In vitro phase i metabolism of cis -zearalenone

Article abstract of DOI:10.1021/tx500312g

The present study investigates the in vitro phase I metabolism of cis-zearalenone (cis-ZEN) in rat liver microsomes and human liver microsomes. cis-ZEN is an often ignored isomer of the trans-configured Fusarium mycotoxin zearalenone (trans-ZEN). Upon the influence of (UV-) light, trans-ZEN isomerizes to cis-ZEN. Therefore, cis-ZEN is also present in food and feed. The aim of our study was to evaluate the in vitro phase I metabolism of cis-ZEN in comparison to that of trans-ZEN. As a result, an extensive metabolization of cis-ZEN is observed for rat and human liver microsomes as analyzed by HPLC-MS/MS and high-resolution MS. Kinetic investigations based on the substrate depletion approach showed no significant difference in rate constants and half-lives for cis- and trans-ZEN in rat microsomes. In contrast, cis-ZEN was depleted about 1.4-fold faster than trans-ZEN in human microsomes. The metabolite pattern of cis-ZEN revealed a total of 10 phase I metabolites. Its reduction products, α- and β-cis-zearalenol (α- and β-cis-ZEL), were found as metabolites in both species, with α-cis-ZEL being a major metabolite in rat liver microsomes. Both compounds were identified by co-chromatography with synthesized authentic standards. A further major metabolite in rat microsomes was monohydroxylated cis-ZEN. In human microsomes, monohydroxylated cis-ZEN is the single dominant peak of the metabolite profile. Our study discloses three metabolic pathways for cis-ZEN: reduction of the keto-group, monohydroxylation, and a combination of both. Because these routes have been reported for trans-ZEN, we conclude that the phase I metabolism of cis-ZEN is essentially similar to that of its trans isomer. As trans-ZEN is prone to metabolic activation, leading to the formation of more estrogenic metabolites, the novel metabolites of cis-ZEN reported in this study, in particular α-cis-ZEL, might also show higher estrogenicity.

Full text of DOI:10.1021/tx500312g

Article
pubs.acs.org/crt
In Vitro Phase I Metabolism of cis-Zearalenone
Sarah S. Drzymala,*,† Antje J. Herrmann,† Ronald Maul,†,‡ Dietmar Pfeifer,† Leif-Alexander Garbe,§
and Matthias Koch†
†Department of Analytical Chemistry; Reference Materials, Federal Institute for Materials Research and Testing (BAM),
Richard-Willstaetter-Strasse 11, 12489 Berlin, Germany
‡Department of Quality, Leibniz-Institute of Vegetable and Ornamental Crops (IGZ), Theodor-Echtermeyer-Weg 1, 14979
Großbeeren, Germany
§Department of Biotechnology, Institute of Bioanalytics, Technische Universitat Berlin, 13353 Berlin, Germany
̈
ABSTRACT: The present study investigates the in vitro phase I
metabolism of cis-zearalenone (cis-ZEN) in rat liver microsomes
and human liver microsomes. cis-ZEN is an often ignored isomer
of the trans-configured Fusarium mycotoxin zearalenone (trans-
ZEN). Upon the influence of (UV-) light, trans-ZEN isomerizes to
cis-ZEN. Therefore, cis-ZEN is also present in food and feed. The
aim of our study was to evaluate the in vitro phase I metabolism of
cis-ZEN in comparison to that of trans-ZEN. As a result, an
extensive metabolization of cis-ZEN is observed for rat and human
liver microsomes as analyzed by HPLC-MS/MS and high-
resolution MS. Kinetic investigations based on the substrate
depletion approach showed no significant difference in rate
constants and half-lives for cis- and trans-ZEN in rat microsomes.
In contrast, cis-ZEN was depleted about 1.4-fold faster than trans-
ZEN in human microsomes. The metabolite pattern of cis-ZEN
revealed a total of 10 phase I metabolites. Its reduction products,
α- and β-cis-zearalenol (α- and β-cis-ZEL), were found as
metabolites in both species, with α-cis-ZEL being a major
metabolite in rat liver microsomes. Both compounds were identified by co-chromatography with synthesized authentic
standards. A further major metabolite in rat microsomes was monohydroxylated cis-ZEN. In human microsomes,
monohydroxylated cis-ZEN is the single dominant peak of the metabolite profile. Our study discloses three metabolic pathways
for cis-ZEN: reduction of the keto-group, monohydroxylation, and a combination of both. Because these routes have been
reported for trans-ZEN, we conclude that the phase I metabolism of cis-ZEN is essentially similar to that of its trans isomer. As
trans-ZEN is prone to metabolic activation, leading to the formation of more estrogenic metabolites, the novel metabolites of cis-
ZEN reported in this study, in particular α-cis-ZEL, might also show higher estrogenicity.
products were found in various animal species and humans,
in particular α- and β-trans-zearalenol (α- and β-trans-ZEL).4−7
The estrogenic activity of α-trans-ZEL exceeds that of trans-
ZEN by approximately a factor of 80, thus linking the toxicity of
trans-ZEN inevitably to its metabolism.8 Several monohydroxy-
lated metabolites were discovered in rat and human in vitro
assays, including one monohydroxylated ZEN discovered in rat
urine in vivo.9−12
Various studies demonstrated that trans-ZEN is subject to
phase II metabolism. Conjugation of trans-ZEN with glucoronic
acid and sulfate proved to be a major metabolic pathway in
different animal species and humans in vitro as well as in
vivo.13−15 These reports also show that phase I metabolites of
trans-ZEN are prone to conjugation reactions.
INTRODUCTION
■
trans-Zearalenone (trans-ZEN) is an estrogenic mycotoxin that
contaminates food and feed worldwide. It is produced by
various molds of the Fusarium genus, which can infest cereal
crops including wheat, rye, oats, soybeans, rice, and maize.
Humans and animals are exposed to trans-ZEN by ingestion of
contaminated food and feed. The spatial structure of trans-ZEN
enables binding to the mammalian estrogen receptor, thus
causing reproductive disorders in numerous animal species and
possibly in humans.1 Thus, maximum levels have been
established for this fungal toxin in the European Union (EU)
and several countries worldwide.2 In the EU, maximum levels
range between 20 and 400 μg/kg, depending on the
commodity.3
Concerning phase I metabolism, several mammalian in vitro
and in vivo studies have shown that trans-ZEN is extensively
metabolized via reduction and hydroxylation. Reduction
Received: July 31, 2014
Published: September 25, 2014
© 2014 American Chemical Society
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Roth (Karlsruhe, Germany). All standard chemicals were of p.a. grade,
and all solvents, HPLC grade.
Although often ignored, ZEN can occur in two config-
urations. The double bond between C11 and C12 may
isomerize from the trans to the cis configuration (Figure 1).
Microsomal Source. Human and rat liver microsomes were
purchased from BioreclamationIVT (Baltimore, MD, USA). Rat liver
microsomes (RLM) were prepared from Sprague−Dawley female rats.
Human liver microsomes (HLM) were derived from a mixed gender
pool of 50 donors. The total protein content, P450 concentrations,
and specific activities of different P450 isoforms were assumed as
provided by the manufacturer. Regarding RLM, the protein
concentration was 23.7 mg/mL, and the total P450 concentration,
0.483 nmol/mg. For HLM, the protein concentration was 20.0 mg/
mL, and the total P450 concentration, 0.411 nmol/mg.
Preparation of Native and 13C-Labeled cis-Zearalenone.
Native cis-ZEN was generated photochemically from trans-ZEN by a
procedure reported earlier.19 In summary, native trans-ZEN is
dissolved in ethyl acetate and irradiated by UV-light (λ = 350 nm)
for 8 h. The irradiated solution is then subjected to preparative HPLC
in order to separate the trans from the cis isomer and to obtain pure
native cis-ZEN. U-[13C18]-cis-ZEN was synthesized as described earlier
following the same procedure as that for the preparation of native cis-
ZEN.19 Purities were determined to be >99% for the native as well as
the 13C-labelled standard, as measured by HPLC-FLD.
Microsomal Incubations. Incubations of ZEN with either RLM
or HLM were carried out at 37 °C in a volume of 0.2 mL. For kinetic
investigations, a mixture containing 1 μM ZEN dissolved in DMSO,
microsomes (final protein concentration 1 mg/mL), 0.1 mM MgCl2,
and 0.1 M potassium phosphate buffer, pH 7.4, was preincubated for 5
min. The fraction of DMSO in the microsomal incubation volume was
not higher than 1%. Reactions were started by adding 6 mM NADPH.
At different time points the reaction was quenched by addition of
equal volumes of ice-cold acetonitrile containing internal standard.
The respective fully 13C-labeled ZEN analogues were used for
quantification of cis- and trans-ZEN. After the reaction was stopped,
the incubation mixtures were centrifugated in a Eppendorf mini Spin
plus centrifuge (Hamburg, Germany) at 11 650g for 5 min. The
supernatant was transferred to a HPLC vial and analyzed by HPLC-
MS/MS. Negative controls lacked NADPH. Vehicle controls
contained NADPH but no substrate. A control containing NADPH
and substrate but without microsomes was performed as well. Control
incubations were performed in duplicate; all other reactions were
performed in triplicate.
Figure 1. Photoisomerization of ZEN.
Routes of entry into the food chain might arise from the
production of cis-ZEN by fungi16,17 or upon exposure of trans-
ZEN contaminated foods to (UV-) light, although the
detection of cis-ZEN in fungi cultures might be a consequence
of exposure to daylight, as biosynthetic pathways are generally
stereoselective. To date, cis-ZEN has been detected in different
commodities like ground maize, sugar beets, and edible oils as
well as in wet ground maize intended for animal feeding.18−20
Despite the evidence of cis-ZEN occurrence in food and feed,
its metabolism has never been investigated. Presumably, the
lacking availability of authentic standards and missing adequate
analytical methods have hampered a comprehensive inves-
tigation of cis-ZEN metabolism. In order to address this
problem, we recently synthesized cis-ZEN and 13C-labeled cis-
ZEN.19,21 Applying these standards, the aim of the present
work is to investigate the in vitro depletion kinetics of cis-ZEN
and to identify novel metabolites in rat and human liver
microsomes.
MATERIALS AND METHODS
■
Terms and Definitions. Throughout the entire article, the term
zearalenone (ZEN) or zearalenol (ZEL) refers to both cis and trans
isomers. When a certain isomer is considered, it will be specified by
either trans or cis.
Chemical Reagents. trans-ZEN (white powder, purity 99.8%) was
purchased from AppliChem GmbH (Darmstadt, Germany). Zear-
alanone (purity 98.0%), α-trans-ZEL (no purity given), and β-trans-
ZEL (no purity given) were obtained from Sigma-Aldrich (Steinheim,
Germany). NADPH tetrasodium salt was obtained from AppliChem
GmbH (Darmstadt, Germany). MgCl2 was purchased from Avantor
Performance Materials (Center Valley, PA, USA). KH2PO4 was
derived from Merck (Darmstadt, Germany) and K2HPO4 from Carl
In order to calculate depletion kinetics, the peak areas of cis- or
trans-ZEN were determined and normalized to the value obtained at t
= 0 min. Rate constants k (min−1) were obtained by linear least-
squares regression of the term ln (A/A0) = −kt, where t is the
incubation time (min), A is the peak area, and A0 is the peak area at t =
0 min. Half-lives were calculated according to the expression t1/2 = ln
2/k, as reported by Obach and colleagues.22
Figure 2. HPLC-DAD (λ = 274 nm) chromatogram of α-/β-trans-ZEL after UV irradiation for 12 h. The mixture of the four isomers can be well-
separated using a C18 column. Retention times differ from the metabolite patterns in Figures 4 and 5 due to the use of a different analytical column
and method.
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In contrast to the kinetic investigations, 10 μM ZEN were used as
the substrate concentration for qualitative analysis to enhance the
signal intensity of the formed metabolites. The reaction was run for a
time period of 120 min and then stopped by addition of 50 μL ice-cold
acetonitrile. Analysis was carried out by high-resolution mass
spectrometry (HRMS). Phase I metabolites were identified by
comparing chromatograms from vehicle control and negative control
incubations to that from incubations in the presence of NADPH.
Furthermore, incubations in the presence of NADPH were compared
to incubations containing NADPH and substrate but without
microsomes. All other conditions were as stated for the kinetic
investigations.
Metabolite Synthesis: α- and β-cis-Zearalenol. In a series of
pretests, standards of α- and β-trans-ZEL were UV-irradiated
separately at λ = 350 nm. Each standard gave rise to one main
irradiation product, which was tentatively assigned α- or β-cis-ZEL,
depending on the configuration of the educt. The maximum cis-ZEL
formation was observed after 24 h for α-trans-ZEL and after 48 h for β-
trans-ZEL. Irradiation in ethyl acetate yielded the highest cis-/trans-
ZEL ratio (α-ZEL, 3.5; β-ZEL, 3.6) when compared to heptane,
acetonitrile, acetone, and methanol, confirming the results of Koppen
et al.19 for ZEN isomerization.
For final synthesis of the cis-ZELs, a two step procedure was
planned using sodium borohydride reduction of trans-ZEN and
subsequent UV radiation. Due to the isomerization equilibrium of the
trans- and cis-ZELs after irradiation, a suitable analytical method was
developed to separate all four ZEL isomers in one run (Figure 2). The
chromatographic system consisted of a SpectraSystem HPLC
(Thermo Fisher Scientific, Waltham, MA, USA) including a pump
P4000, a degasser SCM 1000F, an autosampler AS3000F, a DAD
UV6000LP, and a FLD Surveyor Plus with a Eurospher II 100-3 C18 P
column (150 × 2 mm, 3 μm particle size; Knauer GmbH, Berlin,
Germany). A 50 °C oven temperature, an injection volume of 10 μL, a
flow rate of 0.4 mL/min, and a runtime of 30 min were employed.
Methanol/water 45:55 (v/v) containing 0.1% formic acid was used as
isocratic elution solvent. The DAD was set to λ = 274 nm, and
fluorescence detection was done at λEx = 274 nm and λEm = 456 nm.
Final synthesis of both cis-ZEL isomers was carried out with 38 mg
of NaBH4 (1.0 mmol), which was added to a solution of 25 mg of
trans-ZEN (0.9 mmol) in 10 mL of methanol. After 15 min at room
temperature, the reaction was stopped by dropwise addition of acetic
acid (25% in water) until no further gas formation could be observed.
The completion of the reaction was confirmed by HPLC-FLD
(chromatography conditions as stated above). The ratio of α- to β-
trans-ZEL was calculated from HPLC-DAD peak area (λ = 274 nm) as
33:67. The solution was evaporated to dryness, redissolved in ethyl
acetate, and split into 10 fractions in clear glass HPLC vials, which
were directly exposed to UV light (λ = 350 nm). The exposure was
carried out for 24 h using a universal UV lamp, type TL-900
(CAMAG, Muttenz, Switzerland). The generated cis isomers were
purified from the trans isomers using the chromatographic conditions
described above with the HPLC system connected to an automatic
fraction collector Foxy Jr. (Teledyne Isco, Lincoln, NE, USA). The
purification yielded 5.6 mg (22%) of α-cis-ZEL in a purity > 94% and
7.0 mg of β-cis-ZEL in a purity > 99%. Purities were determined by
HPLC-UV analysis at λ = 274 nm.
α-cis-ZEL. δ (ppm) 1.24−1.82 (m, 10H, H-4, H-5, H-6, H-8, H-9),
1.35 (d, 3H, -CH3, J = 6.2 Hz), 2.28 (m, 2H, H-10), 3.75 (m, 1H, H-
7), 5.15 (m, 1H, H-3, J = 6.2 Hz), 5.57 (ddd, 1H, H-11, J = 11.5, 11.2,
4
3.8 Hz), 6.14 (d, 1H, H-15, J(13,15) = 2.5 Hz), 6.23 (d, 1H, H-13,
4J(13,15) = 2.5 Hz), 6.71 (d, 1H, H-12, J(12,11) = 11.5 Hz).
β-trans-ZEL. δ (ppm) 1.29−1.79 (m, 10H, H-4, H-5, H-6, H-8, H-
9), 1.34 (d, 3H, -CH3, J = 6.2 Hz), 2.28 (m, 2H, H-10), 3.71 (m, 1H,
H-7), 5.12 (m, 1H, H-3, J = 6.3 Hz), 5.96 (ddd, 1H, H-11, J = 15.5,
4
8.4, 6.0 Hz), 6.21 (d, 1H, H-15, J(13,15) = 2.5 Hz), 6.44 (d, 1H, H-13,
4J(13,15) = 2.5 Hz), 6.72 (d, 1H, H-12, J(12,11) = 15.6 Hz).
β-cis-ZEL. δ (ppm) 1.28−1.69 (m, 10H, H-4, H-5, H-6, H-8, H-9),
1.36 (d, 3H, −CH3, J = 6.5 Hz), 2.17 (m, 2H, H-10), 3.60 (m, 1H, H-
7), 5.34 (m, 1H, H-3, J = 6.5 Hz), 5.60 (ddd, 1H, H-11, J = 11.5, 10.1,
4
5.1 Hz), 6.14 (d, 1H, H-15, J(13,15) = 2.5 Hz), 6.22 (d, 1H, H-13,
4J(13,15) = 2.5 Hz), 6.72 (d, 1H, H-12, J(12,11) = 11.5 Hz).
HPLC-MS Analysis. HPLC-MS/MS. Kinetic experiments were
analyzed on an Agilent 1200 series HPLC coupled to an API 4000
QTRAP hybrid mass spectrometer (AB Sciex, Foster City, USA). A
Gemini-NX C18 column (150 mm × 2 mm, 3 μm particle size;
Phenomenex, Aschaffenburg, Germany) was used with a flow rate of
0.25 mL/min and an oven temperature of 50 °C. The mobile phase
consisted of water with 0.1% formic acid (A) and methanol containing
0.1% formic acid (B). A gradient program was used starting at 50%
mobile phase B. Within 17 min, B was raised to 60%, followed by an
increase to 100% at minute 18. Afterwards, the column was re-
equilibrated to starting conditions for 5 min. HPLC-MS/MS runtime
was 24 min per sample, and the injection volume was set to 10 μL.
The mass spectrometer was operated in multiple reaction
monitoring (MRM) mode with negative electrospray ionization
(ESI). For both native ZEN isomers, the monitored transitions were
(m/z) 317.1 → 131.1 (quantifier) and 317.1 → 175.0 (qualifier). For
the two internal standards, U-[13C18]-ZEN (m/z) 335.2 → 140.2 was
monitored. trans- and cis-ZEN were separated by chromatography and
assigned via retention times.
The following ion source parameters were used: ion spray voltage,
−4000 V; desolvation temperature, 450 °C; ion source gas, 1:60; ion
source gas, 2:60; curtain gas, 20. The optimized MRM compound-
specific parameters were (quantifier/qualifier/internal standard):
declustering potential, −65/−65/−65 V; entrance potential, −10/−
10/−10 V; collision energy, −40/−34/−40 V; collision cell exit
potential, −9/−6/−9 V; dwell time, 50/50/50 ms. Data acquisition
was done using Analyst 1.6.2 software (AB Sciex, Foster City, CA,
USA).
HRMS. Qualitative metabolite profiling and accurate mass based
analyses were done on an Agilent 1290 Infinity UPLC coupled to an
Agilent 6230 accurate-mass time-of-flight MS. The column oven was
set to 50 °C. Separation of the metabolites was achieved on an
Ascentis Express F5 column (150 mm × 3 mm, 2.7 μm particle size;
Sigma-Aldrich, Steinheim, Germany). The Ascentis F5 column
separated trans- and cis-ZEN as well as the cis and trans isomers of
α- and β-ZEL using water with 0.1% formic acid (A) and methanol
containing 0.1% formic acid (B) as solvents in a gradient program at a
flow rate of 0.35 mL/min. A linear gradient was applied starting from
50% B to 70% at minute 18. Then, the column was re-equilibrated for
8 min.
For ESI operation, nitrogen was used as the drying gas (350 °C, 10
L/min) and nebulizer gas (35 psi). The capillary, skimmer, and
octapole RF voltages were set at 3.7 kV, 65 V, and 750 V, respectively.
Data was acquired in the negative ionization mode within a range of 75
to 500 m/z. Automatic calibration was enabled by continuous
postcolumn infusion of calibrant solution. Data analysis was done
using MassHunter 6.0. Determination of the elemental composition of
individual metabolites was based on accurate masses, typically better
than 10 ppm mass accuracy.
The accurate mass of the cis-ZELs was determined by HRMS (m/z
319.157 for both) and matches the calculated exact mass for ZEL.
Furthermore, the configuration of the double bond C11−C12 was
resolved by 1H NMR. NMR spectra were recorded on a Bruker DMX-
400 or a Bruker AVANCE III 500 spectrometer (Bruker Daltonik,
Bremen, Germany) in CD3OD. Proton chemical shifts were referenced
to residual native methanol at 3.31 ppm. Complete assignments for
proton resonances were performed in accordance to previously
published works on α- and β-trans-ZEL.23
α-trans-ZEL. δ (ppm) 1.16−1.94 (m, 10H, H-4, H-5, H-6, H-8, H-
9), 1.39 (d, 3H, -CH3, J = 6.1 Hz), 2.32 (m, 2H, H-10), 3.76 (m, 1H,
H-7), 4.96 (m, 1H, H-3), 5.70 (ddd, 1H, H-11, J = 15.4, 9.9, 4.6 Hz),
6.22 (d, 1H, H-15, 4J(13,15) = 2.5 Hz), 6.37 (d, 1H, H-13, 4J(13,15) = 2.5
Hz), 7.12 (d, 1H, H-12, J(12,11) = 15.4 Hz).
RESULTS
■
Depletion Kinetics. In the presence of NADPH, cis-ZEN is
rapidly metabolized by rat liver microsomes (RLM) and human
liver microsomes (HLM), which can be seen by the decrease of
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Figure 3. Depletion profile of 1 μM cis-ZEN (triangle) and trans-ZEN (square) in RLM (a) and HLM (b). The corresponding NADPH-depleted
controls clearly show the dependence of phase I metabolism of cis-ZEN (blank triangle) and trans-ZEN (blank square) on NADPH as cofactor. Error
bars represent the standard deviation (n = 3 for samples containing NADPH; n = 2 for controls).
Table 1. Overview of Compounds, Metabolic Reactions, Retention Times (tR), Elemental Formulas, Observed Masses, and
Errors for cis-ZEN Metabolites Formed by RLM or HLM
compd
metabolic reaction
RLM
HLM
tR
formula
m/z observed
mass error (mDa)
cis-ZEN
M1
15.3
6.5
C18H21O5
C18H21O6
C18H21O6
C18H21O6
C18H23O6
C18H23O6
C18H21O6
C18H21O6
C18H21O6
C18H23O5
C18H23O5
317.142
333.134
333.134
333.134
335.151
335.151
333.134
333.134
333.134
319.157
319.157
+3
0
+O
×
×
×
M2
+O
7.5
0
M3
+O
×
×
×
×
×
×
×
×
8.5
0
M4
+O and +2H
8.5
+1
+1
0
M5
+O and +2H
8.8
M6
+O
×
×
×
×
×
10.0
10.7
10.7
13.1
13.8
M7
+O
0
M8
+O
0
M9
+2H
+2H
+2
+2
M10
Figure 4. Overlay of extracted ion chromatograms that were obtained by analysis of 10 μM cis-ZEN incubated with RLM. The four ion traces, m/z
319.157, m/z 333.134, m/z 335.152, and m/z 317.342, were extracted and combined.
substrate concentration (Figure 3). In the absence of NADPH,
cis-ZEN levels remain constant. In order to investigate if
changes in double bond configuration affect depletion kinetics,
the degradation of cis-ZEN is displayed in comparison to that of
trans-ZEN.
and 20 1 × 10−3 min−1 for trans-ZEN, which is also reflected
in their half-lives (24 1 min for cis-ZEN and 34 3 min for
trans-ZEN). A two-sided t-test (f = 4, P = 95%) confirmed
statistically significant differences for the half-lives of cis- and
trans-ZEN in HLM.
Initial degradation constants and half-lives were calculated in
approximation to first-order decay functions for RLM and
HLM. In RLM, the rate constants for cis- and trans-ZEN are
comparable, with values of 41 3 and 45 3 × 10−3 min−1,
respectively. The half-lives were determined to be 17 1 for
cis-ZEN and 15 1 for trans-ZEN. By contrast, in HLM, cis-
ZEN is metabolized about 1.4-fold faster than is trans-ZEN,
with the rate constants being 29 1 × 10−3 min−1 for cis-ZEN
Metabolite Profiles of cis-ZEN. Analysis of the RLM and
HLM incubations of cis-ZEN by high-resolution mass
spectrometry (HRMS) revealed a total of 10 unknown
metabolites, which are listed in Table 1.
The chromatograms of the metabolite profiles of cis-ZEN
formed by RLM or HLM, as measured by HRMS, are shown in
Figures 4 and 5. Peaks without designation were also observed
in controls. All detected metabolites eluted prior to cis-ZEN.
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Figure 5. Overlay of extracted ion chromatograms that were obtained by analysis of 10 μM cis-ZEN incubated with HLM. The four ion traces, m/z
319.157, m/z 333.134, m/z 335.152, and m/z 317.342, were extracted and combined.
Nine metabolites were detected in RLM incubations (Figure
4). The profile shows two dominating peaks, M6 and M10. The
mass of M6 is increased by 15.992 amu, corresponding to a
monohydroxylation (+O). Apart from M6, various smaller
peaks display the same mass shift, indicating different sites of
hydroxylation (M1, M3, M7, and M8). The second main peak,
M10, displays a mass shift of +2.015 amu, which suggests a
reduction (+2H). The same m/z is observed for the smaller
peak, M9. The remaining signals, M4 and M5, are products of
sequential reduction and monohydroxylation, with mass shifts
of +18.009 amu.
The metabolite pattern of cis-ZEN, as catalyzed by HLM,
shows seven different metabolites (Figure 5). M6, a
monohydroxylated cis-ZEN, is the most abundant peak. Several
smaller peaks (M1, M2, M7, and M8) also show a m/z
corresponding to monohydroxylated cis-ZEN, which suggests
multiple sites of monohydroxylation. The reductive metabolites
M9 and M10 are observed for HLM as well, with both peaks
being less pronounced in HLM assays compared those in RLM.
Overall, three different sets of mass increase can be
concluded from Table 1: reduction (+2.015 amu, +2H),
monohydroxylation (+15.992 amu, +O), and a combination of
reduction and monohydroxylation (+18.009 amu, +O and +2
H). The latter is observed only for RLM.
typical ranges, which are 6−14 Hz for cis and 12−18 Hz for
trans olefinic protons.
DISCUSSION
■
The present study investigates the quantitative and qualitative
phase I metabolism of cis-ZEN. Incubations with rat liver
microsomes (RLM) or human liver microsomes (HLM) show
a depletion of cis-ZEN in the presence of NADPH. In the
absence of NADPH, cis-ZEN is not degraded, implying a strong
dependence on NADPH as cofactor for phase I metabolism of
cis-ZEN. In order to investigate if the isomers differ concerning
their depletion kinetics, the degradation of cis-ZEN was
compared to that of trans-ZEN. As a result, depletion
characteristics were comparable in RLM, whereas HLM
metabolized cis-ZEN 1.4-fold faster than trans-ZEN. Thus, in
HLM, phase I enzymes appear to have a higher affinity toward
the cis isomer.
The microsomal incubations were subjected to HRMS in
order to characterize the resulting metabolites. A multitude of
metabolites was generated, as seen from the metabolite patterns
(Figures 4 and 5), with vast qualitative similarities in RLM and
HLM. Assays using microsomes of both species yield the
reduction products α- and β-cis-ZEL, as identified by a mass
increase of 2.015 amu. α-cis-ZEL was found as a main
metabolite in RLM. By contrast, α- and β-cis-ZEL are only of
minor abundance in HLM. Structure identification was
conducted by co-chromatography with synthesized authentic
Identification of cis-ZEN Metabolites M9 and M10. M9
and M10 share a mass increase of 2.015 amu, which can arise
from two metabolic reactions: reduction of the double bond
between C11 and C12 or reduction of the ketone. The
reduction of the double bond would yield zearalanone, which
was excluded by co-chromatography of M9 and M10 with an
authentic reference standard. The reduction of the keto group
could be confirmed by co-chromatography with synthesized
authentic reference standards of α- and β-cis-ZEL.
1
standards, which were characterized by HRMS and H NMR.
The microsomal transformation of trans-ZEN into the
reduction products α- and β-trans-ZEL has been described in
the literature in vitro and in vivo.4−7 α-trans-ZEL formation can
be regarded as a bioactivation reaction due to a higher binding
affinity to the estrogen receptor than its precursor compound
trans-ZEN. Thus, the estrogenicity of α- and β-cis-ZEL should
be investigated in the future.
Also, for the formation of monohydroxylated metabolites
from cis-ZEN, considerable similarities for RLM and HLM are
observed. Five isobaric metabolites with a mass shift indicating
incorporation of a single oxygen atom are detected for each
microsomal species, suggesting multiple reaction targets for the
metabolic enzymes of RLM and HLM. The site of
hydroxylation cannot be determined due to missing reference
compounds. Nevertheless, monohydroxylation can be con-
cluded to be the major phase I metabolic pathway in HLM with
respect to metabolite abundance and intensity. This is
underpinned by analysis of the same incubation mixtures by
HPLC with UV detection (data not shown), which also showed
Structure identification of the synthetic cis-ZELs was
achieved by 1H nuclear magnetic resonance spectroscopy
(NMR) and HRMS. HRMS showed a mass of m/z 319.157 for
both α/β-isomers, which matches the calculated exact mass. 1H
NMR allows the configuration of the double bond at C11−C12
to be determined because the vicinal coupling constant
between cis proton pairs is smaller than the corresponding
constant between trans proton pairs at chemical double bonds.
Thus, the two olefinic protons in α-trans-ZEL give rise to a
3
doublet with a vicinal coupling constant of JHH,trans = 15.4 Hz.
3
In contrast, α-cis-ZEL shows a coupling constant of JHH,cis
=
11.5 Hz. Likewise, 3JHH,trans = 15.6 Hz for β-trans-ZEL decreases
3
to JHH,cis = 11.5 Hz in β-cis-ZEL. The observed magnitudes of
the vicinal (3JHH) coupling constants are in accordance with the
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dx.doi.org/10.1021/tx500312g | Chem. Res. Toxicol. 2014, 27, 1972−1978

Chemical Research in Toxicology
Article
several monohydroxylated peaks (assigned according to
retention time) with similar relative intensities when compared
to the HPLC-HRMS chromatograms.
ACKNOWLEDGMENTS
■
The authors would like to thank Prof. Dr. Monika Schreiner for
the opportunity to use the HRMS facilities at IGZ Großbeeren.
Due to the similarities of monohydroxylated products
formed by both microsomal species, RLM might be a potential
model for studies of cis-ZEN hydroxylation in humans.
Regarding trans-ZEN, oxidative metabolism was first reported
in 2007. Since then, monohydroxylated derivatives of trans-
ZEN were described repeatedly as metabolites formed by
ABBREVIATIONS
■
HLM, human liver microsomes; HPLC-MS/MS, high-pressure
liquid chromatography tandem mass spectrometry; HRMS,
high-resolution mass spectrometry; NADPH, nicotinamide
adenine dinucleotide phosphate; RLM, rat liver microsomes;
ZEL, zearalenol; ZEN, zearalenone
mammalian microsomes including RLM and HLM.9−12
A
monohydroxylated ZEN derivative was also found in rat urine,
demonstrating the presence of oxidative metabolites in vivo.12 It
should be the subject of further studies to investigate if
differences concerning the positions and stereochemistry of the
newly introduced hydroxyl groups exist between cis- and trans-
ZEN.
REFERENCES
■
(1) EFSA (2011) EFSA panel on contaminants in the food chain.
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A difference in RLM and HLM phase I metabolism of cis-
ZEN can be found in sequential reduction and monhydrox-
ylation. A mass increase of 18.009 amu is seen for two
metabolites in RLM but not in HLM. Regarding the trans
isomer, successive hydroxylation and reduction has been
reported for RLM and also for HLM.9,11 This finding cannot
be attributed to a stereospecific metabolic difference of cis- and
trans-ZEN, as incubations of HLM with trans-ZEN did not
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A further question that can be answered from comparison of
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isomerization does occur during phase I metabolism.
Chromatographic conditions were set up to separate not only
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49-30-8104-5889. E-mail: sarah.drzymala@bam.de.
Funding
This work was funded by the Federal Institute for Materials
Research and Testing.
Notes
The authors declare no competing financial interest.
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