Carbonyl reduction of triadimefon by human and rodent 11β- hydroxysteroid dehydrogenase 1

Article abstract of DOI:10.1016/j.bcp.2013.02.014

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) catalyzes the conversion of inactive 11-oxo glucocorticoids (endogenous cortisone, 11-dehydrocorticosterone and synthetic prednisone) to their potent 11β-hydroxyl forms (cortisol, corticosterone and prednisolone). Besides, 11β-HSD1 accepts several other substrates. Using rodent liver microsomes and the unspecific inhibitor glycyrrhetinic acid, it has been proposed earlier that 11β-HSD1 catalyzes the reversible conversion of the fungicide triadimefon to triadimenol. In the present study, recombinant human, rat and mouse enzymes together with a highly selective 11β-HSD1 inhibitor were applied to assess the role of 11β-HSD1 in the reduction of triadimefon and to uncover species-specific differences. To further demonstrate the role of 11β-HSD1 in the carbonyl reduction of triadimefon, microsomes from liver-specific 11β-HSD1-deficient mice were employed. Molecular docking was applied to investigate substrate binding. The results revealed important species differences and demonstrated the irreversible 11β-HSD1-dependent reduction of triadimefon. Human liver microsomes showed 4 and 8 times higher activity than rat and mouse liver microsomes. The apparent V_max/ K_m of recombinant human 11β-HSD1 was 5 and 15 times higher than that of mouse and rat 11β-HSD1, respectively, indicating isoform-specific differences and different expression levels for the three species. Experiments using inhibitors and microsomes from 11β-HSD1-deficient mice indicated that 11β-HSD1 is the major if not only enzyme responsible for triadimenol formation. The IC₅₀ values of triadimefon and triadimenol for cortisone reduction suggested that exposure to these xenobiotica unlikely impairs the 11β-HSD1-dependent glucocorticoid activation. However, elevated glucocorticoids during stress or upon pharmacological administration likely inhibit 11β-HSD1-dependent metabolism of triadimefon in humans.

Full text of DOI:10.1016/j.bcp.2013.02.014

Biochemical Pharmacology 85 (2013) 1370–1378
Contents lists available at SciVerse ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
Carbonyl reduction of triadimefon by human and rodent
11b-hydroxysteroid dehydrogenase 1
Arne Meyer a, Anna Vuorinen b, Agnieszka E. Zielinska c, Thierry Da Cunha a,
Petra Strajhar a, Gareth G. Lavery c, Daniela Schuster b, Alex Odermatt a,
*
a Swiss Center for Applied Human Toxicology and Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel,
Klingelbergstrasse 50, 4056 Basel, Switzerland
b Institute of Pharmacy/Pharmaceutical Chemistry and Center for Molecular Biosciences Innsbruck – CMBI, University of Innsbruck, Innrain 80/82, 6020
Innsbruck, Austria
c Centre for Endocrinology Diabetes and Metabolism (CEDAM), Institute of Biomedical Research, Medical School Building, School of Clinical and Experimental
Medicine, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
A R T I C L E I N F O
A B S T R A C T
-Hydroxysteroid dehydrogenase 1 (11b-HSD1) catalyzes the conversion of inactive 11-oxo
Article history:
11
glucocorticoids (endogenous cortisone, 11-dehydrocorticosterone and synthetic prednisone) to their
potent 11 -hydroxyl forms (cortisol, corticosterone and prednisolone). Besides, 11 -HSD1 accepts
several other substrates. Using rodent liver microsomes and the unspecific inhibitor glycyrrhetinic acid,
it has been proposed earlier that 11 -HSD1 catalyzes the reversible conversion of the fungicide
triadimefon to triadimenol. In the present study, recombinant human, rat and mouse enzymes together
with a highly selective 11 -HSD1 inhibitor were applied to assess the role of 11 -HSD1 in the reduction
of triadimefon and to uncover species-specific differences. To further demonstrate the role of 11 -HSD1
in the carbonyl reduction of triadimefon, microsomes from liver-specific 11 -HSD1-deficient mice were
employed. Molecular docking was applied to investigate substrate binding. The results revealed
important species differences and demonstrated the irreversible 11 -HSD1-dependent reduction of
triadimefon. Human liver microsomes showed 4 and 8 times higher activity than rat and mouse liver
microsomes. The apparent Vmax/Km of recombinant human 11 -HSD1 was 5 and 15 times higher than
that of mouse and rat 11 -HSD1, respectively, indicating isoform-specific differences and different
expression levels for the three species. Experiments using inhibitors and microsomes from 11 -HSD1-
deficient mice indicated that 11 -HSD1 is the major if not only enzyme responsible for triadimenol
formation. The IC50 values of triadimefon and triadimenol for cortisone reduction suggested that
exposure to these xenobiotica unlikely impairs the 11 -HSD1-dependent glucocorticoid activation.
However, elevated glucocorticoids during stress or upon pharmacological administration likely inhibit
11 -HSD1-dependent metabolism of triadimefon in humans.
b
Received 22 December 2012
Accepted 8 February 2013
Available online 16 February 2013
b
b
b
Keywords:
Triadimefon
b
b
11b-Hydroxysteroid dehydrogenase
Metabolism
b
b
Liver microsomes
Azole fungicide
Molecular docking
b
b
b
b
b
b
b
ß 2013 Elsevier Inc. All rights reserved.
1. Introduction
emphasizes the need to investigate both the environmental fate
and the potentially hazardous effects on animals and humans.
Toxicological studies revealed neurotoxic effects of triadimefon
and triadimenol in rats, mice and rabbits [1]. Teratogenic effects
were observed at very low concentrations in experiments using rat
embryos [5]. Furthermore, triadimefon and triadimenol were
shown to cause thyroid and liver tumors in rats, and they are
considered as potential human carcinogens [1]. They act by
Triadimefon and to a lesser extent the active metabolite
triadimenol are used as broad-spectrum fungicides in agriculture
and landscaping, with annual application rates of about 135,000
and 24,000 lbs/year, respectively [1]. Humans can be exposed
through consumption of foods containing triadimefon or triadi-
menol residues [2]. More critical is occupational exposure through
dermal contact and inhalation of sprays by field workers applying
these fungicides [3]. The wide use of triadimefon and its long half-
life of around 23 days under controlled laboratory conditions [4]
inhibiting the activity of fungal lanosterol-14a-demethylase, a
cytochrome P450 enzyme (CYP51), thereby blocking ergosterol
biosynthesis which is essential for fungal cell wall integrity [6].
Like other azole fungicides, triadimefon and triadimenol can
inhibit some of the mammalian cytochrome P450 enzymes
involved in steroidogenesis, which may lead to endocrine
disturbances [7].
* Corresponding author. Tel.: +41 61 267 1530; fax: +41 61 267 1515.
E-mail address: alex.odermatt@unibas.ch (A. Odermatt).
0006-2952/$ – see front matter ß 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bcp.2013.02.014

A. Meyer et al. / Biochemical Pharmacology 85 (2013) 1370–1378
1371
According to conclusions by the US Environmental Protection
Agency (EPA), the mechanisms of toxicity of triadimefon and
triadimenol differ from those of other azole fungicides [1]. Kenneke
et al. proposed that differences in the metabolism of triadimefon
compared with other azole fungicides may be involved [8].
Experiments by Barton et al. with liver microsomes revealed that
triadimefon can be metabolized by CYPs, whereby CYP2B6,
CYP2C19 and CYP3A4 were the most active enzymes in human
liver [9]. The authors mentioned very low formation of triadime-
nol; however, they used assay conditions that do not allow to
measure luminal carbonyl reductase activity. Kenneke et al., using
rat liver microsomes and the unselective inhibitor glycyrrhetinic
acid, then provided evidence that triadimefon is mainly metabo-
glycyrrhetinic acid (GA) and all other chemicals were from
Sigma–Aldrich Chemie GmbH (Buchs, Switzerland). The solvents
were of analytical and high performance liquid chromatography
grade and the reagents of the highest grade available.
2.2. Cell culture, transfection and enzyme expression
HEK-293 cells were cultivated in Dulbecco’s modified Eagle
medium (DMEM) containing 4.5 g/L glucose, 10% fetal bovine
serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 1Â MEM
non-essential amino acids and 10 mM HEPES buffer, pH 7.4. Cells
were incubated at 37 8C in a humidified 5% CO2 atmosphere. Cells
were transiently transfected by the calcium phosphate transfec-
tion method with plasmids for C-terminally FLAG-tagged human,
lized to triadimenol and that this reaction is catalyzed by 11
b-
hydroxysteroid dehydrogenase 1 (11 -HSD1, SDR26C1) [8,10,11].
Interestingly, in a follow-on study they reported the conversion of
triadimefon to triadimenol by rainbow trout microsomes [12],
b
rat or mouse 11
HEK-293 cells at 70% confluence on a 10 cm2 dish with 10 mL of
culture medium were transfected with 10 g plasmid. The plasmid
was diluted in 430 L sterile water, followed by drop wise addition
of 62.5 L of 2 M CaCl2. This mixture was then added drop wise to
500 L BEST buffer (500 mL H2O containing 8.0 g NaCl, 0.198 g
b-HSD1 [27], or human 11b-HSD2 [28]. Briefly,
m
although it is known that the gene encoding 11
b
-HSD1 is absent in
m
teleost species [13], thus suggesting the involvement of another
enzyme.
m
m
11
b
-HSD1 plays a pivotal role in the regulation of energy
Na2HPO4-heptahydrate, 5.3 g BES (N,N-bis[2-hydroxyethyl]-2-
amino ethane sulfonic acid), pH 7.0). After incubation for 10 min
at room temperature, this mixture was added to the cells. Medium
was changed at 6 h post-transfection. The transfection efficiency
was approximately 20%. Cells were trypsinized 48 h post-
transfection, followed by centrifugation at 900 Â g for 4 min. Cell
pellets (4 pellets per 10 cm2 dish) were immediately shock frozen
on dry ice and stored at À80 8C. Upon determination of the protein
concentration using the Pierce BCA protein assay kit (Thermo
metabolism through the activation of endogenous glucocorticoids
in tissues such as liver, adipose and skeletal muscle [14]. Moreover,
it essentially regulates the balance of mineralocorticoid receptor
(MR)- and glucocorticoid receptor (GR)-mediated modulation of
inflammatory parameters in macrophage-derived cells [15–17].
11
and prednisone, which do not bind to corticosteroid receptors.
Since 11 -HSD1 is considered as a promising target for the
treatment of metabolic disorders, there is great interest in the
development of 11 -HSD1 inhibitors [14,18]. Besides its role in
glucocorticoid activation, 11 -HSD1 catalyzes the carbonyl
b-HSD1 is required for the pharmacological effect of cortisone
b
Fisher Scientific Inc., Rockford, IL, USA), 20
mg of total protein were
b
loaded onto SDS-PAGE and expression of FLAG-tagged enzymes
was semi-quantitatively analyzed by Western blotting and
immune-detection using mouse monoclonal M2 anti-FLAG anti-
body (Sigma–Aldrich Chemie GmbH) and horseradish peroxidase-
conjugated secondary antibodies as described previously [29].
b
reduction of several endogenous oxidized sterols such as 7-
ketocholesterol [19,20], 7-ketodehydroepiandrosterone [21] and
the secondary bile acid 7-oxolithocholic acid [22], as well as that of
several xenobiotics including oracin [23], metyrapone [24],
ketoprofen [25], 4-(methylnitrosamino)-1-(3-pyridyl)-1-buta-
none (NNK) [26], and as mentioned above, triadimefon [8,10,11].
b
-Actin was used as a loading control and was detected using
rabbit anti-actin IgG from Santa Cruz Biotechnology Inc. (Santa
Cruz, CA, USA).
Since the evidence for a role of 11b-HSD1 in the metabolism of
triadimefon was based on rat microsomal activities and inhibition
by the unselective inhibitor glycyrrhetinic acid (GA), we aimed in
the present study to (1) optimize the assay conditions to
distinguish between luminal enzymes and microsomal enzymes
facing the cytoplasm, (2) compare carbonyl reduction activity in
human, rat and mouse liver microsomes in the presence and
absence of a selective 11b-HSD1 inhibitor, (3) assess whether
other enzymes contribute to the carbonyl reduction of triadimefon
in human, rat and mouse liver microsomes, (4) assess activities of
2.3. Preparation of liver microsomes
Sprague-Dawley rats were obtained from Charles River, Paris,
France, and housed in the breeding facility of the Biocenter,
University of Basel, in groups of four in a 12:12-h light–dark cycle
with standard laboratory chow and tap water ad libitum. Mice on a
mixed C57BL/6J/129vJ background and liver-specific knock-out
mice (LKO) generated by crossing albumin-Cre transgenic mice on
a C57BL/6J background with floxed homozygous HSD11B1 mice on
a mixed C57BL/6J/129SvJ background were bred at the breeding
facility of the University of Birmingham, UK, as described earlier
[30]. Pooled microsomes were prepared from the livers of three
adult male Sprague-Dawley rats or three C57BL/6J/129vJ parental
mice or LKO mice. Liver pieces were homogenized in solution A
(0.3 M sucrose, 10 mM imidazole, pH 7.0; 2 mL per 100 mg tissue)
with a Potter-Elvehjem PTFE pestle with 10–12 strokes and at
220 rpm. Debris and nuclei were removed by two centrifugation
steps for 10 min at 1000 Â g. The supernatant was centrifuged
twice for 10 min at 12,000 Â g to remove mitochondria, followed
by ultracentrifugation for 1 h at 100,000 Â g to obtain microsomes.
The pellet was resuspended in solution B (0.6 M potassium
the corresponding recombinant 11b-HSD1 enzymes, and (5)
investigate the binding of triadimefon to 11
modeling.
b-HSD1 by molecular
2. Materials and methods
2.1. Chemicals and reagents
Human liver microsomes were purchased from Celsis In Vitro
Inc. (Baltimore, MD) and were obtained from a 77 year old male
Caucasian. Human embryonic kidney (HEK-293) cells from ATCC
(No. CRL-1573) were obtained through LGC Standards S.a.r.l.,
Molsheim Cedex, France. Cell culture medium was from Invitrogen
(Carlsbad, CA) and 5H-1,2,4-triazolo(4,3-a)azepine,6,7,8,9-tetra-
hydro-3-tricyclo(3Á3Á1Á13Á7)dec-1-yl (T0504) from Enamine (Kiev,
Ukraine). BNW16 was kindly provided by Dr. Thomas Wilckens,
BioNetWorks GmbH, Munich, Germany. Steroids were purchased
from Steraloids (Newport, RI). Triadimefon, triadimenol,
chloride, 0.3 M sucrose, 20 mM Tris–maleate, pH 7.0; 500
100 mg tissue) and the ultracentrifugation step was repeated. The
final pellet was resuspended in solution C (0.15 M potassium
chloride, 0.25 M sucrose, 10 mM Tris–maleate, pH 7.0; 200
100 mg tissue). The microsomes were then aliquoted, shock frozen
mL per
mL per
on dry ice and stored at À80 8C until further use. The microsomal

1372
A. Meyer et al. / Biochemical Pharmacology 85 (2013) 1370–1378
protein concentration was measured using the Pierce BCA protein
assay kit. The quality of the microsomal preparations was analyzed
using the cytochrome C reductase assay kit (Sigma–Aldrich Chemie
2.6. Liquid chromatography–tandem mass spectrometry
measurements
GmbH) and by assessing the latent activity of the 11
b
-HSD1-
All chromatographic separations (HPLC) were performed using
dependent oxoreduction of cortisone in the presence of glucose-6-
phosphate (G6P).
an Atlantis T3 column (3
m
m, 2.1 mm  150 mm, Waters) and an
Agilent 1200 Infinity Series chromatograph (Agilent Technologies,
Basel, Switzerland). The mobile phase consisted of solvent A
(water:acetonitrile, 95:5 (v/v), containing 0.1% formic acid) and
solvent B (water:acetonitrile, 5:95 (v/v), containing 0.1% formic
2.4. Determination of enzyme activities using microsomal
preparations
acid), at
a total flow rate of 0.4 mL/min. Triadimefon and
In order to measure the oxoreduction of cortisone, micro-
somes of human liver (final concentration (f.c.) 0.5 mg/mL), rat
liver (f.c. 0.25 mg/mL), mouse liver (f.c. 0.5 mg/mL) and LKO
mouse liver (f.c. 0.5 mg/mL) were incubated in a final reaction
triadimenol were separated using 25% solvent B for 1 min,
followed by a linear gradient from 1 to 20 min to reach 70%
solvent B, and then 100% solvent B for 3 min. The column was then
re-equilibrated with 25% solvent B. Cortisone and cortisol were
resolved using 30% solvent B from 0 to 4 min, followed by a linear
gradient from 30% solvent B to 40% solvent B from 4 to 7 min,
solvent B was then increased to 100% from 7 to 7.5 min and then
continued for another 2.5 min, followed by re-equilibration with
30% solvent B for 3 min.
The LC was interfaced to an Agilent 6490 triple quadropole
tandem mass spectrometer (MS/MS).
The entire LC–MS/MS system was controlled by Mass Hunter
workstation software (version B.01.05). The injection volume of
volume of 22
EDTA, 1 mM MgCl2, 250 mM sucrose, 20 mM Tris–HCl, pH 7.4),
supplemented with 1 M cortisone and either 1 mM G6P or
1 mM NADPH in the presence or absence of 20 M of the
selective 11 -HSD1 inhibitor T0504 for 15 min at 37 8C. For
measuring the metabolism of triadimefon, 1 M triadimefon and
mL of TS2 buffer (100 mM NaCl, 1 mM EGTA, 1 mM
m
m
b
m
rat liver microsomes (f.c. 1 mg/mL), mouse liver microsomes (f.c.
1 mg/mL) or human liver microsomes (f.c. 0.2 mg/mL) were
incubated for 1 h at 37 8C. Reactions were stopped by the
addition of 200
m
L 0.3 M zinc sulfate in a 1:1 (v/v) mixture of
each sample was 10 mL. The mass spectrometer was operated in
water and methanol. The internal standard (atrazine for
triadimefon and deuterized d4-cortisol for cortisone) was added
at a final concentration of 50 nM, followed by vortexing for 10 s
and centrifugation for 10 min at 12,000 Â g on a table top
electrospray ionization (ESI) positive ionization mode, with the
source temperature of 350 8C, with nebulizer pressure of 20 psi.
The capillary voltage was set at 4000 V.
The compounds were analyzed using multiple-reaction moni-
toring (MRM) and identified by comparing their retention time and
mass to charge ratio (m/z) with those of authentic standards. The
transitions, collision energy and retention time were m/z 294.8/
197, 12 V, 13 min for triadimefon; m/z 296.8/70, 12 V, 11.0 and
11.5 min (R/S enantiomer) for triadimenol; m/z 216/174, 16 V,
5 min for atrazine; m/z 361/163, 25 V, 4.6 min for cortisone; m/z
363/121, 26 V, 4.3 min for cortisol; and m/z 367.2/121.1, 36 V,
4.3 min for the internal standard d4-cortisol.
The LC–MS/MS method was validated for accuracy, precision,
sensitivity, stability, recovery, and calibration range. Acceptable
inter-day assay precision (ꢀ5.2%) and accuracy (95.0–103.9%)
were achieved over a linear range of 50–5000 nM for both
triadimefon and triadimenol. Recovery of triadimefon was close to
100% and that of triadimenol >60% in all solid phase extractions.
For each experiment a new calibration curve was determined.
centrifuge. Supernatants (180
mL) were transferred onto solid
phase extraction columns (Oasis HBL 1 cc (30 mg) Waters
WAT094225, Waters, Milford, MA, USA) pre-conditioned with
1 mL of methanol and 1 mL of distilled water. After washing
with 1 mL water, compounds were eluted with 1 mL methanol.
The eluate was evaporated to dryness, reconstituted in 100
mL
methanol and stored at À20 8C until analysis by liquid
chromatography–tandem mass spectrometry (LC–MS/MS)
(Section 2.6).
2.5. Determination of enzyme activities using lysates of transfected
HEK-293 cells
Frozen pellets of HEK-293 cells transiently transfected with
human, rat or mouse 11b-HSD1 were resuspended in TS2 buffer
and immediately sonicated at 4 8C using a Branson sonicator
(5 pulses, output 2, and duty cycles 20). Lysates were incubated
for 1 h at 37 8C in the presence of 1 mM NADPH and different
2.7. Molecular modeling
concentrations of triadimefon (8
m
M,
4
m
M,
2
m
M,
1
m
M,
Triadimefon and triadimenol were docked to the X-ray crystal
500 nM, 250 nM and 125 nM) in a final volume of 22
m
L to
structure of 11b-HSD1 using AutoDock4 [31]. The 3D-structures of
estimate apparent Km and apparent Vmax values for the three
species. Substrate conversion in all experiments was kept below
25%. Reactions were stopped and processed as described in
Section 2.4.
Alternatively, the oxidation of triadimenol was assessed by
incubating lysates of cells, transiently transfected with human
the ligands were downloaded from PubChem [32] (CID-codes:
39385 for triadimefon and 41368 for triadimenol, respectively),
and the structure of the protein was obtained from Protein Data
Bank (PDB, www.pdb.org [33], entry: 2BEL [34]). The selected
protein structure contains the tetrameric form of the protein;
however, the docking studies were performed only with chain A.
The protein was prepared for docking by removing the cocrys-
tallized ligand carbenoxolone and water molecules from the
protein structure as well as by adding hydrogens. The atom types
of the protein and the ligands were automatically created by the
program. During the docking, the ligand conformations were set
flexible (with five rotatable bonds for triadimefon and six for
triadimenol, respectively) and the protein was handled as rigid.
The binding site was defined as a 3D-grid, centered at the binding
site point X = 8.858, Y = 22.143, and Z = 15.547, with 30, 40, and 30
points in the respective dimensions. The grid spacing was set to
11
1 mM NADP+ to measure the oxidation capacity of 11
with 1
M triadimenol and 1 mM NAD+ to measure 11
activity. The conversion of cortisol (at a concentration of 1
was determined as a positive control.
b
-HSD1 or 11
b
-HSD2 (SDR9C3), with 1
m
M triadimenol and
-HSD1, or
-HSD2
M)
b
m
b
m
For determination of the reductase activity of human 11
HSD1, cell lysates were incubated in the presence of 1
cortisone or 1 M triadimefon as substrate and various concen-
b-
m
M
m
trations of either triadimefon and triadimenol or cortisone as the
respective inhibitor. IC50 values were calculated by non-linear
regression using four parametric logistic curve fitting (GraphPad
Prism software).
˚
0.375 A. The genetic algorithm was selected as search method with
default settings, except for the maximum number of evaluations,

A. Meyer et al. / Biochemical Pharmacology 85 (2013) 1370–1378
1373
which was set to short (250,000). The default settings for docking
run were kept, with one exception: the RMS cluster tolerance was
˚
set to 1.0 A. Using these settings, the docking program was able to
reproduce the binding orientation of the cocrystallized ligand,
carbenoxolone, which validated the docking settings.
3. Results
3.1. Optimization of assay conditions and measurement of cortisone
reduction in liver microsomes
In a first step, the assay conditions were optimized in order to
distinguish between NADPH-dependent activities of microsomal
enzymes facing the cytoplasm and enzymes facing the ER-lumen.
The preparation employed in the present study yielded micro-
somes with approximately 90% inside-out orientation, based on
the latency of 11b-HSD1-dependent reduction of cortisone as well
as the latent activity of hexose-6-phosphate dehydrogenase
(H6PDH) [35]. Thus, the luminal compartment is protected by
the microsomal membrane, and enzymes with a cytoplasmic
orientation such as CYPs and 17b-HSD1 or 17b-HSD3 can be
readily measured upon addition of NADPH to the reaction mixture
[36]. A NADPH regenerating system using bacterial G6PDH and
G6P, widely used for measurements of CYP activities, further
stimulates microsomal enzymes with cytoplasmic orientation
Fig. 1. Conversion of cortisone to cortisol by liver microsomes. Human liver
microsomes (HLM, black bars, f.c. 0.5 mg/mL), rat liver microsomes (RLM, gray bars,
f.c. 0.25 mg/mL) and mouse liver microsomes (MLM, white bars, f.c. 0.5 mg/mL)
were incubated for 15 min at 37 8C in the presence of 1
glucose-6-phosphate, in the absence or presence of 20
inhibitor T0504. The amount of cortisone and cortisol was then quantitated. Lack of
activity of liver microsomes from 11 -HSD1-deficient mice is indicated by KO. Data
m
M cortisone and 1 mM
m
M of the 11 -HSD1
b
when high substrate concentrations (>10
mM) are applied. In
contrast, carbonyl reductases such as 11 -HSD1 that protrude into
b
b
the ER-lumen are dependent on the NADPH pool present in the
microsomal vesicle [37–39]. The high endogenous expression of
H6PDH in the liver represents an endogenous NADPH regenerating
system, and we found that the addition of G6P to the assay mixture
(mean Æ SD) were obtained from at least three independent experiments using pooled
samples. Repeated measures ANOVA found significant species differences in cortisone
reduction. Post hoc analysis by Tukey test was used for multiple comparisons.
***p < 0.001.
was required and sufficient to stimulate 11b-HSD1 reductase
activity. Due to the relatively small vesicle volume, the capacity of
this endogenous regenerating system is limited, however, and
microsomes of liver-specific 11b-HSD1 knock-out mice showed no
conversion of triadimefon to triadimenol.
substrate concentrations have to be kept below 5–10
m
M.
A species comparison revealed about 4-fold higher triadimefon
carbonyl reductase activity of human liver microsomes compared
with rat liver microsomes (p < 0.001) and 8-fold higher activity
Therefore, a substrate concentration of 1
experiments with liver microsomes.
mM was chosen for the
A comparison of the cortisone reduction in human, rat and
mouse liver microsomes yielded comparable activities of human
and mouse liver microsomes and approximately 2-fold higher
activity of rat microsomes (p < 0.001) (Fig. 1). The latency of
11b-HSD1 activity was about 90% for rat and mouse microsomes
and about 75% for the commercially available human liver
microsomes (data not shown). To compare the activity of liver
microsomes from wild-type and 11b-HSD1-deficient mice,
cytochrome C reductase activity was determined. Comparable
activities were obtained for microsomes of wild-type and knock-
out mice with 3.35 U/mL and 3.13 U/mL, respectively. Impor-
tantly, microsomes of 11b-HSD1-deficient mice were devoid of
cortisone reductase activity as expected, and cortisone reductase
activity in hepatic microsomes from wild-type mice was
completely blocked upon coincubation with the selective 11
HSD1 inhibitor T0504.
b-
3.2. Reduction of triadimefon in liver microsomes
In the presence of G6P triadimefon was efficiently converted to
triadimenol by mouse liver microsomes (Fig. 2). In contrast, much
lower activity was detected when microsomes were incubated
with NADPH (p < 0.001), an activity corresponding to the low
percentage of right-side out vesicles. Importantly, the conversion
of triadimefon to triadimenol could be completely blocked with
Fig. 2. Conversion of triadimefon to triadimenol by mouse liver microsomes.
Microsomes (1 mg/mL), prepared from wild-type (wt) and liver-specific 11
knock-out mice (ko), were incubated for 1 h at 37 8C in the presence of 1
triadimefon and either 1 mM of NADPH or 1 mM of glucose-6-phosphate (G6P), in
the absence or presence of 20 M T0504 or glycyrrhetinic acid (GA). Data represent
b-HSD1
mM
m
the specific 11b-HSD1 inhibitors T0504 (Fig. 2) and BNW16 (not
mean Æ SD from at least three independent experiments using pooled samples.
Repeated measures ANOVA found significant differences in the groups. Post hoc
analysis by Tukey test was used for multiple comparisons. **p < 0.01, ***p < 0.001,
ns = not significant.
shown) as well as with the unspecific inhibitor glycyrrhetinic acid
(GA). Further excluding the possibility that other enzymes might
be involved in the observed carbonyl reduction of triadimefon,

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A. Meyer et al. / Biochemical Pharmacology 85 (2013) 1370–1378
Fig. 4. Inhibition of 11
b-HSD1-dependent triadimefon reduction by cortisone.
Inhibition of the 11 -HSD1-dependent conversion of triadimefon to triadimenol by
b
various concentrations of cortisone was measured in lysates of HEK-293 cells
transfected with the human enzyme as described in Section 2. Lysates were
simultaneously incubated with triadimefon (1
mM) and cortisone for 60 min at
37 8C. Data were normalized to vehicle control (0.05% DMSO) and represent
mean Æ SD from three independent experiments.
Fig. 3. Triadimenol formation in liver microsomes. Human liver microsomes (HLM,
black bars, f.c. 0.2 mg/mL), rat liver microsomes (RLM, gray bars, f.c. 1 mg/mL) and
mouse liver microsomes (MLM, white bars, f.c. 1 mg/mL) were incubated for 1 h at
37 8C with 1
mM triadimefon and 1 mM G6P, in the absence or presence of 20
mM
T0504. Lack of activity of MLM of 11
b
-HSD1-deficient mice is indicated by KO. Data
reduction revealed a 3–4-fold higher affinity of human compared
(mean Æ SD) were obtained from at least three independent experiments using pooled
samples. Repeated measures ANOVA found significant species differences in
triadimefon reduction. Post hoc analysis by Tukey test was used for multiple
comparisons. ***p < 0.001.
with rat and mouse 11
b-HSD1 (Table 1). The expression levels of
11 -HSD1 in transiently transfected cells were semi-quantita-
b
tively analyzed by Western blotting and densitometry and did
not vary significantly between species (data not shown). Human
11
and 5-fold and 15-fold higher Vmax/Km values than mouse and rat
11 -HSD1, respectively (Table 1).
b-HSD1 was most active with 2-fold and 4-fold higher Vmax
than mouse liver microsomes (p < 0.001) (Fig. 3). The fact that the
selective inhibitor T0504 completely abolished triadimefon
b
reductase activity indicated that 11
b-HSD1 is the major if not
only microsomal enzyme catalyzing this reaction.
3.4. Inhibition of 11b-HSD1-dependent cortisone reduction by
triadimefon and vice versa
3.3. Reduction of triadimefon by recombinant 11b-HSD1 measured in
cell lysates
In order to estimate the potential of triadimefon and
triadimenol to interfere with glucocorticoid activation, inhibition
The different microsomal activities can potentially be due to
differences in 11 -HSD1 expression levels, differences in the
expression of H6PDH and/or its interaction with 11 -HSD1, or
species-specific differences in the kinetic properties of 11 -HSD1.
Significant species-specific differences in the substrate and
inhibitor specificity of 11 -HSD1 have been reported [27,40].
Therefore, in a next step, the carbonyl reduction of triadimefon by
recombinant human, rat and mouse 11 -HSD1 was measured in
of human 11b-HSD1-dependent cortisone reduction by the azole
b
fungicides was measured. IC50 values of 15.3 Æ 7.0 mM and
56 Æ 14 mM were obtained for triadimefon and triadimenol, respec-
tively (Fig. 4). The 11b-HSD1-dependent reduction of triadimefon
was inhibited by cortisone with an IC50 of 289 Æ 54 nM (Fig. 5).
b
b
b
3.5. 11
b-HSD1 and 11b-HSD2 do not catalyze the oxidation of
b
triadimenol
lysates of transiently transfected HEK-293 cells. HEK-293 cells
were chosen because they do not express endogenous steroid-
metabolizing enzymes and to be able to compare the enzymes of
the three species in the same cellular background. Because HEK-
293 cells express no or very low H6PDH levels [37], lysates were
prepared by sonication, which leads to microsomal vesicles with
11
interconversion of cortisone/cortisol, 11-dehydrocorticosterone/
corticosterone, prednisone/prednisolone, 7 -hydroxycholesterol/
7-oxocholesterol, and 7 - and 7 -hydroxydehydroepiandroster-
one/7-oxodehydroepiandrosterone [41]. However, we reported
recently that 11 -HSD1 irreversibly catalyzes the reduction of
b-HSD1 is a reversible enzyme in vitro and catalyzes the
b
a
b
mixed orientation and allows measuring 11
b
-HSD1 activity in
b
the presence of NADPH. Lysates of untransfected HEK-293 cells did
not metabolize triadimefon. A comparison of the triadimefon
the secondary bile acid 7-oxolithocholic acid to chenodeoxycholic
acid [22]. Therefore, the potential oxidation of triadimenol by
Table 1
Kinetic parameters of 11b-HSD1-dependent carbonyl reduction of triadimefon. HEK-293 cells were transiently transfected with either human, rat or mouse 11b-HSD1,
followed by measuring the carbonyl reduction of triadimefon and determination of apparent Vmax and apparent Km values as described in Section 2. The appVmax values are
expressed relative to total protein concentration of the lysates used. Data were calculated by non-linear regression using four parametric logistic curve fitting (GraphPad
Prism) and represent mean Æ SD of three independent experiments. One-way ANOVA found significant differences (p < 0.01) in appVmax values, post hoc analysis by Tukey test was
used for multiple comparisons. Human appVmax value was significant higher than rat appVmax (p < 0.01) and mouse appVmax (p < 0.05). Other comparisons were not significant.
11b-HSD1
appVmax
appKm
appVmax/appKm
Human
Rat
0.54 Æ 0.060 nmol  mgÀ1  hÀ1
0.14 Æ 0.031 nmol  mgÀ1  hÀ1
0.31 Æ 0.129 nmol  mgÀ1  hÀ1
3.5 Æ 0.8 mM
12.8 Æ 4.2 mM
10.4 Æ 6.5 mM
154  10À6 l  mgÀ1  hÀ1
11  10À6 l  mgÀ1  hÀ1
30  10À6 l  mgÀ1  hÀ1
Mouse

A. Meyer et al. / Biochemical Pharmacology 85 (2013) 1370–1378
1375
Fig. 7. The binding orientations of triadimefon (cyan) and triadimenol (magenta) in
the 11 -HSD1 binding site. The carbonyl-oxygen of triadimefon is facing toward
catalytic residues, with a distance of the hydroxyl on Tyr183 to the carbonyl-oxygen
Fig. 5. Inhibition of 11
b
-HSD1-dependent cortisone reduction by triadimefon and
b
triadimenol. Inhibition of the 11
b-HSD1-dependent conversion of cortisone to
cortisol by various concentrations of triadimefon and triadimenol was measured in
lysates of HEK-293 cells transfected with the human enzyme as described in Section
˚
of 3.12 A. Triadimenol is predicted to have a flipped binding orientation, where the
reduced carbonyl-oxygen faces away from the catalytic residues, with a distance of
2. Lysates were simultaneously incubated with cortisone (1
mM) and triadimefon or
˚
the hydroxyl on Tyr183 to the carbonyl-oxygen of 6.96 A.
triadimenol for 15 min at 37 8C. Data were normalized to vehicle control (0.05%
DMSO) and represent mean Æ SD from three independent experiments.
11
b
-HSD1 was tested in the presence of the cofactor NADP+.
-HSD1 (Fig. 6). As a control to
of triadimefon is comparable to that reported by Mazur et al. [10],
while triadimenol is observed in the binding pocket in a flipped
way compared with triadimefon (Fig. 7). Triadimefon is located in
the binding pocket with the carbonyl-oxygen facing toward the
catalytic amino acids Tyr183 and Ser170, and forming hydrogen
bonds with them (Fig. 8A and B). In contrast, triadimenol is located
in the same area with the alcohol group pointing away from Tyr183
and Ser170 (Fig. 8A, C and D). Instead, the alcohol group forms a
hydrogen bond with the cofactor molecule.
Triadimenol was not oxidized by 11
b
verify enzyme activity, the reduction of triadimefon was measured,
resulting in efficient formation of triadimenol, with 70% substrate
conversion. Furthermore, incubation of triadimenol with lysates of
cells expressing 11b
-HSD2 in the presence of NAD+ did not result
in the formation of any triadimefon. Under similar conditions,
cortisol was converted by 90% to cortisone (not shown).
3.6. Analysis of the binding of triadimefon and triadimenol to 11
HSD1 by molecular modeling
b-
4. Discussion
Using molecular docking, the binding orientations were
predicted for triadimefon and triadimenol. The binding orientation
Almost all studies on the assessment of NADPH-dependent
enzyme activities reported in the literature so far used either
NADPH or an NADPH-regenerating system (NRS), consisting of
NADP+, G6P and purified bacterial G6PDH. Mazur et al. compared
different conditions to measure 11b-HSD1 reductase activity and
observed highest activity upon incubation of microsomes with an
NRS in the presence of the pore forming agent alamethicin [10].
However, in this setting NADPH is produced in the extra-vesicular
space and can be readily utilized by cytochrome P450 enzymes.
In the present study, optimized assay conditions have been
applied to distinguish between activities of NADPH-dependent
microsomal enzymes facing the cytoplasm and enzymes protrud-
ing into the ER luminal compartment. Intact liver microsomes
contain an endogenous NRS, consisting of the luminal pyrimidine
nucleotide pool, the glucose-6-phosphate translocase (G6PT) and
H6PDH. Because of the neglectible permeability of the ER
membrane for pyridine nucleotides, the NADPH generated by
H6PDH upon addition of G6P into the assay buffer is exclusively
available for ER luminal enzymes. The intactness of microsomal
vesicles and the percentage of inside-out vesicles (approximately
90% in the protocol used) can be tested by comparing 11b-HSD1-
dependent cortisone reduction in the presence of either NADPH or
G6P. The quality of microsomal preparations can be further
assessed by measuring cytochrome C reductase activity. This
approach should be valuable for the characterization of enzymatic
conditions of other luminal carbonyl reductases.
In mouse liver microsomes the NADPH-dependent conversion
of triadimefon to metabolites other than triadimenol was almost
two times higher than the G6P-dependent formation of triadime-
nol. This ratio was significantly different in rat and human liver
microsomes, where the carbonyl reduction of triadimefon was
Fig. 6. Triadimenol is not oxidized by 11
human 11 -HSD1 and 11 -HSD2 were expressed in HEK-293 cells. Cells were lysed
by sonication to obtain vesicles with mixed orientation. 11 -HSD1 activity was
measured by incubation of lysates for 1 h at 37 8C with 1 M triadimefon and 1 mM
NADPH or with 1 -HSD2 activity was
M triadimenol and 1 mM NADP+. 11
measured in the presence of 1
b-HSD1 and 11b-HSD2. Recombinant
b
b
b
m
m
b
m
M triadimenol and 1 mM NAD+. Data (mean Æ SD)
were obtained from at least three independent experiments. Repeated measures
ANOVA found significant differences. Post hoc analysis by Tukey test was used for
multiple comparisons. ***p < 0.001.

1376
A. Meyer et al. / Biochemical Pharmacology 85 (2013) 1370–1378
Fig. 8. The predicted binding orientations of triadimefon (A and B) and triadimenol (C and D) in 11b-HSD1. The ligand–protein interactions are color-coded: hydrogen bond
acceptor – red arrow, hydrophobic – yellow sphere. The ligand binding pocket is colored by aggregated lipophilicity. The catalytic amino acids are highlighted in ball- and
stick style and the cofactor NADPH in stick style.
2- and 8-fold higher than in mice. The cytochrome P450-mediated
metabolism of triadimefon has been described earlier [9,42].
Barton et al. reported a role for cytochrome P450 subfamilies 2C
and 3A in the hydroxylation of triadimefon by rat liver microsomes
[9]. Iyer et al. identified the two metabolites 1-(4-chlorophenoxy)-
4-hydroxy-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone
species specificity of available antibodies. The present study
suggests that rats and mice are of limited use to study the possible
consequences of impaired carbonyl reduction of triadimefon for
humans; however, 11b-HSD1-deficient mice turned out to be very
useful for solving mechanistic questions.
Crowell et al. recently developed a physiologically based
pharmacokinetic model for triadimefon and triadimenol in rats
and humans [43]. The model showed good results for peak blood
and tissue levels, but the clearance of both compounds was over
estimated. Better results were obtained by a reverse metabolism
(kwg1323) and
b-(4-chlorophenoxy)-a-(1,1-dimethylethyl)-1H-
1,2,4-triazole-1-ethanol (desmethyl kwg1342) in experiments
using cultured rat hepatocytes.
The use of selective 11
the carbonyl reduction of triadimefon is catalyzed exclusively by
11 -HSD1. This is further substantiated by the fact that no
triadimenol formation could be observed in microsomes from
livers of 11 -HSD1-deficient mice. The analysis of the kinetic
properties of recombinant 11 -HSD1 revealed clearly higher
b-HSD1 inhibitors demonstrates that
model, based on the assumption that 11
11 -HSD2, might catalyze the oxidation of triadimenol. However,
our results revealed that neither 11 -HSD1 nor 11 -HSD2
catalyze the oxidation of triadimenol. Previous studies demon-
strated that 11 -HSD1 is a reversible enzyme that catalyzes
b-HSD1, or alternatively
b
b
b
b
b
b
b
triadimefon reductase activity of the human isoform compared
with the rodent isoforms. Although it must be taken into
consideration that the rat and mouse enzymes were expressed
in a human cell line, and that it cannot be fully excluded that the
lower activities might emerge from protein folding disturbances,
or the lack of some mouse- or rat-specific factors in human cells,
comparable cortisone reductase activities for the three enzymes
have been observed in this cell system in previous experiments
[27]. The present study revealed similar affinities for triadimefon of
the interconversion of endogenous glucocorticoids as well as
7-oxigenated cholesterol and 7-oxigenated DHEA in vitro, and
molecular modeling revealed the close proximity of the carbonyl
and the respective hydroxyl on C7 and C11 of the steroid backbone
to the catalytic Tyr183 [21,44,45]. However, a recent study
reported the irreversible reduction of 7-oxolithocholic acid by
11b-HSD1, whereby molecular modeling suggested that only 7-
oxolithocholic acid has optimal binding of substrate and cofactor
to Tyr183 and Lys187, thus allowing electron transfer with the
cofactor [22]. Similarly, the docking studies of the present study
support our experimental findings that triadimenol is not oxidized
rat and mouse 11b-HSD1. The fact that the recombinant mouse
enzyme had 3-fold higher catalytic efficiency (Vmax/Km) than the
rat enzyme but rat microsomes were twice as active as mouse
microsomes (in line with a previous study by Crowell et al. [11])
by 11b-HSD1 (Figs. 7 and 8). Triadimefon binds to 11b-HSD1 in an
orientation, where the carbonyl-oxygen is pointing toward the
catalytic amino acids Tyr183 and Ser170, and forming hydrogen
bonds with them. This orientation is essential, since in the
reduction reaction, the hydrogen is transferred from Tyr183 to the
substrate [46]. Thus, the binding orientation of triadimefon allows
the reduction reaction to take place. In contrast, triadimenol has a
suggests
a higher expression of 11b-HSD1 in rats. Indeed,
approximately two times higher cortisone reductase activity
was obtained in rat liver microsomes compared with mouse liver
microsomes. A reliable comparison of 11b-HSD1 protein expres-
sion levels in human, rat and mouse is difficult due to significant

A. Meyer et al. / Biochemical Pharmacology 85 (2013) 1370–1378
1377
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flipped binding mode compared to triadimefon, suggesting why
this compound is not oxidized by 11 -HSD1. These findings
b
suggest that after reduction of triadimefon to triadimenol, the
compound rotates away from the catalytic amino acids, thus
preventing its oxidation. However, the fact that triadimenol fits to
the binding pocket and forms hydrogen bonds with the catalytic
amino acid Ser170 and the cofactor, could explain the weak
inhibitory activity of this compound.
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triadimenol might affect 11b-HSD1-dependent glucocorticoid
activation, we determined IC50 values of the two fungicides for
cortisone reduction. Regarding the expected exposure levels upon
intake of contaminated food or water or upon occupational
exposure of field workers and uptake through skin, it is highly
unlikely that concentrations as high as 10
mM are reached to
significantly inhibit 11 -HSD1-dependent cortisone reduction. On
b
the other side, cortisone efficiently inhibited the carbonyl
reduction of triadimefon. Under the conditions applied, an
apparent Km of 300–400 nM for cortisone reduction has been
obtained [47]. Thus, the IC50 of about 300 nM obtained in the
present study suggests that at elevated concentrations of 11-
oxoglucocorticoids, i.e. during stress situations or therapeutic
treatment, the carbonyl reduction of triadimefon may be
significantly lowered. The competition of cortisone (or 11-
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HSD1 may explain the lower than expected clearance of
triadimefon based on the physiologically based pharmacokinetic
model in the study by Crowell et al. [43]. The observation suggests
that the circadian rhythm of glucocorticoids should be considered
for estimation of the clearance of triadimefon.
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ty. J Steroid Biochem Mol Biol 2010;120:67–8.
In conclusion, the use of recombinant enzymes demonstrated
the ability of 11
reduction of triadimefon. Comparison of human, rat and mouse
11 -HSD1 revealed at least five times higher catalytic efficiency of
the human compared with the rodent enzymes, which is relevant
regarding an improved cross-species extrapolation for risk
assessment. Absence of triadimenol formation upon incubation
b
-HSD1 to irreversibly catalyze the carbonyl
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tant regulator at the interface of obesity and inflammation. J Steroid Biochem
Mol Biol 2010;119:56–72.
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et al. Hexose-6-phosphate dehydrogenase modulates 11beta-hydroxysteroid
dehydrogenase type 1-dependent metabolism of 7-keto- and 7beta-hydroxy-
neurosteroids. PLoS ONE 2007;2:e561.
[22] Odermatt A, Da Cunha T, Penno CA, Chandsawangbhuwana C, Reichert C, Wolf A,
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b
of microsomes from livers of 11
microsomal preparations with selective 11
indicate that 11 -HSD1 is the major if not only enzyme catalyzing
b
-HSD1-deficient mice and of liver
b
-HSD1 inhibitors
b
the conversion of triadimefon to triadimenol. Finally, inhibition
studies suggest that the carbonyl reduction of triadimefon is
impaired by elevated cortisone levels.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
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This work was supported by the Swiss National Science
Foundation (PDFMP3_127330) to Alex Odermatt and a BBSRC
David Philips fellowship (BB/G023468/1) to Gareth G. Lavery. Alex
Odermatt has a Chair for Molecular and Systems Toxicology by the
Novartis Research Foundation. Anna Vuorinen thanks the Univer-
sity of Innsbruck, Nachwuchsfo¨rderung for financial support.
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