Article pubs.acs.org/crt
Investigation of the Hepatic Glucuronidation Pattern of the Fusarium Mycotoxin Deoxynivalenol in Various Species Ronald Maul,*,† Benedikt Warth,‡ Jill-Sandra Kant,† Nils Helge Schebb,§ Rudolf Krska,‡ Matthias Koch,† and Michael Sulyok‡ †
Division of Food Analysis, Federal Institute for Materials Research and Testing (BAM), Richard-Willstätter-Straße 11, 12489 Berlin, Germany ‡ Center for Analytical Chemistry, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad-Lorenz-Straße 20, 3430, Tulln, Austria § Institute of Food Toxicology and Chemical Analysis, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany S Supporting Information *
ABSTRACT: Deoxynivalenol (DON) is one of the most abundant mycotoxins contaminating food and feed worldwide. Upon absorption, the major portion of the toxin is excreted by humans and animal species as glucuronide. However, consistent in vitro data on DON glucuronidation are lacking. In the present study, the metabolism of DON was investigated using liver microsomes from humans and six different animal species. It was shown that all animal and human liver microsomes led to the formation of up to three different mono-O-glucuronides with significant interspecies differences. While the activity of human liver microsomes was low (0.8 to 2.2 pmol·min−1·mg−1), bovine liver and rat liver microsomes conjugated DON with activities of 525 pmol·min−1·mg−1 and 80 pmol·min−1·mg−1, respectively.
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INTRODUCTION The trichothecene deoxynivalenol (DON) is one of the most prevalent mycotoxins in grains and cereal products. The toxin is mainly produced by Fusarium species causing preharvest contamination. Because of its acute toxicity, DON is regulated by legal limits in many countries.1 While in humans only symptoms of acute toxicity like nausea, diarrhea, and vomiting were observed, a decreased weight gain, anorexia, decreased nutritional efficiency, and altered immune function additionally became obvious in animal studies using pigs and rats.2 DON is efficiently absorbed in humans and animals, and the toxin is dominantly excreted as a glucuronide.3,4 In human urine, DON is almost exclusively excreted as a glucuronide accounting for about 91% of total DON.5 As a consequence, DON-3glucuronide (DON-3-GlcA) was suggested as a biomarker for the assessment of human dietary exposition to this toxin.6,7 While in rat urine two different glucuronic acid conjugates occur, only one DON-glucuronide was detected in human urine. In rat urine, the predominating glucuronide was suggested to be the DON-3-GlcA.8 However, recently Warth et al. described a second glucuronide dominating in human urine, tentatively assigned to the 15-glucuronide (DON-15GlcA).3 Despite various in vivo data, no consistent results could be obtained for the DON metabolism in in vitro studies. In a microsomal study using rat and pig liver microsomes, no hepatic metabolism of DON was observed.9 By contrast, a © 2012 American Chemical Society
similar study using human, rat, and minipig microsomes led to the qualitative detection of up to three glucuronides.10 These in vivo and in vitro results imply that the DON glucuronidation may be species dependent and show interindividual variation. Thus, enhanced knowledge about the pattern of all formed glucuronides is crucial to enable proper backward calculation of ingested DON amounts from food and feed. Moreover, knowledge about the conjugative metabolism of xenobiotics is crucial for estimation of toxic effects and intrinsic clearance in humans but also for farm animals fed with contaminated cereal based products. Therefore, the aim of this work was to clarify the microsomal formation of DON glucuronides by human and various animal microsomal preparations and to examine differences in the pattern and extent between species.
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MATERIALS AND METHODS
Human liver microsomes (HLM) were obtained from Xenotech (Offenbach, Germany) and Advancell (Barcelona, Spain). All other animal liver fractions were prepared in house as described by Lake.11 Deoxynivalenol-3-O-glucuronide (DON-3-GlcA) was synthesized by an optimized Kö nigs-Knorr procedure, and the structure was confirmed by NMR.12 DON-15-GlcA was isolated from a naturally contaminated human urine sample by fractionation as described Received: August 3, 2012 Published: October 29, 2012 2715
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elsewhere in detail.3 DON was purchased from Romer Labs (Tulln, Austria). The glucuronidation assay was carried out as described previously in a total volume of 200 μL.13 For the in vitro incubation, various optimized protein concentrations for the 10 types of microsomes were used. The substrate DON was prepared in methanol/water 50/50 resulting in a final DON concentration of 3.75 μM (final methanol concn 1.0%), and the incubation time was 30 min (RLM, carp-LM, and BLM) or 60 min (HLM, CLM, PLM, and trout-LM). The assays were terminated by adding 200 μL of iced ACN, evaporated to dryness, and redissolved in ACN/water (10/90, v/v) for analysis. All experiments were carried out in duplicate. The recovery of DON-3GlcA was evaluated by spiking substrate free microsomal incubations with 0.4 μM DON-3-GlcA and the subsequent preparation as described above. Using two different representative microsomal preparations (i-RLM and carp-LM; n = 3), a recovery rate of 89.8% ± 2.9% was determined and included in the calculation of all experimental DON glucuronidation results (Supporting Information, Table S1). DON glucuronide analysis was performed by LC-MS/MS as described previously.7 ESI-MS/MS was done in selected reaction monitoring (SRM) mode, and three transitions were monitored for DON-GlcA (471.0 [M − H]− → 265.2/175.2/441.0). The limit of detection (LOD) and limit of quantification (LOQ) values were calculated from chromatograms of spiked microsomal blank incubations omitting DON as substrate, respectively, as 0.3/1.0 μg/ L (DON-3-GlcA) and 0.2/0.7 μg/L (DON-15-GlcA). In order to characterize the glucuronidation activity of the used microsomal preparations, the activity for the glucuronidation of 4-trifluoromethylumbelliferone (TFMU) as a reference substrate was measured by HPLC-FLD as described elsewhere.14 The substrate concentration was 100 μM, and quantification was accomplished by external matrix matched calibration with TFMU and its glucuronide using umbelliferone as internal standard.
Table 1. Species-Dependent Glucuronidation Activity (in pmol·min−1·mg Protein−1) of Deoxynivalenol (DON) and the Reference Substrate Trifluoromethylumbelliferone (TFMU)a type/species of microsomes induced female rat liver induced male rat liver noninduced rat liver bovine liver porcine liver chicken liver carp liver trout liver human liver (xenotech) human liver (advancell)
DON3-GlcA
DON15-GlcA
3rd DONGlcA*)
TFMUGlcA
i-RLM female i-RLM male
120.4
nd
low
54400
217.6
nd
+
46700
RLM
79.9
nd
low
12600
BLM PLM CLM carp-LM trout-LM HLM-x
524.8 4.3 0.1 14.0 5.8 0.2
nd nd nd nd nd 0.6
+ nd nd low + nd
33300 41700 200 2300 2800 30700
HLM-a
1.0
1.2
low
34200
abbreviation
a
For the 3rd DON-GlcA, only a semi-quantitative estimation of the activity based on the peak area in LC-MS is given. nd, not detected. *), formation of the 3rd DON-GlcA is ranked based on peak area in LCMS: low, low amounts; +, considerable amounts
intensity may have an impact on the toxicity of DON to the investigated species. While a rapid metabolic conjugation coincides with a rapid detoxification, intrinsic clearance, and therefore decreased time of exposure of free toxin to the body, in those species with a low metabolic activity the same dose of DON may lead to toxic symptoms. The fast glucuronidation may in part explain prior findings that already low levels of DON lead to vomiting in pigs, while rats or ruminants seem to be less sensitive.1 Additionally, DON is partly de-epoxidized by rumen microbiota causing detoxification in ruminants.2 In chicken, the fact that only very low amounts of DON are resorbed may explain the insensitivity.15 In addition to DON-3GlcA, HLM formed DON-15-GlcA (Table 1). This metabolite showed a retention time (RT) only few seconds different for DON-3-GlcA in LC-MS analysis. The independent detection of both coeluting metabolites was feasible by monitoring different specific SRM transitions (Supporting Information Figure S1). With a conjugation rate of 0.6 and 1.0 pmol·min−1·mg protein−1, DON-15-GlcA formation exceeded the formation of DON-3-GlcA in both HLM preparations tested. By contrast, none of the animal LM tested showed the formation of DON15-GlcA. Incubations with RLM, BLM, and the HLM additionally gave rise to a third peak of moderate intensity at the mass transitions of the DON-GlcA. In RP-LC, this DONmetabolite eluted 30 s after DON-3-GlcA, and the MS fragment spectrum showed large similarities to the spectra of the other DON-GlcAs (Supporting Information, Figures S1 and S2). As the only additional functional group of the DON molecule is the hydroxyl group in position C-7, it is likely that this product is the DON-7-GlcA. However, the confirmation of identity of this metabolite requires further investigation.
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RESULTS AND DISCUSSION All microsomal fractions showed good activity for the TFMU conjugation with most of the enzymatic preparations exerting comparable glucuronidation rates between 13 and 54 nmol·min−1·mg protein−1. Only the CLM, carp-LM, and trout-LM possess an activity that was at least 5- to 10-fold lower (Table 1). For all microsomes, the formation of both known glucuronidation products, the DON-3-GlcA as well as the second product, tentatively identified as the DON-15-GlcA, were quantified. With the exception of HLM, in all cases the DON-3-GlcA is the clearly prevailing product (Table 1). Notably, the conjugation activities varied strongly between the species with the CLM and HLM possessing the lowest capacity for DON glucuronidation. BLM and i-RLM showed the highest activity for the conversion of DON, almost exclusively forming the DON-3-GlcA. No sex specific differences could be detected for i-RLM. Also, the noninduced RLM show a high activity of 80 pmol·min−1·mg protein−1; however, the inductive treatment results in a significant increase in DON glucuronidation activity. Moderate conjugation activity was found for trout-LM, carpLM, and PLM. For the fish microsomes, the intense difference to the TFMU conversion is maybe due to different UGT compositions. By contrast, HLM possess an extremely low DON converting activity being approximately a factor of 100 lower than the activity detected for the different RLM or the BLM and a factor of 10 lower than the fish LM. However, the intense glucuronidation of the reference substrate TFMU confirms the integrity of both HLM. Regarding the TFMU conjugating activities of the other microsomes, it becomes obvious that large interspecies differences in specificity exist. However, all microsomal preparations are generally active. The pronounced difference in the glucuronidation pattern and
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CONCLUSIONS These results demonstrate for the first time not only that the activity to conjugate DON varies between species but also that the pattern of glucuronides is significantly different. While human liver UGT form up to three different DON glucuronides, rat bovine and carp liver UGTs catalyze the 2716
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to assess human exposure to mycotoxins. Rapid Commun. Mass Spectrom. 26, 1533−1540. (8) Lattanzio, V. M., Solfrizzo, M., De Girolamo, A., Chulze, S. N., Torres, A. M., and Visconti, A. (2011) LC-MS/MS characterization of the urinary excretion profile of the mycotoxin deoxynivalenol in human and rat. J. Chromatogr., B 879, 707−715. (9) Cote, L. M., Buck, W., and Jeffery, E. (1987) Lack of hepatic microsomal metabolism of deoxynivalenol and its metabolite, DOM-1. Food Chem. Toxicol. 25, 291−295. (10) Faeste, C. K., Ivanova, L., and Uhlig, S. (2012) Species-Specific in Vitro Glucuronidation of the Mycotoxin Deoxynivalenol, Proceedings of the 12th European Regional ISSX Meeting. (11) Lake, B. G. (1987) Preparation and Characterization of Microsomal Fractions for Studies on Xebobiotic Metabolism, in Biochemical Toxicology: A Practical Approach (Snell, K., and Mullock, B., Eds.) pp 183−215, Oxford University Press, Oxford, U.K. (12) Fruhmann, P., Warth, B., Hametner, C., Berthiller, F., Horkel, E., Adam, G., Sulyok, M., Krska, R., and Fröhlich, J. (2011) Synthesis of deoxynivalenol-3-ß-D-O-glucuronide for its use as biomarker for dietary deoxynivalenol exposure. World Mycotoxin J. 5, 127−132. (13) Maul, R., Siegl, D., and Kulling, S. E. (2011) Glucuronidation of the red clover isoflavone irilone by liver microsomes from different species and human UDP-glucuronosyltransferases. Drug Metab. Dispos. 39, 610−616. (14) Schebb, N. H., Franze, B., Maul, R., Ranganathan, A., and Hammock, B. D. (2012) In Vitro Glucuronidation of the antibacterial triclocarban and its oxidative metabolites. Drug Metab. Dispos. 40, 25− 31. (15) Prelusky, D. B., Hamilton, R. M. G., Trenholm, H. L., and Miller, J. D. (1986) Tissue distribution and excretion of radioactivity following administration of 14C-labeled deoxynivalenol to white Leghorn hens. Toxicol. Sci. 7, 635−645.
formation of two conjugates. The liver enzymes of the other species tested seem to exclusively form DON-3-GlcA. This varying pattern of the formed glucuronides between the species makes it necessary to be specific when suitable exposure biomarkers for blood and urine are selected. In order to obtain detailed information about the distinct enzymes responsible for the DON conjugation and enable the calculation of enzyme kinetic parameters, additional investigations using other tissue microsomes and purified recombinant UGT enzymes are currently being carried out. These experiments will help to gain a deeper insight into the influence of metabolism on DON impact on human and animal health.
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ASSOCIATED CONTENT
S Supporting Information *
HPLC-MS/MS chromatograms of the microsomal incubations using i-RLM and HLM; MS/MS spectrum of the 3rd glucuronide; recovery rate for DON-GlcA for the applied sample preparation method. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +49 (0) 30 81045960. E-mail:
[email protected]. Funding
We thank the EC (KBBE-2007-22269-2 MYCORED), the Lower Austrian Government, the FWF (project L255-B11), and the graduate school program Applied Bioscience Technology (AB-Tec) of the Vienna University of Technology and the University of Natural Resources and Life Sciences Vienna for financial support. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS ACN, acetonitrile; DON, deoxynivalenol; TFMU, 4-trifluoromethylumbelliferone; GlcA, glucuronide; RT, retention time; UGT, UDP-glucuronosyltransferase
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REFERENCES
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