M. Devreese). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.11.005
icity and death occurred only after application of 10–40 mg/kg BW per day intraperitoneally over 6 days (McKee et al., 1997), whereas oral dosing of 0.5–1 mg/kg BW over 6 days to mice and single doses of up to 50 mg/kg BW in rats, did not produce toxic effects (Gäumann et al., 1950; Bosch et al., 1989). To date, no in vivo toxicity data of ENNs are available for pigs. For a better understanding of the in vivo toxic effects and correlating in vitro toxicity data to in vivo, elucidating the toxicokinetic characteristics of ENNs is crucial. The goal of the present study was to gain more information regarding the absorption, distribution, metabolisation and excretion (ADME) of ENN B1 in pigs. 2. Materials and methods 2.1. Chemicals, products and reagents The analytical standard of ENN B1 (molecular weight: 653.85, purity P 95%, stored at 6 15 °C), used for both plasma analysis and the animal experiment was obtained from Sigma–Aldrich (Bornem, Belgium). The internal standard (IS) maduramicin (MAD) was a kind gift from Alpharma (Wilrijk, Belgium) and was stored at 2–8 °C. Water, methanol and acetonitrile (ACN) used for the plasma analysis were of LC–MS grade and obtained from Biosolve (Valkenswaard, The Netherlands). Glacial acetic acid for analytical experiments and water and ethanol for the animal experiment were of analytical grade and obtained from VWR (Leuven, Belgium). 2.2. Animals and experimental procedure Five piglets (BW = 21.7 ± 1.2 kg BW) were housed together and acclimatized for one week. Twelve hours before the start of the experiment, the animals were famished. After this period, all animals were administered ENN B1 (0.05 mg/kg BW), either by oral gavage (pig 1–3, PO) or by intravenous (IV) injection in the ear vein
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Fig. 1. Chemical structure (insert) and plasma concentration–time profile of enniatin B1 after oral (PO) and intravenous (IV) administration (0.05 mg/kg BW) to pigs (n = 5). Results are expressed as mean + SD.
(pig 4–5). This dose resembles a feed contamination level of 1 mg/kg, since the feed intake of a 20 kg weighing pig is about 1 kg/day. This theoretical feed contamination level was chosen arbitrarily as it is included in the wide range of ENN B1 contamination levels that have been detected in feed (±10 lg/kg to P5 mg/kg) (Jestoi, 2008; Streit et al., 2013). The oral bolus solution was prepared instantly before administration by dissolving the ENN B1 standard in ethanol (1 mg/mL) and further diluted with 50 mL of water. This solution was given by gavage using an intragastric tube. The tube was rinsed sufficiently with water to assure complete delivery in the stomach. For IV injection, a stock solution 10 mg ENN B1/mL ethanol was prepared and diluted with physiological saline to obtain a final concentration of 0.25 mg/mL. Following the oral and intravenous administration, blood was drawn at various time points: 0 (just before administration) and 5, 10, 20, 30, 40, 60, 90 and 120 min post-administration. Samples were centrifugated (2851g, 10 min, 4 °C). Aliquots of plasma (250 lL) were stored at 615 °C until analysis. After a threeday wash-out period, pig 1–3 received an intravenous injection of the mycotoxin and pig 4–5 an oral bolus (two-way cross-over design). The dosing, blood collection and sample storage was performed in the same way as the first administration. This animal experiment was approved by the Ethical Committee of Ghent University (case number 2013/27).
accuracy was 4.7%, 3.5% and 2.9%, respectively. The between-run precision (reproducibility) and accuracy were determined by analyzing quality control samples (1, 10 and 100 ng/mL) together with each analytical batch of samples, run on three different days. The results for reproducibility were 7.0%, 7.0% and 7.4%, respectively, and for between-run accuracy 6.3%, 1.9% and 5.9%, respectively. All the results for within- and between-run accuracy and precision fell within the acceptability ranges specified by Devreese et al. (2013). The limit of quantification (LOQ) was the lowest concentration of the analyt for which the method was validated with an accuracy and precision that fell within the recommended ranges. The LOQ was also established as the lowest point of the calibration curve. The LOQ was determined by analyzing six samples spiked at 0.2 ng/mL, on the same day, and fell within the acceptability ranges for accuracy and within- and between-run precision, 50% to +20% and 30.2 and 45.3%, respectively. The limit of detection (LOD) was defined as the lowest concentration of ENN B1 that could be recognized by the detector with a signal-to-noise (S/N) ratio of P3. The LOD was 0.08 ng/mL. For the recovery experiments, two types of matrix-matched calibration curves were prepared, namely by spiking the blank calibrator samples before (spiked) and after extraction (spiked extract). One calibration curve was prepared using standard solutions. The slopes of the resulting linear, 1/x weighted, calibration curves with spiked and spiked extracts samples were compared with the related slopes of the calibration curves with spiked extracts and standard solution. The recovery of the extraction step (RE) was 88.31% and the signal suppression/enhancement (SSE) 92.26%. 2.4. Toxicokinetic analysis Toxicokinetic analysis was performed with WinNonlin 6.3. (Pharsight, St-Louis, MI, USA). The most important toxicokinetic parameters were calculated for IV and PO administration (Table 1): maximal plasma concentration (Cmax), plasma concentration at time 0 (C0), time to maximal plasma concentration (Tmax), area under the plasma concentration–time curve from time 0 to infinite (AUC0-inf), absorption rate constant (ka), absorption half-life (T1/2a), distribution rate constant (kela), distribution half-life (T1/2ela), elimination rate constant (kelb), elimination half-life (T1/2elb), clearance (Cl), volume of distribution in the central (Vc) and peripheral (Vp) compartment. The absolute oral bioavailability (OBB) was calculated according to the formula:
OBB ¼ AUC0inf PO =AUC0inf IV
2.5. Statistical analysis All toxicokinetic parameters were compared between both routes of administration with a Student’s t-test (SPSS 19, IBM, USA). The level of significance was set at 0.05 and indicated with ⁄.
3. Results and discussion 2.3. LC–MS/MS analysis Sample treatment and ENN B1 quantification in plasma was performed as previously described by Devreese et al. (2013). Briefly, to 250 lL of plasma were added 12.5 lL of the IS working solution and 750 lL of ACN, followed by a vortex mixing (15 s) and centrifugation step (8517g, 10 min, 4 °C). Next, the supernatant was transferred to another tube and evaporated using a gentle nitrogen (N2) stream (45 ± 5 °C). The dry residue was reconstituted in 200 lL of ACN/water (80/20, v/ v). After vortex mixing (15 s), the sample was transferred into an autosampler vial. An aliquot (5 lL) was injected onto the LC–MS/MS instrument. Chromatographic separation was achieved on a Hypersil Gold column (50 mm 2.1 mm i.d., dp: 1.9 lm) in combination with a guard column of the same type (10 mm 2.1 mm i.d., dp: 3 lm), both from ThermoFisher Scientific (Breda, The Netherlands). Mobile phase A consisted of 0.1% glacial acetic acid in water whereas mobile phase B was ACN. Following gradient elution program was run: 0–0.5 min (70% A, 30% B), 0.5–2.5 min (linear gradient to 20% A), 2.5–8.5 min (20% A, 80% B), 8.5–10.0 min (linear gradient to 70% A), 10.0–12.0 min (70% A, 30% B). Flow rate was set at 300 lL/min. The LC column effluent was interfaced to a TSQÒ Quantum Ultra triple quadrupole mass spectrometer, equipped with a heated electrospray ionization (h-ESI) probe operating in the positive ionization mode (all from ThermoFisher Scientific). Acquisition was performed in the selected reaction monitoring (SRM) mode. Following transitions (m/z) were monitored for qualification and quantification, respectively, for ENN B1: 654.46 > 214.07 and 654.46 > 196.08 and for MAD: 934.49 > 393.14 and 629.23 (Fig. 2). The method was validated by a set of parameters previously described by De Baere et al. (2011). Linearity was evaluated by preparing matrix-matched calibration curves (range 0.2–200 ng/mL). The correlation coefficient (r) and goodnessof-fit coefficient (g) were 0.9992 and 11.31%, respectively. Within-run precision (repeatability) and accuracy were determined by analyzing six blank samples spiked at 1, 10 and 100 ng/mL in the same run. The repeatability, expressed as relative standard deviation (RSD) was 3.4%, 5.7% and 4.2%, respectively. Within-run
Graphically, the plasma concentration–time profiles of ENN B1 (Fig. 1) fitted a two-compartmental model. This two-compartmental Table 1 Main toxicokinetic parameters of enniatin B1 after oral (PO) and intravenous (IV) administration (0.05 mg/kg BW) to pigs (n = 5).
Cmax (ng/mL) C0 (ng/mL) Tmax (h) AUC0-inf (h ng/mL) ka (h1) T1/2a (h) Cl (L/h/kg) kel a (h1) kel b (h1) T1/2el a (h) T1/2el b (h) Vc (L/kg) Vp (L/kg) OBB (%)
PO
IV
29.69 ± 5.904 – 0.24 ± 0.063 25.28 ± 8.050 4.66 ± 0.239 0.15 ± 0.008 2.00 ± 0.637 4.60 ± 0.386 0.57 ± 0.272 0.15 ± 0.013 1.57 ± 0.746 0.60 ± 0.170 0.86 ± 0.395 90.9 ± 12.85
– 89.51 ± 22.693 – 27.80 ± 6.670 – – 1.91 ± 0.457 5.03 ± 1.557 0.92 ± 0.524 0.15 ± 0.047 1.13 ± 0.645 0.57 ± 0.172 0.69 ± 0.373 –
Cmax: maximal plasma concentration, C0: plasma concentration at time 0, Tmax: time to maximal plasma concentration, AUC0-inf: area under the plasma concentration– time curve from time 0 to infinite, ka: absorption rate constant, T1/2a: absorption half-life, Cl: clearance, kel a: distribution rate constant, kel b: elimination rate constant, T1/2el a: distribution half-life, T1/2el b: elimination half-life, Vc: volume of distribution in the central compartment, Vp: volume of distribution in the peripheral compartment, OBB: absolute oral bioavailability.
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Fig. 2. LC–MS/MS chromatogram showing the SRM traces of enniatin B1 (ENN B1) and the internal standard maduramicin (MAD) for the analysis of a pig plasma sample that was taken 5 min after oral (a) and intravenous (b) administration of 0.05 mg ENN B1/kg BW, and a blank control sample (c).
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Fig. 2 (continued)
fitting was confirmed by toxicokinetic analysis, with a correlation coefficient of 0.97 (±0.058). The results revealed that ENN B1 is rapidly absorbed after oral intake (ka = 4.66 h1, Tmax = 0.24 h) and the OBB is remarkably high (90.9%). Furthermore, the mycotoxin is rapidly distributed and eliminated as well (T1/2ela,PO = 0.15 h; T1/2elb,PO = 1.57 h). After IV administration, ENN B1 was distributed and eliminated rapidly (T1/2ela,IV = 0.15 h; T1/2elb,IV = 1.13 h), in accordance with oral administration. A good level of agreement was also found for plasma clearance (ClPO = 2.00 L/h/kg; ClIV = 1.91 L/h/kg). No significant differences in the toxicokinetic parameters were found between PO and IV administration. Due to the discrepancy of significant in vitro but low in vivo acute toxicity of ENNs, several authors attributed this to a low bioavailability and/or rapid elimination of these mycotoxins after oral uptake (Faeste et al., 2011; Ivanova et al., 2011). In a recent study, the intestinal biovailability of ENNs was assessed in vitro with Caco-2 cells (Meca et al., 2012). For ENN B1, the apparent bioavailability was 55–66%. The present in vivo trial demonstrated an even higher OBB of ENN B1 in pigs of 91%. On the other hand, rapid metabolisation and elimination might explain the low acute in vivo toxicity of these compounds, which was confirmed by the present study. The metabolisation pathways of ENNs in vivo remain unclear, only in vitro data for ENN B is available. After incubating ENN B with human liver microsomes, the toxin undergoes extensive metabolisation, mainly by CYP3A4 (Faeste et al., 2011), resulting in oxidation and N-demethylation products (Ivanova et al., 2011). Furthermore, it was suggested that CYP3A and CYP1A might also be relevant in rats and dogs (Faeste et al., 2011). Bearing this in vivo and previous in vitro studies in mind, future research should focus on elucidating the phase I and II metabolisation pathways of ENNs and the toxicity of these metabolites. Next,
toxicokinetic studies of ENN B1, and other ENNs, should be performed in other animal species to compare mycotoxin and species dependent differences in toxicity and sensitivity. Conflict of Interest None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgments The authors would like to thank the ‘Agency for Innovation by Science and Technology’ (IWT, Brussels, Belgium) for its financial support (SB Grant 2010 No. 101301). References Bosch, U., Mirocha, C.J., Abbas, H.K., Dimenna, M., 1989. Toxicity and toxin production by Fusarium isolates from New Zealand. Mycopathologia 108, 73– 79. De Baere, S., Goossens, J., Osselaere, A., Devreese, M., Vandenbroucke, V., De Backer, P., Croubels, S., 2011. Quantitative determination of T-2 toxin, HT-2 toxin, deoxynivalenol and de-epoxy-deoxynivalenol in animal body fluids using LC– MS/MS detection. J. Chromatogr. B 879, 2403–2415. Devreese, M., De Baere, S., De Backer, P., Croubels, S., 2013. 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 106, 212–219. Faeste, C.K., Ivanova, L., Uhlig, S., 2011. In vitro metabolism of the mycotoxin enniatin B in different species and cytochrome P450 enzyme phenotyping by chemical inhibitors. Drug Metab. Dispos. 39, 1768–1776. Gäumann, E., Naef-Roth, S., Ettlinger, L., 1950. Zur gewinnung von enniatinen aus dem myzel verschiedener Fusarien. J. Phytopathol. 16, 289–299. Ivanova, L., Faeste, C.K., Uhlig, S., 2011. In vitro phase I metabolism of the depsipeptide enniatin B. Anal. Bioanal. Chem. 400, 2889–2901.
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