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Arch ToxicolDOI 10.1007/s00204-014-1286-7

TOxIcOkIneTIcs AnD MeTAbOlIsM

In vitro glucuronidation kinetics of deoxynivalenol by human and animal microsomes and recombinant human UGT enzymes

Ronald Maul · Benedikt Warth · Nils Helge Schebb · Rudolf Krska · Matthias Koch · Michael Sulyok

Received: 24 February 2014 / Accepted: 28 May 2014 © springer-Verlag berlin Heidelberg 2014

for UGT2b7 the DOn-3-O-glucuronide (DOn-3GlcA) metabolite prevailed. For human UGTs, liver, and intestinal microsomes, the glucuronidation activities were low. The estimated apparent intrinsic clearance (clapp,int) for all human UGT as well as tissue homogenates was <1 ml/min mg protein. For the animal liver microsomes, moderate clapp,int between 1.5 and 10 ml/min mg protein were calculated for carp, trout, and porcine liver. An elevated glucuronidation activity was detected for rat and bovine liver microsomes leading to clapp,int between 20 and 80 ml/min mg protein. The obtained in vitro data points out that none of the animal models is suitable for estimating the human DOn metabo-lism with respect to the metabolite pattern and formation rate.

Keywords Deoxynivalenol · Glucuronidation · Uridine-diphosphoglucuronyltransferases (UGT) · Human recombinant UGT · Trichothecene · Phase II metabolism

Introduction

The mycotoxin DOn represents one of the most important contaminants in grains worldwide. The toxin is formed by different Fusarium species such as F. graminearum and F. culmorum infecting the grains already during growth in the field. severe infection may lead to visible plant disease and decreased crop yield. However, significant amounts of moderately infected grains enter the food and feed pro-duction chain (Rocha et al. 2005). The frequency of DOn contamination of grain-based products turns the toxin into a serious matter of concern for the consumers’ health even though the single food commodity may comply with legal limits. A recent survey provides proof for the contamina-tion of almost every grain-based product on the eU/Us food market, with wheat and maize products showing

Abstract The mycotoxin deoxynivalenol (DOn), formed by Fusarium species, is one of the most abundant mycotox-ins contaminating food and feed worldwide. Upon ingestion, the majority of the toxin is excreted by humans and animal species as glucuronide conjugate. First in vitro data indicated that DOn phase II metabolism is strongly species dependent. However, kinetic data on the in vitro metabolism as well as investigations on the specific enzymes responsible for DOn glucuronidation in human are lacking. In the present study, the DOn metabolism was investigated using human micro-somal fractions and uridine-diphosphoglucuronyltransferases (UGTs) as well as liver microsomes from five animal spe-cies. Only two of the twelve tested human recombinant UGTs led to the formation of DOn glucuronides with a different regiospecificity. UGT2b4 predominantly catalyzed the for-mation of DOn-15-O-glucuronide (DOn-15GlcA), while

R. Maul · M. koch Division of Food Analysis, bAM - Federal Institute for Materials Research and Testing, Richard-Willstätter-straße 11, 12489 berlin, Germany

R. Maul (*) Department of Quality, leibniz-Institute of Vegetable and Ornamental crops Großbeeren/erfurt e.V., Theodor-echtermeyer-Weg 1, 14979 Großbeeren, Germanye-mail: maul@igzev.de

b. Warth · R. krska · M. sulyok Department for Agrobiotechnology (IFA-Tulln), center for Analytical chemistry, University of natural Resources and life sciences, Vienna (bOkU), konrad-lorenz-straße 20, 3430 Tulln, Austria

n. H. schebb Institute for Food Toxicology and Analytical chemistry, University of Veterinary Medicine Hannover, bischofsholer Damm 15, 30173 Hannover, Germany

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the highest concentration levels (De boevre et al. 2012). Products based on barley are widely affected by DOn contamination as revealed by a recent study investigating many different types of beer (Varga et al. 2012). besides the mycotoxin in its free form, conjugates of DOn (e.g., DOn-3-glucoside or acetylated DOn), so-called masked mycotoxins, have to be considered as an additional source of exposure (berthiller et al. 2013). In particular, certain processed cereal products like beer exhibit a high preva-lence of DOn-glucoside (Maul et al. 2012a; Varga et al. 2012). Thus, potential systemic effects of the conjugates in humans need to be considered. The main toxic effect of DOn on the cellular level is the inhibition of protein syn-thesis (Pestka 2010; Rocha et al. 2005). The visible acute symptoms observed in humans are nausea, diarrhea, and vomiting. In chronic animal studies using rats and pigs, a decreased weight gain, anorexia, decreased nutritional effi-ciency, and altered immune function additionally became obvious (Pestka 2007). DOn potentially triggers geno-toxicity in human lymphocytes probably via inhibition of DnA repair (Yang et al. 2014). However, a clear classifi-cation as carcinogen cannot be made as the studies carried out so far are quite contradictory (Ma and Guo 2008). Pri-marily due to the acute toxicity in humans, DOn content in foodstuffs is regulated by legal limits in many countries. In the eU, commodity-dependent legal limits between 200 and 1,750 µg/kg have been established derived from a tol-erable daily intake for DOn of 1 µg/kg body weight (bw) which was established in 2002 (ec 2007; scF 2002).

Upon ingestion of contaminated food by humans, typi-cally more than 75 % of the excreted DOn is present in the glucuronidated form (Turner et al. 2011; Warth et al. 2012a, 2013). Although the glucuronidation represents an important metabolic route for DOn in humans as well as in animals, no detailed in vitro evaluation of glucuronida-tion or investigation on the distinct enzymes responsible for the conversion has been carried out. To date, more than 20 uridine-diphosphoglucuronyltransferases (UGT) enzymes have been characterized whereof 12 are commercially available enabling an UGT reaction phenotyping and thus the identification of potential health hazards arising from environmental toxins (luo et al. 2012). The expression of the UGTs varies strongly between the different human tis-sues. Thus, the assignment of a glucuronide formation to any distinct UGT also enables an estimation of the tissue(s) responsible for the conjugation reaction in vivo (king et al. 2000).

In a preliminary study, we demonstrated that the glucu-ronidation of DOn at 3.75 µM substrate concentration is highly species specific (Maul et al. 2012b). With respect to both activity and product pattern, strong differences were observed among animal (i.e., rat, fish, bovine, and porcine) and human liver microsomes (HlM). Two main

glucuronides were formed, with the animal microsomes dominatingly giving rise to the DOn-3-O-glucuronide (DOn-3GlcA) conjugate, while HlM led to DOn-15-O-glucuronide (DOn-15GlcA) as the prevailing metabolic product (Fig. 1). A third glucuronide, tentatively assigned to the DOn-7GlcA, was also formed by several microsomes in trace amounts and recently confirmed in Ms/Ms experi-ments (Šarkanj et al. 2013). Additionally, the formation of a DOn-8GlcA has been shown for Wistar RlM recently (Uhlig et al. 2013). An in vivo case study provided proof for the dominant urinary excretion of the DOn-15GlcA in humans (Warth et al. 2013), while in rats, the DOn-3GlcA excretion prevails in in vivo experiments (nagl et al. 2012; Veršilovskis et al. 2012). However, the conjugation kinet-ics of DOn has not been investigated in detail. such in vitro data would allow for a better understanding of the metabolism reaction found in vivo. Moreover, these data also would allow for estimation of the capacity of an organ or organism to metabolize a certain xenobiotic substance based on the calculated apparent intrinsic clearance.

The aim of the present study was to identify the specific human UGT enzymes responsible for the DOn glucuroni-dation and the characterization of glucuronidation kinetic parameters of DOn by different animal microsomes.

Materials and methods

chemicals and reagents

UDP-glucuronic acid (UDPGA) and alamethicin were purchased from sigma-Aldrich (Taufkirchen, Germany). Deoxynivalenol-3-O-glucuronide (DOn-3GlcA) was syn-thesized by an optimized königs–knorr procedure and its structure successfully confirmed by nMR (Fruhmann et al. 2012). DOn-15GlcA was isolated from a naturally contam-inated human urine sample by fractionation as described in detail elsewhere (Warth et al. 2012a). DOn was purchased from Romer labs (Tulln, Austria). A reference standard of DOn-8GlcA was kindly provided by Dr. silvio Uhlig (Uhlig et al. 2013). All animal liver fractions were prepared according to the protocol established by lake with slight modifications (lake 1987) except pooled human liver microsomes (mixed gender, pool of 50 individuals) which were obtained from xenotech (Offenbach, Germany). Induced rat liver microsomes (iRlM) were prepared from male sprague–Dawley rats induced with phenobarbital/β-naphthoflavone. The measurement of the protein content of the self-prepared microsomes was carried out according to the method of bradford using bovine serum albumin as reference for calibration. Recombinant human UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2b4, UGT2b7, UGT2b10, UGT2b15,

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and UGT2b17 expressed in baculovirus-infected insect cells (supersomes) as well as human intestinal microsomes (HIM) were purchased from bD Gentest (Heidelberg, Ger-many). solvents for the liquid chromatography (lc) were obtained either from Merck (Darmstadt, Germany; metha-nol (lc gradient grade) and glacial acetic acid (p.a.)) or from VWR (leuven, belgium; acetonitrile (lc gradient grade)). All other chemicals and solvents were from sigma-Aldrich (Taufkirchen, Germany) and of the highest grade commercially available.

Microsomal incubations

A typical incubation mixture (200 µl of total volume) contained 100 mM potassium phosphate buffer (pH 7.4), 5 mM Mgcl2, 2.5 mM UDPGA, and 25 µg/ml alameth-icin. The concentrations of microsomal protein are given in Table 1. The DOn working solutions were prepared in methanol/water 50/50 (v/v) resulting in a methanol con-centration of 1.0 % in the final assay. For the incubations,

DOn concentrations of 0.4–50 µM were prepared. The incubation time was 30 min (RlM, carp-lM, and blM) or 60 min (HlM, PlM, and trout-lM). experiments investi-gating the recombinant UGTs were conducted for 60 min using 1.0 mg/ml of protein. The reactions were prepared and preincubated at 37 °c for 5 min before the reaction was initiated by the addition of UDPGA and further incu-bated at 37 °c. The reactions were terminated by addition of 200 µl acetonitrile. After centrifugation at 14,000×g for 5 min, 200 µl of the supernatant was evaporated to dry-ness in a vacuum concentrator and re-dissolved in 200 µl water/acetonitrile 90/10 (v/v) for lc–Ms/Ms analysis. Ali-quots of 10 µl were injected into the lc–Ms/Ms system using an Ab sciexQTrap 5500 Ms system (Foster city, cA, UsA) equipped with a Turbo V electrospray ioniza-tion (esI) source interfaced with an Agilent 1290 series lc system (Waldbronn, Germany). For control of the lc–Ms/Ms system and data evaluation, Analyst software (version 1.5.2) was used. All microsomal experiments were carried out in duplicate.

Fig. 1 Deoxynivalenol and the known glucuronides described for in vitro experiments (Maul et al. 2012b; Uhlig et al. 2013)

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lc–Ms/Ms conditions

The analytes were separated on a c18-reversed-phase Atlantis T3 column (3.0 × 150 mm, Waters, Wexford, Ireland) with 3-µm particle size and a c18 security guard cartridge. eluent A was water, while eluent b was ace-tonitrile, both containing 0.1 % acetic acid. After an initial time period of 2.0 min at 95 % A, the percentage of b was linearly raised to 30 % until min 10.0. Then, eluent b was raised to 96 % until min 14.0 followed by a hold time of 1.0 min and subsequent 2.25-min col-umn re-equilibration at 95 % A. The flow rate was set to 600 µl/min. esI–Ms/Ms using negative-ion mode was performed in selected reaction monitoring (sRM) mode. Three individual transitions were monitored for the DOn glucuronides since the third transition (m/z 471.0 [M-H]− to 441.0) is crucial to discrimination between DOn-15GlcA on the one hand and DOn-3GlcA as well as the third glucuronide on the other (Warth et al. 2012b). The optimized Ms/Ms parameters for the tree transi-tions were as follows: precursor 471.0 [M–H]−; DP -135; 265.2/175.2/441.0; ce -38/-40/-30; cxP -9/-5/-9. The esI source parameters were used as described elsewhere (Warth et al. 2012b). The values for the limit of detection (lOD) and the limit of quantification (lOQ) were calcu-lated from chromatograms of spiked microsomal blank incubations omitting DOn as substrate, respectively, as 0.6/2.1 nM (DOn-3GlcA) and 0.4/1.4 nM (DOn-15GlcA). For DOn-7GlcA, only a semi-quantitative esti-mation of the concentration based on the peak area was feasible assuming the same molar response as for DOn-3GlcA in esI–sRM–Ms.

kinetic data analysis

For DOn-3GlcA and DOn-15GlcA, the apparent kinetic parameters Km and Vmax were calculated from the untrans-formed data by least square regression using sigmaPlot 12.3 (systat software, Inc., san Jose, cA, UsA). Data were fitted to the equations of the following kinetic models:

The Michaelis–Menten equation,

where v is the rate of reaction, Vmax is the maximum veloc-ity, Km is the Michaelis–Menten constant (substrate con-centration at 0.5 Vmax), and S is the substrate concentration.

The Hill equation, which describes sigmoidal kinetics, is

where S50 is the substrate concentration resulting in 50 % of Vmax in Hill kinetic profiles and n is the Hill coefficient.

The substrate inhibition model equation is,

where Ksi is the constant describing the substrate inhibition interaction. Graphical fitting was done using the enzyme kinetics module of sigmaPlot.

For reactions exhibiting Michaelis–Menten as well as substrate-inhibited kinetics, intrinsic clearance (clint) was calculated as Vmax/Km. In the case of Hill kinetics, S50 is used instead of Km.

The quality of the fit was determined by comparison of the regression coefficient derived from least square regres-sion as well as the standard errors of the various parameter estimates.

For the formation of the putative DOn-7GlcA, a calcu-lation of any apparent kinetic parameters is not possible, as no quantitation of the amount of the glucuronide can be carried out without an authentic reference standard. Tenta-tively, values were calculated based on the assumption of a response identical to DOn-3GlcA in sRM–Ms detection.

As reproducible and satisfactory recovery rate of 89.8 ± 2.9 % from RlM and clM for DOn-3GlcA has been demonstrated before (Maul et al. 2012b), only the recovery from assays using human recombinant UGT was tested. For this purpose, blank samples containing UGT1A1

v =Vmax × S

Km + S

v =Vmax × S

n

Sn

50+ Sn

v =Vmax × S

Km + S ×

(

1 +S

Ksi

)

Table 1 Optimized protein concentration and incubation time used for the glucuronidation assays

a According to the suppliers’ information

enzyme preparation Protein concentration (mg/ml) Incubation time (min)

carp-lM (clM) 0.94 30

Trout-lM (TlM) 0.78 60

bovine-lM (blM) 0.02 30

Porcine-lM (PlM) 0.20 60

Induced rat-lM (iRlM) 0.05 30

Rat-lM (RlM) 0.05 30

Human-lM (HlM) 1.0a 60

Human intestinal microsomes (HIM) 1.0a 60

UGTs 1.0a 60

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or UGT1A6 (1 mg protein/ml) without UDPGA, were spiked with 0.4 µM DOn-3 GlcA and treated as described above prior to HPlc–Ms measurement. Very similar to the microsomal assays, a recovery rate of 91.7 ± 7.4 % was detected. All results presented in the following are corrected according to the mean microsomal recovery rate of 89.8 % and the determined UGT assay recovery rate, respectively.

Results

Microsomal experiments

All microsomal preparations were screened for their abil-ity to glucuronidate DOn in order to identify of appropriate enzyme concentration and incubation time assuring the for-mation of detectable amounts of metabolite formation and yet a substrate depletion of less than 10 %. The results are given in Table 1. In the subsequent main experiments, up to three DOn-O-glucuronides were detected in the incuba-tions using the different enzymatic preparations in the pres-ence of UDPGA (Fig. 2). The two dominating glucuronida-tion products could be clearly assigned to DOn-3GlcA and DOn-15GlcA by comparison with available reference sub-stances. Additionally, in all RlM, blM, and the fish-lM, the formation of DOn-7GlcA was detected.

For all investigated non-human species, the formation of DOn-3GlcA is dominating, while the second glucuro-nide is only of minor importance (Fig. 2). For PlM DOn-15GlcA is the minor metabolite while in RlM, blM, and

fish-lM, this metabolite is absent and DOn-7GlcA repre-sents the less abundant metabolite. none of the investigated lM led to the simultaneous formation of all three glucu-ronides in detectable amounts. Within the investigated set of six animal microsomal preparations, large differences in the observed activity at 30 µM substrate concentration were found comprising almost two orders of magnitude between fish-lM (lowest activity) and blM (highest activity).

For the investigation of human DOn glucuronidation, two different tissue fractions were included. In addition to the liver microsomes, preparations derived from intestinal tissue were used as well in order to obtain further infor-mation on the tissue specific distribution of human DOn metabolism. The DOn glucuronidation activity in both tis-sue fractions is at least an order of magnitude lower than for all animal lM investigated (Fig. 2). Interestingly, the product pattern of the two different human organs is signifi-cantly different. While in human liver incubations, DOn-15GlcA clearly dominates over DOn-3GlcA, in HIM the preference in product formation in inverted. Additionally, the formation of the DOn-7GlcA is also observed in trace amount for HIM, but it is absent in HlM.

In addition, a set of 12 commercially available human recombinant UGTs was investigated for the ability to trans-form DOn into any of the four glucuronides described so far by Šarkanj et al. (2013). Incubations with ten of the enzymatic preparations did not lead to the formation of any DOn glucuronide. However, the enzymes UGT2b4 and UGT2b7 led to the formation of one dominating glucuro-nide each, when incubated in the presence of 30 µM DOn

Fig. 2 DOn glucuronidation activity of different animal liver microsomes (lM) and human tissue fractions as well as human recombinant UGTs at 30 µM substrate concentra-tion (50 µM for PlM). The activity for the formation of the products DOn-3-O-glucuronide (DOn-3GlcA) and DOn-15-O-glucuronide (DOn-15GlcA) is independently calculated and given in logarithmical scale. For DOn-7-O-glucuronide (DOn-7GlcA), the activity could only be semi-quantified

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(Fig. 2). UGT2b4 predominantly catalyzed the formation of DOn-15GlcA, while UGT2b7 mainly formed DOn-3GlcA. The activities of both recombinant enzymes for the formation of their respective main glucuronides are low. Additionally, for both enzymes, the formation of the main product of the other was observed to a minor extend. Fur-thermore, UGT2b7 is capable of forming DOn-7GlcA in trace amounts.

enzyme kinetic evaluation

For all microsomes as well as for those UGTs tested posi-tive for the formation of DOn glucuronides, the enzymatic activity for the formation of the three potential conju-gates was measured in a concentration range between 0.4 and 50 µM. The obtained activities are plotted against the

respective concentration (Figs. 3, 4). Additionally, the data were submitted to a mathematical fitting process in order to estimate the most suitable kinetic model by applying least square regression. The estimated enzyme kinetic parame-ters (Vmax, Km, S50, and clint,app) derived from the best fit are given in Table 2.

by plotting of the unprocessed data, it became visible that only for PlM and clM both formed glucuronides contribute significantly to the DOn conjugation, while for the other microsomes, one product is clearly dominating over the whole observed substrate concentration range. Also, for most of the formed glucuronides, the data fit reveals a tendency toward a slight sigmoid kinetic progres-sion. However, the sigmoidity is not very pronounced as indicated by Hill coefficients always less than 2.5. This is also reflected in the eadie–Hofstee plots where no clear

Fig. 3 Glucuronidation kinetics for the DOn conjugation by various pooled animal micro-somes. Data were fit according to the kinetic model leading to the best fit defined as the equation showing the lowest errors derived from least square regression. each data point represents the mean of duplicate determinations and their devia-tions

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indications for atypical sigmoid kinetic progression can be detected (Fig. 5). none of the enzyme preparations followed a substrate-inhibited kinetic progression. More pronounced differences become obvious for the further enzyme kinetic parameters Vmax and Km or S50, respec-tively, between the various species (Table 2). TlM pos-sess a high substrate affinity, i.e., a relatively low apparent

Km of 12 and 16 µM for the formation of DOn-7GlcA and DOn-3GlcA, respectively. by contrast, blM and iRlM exhibit relatively weak affinities of 120.8 and 159.4 µM to DOn with respect to the formation of the main glucuro-nide. nevertheless, these two microsomal fractions show a relatively efficient apparent DOn clearance due to the high estimated Vmax of approx. 5 nmol/min/mg protein and

Fig. 4 Glucuronidation kinetics for the DOn conjugation by human liver microsomes and the two human recombinant UGTs which are involved in DOn metabolism. each data point represents the mean of duplicate determinations and their deviations

Table 2 enzyme kinetic parameters were estimated by fitting to the Michaelis–Menten or Hill kinetics equation using 0.4–50 µM DOn as sub-strate

The best fit was determined by choosing the fitting curve leading to the lowest sum of squares. In cases where fitting according to the Hill equa-tion led to the best fit, the Hill coefficient “n” is given. Data are expressed as mean ± sD (for Vmax, Km, S50, and n) or the mean of triplicate samples either given for each single glucuronide or as sum of all glucuronides formed by an enzymatic preparation (for the apparent intrinsic clearance)a Km applies to the Michaelis–Menten type kinetics (n = 1), S50 refers to Hill kinetics (n > 1); b only semi-quantitation of DOn-7GlcA was fea-sible, the presented values represent an estimation based on the assumption of identical response in sRM–Ms detection

Glucuronidation site

Km or S50a (µM)

± Vmax (nmol/min/mg protein)

± n ± sum of squares R2 clint,app (ml/min/mg)

Σ clint,app (ml/min/mg)

CLM

3 36.4 5.6 0.20 0.02 1 4.52e−04 0.984 5.72 7.06

7b 30.6 5.7 0.041 0.004 1 3.38e−05 0.974 1.33

TLM

3 16.0 2.4 0.024 0.002 1.38 0.14 9.03e−06 0.989 1.51 1.55

7b 11.9 3.5 0.0005 0.0001 2.00 0.91 5.51e−08 0.856 0.04

BLM

3 120.8 21.0 9.04 1.21 1 6.61e−02 0.995 74.82 78.19

7b 23.0 3.9 0.078 0.009 1.60 0.21 4.45e−05 0.989 3.37

PLM

3 33.2 21.5 0.073 0.029 1.36 0.36 1.72e−04 0.956 2.19 2.87

15 30.5 18.1 0.021 0.008 1.44 0.40 1.81e−05 0.950 0.68

iRLM

3 159.4 35.3 4.66 0.84 1 1.26e−02 0.995 29.26 30.75

7b 36.5 10.6 0.054 0.010 1.24 0.13 1.34e−05 0.992 1.49

RLM

3 47.7 24.5 1.09 0.42 1.44 0.26 1.51e−02 0.976 22.95 23.59

7b 35.1 12.2 0.022 0.007 2.22 0.67 1.48e−05 0.957 0.63

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9 nmol/min/mg protein. For the estimated clapp,int, differ-ences of factor 50 are resulting between TlM and blM. While the iRlM show a similar or even higher Km com-pared to untreated RlM, the Vmax is significantly elevated. As a consequence, the clint,app for iRlM is higher than for RlM. However, induction of UGTs by phenobarbital and β-naphthoflavone seems to have only moderate influence on the DOn glucuronidation.

Figure 4 presents the progression of the metabolite for-mation (glucuronide formation plotted against substrate concentration) for HlM as well as the two UGTs that are capable of forming DOn glucuronides. Under the selected experimental conditions, the glucuronidation activities rise with increased substrate concentration in an almost lin-ear manner. Thus, an enzyme kinetic evaluation is ham-pered. As expected from the results obtained for the first

Fig. 5 eadie–Hofstee plots obtained for the formation of the DOn-3-glucuronides repre-senting the most abundant by all pooled animal liver microsomes

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experiments using the 30 µM substrate concentration, the overall glucuronide formation is very low for all human enzyme preparations. In all cases, estimated Km values clearly above 100 µM have to be expected. eadie–Hofstee plotting of the data does also not lead to any valid addi-tional information as the resulting scatter plot allows no estimation of linear or any other characteristic nonlinear dependency (data not shown). Thus, a kinetic evaluation of the obtained data using DOn concentrations between 0.4 and 50 µM is not feasible.

Discussion

Qualitatively and quantitatively, the experiments pointed out vast differences for the DOn glucuronidation intensity between human and animal liver homogenates. Also with respect to the regioselectivity of the glucuronidation, ani-mal and human enzymatic preparations, mainly derived from liver tissue, large variations were observed. While the identification and quantification of the two most abundant glucuronides formed by different microsomes and recom-binant UGTs was feasible based on synthesized or iso-lated reference substances, the third glucuronide was very recently assigned to the DOn-7GlcA based on mass spec-trometric fragmentation data (Šarkanj et al. 2013).

With respect to human metabolism, the UGTs 2b4 and 2b7 could be identified as main enzymes involved in DOn glucuronidation. To date the substrate specificity of those enzymes was predominantly attributed to steroids such as bile acid (e.g., hyodeoxycholic acid) or steroid hormones (barre et al. 2007). Those substances do not possess much structural similarities to DOn; however, for UGT2b7, also the ability to conjugate more structurally related natural products and drugs like morphine is reported (coffman et al. 1997; stone et al. 2003). In vivo, potential bioac-tive substances being natural substrates for the UGTs 2b4 and 2b6 might be conjugated less efficiently in the pres-ence of high amounts of DOn. In our experiment, the two enzymes showed marked differences for the DOn glucu-ronidation with 2b4 preferentially forming DOn-15GlcA while 2b7 catalyzes the DOn-3GlcA formation. Although regiospecific preferences have been described for UGT2b7 recently (sneitz et al. 2013), it is surprising that such dif-ferences are that pronounced between 2b4 and 2b7 in spite of the fact that the enzymes are largely homologous (barre et al. 2007). The observed specificity is also reflected in the human microsomal experiments. While in human liver DOn-15GlcA clearly dominates over DOn-3GlcA, in HIM the preference in product formation is inverted. both, UGT2b4 and UGT2b7, genes are expressed in liver tissue; however, UGT2b4 has been detected in a tenfold higher amount at the RnA level (nakamura et al. 2008; Ohno

and nakajin 2009). Thus, a dominating presence of the UGT2b4 enzyme can be expected. This may explain the similar metabolite pattern with a dominating DOn-15GlcA occurrence in HlM and UGT2b4. The observed predomi-nant formation of DOn-15GlcA in HlM is not as strong as in recombinant UGT2b4 as in the microsomes also DOn-3GlcA forming UGT2b7 is present. by contrast, the UGT2b4 gene is not expressed in intestinal tissue. conse-quently, the metabolite pattern observed for HIM is similar to that of UGT2b7, indicating and exclusive conjugation by this isoform.

With respect to the renal contribution to the overall human DOn glucuronidation capacity, it seems possi-ble that both glucuronides may be formed in the kidney. However, the renal contribution to xenobiotica glucuroni-dation is not very pronounced as the kidney is not part of the first pass metabolism where many substances are read-ily metabolized. Data on the isoform specific gene expres-sion concerning UGTs 2b4 and 2b7 in the kidney show that 2b7 is much more abundant than 2b4 (nakamura et al. 2008; Ohno and nakajin 2009). Thus, in the case of DOn, the renal contribution could lead to a slight enhancement of the proportion of DOn-3GlcA in vivo. However, our findings for the regiospecificity of the DOn glucuronida-tion by HlM and HIM are consistent with in vivo data for human urine. A pronounced involvement of the renal tis-sue and thus formation of DOn-3GlcA was not observed. DOn-15GlcA was the clearly dominating excretion prod-uct with DOn-3GlcA as a minor conjugate in several euro-pean and African populations (Abia et al. 2013; shephard et al. 2013; Warth et al. 2012a) while DOn-7GlcA was only detected in trace amounts in highly contaminated sam-ples (Šarkanj et al. 2013; Warth et al. 2013). While neither HIM nor HlM led to the formation of the DOn-7GlcA, the UGT2b7 showed a small yet detectable signal also for this glucuronidation product at substrate levels above 10 µM corresponding to the in vivo occurrence only in some highly contaminated samples.

As shown in our previous study, the metabolite pat-tern is subject to strong inter-individual variation (Maul et al. 2012b). Although only a single concentration level of 3.75 µM was used in this preliminary study, these data clearly showed the dominating formation of DOn-3GlcA in most animals, while in humans, the DOn-15GlcA pre-vailed. For HlM derived from a donor pool of 50 mixed gender individuals, no detectable signal for the DOn-7GlcA was present, whereas a second mixed gender micro-somal preparation derived from an unspecified number of donors catalyzed the formation of this metabolite addition-ally to the dominating DOn-15GlcA and the minor metab-olite DOn-3GlcA.

The investigation of the qualitative DOn glucuronide formation using six enzymatic liver preparations derived

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from five animal species shows that the dominating metab-olite is DOn-3GlcA, which is a remarkable difference in regioselectivity to the experiments using HlM or regard-ing the human urinary excretion profile. It becomes obvi-ous that none of the very frequently used animal species (i.e., rat or pig) would have had the capability to predict the human metabolism (Pestka 2007). However, the metabolite pattern of PlM, representing a less frequently used species, shows the highest similarly to HlM.

some of the enzyme kinetic evaluations including those concerning the human enzymatic preparations suf-fer from the limited substrate concentration range that was used for the metabolic assays. As a consequence, fitting according to Michaelis–Menten or Hill equation shows relatively large uncertainties. Particularly, for both RlM assays, the resulting Km value lies outside the range where the fitted curve is supported by valid data points. For all human enzymatic fractions, the metabolic activity is even lower and as a consequence a reasonable fitting could not be carried out in the investigated DOn concen-tration range. The low glucuronidation capacity of the HlM was specific for DOn as in previous experiments, we could show for the identical set of microsomes that the glucuronidation activity for the conjugation of TFMU as reference substrate is in the same range as for the other mammal microsomes (Maul et al. 2012b). The signifi-cance of the exact figures presented for e.g. RlM, must therefore be regarded as limited. Additionally, it can be concluded that most of the UGTs in rat and human have a very limited substrate affinity for DOn with Km higher than 50 µM DOn representing a toxin level which is very unlikely to occur in vivo.

In contrast to the human enzymes possessing a very low capacity for DOn glucuronidation, some of the investi-gated animal species show moderate values for the apparent intrinsic clearances. Despite of the existing uncertainties regarding the accuracy of the enzyme kinetic parameters, the values presented in Table 2 enable a classification of the various species. Three clusters of species can be formed with RlM and blM possessing high clint,app values ≫10 as first group. Into the second cluster, the PlM and the fish-lM are belonging with clint,app values between 1 and 10, while human enzymatic preparations form the third cluster with low clint,app values ≪1.

Furthermore, the data show a moderate effect of enzyme induction by treating rats with phenobarbital/β-naphthoflavone with respect to DOn glucuronidation. Interestingly, for both glucuronides formed by RlM, the calculated Vmax was clearly elevated by at least factor two in the induced microsomes. However, also the slope of the Michaelis–Menten plots is lower which reflects a sig-nificant increase in the concentration of the half maximum velocity. As a consequence, the calculated clint,app does not

differ strongly. Additionally, the weak sigmoidity of the RlM DOn-3GlcA formation is no longer detectable for iRlM. experiments investigating the UGT enzyme induc-tion caused by phenobarbital treatment in the rat showed only a limited impact on the UGT gene expression. Par-ticularly, for the UGT2 family, inductive effects could only be observed for UGTb1 and UGTb12 (Ritter 2000). It seems likely that the treatment using phenobarbital/β-naphthoflavone has only a weak inductive effect on the enzymes responsible for DOn glucuronidation which could explain the limited differences between RlM and iRlM in our study.

In most cases, the fit reveals Michaelis–Menten type kinetics or an only weak sigmoid progression for the glu-curonide formation. Only in few cases, the kinetic data fit reveals a clear sigmoid progression. This kind of kinetic progression always applies to metabolites formed in minor amounts with another glucuronide being formed dominat-ingly following a Michaelis–Menten kinetic. Thus, for the DOn glucuronidation, the consideration of atypical kinetic progression seems to be negligible. However, as micro-somes represent mixtures of different UGTs, the estimation of one distinct kinetic progression model for the metabolite formation is difficult as the various underlying metaboli-cally active enzymes may follow different kinetic models. This might also explain the ambiguous outcome of the eadie–Hofstee plotting which often could not provide clear evidence for one distinct kinetic model.

From the results obtained for the animal lM, it might be concluded that those species being well adapted to grains and grasses as important feed source (e.g., cow or rats) show particularly high clint,app, while those animals that traditionally have other primary feed sources (e.g., fish or pork) do not possess a rapid clearance. Also in human nutrition, leafy vegetables and fruits were primar-ily used in ancient times, while (cultivated) grains are rela-tive new components of the human diet (bocquet-Appel et al. 2012). Thus, the very low human clint,app fits in this theory as human metabolism is confronted with grains and the accompanying contaminants only since few thousands of years representing a short period for metabolic adapta-tion. Generally, it becomes obvious that for DOn glucu-ronidation, it is difficult to refer the obtained data to any specific animal habit or physiological background. Thus, it is not possible to make any predictions for further spe-cies not tested yet. With respect to the animal sensitivity toward DOn-contaminated feed also, no direct correlation can be established. Those animals being relatively sensi-tive to the toxic effects of DOn (i.e., pigs or rats) do not possess a particularly slow DOn metabolism that might lead to a prolonged exposure to the free toxin (Pestka 2007). However, blM were the most effective preparation for the apparent DOn clearance and belong to the group

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of ruminants which have been reported to be particu-larly insensitive. This insensitivity can also be attributed to ruminal DOn detoxification to a non-toxic deepoxy metabolite, and therefore, such a hypothesis would have to be evaluated for a larger set of animal microsomes and animal species.

Conclusion

In the present study, we were able to identify UGTs 2b4 and 2b7 as main human enzymes that catalyze the glucu-ronidation of DOn. Thus, a high DOn exposure may inter-fere with the metabolism of endogenous substrates or xeno-biotics (e.g., hormons or morphine analogues) transformed by UGT2b4 or 2b7 in vivo. The in vitro DOn glucuroni-dation catalyzed by these enzymes is a relatively slow pro-cess. This corresponds to the low glucuronidation activ-ity observed using HlM and HIM indicating a prolonged excretion of DOn from the human body. by contrast, the investigation of different animal lM showed strongly het-erogeneous glucuronidation activities. experiments using bovine and rat-lM led to relatively rapid apparent intrin-sic clearances, while fish and porcine-lM showed moder-ate activity. In all cases, the formation of DOn-3GlcA pre-vailed over DOn-7GlcA (or DOn-15GlcA in the case of PlM). These results for the animal lM are in contrast to the results obtained for human enzyme preparations where DOn-15GlcA plays a dominant role. neither qualitatively nor quantitatively was the metabolism of the investigated animal lM comparable to the pattern and intensity of the DOn metabolites formed by in human microsomes or recombinant UGTs. These findings may impact on the gen-eral toxicological evaluation of the mycotoxin DOn as its toxicity assessment is based on animal studies in general. In the case of DOn, it seems likely that results from, e.g., common rodent experiments leading to lD50 or nOAel values are critical with respect to estimating the situation in human as the factor of 10 usually included for compen-sation of inter-species differences might not be sufficient. However, our findings on the in vitro glucuronidation should be verified in in vivo studies investigating the excre-tion profile and kinetics of DOn metabolism in different species.

Acknowledgments The authors thank the ec (kbbe-2007-22269-2 MYcOReD), the lower Austrian Government, the FWF (project l255-b11), and the graduate school program Applied biosci-ence Technology (Ab-Tec) of the Vienna Universities of Technology (TU-Wien) and natural Resources and life sciences (bOkU Wien) for financial support. Furthermore, the authors would like to express their gratitude toward Dr. silvio Uhlig (norwegian Veterinary Insti-tute, Oslo) for providing DOn-8GlcA reference standard. Jill-sandra kant is acknowledged for assistance with the in vitro glucuronidation assays.

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