Endogenous boldenone-formation in cattle: Alternative invertebrate organisms to elucidate the enzymatic pathway and the potential role of edible fungi on cattle's feed

Article abstract of DOI:10.1016/j.jsbmb.2010.02.020

Although β-boldenone (bBol) used to be a marker of illegal steroid administration in calves, its endogenous formation has recently been demonstrated in these vertebrates. However, research on the pathway leading to bBol remains scarce. This study shows the usefulness of in vivo invertebrate models as alternatives to vertebrate animal experiments, using Neomysis integer and Lucilia sericata. In accordance with vertebrates, androstenedione (AED) was the main metabolite of β-testosterone (bT) produced by these invertebrates, and bBol was also frequently detected. Moreover, in vitro experiments using feed-borne fungi and microsomes were useful to perform the pathway from bT to bBol. Even the conversion of phytosterols into steroids was shown in vitro. Both in vivo and in vitro, the conversion of bT into bBol could be demonstrated in this study. Metabolism of phytosterols by feed-borne fungi may be of particular importance to explain the endogenous bBol-formation by cattle. To the best of our knowledge, it is the first time the latter pathway is described in literature.

Full text of DOI:10.1016/j.jsbmb.2010.02.020

Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 161–170
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
Endogenous boldenone-formation in cattle: Alternative invertebrate organisms
to elucidate the enzymatic pathway and the potential role of edible fungi on
cattle’s feed
K. Verheydena,∗, H. Noppea, H. Zornb, F. Van Immerseelc, J. Vanden Busschea, K. Willea,
K. Bekaerta, C.R. Janssend, H.F. De Brabandera, L. Vanhaeckea
a Ghent University, Faculty of Veterinary Medicine, Research Group of Veterinary Public Health and Zoonoses,
Laboratory of Chemical Analysis, Salisburylaan 133, B-9820 Merelbeke, Belgium
b Justus Liebig University Giessen, Institute of Food Chemistry and Food Biotechnology, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany
c Ghent University, Faculty of Veterinary Medicine, Research Group of Veterinary Public Health and Zoonoses,
Department of Bacteriology, Salisburylaan 133, B-9820 Merelbeke, Belgium
d Ghent University, Faculty of Bioscience Engineering, Department of Applied Ecology and Environmental Biology,
Laboratory of Environmental Toxicology and Aquatic Ecology, Jozef Plateaustraat 22, B-9000 Ghent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Although ␤-boldenone (bBol) used to be a marker of illegal steroid administration in calves, its endoge-
nous formation has recently been demonstrated in these vertebrates. However, research on the pathway
leading to bBol remains scarce. This study shows the usefulness of in vivo invertebrate models as alter-
natives to vertebrate animal experiments, using Neomysis integer and Lucilia sericata. In accordance with
vertebrates, androstenedione (AED) was the main metabolite of ␤-testosterone (bT) produced by these
invertebrates, and bBol was also frequently detected. Moreover, in vitro experiments using feed-borne
fungi and microsomes were useful to perform the pathway from bT to bBol. Even the conversion of phy-
tosterols into steroids was shown in vitro. Both in vivo and in vitro, the conversion of bT into bBol could be
demonstrated in this study. Metabolism of phytosterols by feed-borne fungi may be of particular impor-
tance to explain the endogenous bBol-formation by cattle. To the best of our knowledge, it is the first
time the latter pathway is described in literature.
Received 2 November 2009
Received in revised form 21 February 2010
Accepted 22 February 2010
Keywords:
Steroids
Testosterone
Phytosterols
LC–MS–MS
Model organism
Animal feed
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
related to bBol and bT are the bBol epimer ␣-boldenone (aBol),
androstadienedione (boldione, ADD) and androstenedione (AED)
Because of their muscle-building and growth-enhancing prop-
erties [1–3], steroid hormones are still illegally administered to
food-producing animals. Nevertheless, the possible risks to human
health from residues of growth-promoting hormones in food prod-
ucts of animal origin resulted in the ban of hormonal substances
in animal production [4]. This European Union legislation applies
equally to novel steroids, not existing in nature, and synthetically
produced versions of naturally occurring steroid hormones, i.e.
exogenous and synthetic endogenous hormones, respectively.
As an anabolic steroid with low androgenic activity, boldenone
(Bol) has frequently been subject of discussion in residue analysis.
␤-Boldenone (bBol), also called 1-dehydrotestosterone, only differs
from the major circulating androgen ␤-testosterone (bT) by one
double bond at the 1-position (Fig. 1). Important steroids closely
(Fig. 1) [5]. Especially because of the structure-similarity of Bol and
testosterone (T), Bol is known to be administered to cattle to pro-
mote growth [2]. However, this analyte has been shown to be a
metabolite of other steroid hormones, such as T [6]. The incidence
of Bol in biological samples has long been considered of exogenous
origin. An increase in the amount of Bol-positive samples in the last
decades has suggested the origin of Bol in cattle to be endogenous
[7–10].
For some time now it has been possible to prove the abuse
of exogenous steroid hormones in livestock using sophisticated
analytical GC–MSn or LC–MSn techniques [3,9–19]. Unfortunately,
these techniques do not allow to distinguish between synthetically
manufactured endogenous steroids and the chemically identi-
cal endogenous hormones which occur naturally in the animal’s
body. Recently many laboratories have been focussing on the
development of new analytical strategies to distinguish between
synthetically endogenous and naturally endogenous Bol to prove
or disprove the illegal use of this steroid [20–23].
∗
Corresponding author. Tel.: +32 9 2647340; fax: +32 9 2647492.
E-mail address: Karolien.Verheyden@UGent.be (K. Verheyden).
0960-0760/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jsbmb.2010.02.020

162
K. Verheyden et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 161–170
(Mysidaceae; Crustacea) and larvae of the greenbottle fly L. sericata
(Arthropoda; Diptera). Secondly, the enzymatic activity of feed-
borne fungal species, and more specifically microsomes of Pleurotus
sapidus (Basidiomycota; Agaricales), to convert testosterone into
steroid metabolites was evaluated in vitro.
2. Materials and methods
2.1. Reagents and chemicals
␤-Testosterone (androst-4-ene-17␤-ol-3-one, bT, purity
≥98%),
methyltestosterone
MeT, purity
(17␣-methyl-4-androstene-
≥97%), androstadienedione
17␤-ol-3-one,
(androsta-1,4-diene-3,17-dione, ADD, purity ≥98%), stigmas-
tanol (24␣-ethyl-5␣-cholestan-3␤-ol, purity ≥95%), ␤-sitosterol
(5-stigmasten-3␤-ol, purity ≥70%, residual campesterol and
␤-sitostanol) and ␣-pinene (purity ≥98%) were obtained from
Sigma–Aldrich Corp. (St-Louis, MO, USA). Androstenedione
(androst-4-ene-3,17-dione, AED, purity ≥96%), ␤-boldenone
(androsta-1,4-diene-17␤-ol-3-one, bBol, purity ≥98%), ␣-
testosterone (androst-4-ene-17␣-ol-3-one, aT, purity ≥98%),
dihydrotestosterone (5␣-androstan-17␤-ol-3-one, DHT, purity
≥98%) and different testosterone metabolites (4-androstene-[2␣-,
6␣-, 7␣-, 11␣-, 15␣-, 16␣-, 11␤-],17␤-diol-3-one, purity ≥98%)
were purchased from Steraloids (Newport, USA). Formic acid
(HCOOH), ethyl acetate and ethanol were of analytical grade,
while methanol (MeOH) and water were of HPLC grade quality.
All solvents and reagents were purchased from VWR International
(Merck, Darmstadt, Germany). Standard stock solutions were
Fig. 1. Mutual reductive (bold arrows) and oxidative (dotted arrows) conversions
of androstenedione, ␤-testosterone, androstadienedione and ␤-boldenone, as has
been demonstrated in the exposure medium of Neomysis integer (adapted from
Poelmans et al. [30]).
The origin of endogenous Bol and related substances in cat-
tles’ urine and faeces has often been linked to the conversion of
phytosterols [8,20,21,24–26]. Animal feed is a natural source of
phytosterols, such as ␤-sitosterol, especially since vegetable fat has
been used commonly as an additive to animal feed in response
to the crises caused by bovine spongiform encephalopathy, better
known as mad cow disease, and polychlorobiphenyls (PCBs) food
contamination [8,27]. Phytosterols only differ from steroid hor-
mones by their side chain [28,29], and their conversion to steroids
has been frequently reported [30–32].
prepared in absolute ethanol at a concentration of 200 ng ␮L−1
.
Working standard solutions were prepared by appropriate dilu-
tion of the stock solutions in ethanol. All standard solutions were
stored at 4 ◦C following the quality assurance instructions of Belac
accreditation (EN17025).
2.2. Sampling
However, the pathway leading to endogenously formed Bol
in cattle has not been studied extensively. Vertebrate animal tri-
als are time consuming, have serious cost implications, and are
often associated with ethical constraints [33,34]. For these rea-
sons in vivo invertebrate models have been proposed to simulate
vertebrate pathways. In common with Bol, ADD, AED and T have
also been identified in several invertebrate species [32,33,35]. In
some invertebrates these steroid hormones even seem to exert
functions analogous to these of their vertebrate equivalents [36].
Moreover, the metabolic reactions involved in Bol-formation, have
been shown in invertebrate as well as in vertebrate animals [33,34].
Over the last decade, our laboratory has built up a solid expertise
in the use of the mysid shrimp Neomysis integer (Mysidaceae; Crus-
tacea) as invertebrate model partly replacing vertebrate animals in
metabolism studies. Based on this experience, experiments using
N. integer are reported in this publication [8,31–33,36]. In addition,
larvae of the greenbottle fly Lucilia sericata (Arthropoda; Diptera)
are considered of potential use in metabolism experiments because
of their value as reliable substrates for qualitative analysis of drugs
in human tissues, in order to provide post-mortem data for badly
decomposed bodies [37]. In addition to in vivo invertebrate models,
in vitro biotransformation studies using microsomal preparations
also appear a specifically useful tool to characterise metabolites of
particular analytes [38–42].
N. integer populations were initially collected from the brack-
ish lake Galgenweel (Antwerp, Belgium). After a 24 h acclimation
period to the maintenance temperature of 15 1 ◦C, the organisms
were transferred to 200-L glass aquaria filled with artificial sea-
water (Instant Ocean®, Aquarium Systems, France) diluted with
deionised carbon-filtered tap water to a final salinity of 5‰. Cul-
tures were fed daily with 24–48 h old Artemia nauplii ad libitum
[36].
Maggots of L. sericata were obtained through a generous gift
from PRO fishing (Oetingen, Belgium).
Fungal species were isolated from grinded corn and inocu-
lated on Sabouraud Dextrose agar in Petri dishes as performed by
The Department of Pathology, Bacteriology and Poultry Diseases
(Faculty of Veterinary Medicine, Ghent University, Belgium). Sub-
sequently the plates were incubated at 26 ◦C for 4–7 days until
harvesting.
Precultures of P. sapidus (8266 DSM) were prepared by
The Technical Biochemistry Workgroup (Faculty of Biochemical
and Chemical Engineering, Dortmund University, Germany) as
described by Zorn et al. [43] and made available for the pur-
pose of this article. As an up-regulation of several enzymes was
shown when ␣-pinene was present in the growing medium, both
␣-pinene-induced (by addition of 1 mM ␣-pinene daily) and non-
induced cultures were grown.
The aim of this study was to provide evidence for the natural
endogenous formation of Bol by cattle based on both alternative
in vivo and in vitro experiments. Firstly, the feasibility of using
invertebrates as models for vertebrate metabolism studies was
evaluated by examining the metabolic pathway of testosterone
in vivo in two invertebrate species: the mysid shrimp N. integer
2.3. Experimental setup
2.3.1. N. integer
Juvenile N. integer (Mysidaceae; Crustacea) were individually
exposed to an excess of 3.5 ␮M of ␤-testosterone for 6 h in 2 mL of

K. Verheyden et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 161–170
163
medium (water with a salinity of 5‰, artificial sea water (Instant
Ocean®) diluted with deionised carbon-filtered tap water). Dur-
ing exposure, test organisms were placed in 5-mL glass tubes in
a temperature-controlled chamber (15 ◦C, Liebher®, Laborimpex,
Brussels) [44–46].
grinded under liquid nitrogen, resuspended in phosphate buffer
(pH 6.0) and centrifuged. The microsomal fraction was pelleted
by high speed centrifugation, resuspended in phosphate buffer
(pH 6.0) containing 10% (v/v) glycerol, and diluted in Hepes
buffer (200 mM, pH 5.5) to reach a final protein-concentration of
0.2 mg mL−1 as determined by means of the Popov assay. In the case
of freezing samples before analyses, storage occurred at −20 ◦C. To
perform metabolism experiments, a 1 mL aliquot of the microsomal
fraction was incubated with 20 ␮M of an analyte for 22 h at 24 ◦C
while shaking at 150 rpm.
2.3.2. L. sericata
Maggots of L. sericata (Arthropoda; Diptera) were individually
placed into 5-mL glass tubes containing 2 mL of medium (water
with a salinity of 5‰, artificial sea water (Instant Ocean®) diluted
with deionised carbon-filtered tap water) at room temperature.
Test species were exposed to an excess of 3.5 ␮M of ␤-testosterone
or 2.4 ␮M of a phytosterol (␤-sitosterol or stigmastanol) for 4 h [32].
2.4. Extraction
2.3.3. Unidentified fungal species
N. integer and maggots of L. sericata were isolated from their
exposure medium prior to extraction of analytes from these media.
Analytes were extracted from exposure media of fungal species
and P. sapidus without removal of any residual organic material.
Methyltestosterone (MeT) at a concentration of 19.8 nM was added
to the medium as internal standard before extraction, except for
samples of P. sapidus to which a 10-fold of this concentration
was added. Extracts were analysed for the following phase I
metabolites: 2␣, 6␣, 7␣, 11␣, 15␣, 16␣, 11␤-hydroxytestosterone
(OHT), ␣-testosterone (aT), androstenedione (AED), androstadi-
enedione (ADD), ␤-boldenone (bBol) and dihydrotestosterone
(DHT). Extraction and clean-up are based on the method described
by De Wasch et al. [33] for N. integer. In short, metabolites were
extracted from the medium using 4 mL ethyl acetate and after
centrifugation (5 min, 14,000 × g, 4 ◦C) the organic phase was
withdrawn. Ethyl acetate fractions were combined and vacuum
evaporated to dryness (Speedvac Plus SC210A, Savant Instruments,
Inc., Farmingdale, USA).
All media employed in fungal experiments were autoclaved
prior to use and standard sterile techniques were applied through-
out the procedure in accordance with the specific laboratory
guidelines. Samples of fungal mycelium were transferred into
15-mL plastic screw-cap bottles. Subsequently samples were
homogenised in HPLC-grade water and the mycelium was sep-
arated from the extracellular enzyme containing supernatant by
centrifugation. Phosphate buffered saline (PBS), pH 7, was prepared
(8 g NaCl, 0.134 g KH2PO4, 1.12 g K2HPO4) and 0.5 mL PBS buffer
was added to 0.5 mL of supernatant and incubated with an excess
of 3.5 ␮M of an analyte at 37 ◦C for 1 h.
2.3.4. P. sapidus
As described by Zorn et al. [43], mycelia of the edible basid-
iomycete P. sapidus were collected by centrifugation and the culture
supernatants were discarded. Mycelia were washed once with dis-
tilled water and frozen in liquid nitrogen. Frozen mycelia were
Fig. 2. Chromatograms and spectra of a standard solution of steroids. A concentration of 2 ng was brought on column. Abbreviations: OHT, hydroxytestosterone; AED,
androstenedione; bBol, ␤-boldenone; bT, ␤-testosterone; aT, ␣-testosterone; DHT, dihydrotestosterone; MeT, methyltestosterone; ADD, androstadienedione.

164
K. Verheyden et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 161–170
Table 1
the chromatographic devices (Fig. 2). Different metabolites were
identified based on their relative retention time, relative to the
internal standard methyltestosterone, and on the ion ratio of their
product ions as described in the performance criteria for analyti-
cal residue methods defined in Commission Decision 2002/657/EC
[47]. The instrument’s limit of detection, determined by stan-
dard injection with a signal-to-noise ratio of at least 3, was 0.1 ng
on column for OHT, AED, bT, aT, MeT and 0.2 ng on column for
ADD, bBol and DHT. All specified product ions were used for peak
integration for quantification purposes (Table 2). Quantification
Optimised working parameters of the LCQ Ion Trap Ion Source for ionisation of
specific steroids.
Ionisation source
APCI
Detection mode
Positive
200
450
80
Capillary temperature (◦C)
Source heater temperature (◦C)
Sheath gas flow (au)
Auxillary gas flow (au)
3
2.5. Liquid chromatography–mass spectrometry
occurred by fitting the metabolites’ area ratio in
a calibra-
tion curve established either in standard solution or in spiked
matrix.
The HPLC apparatus comprised of a HP 1100 series pump, an
AS3000 autosampler and a HP vacuum degasser (Agilent, Palo
Alto, USA). Chromatographic separation was achieved using a
Symmetry C18 column (5 ␮m, 150 mm × 2.1 mm, Waters, Milford,
USA), which is illustrated in Fig. 2. For separation of the different
compounds, a linear gradient was used starting with a mixture
of 60% 0.02 M aqueous HCOOH and 40% MeOH. The methanol
percentage increased from 40 to 80% in 25 min. The flow rate
was set at 0.300 mL min−1. Between each sample the column was
allowed to equilibrate at initial conditions (8 min). Analysis was
carried out using an LCQDECA Ion Trap Mass Analyser (Thermo
Electron, San Jose, USA) equipped with an atmospheric pressure
chemical ionisation (APCI) interface (Thermo Electron). In Table 1
the optimised working parameters for ionisation of the steroids
under investigation are presented. The compounds were detected
in positive ion mode MS full scan and MS–MS scan. For each
specified steroid, Table 2 shows precursor and product ions as well
as the optimal MS–MS conditions. The residue was reconstituted
in 30 ␮L MeOH and 90 ␮L 0.02 M aqueous HCOOH, except for
residues originating from P. sapidus which were reconstituted
in 300 ␮L MeOH and 900 ␮L 0.02 M HCOOH. A volume of 50 ␮L
was injected on the HPLC-system. This method was originally
developed by Verslycke et al. [36] and modified as described by De
Wasch et al. [33]. Data processing was performed using Xcalibur®
2.0 software (Thermo Electron).
To account for the presence of any of the targeted metabolites in
the exposure medium, extracts as such were run (Fig. 3). Extracts
of media in which the analyte of exposure was added were run
to account for microbial transformation or impurity of the stan-
dard solution in the absence of the test species. The influence of
the dilution solvent, ethanol, on the metabolism pattern of the test
species was accounted for by integrating a solvent control. Extracts
of media in which the test species were not exposed to an analyte
were analysed to determine the endogenous excretion of metabo-
lites by the test species.
3. Results
3.1. Method development
For the metabolism experiments using N. integer and several
fungal species, calibration curves were set up by injecting stan-
dard solutions of all targeted metabolites several times at relevant
concentrations. For experiments using L. sericata and microsomal
preparations of P. sapidus, extracts of standard solutions spiked in
the appropriate exposure medium, were injected several times to
set up the calibration curves. In all cases, linearity was achieved
in the range of 0.3–25.0 ng mL−1 for all analytes. The limit of
quantification, determined by a signal-to-noise ratio of at least
6, was set at the lowest concentration of the calibration curve
(0.3 ng mL−1).
2.6. Quality assurance
Prior to sample analysis, standard mixtures of the targeted
metabolites were injected to check the operation conditions of
Table 2
Precursor and product ions of several steroids and their optimal APCI(+) MS–MS conditions expressed by the relative retention time (tr, min) and collision energy (eV).
Analyte
Relative retention time, tr (min)
Precursor ion (m/z)
Product ions (m/z)
Collision energy (eV)
27
Hydroxytestosterone
0.38–0.49–0.59–0.65–0.70–0.72
305.2
251
269
287
Androstadienedione
0.68
0.80
285.2
287.2
121
147
267
25
27
␤-Boldenone
121
135
147
173
Androstenedione
␤-Testosterone
0.82
0.92
1.00
1.01
1.06
287.2
289.2
303.0
289.2
291.2
109
251
27
28
27
28
30
253
271
Methyltestosterone*
␣-Testosterone
267
285
253
271
Dihydrotestosterone
255
273
*
Internal standard.

K. Verheyden et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 161–170
165
Fig. 3. Chromatograms of (left) a standard solution, (center) a blank exposure medium, and (right) a sample. All were spiked with the internal standard at a concentration
of 5 ng on column. A standard solution of steroids was brought on column at a concentration of 2 ng. The sample was an extract of ␤-testosterone-metabolism by L. sericata.
Abbreviations: ADD, androstadienedione; AED, androstenedione; bBol, ␤-boldenone; bT, ␤-testosterone; MeT, methyltestosterone.
3.2. N. integer
3.3. L. sericata
In vivo testosterone-metabolism by N. integer collected from
the Scheldt estuary revealed that, according to mammalian exper-
iments, AED was the main metabolite (Table 3). Testosterone was
also converted into several hydroxy-metabolites and DHT (Table 3).
Even bBol was detected as metabolite, albeit in low concentra-
tions up to 23 11 ng g−1 ww (Table 3). Nevertheless, the variability
in excreted concentrations was high between organisms, for AED
ranging from 151 74 ng g−1 ww to 11511 3321 ng g−1 ww. This
batch dependency was confirmed by experiments performed in
the laboratory. Although organisms were acclimatised under con-
trolled environmental conditions (Section 2.3), hormone levels
between different replica of this species upon testosterone expo-
sure remained highly variable, marked by AED-concentrations from
479 218 ng g−1 ww up to 15000 10500 ng g−1 ww (Table 4).
Upon bT-metabolism, maggots of the greenbottle L. sericata
mainly excreted AED in the medium, at mean concentra-
tions of 2831.3 1206.0 ng g−1 ww (Table 5). ADD and bBol
were also detected in the medium, at concentrations of
170.5 96.3 ng g−1 ww and 452.2 120.1 ng g−1 ww, respectively
(Table 5). This indicates the metabolic capability of L. sericata mag-
gots (Fig. 3).
3.4. Fungal species
Several unidentified fungal species, grown on corn, converted
bT into several hydroxy-metabolites at minor concentrations, and
aT and AED at a mean concentration of 72.0 24.9 ng g ww−1 and
64.1 25.4 ng g ww−1, respectively (Table 6).
Table 3
Metabolite excretion by Neomysis integer, sampled at different locations in the Scheldt estuary, after exposure to ␤-testosterone (mean S.D., ng g−1 ww) (adapted from
Verslycke et al. [53]). Abbreviations: OHT, hydroxytestosterone; aT, ␣-testosterone; bT, ␤-testosterone; AED, androstenedione; ADD, androstadienedione; bBol, ␤-boldenone;
DHT, dihydrotestosterone.
OHT
aT
bT
AED
ADD
bBol
DHT
a
a
a
a
a
a
a
a
a
Sampling point 1
Sampling point 2
Sampling point 3
Sampling point 4
Sampling point 5
Sampling point 6
Sampling point 7
Sampling point 8
Sampling point 9
50 14
98 25
13 10
120 35
63 20
na
na
na
na
na
na
na
na
na
2258 719
11511 3321
368 252
2515 1358
924 288
151 74
2524 1512
1180 153
33 28
na
na
na
na
na
na
na
na
na
6
6
693 585
–
–
663 576
–
–
18 17
–
–
23 11
22 21
7
10
–
7
7
4
9
29
9
139 27
81 17
9
22
4
4
yb
na: no data available.
a
Analyte added in excess to exposure medium, value not quantified.
y: metabolite was only detected in one replicate sample.
b

166
K. Verheyden et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 161–170
Table 4
Overview of metabolite excretion by N. integer after exposure to ␤-testosterone (mean S.D., ng g−1 ww). Abbreviations: OHT, hydroxytestosterone; aT, ␣-testosterone; bT,
␤-testosterone; AED, androstenedione; ADD, androstadienedione; bBol, ␤-boldenone; DHT, dihydrotestosterone.
OHT
aT
bT
AED
2005 1447
1137 933
1027 298
479 218
ADD
bBol
DHT
a
a
a
a
a
bT (n = 10)b [41]
bT (n = 10)b [42]
bT (n = 10)b [42]
bT (n = 10)b
45 14
39 14
51 23
143 54
61 25
na
na
na
na
na
na
na
na
–
25 12
–
–
–
358 453
–
–
–
bT (n = 7)b [43]
12,109 6774
5171 2329
1113 810
na
na: no data available.
a
Analyte added in excess to exposure medium, value not quantified.
n: number of replicates analysed.
b
Table 5
Metabolite excretion by maggots of L. sericata after exposure to ␤-testosterone, ␤-sitosterol or stigmastanol (mean S.D., ng g−1 ww). Abbreviations: OHT, hydroxytestos-
terone; aT, ␣-testosterone; bT, ␤-testosterone; AED, androstenedione; ADD, androstadienedione; bBol, ␤-boldenone DHT, dihydrotestosterone.
OHT
aT
bT
AED
ADD
bBol
DHT
Endogenous
bT
␤-Sitosterol
Stigmastanol
na
na
na
na
na
na
na
na
4.6 2.0 (10/10)b
5.8 2.2 (10/10)
2831.3 1206.0 (9/10)
– (0/10)
– (0/10)
– (0/10)
452.2 120.1 (10/10)
– (0/10)
na
na
na
na
a
170.5 96.3 (9/10)
31.7 8.3 (8/10)
19.4 5.8 (10/10)
– (0/10)
3.2 1.8 (10/10)
– (0/10)
– (0/10)
na: no data available.
a
Analyte added in excess to exposure medium, value not quantified.
(x/n) with x: number of replicates in which the metabolite was detected and n: total number of replicates analysed.
b
Table 6
Metabolite excretion by fungal species after incubation with ␤-testosterone, androstenedione, androstadienedione, ␤-sitosterol or stigmastanol (mean S.D., ng g−1 ww).
Abbreviations: OHT, hydroxyl-testosterone; aT, ␣-testosterone; bT, ␤-testosterone; AED, androstenedione; ADD, androstadienedione; bBol, ␤-boldenone; DHT,
dihydrotestosterone.
OHT
aT
bT
AED
ADD
bBol
DHT
Endogenous
bT
AED
ADD
␤-sitosterol
Stigmastanol
– (0/5)b
9.9 14.4 (13/30)
3.0 0.7 (3/30)
– (0/30)
– (0/30)
– (0/30)
– (0/5)
72.0 24.9 (5/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
87.9 20.1 (5/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
a
64.1 25.4 (4/5)
a
691.6 330.0 (5/5)
288.6 289.4 (5/5)
– (0/5)
247.5 163.1 (5/5)
a
678.7 259.6 (5/5)
136.6 67.6 (5/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
– (0/5)
a
Analyte added in excess in incubation medium, value not quantified.
(x/n) with x: number of replicates in which the metabolite was detected and n: total number of replicates analysed.
b
Upon exposure to ␤-sitosterol, AED was mainly produced at
Besides aBol, bBol has even been detected in urine samples of
untreated animals but not as frequently and in concentrations up to
7.0 ng mL−1. Both Bol isomers have also been reported to be present
in the faeces of untreated cattle (Table 8). Although bBol-positive
urine samples have been used to indicate illegal animal treatment
until the late 1990s, Bol may now be considered as an endoge-
nous analyte as well [7,8]. In this study, the pathway leading to
endogenous Bol in cattle was simulated by in vivo experiments
with invertebrate species as alternative models for vertebrates.
Moreover, feed-borne fungal species were shown to convert the
phytosterol ␤-sitosterol into AED, potentially contributing to an
increase of steroid hormones in the animal feed hence increasing
the chance of endogenous Bol-formation.
Several authors have suggested that a combination of high per-
formance liquid chromatography (HPLC) and mass spectrometry
(MS) is the most powerful analytical technique to study drug-
metabolism [17,53]. In this study, identification and quantification
of known metabolites was required. For this purpose, an ion-trap
mass spectrometer with a high detection sensitivity, offering real
MSn capability, was used in atmospheric pressure chemical ioniza-
tion (APCI) mode (Section 2.5).
a mean concentration of 136.6 67.6 ng g ww−1 (Table 6). No
ADD, OHT, aT, bT, bBol or DHT could be detected upon ␤-
sitosterol-exposure of fungi in our study (Table 6). Nevertheless,
exposure to AED resulted in the production of ADD at a concen-
tration of 247.5 163.1 ng g ww−1 (Table 6). Moreover, exposure
to ADD led to the production of bBol at a concentration of
87.9 20.1 ng g ww−1 (Table 6).
3.5. P. sapidus
Results of these experiments are presented in Table 7. Irre-
spective of the experimental circumstances (fresh or frozen,
induced or non-induced), AED was the main metabolite of bT with
concentrations ranging from 20.6 3.1 to 583.1 ng mg−1 protein
solution. The only hydroxy-metabolite in the medium, detected
at a maximum concentration of 113.3 ng mg−1 protein solution,
was 6␣-OHT.
Metabolism of ␤-sitosterol or stigmastanol was evaluated as
well for fresh as for frozen non-induced microsomal preparations
isolated from P. sapidus. However, none of the investigated metabo-
lites (AED, ADD, bBol, aT, bT, P, OHP) could be detected.
Most publications dealing with in vivo trials on Bol-formation
focused on the metabolism- and excretion-profiles upon oral
or intramuscular steroid administration to vertebrate animals
[10,17,22,41,54]. However, to downscale expensive and time-
consuming vertebrate animal experiments, intensive research on
the use of alternatives has been conducted during the last decade
[33,34,55].
4. Discussion
Research by several laboratories in Europe on the prevalence
of Bol in untreated cattle indicates the frequent detection of aBol
in urine samples up to a concentration of 80.0 ng mL−1 (Table 8).

K. Verheyden et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 161–170
167
Table 7
Metabolite production by microsomes isolated from Pleurotus sapidus activated with ␤-testosterone (mean S.D., ng mg−1 protein solution). Abbreviations: OHT, hydrox-
ytestosterone; aT, ␣-testosterone; bT, ␤-testosterone; AED, androstenedione; ADD, androstadienedione; bBol, ␤-boldenone; DHT, dihydrotestosterone.
6␣-OHT
aT
bT
AED
ADD
bBol
DHT
Frozen, non-induced
Non-activated
bT-activated
1.3 0.2 (2/2)b
12.5 1.5 (2/2)
– (0/2)
– (0/2)
– (0/2)
41.6 1.3 (2/2)
65.7 5.8 (2/2)
– (0/2)
– (0/2)
– (0/2)
– (0/2)
– (0/2)
– (0/2)
a
Frozen, induced
Non-activated
bT-activated
0.9 0.1 (2/2)
9.0 1.5 (2/2)
– (0/2)
– (0/2)
– (0/2)
22.5 5.5 (2/2)
89.3 29.6 (2/2)
– (0/2)
– (0/2)
– (0/2)
– (0/2)
– (0/2)
– (0/2)
a
Fresh, non-induced
Non-activated
bT-activated
1.1 0.2 (2/2)
21.1 3.7 (2/2)
– (0/2)
– (0/2)
– (0/2)
20.6 3.1 (2/2)
159.1 15.0 (2/2)
– (0/2)
– (0/2)
– (0/2)
– (0/2)
– (0/2)
– (0/2)
a
Fresh, induced
Non-activated
bT-activated
1.2 0.0 (2/2)
113.3 (1/1)
– (0/2)
– (0/1)
– (0/2)
– (0/2)
583.1 (1/1)
– (0/2)
– (0/1)
– (0/2)
– (0/1)
– (0/2)
– (0/1)
a
a
Analyte added in excess in incubation medium, value not quantified.
(x/n) with x: number of replicates in which the metabolite was detected and n: total number of replicates analysed.
b
Experiments with the mysid N. integer, performed at our labora-
iments with N. integer, revealed that testosterone metabolites are
preferably excreted in the medium rather than maintained in the
organism’s body [36]. Nevertheless, future experiments investigat-
ing extraction of metabolites from fly larval bodies might provide
additional information on Bol-formation. bT-Metabolism by mag-
gots of the greenbottle L. sericata mainly resulted in the excretion
of AED in the medium. This reaction, comprising the common
oxidation of the enol- to the keto-form, has been described as
the most important conversion of bT for N. integer as well. The
similarity of its metabolic pathways to these described for N.
integer, suggests the potential value of L. sericata as invertebrate
model in animal trials (Tables 3–5). Nevertheless, this observa-
tion requires further confirmation to assure the reproducibility of
the conversion of bT into bBol and consequently the value of this
organism for in vivo metabolism experiments. Until now, excreted
steroid-concentrations upon bT-exposure were highly variable for
fly larvae of different origin, e.g. Belgium, Great Britain, The Nether-
lands and Italy (data not shown). It should be noted that the
conversion of bT into ADD and bBol was only observed in maggots
originating from Italy, as shown in Table 5. As for N. integer, this
variability in invertebrate metabolism of bT might be explained by
the intestinal system of the invertebrates. Within the purpose of
this article no distinction was made however between the enzy-
matic and the intestinal metabolism of invertebrates. In calves’
faeces, the most likely origin of 1-dehydro compounds such as Bol
is believed to be through conversion of specific precursors, such
as phytosterols or other steroids, by gut bacteria [17,51]. However,
the present results indicate the potential relevance of the presence
tory, suggest similarity between the enzymatic steroid metabolism
pathway of these crustaceans and mammals [33,36]. The vertebrate
type steroid ␤-testosterone was used to study metabolic pathways
dependent on cytochrome P450 enzymes, the latter having a major
role in the oxidative metabolism of drugs in mammals [55]. More-
over, it was demonstrated by Poelmans et al. [31] that exposure of
N. integer to bT primarily leads to AED-production, subsequently
followed by the formation of bBol, with ADD as intermediary prod-
uct (Fig. 1). Field as well as laboratory experiments proved that
AED was the main metabolite (Tables 3 and 4). These experiments,
however, indicated both spatial (Table 3) and seasonal (Table 4)
variability in excreted concentrations [56], forming a serious draw-
back in using N. integer as an invertebrate model organism to
investigate hormonal pathways. Especially with regard to bBol,
reproducibility of in vivo exposure experiments with N. integer has
been reported by De Wasch et al. [33] to be poor. In contrast, the
use of N. integer’s testosterone-metabolism has proven its use in
eco-toxicological studies [44–46,57]. For the purpose of simulat-
ing vertebrate metabolic pathways however, another invertebrate
L. sericata, was evaluated as model organism [32].
The study of drugs in insects, entomo-toxicology, has become
an established approach in toxicological studies since 1980 [58].
In fly larvae, detection of controlled substances and drugs has
been carried out by different research groups [58,59]. It should
be noted, however, that in these experiments analyses were done
on whole organism extracts whereas in our study metabolites
were determined upon excretion in the medium, as initial exper-
Table 8
Literature overview of ␣-boldenone and ␤-boldenone findings in urine (ng mL−1) and faeces (ng g−1) of untreated cattle. Abbreviations: aBol, ␣-boldenone; bBol, ␤-boldenone.
Urine (ng mL−1
aBol
)
Faeces (ng g−1
aBol
)
bBol
bBol
Arts et al. [7]
De Brabander et al. [8]
Rossi et al. [48]
<0.1–2.6
nd–>80.0
≤7.9
nd
nd
na
<0.1
nd–≤7.0
≤0.2b
nd
nd
na
na
na
1.0–10.0a
na
0.1–2.0a
na
Nielen et al. [9]
Sangiorgi et al. [10]
Arioli et al. [49]
Arioli et al. [49]
Draisci et al. [50]
Pompa et al. [51]
Pompa et al. [51]
Nielen et al. [25]
Gallina et al. [52]
0.1–2.0a
–
0.1–2.0a
–
nd–10.0
nd
168.7–124664.0a
331.5a
na
nd–0.9
nd
nd
nd
na
nd–5.9
nd–3090.0a
100.0a
na
27.6–89.0
nd–482.0a
4.0a
nd–2.7
<1.0
nd
nd
na
nd: not detected; na: no data available.
a
Analyte was only detected in dried faeces, not in fresh rectal faeces.
Analyte was only detected in urine after faecal contamination.
b

168
K. Verheyden et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 161–170
of maggots living on faecal material to the origin of Bol-positive
faeces samples. Moreover, this mechanism could possibly explain
the observed increase in Bol-detection in dried out faeces (Table 8),
being more exposed to flies and consequently maggots than fresh
faeces.
rations isolated from P. sapidus. However, none of the investigated
metabolites (AED, ADD, bBol, aT, bT, P, OHP) could be detected. Con-
sidering only the fresh microsomal preparations, induction with
␣-pinene resulted in a 3-fold increase of AED, 583.1 compared
to 159.1 15.0 ng mg−1 protein solution (Table 7). Consequently,
these results clearly indicate the importance of a cofactor, e.g. ␣-
pinene, to up-regulate enzyme activity. Although the enzymatic
activity of fresh induced microsomes isolated from the edible fun-
gus P. sapidus was demonstrated, these results are preliminary and
further confirmation is needed.
Even though the role of phytosterols in the formation of Bol and
other steroids has often been suggested, it has only been described
in a few studies [50,52,60,61]. In this study, the ability of edible
fungi – potentially growing on animal feed – to convert phytos-
terols into steroids was hypothesised. Evidence is existing on the
occurrence of fungal species in corn [62]. However, this has mainly
been linked to contamination of corn, and consequently animal
feed, with mycotoxins [63–65]. To the best of our knowledge, inter-
ference of feed-borne fungi in steroid-formation has so far not been
considered. The hormonal activity of animal feed ingredients, was
taken into account in one study analysing steroid hormone excre-
tion in veal calves’ urine and faeces [25]. Unfortunately, only the
estrogenic activity of animal feed was evaluated during this study.
Exposure of several fungal species to ␤-sitosterol mainly
resulted in AED detection (Table 6). This is consistent with a study
performed by Lin et al. [30] in which AED was the main transfor-
mation product of phytosterols in corn flour (mainly containing
␤-sitosterol) produced by an isolated Fusarium strain. The produc-
tion of ADD could not be proven during their study. Accordingly, no
ADD could be detected upon ␤-sitosterol-exposure in our exper-
iment. Nevertheless, exposure to AED was shown to result in
ADD-production, and ADD-exposure led to the production of bBol
by several fungi. These data support the assumption that isolated
fungal species are able to transform phytosterols into bBol (Fig. 1),
which strengthens the hypothesis that animal feed rich in phytos-
terols may be a potential source of bBol or its precursors. Important
in this context is the fact that during this study fungal species
were exposed to micrograms of phytosterols only. A more real-
istic approach would be exposure to grams of phytosterols. Indeed,
cattle at an age of 8–12 months old can be fed approximately 15 kg
corn-rich animal feed [66]. Depending on the specific type of ani-
mal feed and/or the addition of vegetable oils, this equals to about
20 g of ␤-sitosterol [67]. Further research should therefore focus on
growing fungal strains under laboratory conditions on real animal
feed samples to establish steroid hormone production on feed.
To further investigate the P450 enzymatic activity of fungal
species, in vitro evaluation of their testosterone-metabolism was
performed. This resulted in the detection of several hydroxytestos-
terone, aT and AED (Table 6), in accordance with literature on
testosterone biotransformation by fungi [68,69]. Furthermore, T-
conversion by specific microsomal preparations isolated from the
edible fungus P. sapidus was evaluated in vitro. In accordance with
the above-mentioned experiments, AED was the main metabolite
of bT. The only hydroxy-metabolite of bT detected was 6␣-OHT.
According to Van Puymbroeck et al. [41], hydroxylation in posi-
tion 6 of steroid hormones appears to be a common metabolic
pathway by microsomal preparations. Additionally, these authors
reported some interesting advantages for the use of microsomes
as an in vitro tool for metabolism experiments. The easy prepa-
ration of microsomes combined with the relatively low costs and
the possibility to preserve microsomal material for years are clear
advantages. This is in contradiction with our results, since the AED-
concentration of fresh induced bT-activated microsomes was about
6-fold the concentration of their frozen counterparts: i.e. 583.1
and 89.3 29.6 ng mg−1 protein solution, respectively (Table 7).
Possibly this could have been prevented by cryopreserving the
microsomal preparations which appears to be a recognised way
of conserving [55]. Since the applicability of fungal microsomes
to predict the metabolic pathway of bT was demonstrated in this
study, the metabolism of ␤-sitosterol or stigmastanol was also
evaluated using fresh and frozen non-induced microsomal prepa-
5. Conclusion
While many laboratories are focussing on developing novel ana-
lytical methods to distinguish between exogenous and endogenous
Bol in cattle, this study examined the potential pathways of endoge-
nous Bol-formation.
Vertebrate enzymatic activity was simulated in vivo using the
invertebrates N. integer and L. sericata. However, model organ-
ism variability was too high to consider these invertebrate species
as alternative biotransformation models. Besides cattles’ natu-
ral enzymatic metabolic pathway, this study also proposes a
novel mechanism of endogenous Bol-formation. Feed-borne fungi,
potentially living on animal feed, were shown capable to con-
vert phytosterols into steroids. Consequently, increased amounts of
steroid hormones, probable precursors of Bol, may unintentionally
be offered to cattle through their feed.
To consider the prevalence and the amount of Bol residues
in cattle’s urine or faeces as proof of illegal animal treatment, a
more integrated approach is required, taking into account, e.g. the
phytosterol-concentration in animal feed, the environmental cir-
cumstances and the ecology of gut microbiota.
Acknowledgements
The authors wish to thank the Special Research Fund (BOF) for
financial support. Furthermore, A. Van Bastelaere and V. Mortier
are gratefully acknowledged for their practical assistance in the
laboratory.
References
[1] H. De Brabander, H. Noppe, K. Verheyden, J. Vanden Bussche, K. Wille, L. Oker-
man, L. Vanhaecke, W. Reybroeck, S. Ooghe, S. Croubels, Review: Residue
analysis: future trends from a historical perspective, J. Chromatogr. A 1216
(2009) 7964–7976.
[2] G.B. Forbes, The effect of anabolic-steroids on lean body-mass: the dose
response curve, Metab. Clin. Exp. 34 (6) (1985) 571–573.
[3] H. Noppe, B. Le Bizec, K. Verheyden, H.F. De Brabander, Novel analytical meth-
ods for the determination of steroid hormones in edible matrices, Anal. Chim.
Acta 611 (2008) 1–16.
[4] Council Directive 96/23/EC, Council Directive 96/23/EC of 29 April 1996 on
measures to monitor certain substances and residues thereof in live animals
and animal products and repealing Directives 85/358/EEC and 86/469/EEC and
Decisions 89/187/EEC and 91/664/EEC, Off. J. Eur. Comm. L125 (1996) 10.
[5] A.G. Fragkaki, Y.S. Angelis, A. Tsantili-Kakoulidou, M. Koupparis, C. Geor-
gakopoulos, Schemes of metabolic patterns of anabolic androgenic steroids for
the estimation of metabolites of designer steroids in human urine, J. Biochem.
Mol. Biol. 115 (2009) 44–61.
[6] I.M. Bird, A.J. Conley, Steroid biosynthesis: enzymology, integration and control,
in: J.I. Mason (Ed.), Genetics of Steroid Biosynthesis and Function, Routledge,
USA, 2003, p. 479.
[7] C.J.M. Arts, R. Schilt, M. Schreurs, L.A. van Ginkel, Boldenone is a naturally occur-
ring (anabolic) steroid in cattle, in: Proceedings of the Euroresidue III, 1996,
pp. 212–217.
[8] H.F. De Brabander, S. Poelmans, R. Schilt, R.W. Stephany, B. Le Bizec, R. Draisci, S.
Sterk, L. van Ginkel, D. Courtheyn, N. Van Hoof, A. Macri, K. De Wasch, Presence
and metabolism of the anabolic steroid boldenone in various animal species: a
review, Food Addit. Contam. 21 (2004) 1–11.
[9] M.W.F. Nielen, P. Rutgers, E.O. van Bennekom, J.J.P. Lasaroms, J.A.H. van Rhijn,
Confirmatory analysis of 17b-boldenone, 17a-boldenone and androsta-1,4-
diene-3,17-dione in bovine urine, faeces, feed and skin swab samples by liquid

K. Verheyden et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 161–170
169
chromatography-electrospray ionisation tandem mass spectrometry, J. Chro-
matogr. B 801 (2004) 273–283.
of phytosterols to boldenone, in: Proceedings of the Euro. Food Chem., vol. XII,
2003, pp. 74–78.
[10] E. Sangiorgi, V. Polignano, S. Gardini, Boldenone and related hormones quantifi-
cation by liquid chromatography–mass spectrometry in urine and faeces after
calf administration of boldenone undecanoate, Anal. Chim. Acta 529 (2005)
239–248.
[32] K. Verheyden, H. Noppe, V. Mortier, J. Vercruysse, E. Claerebout, F. Van
Immerseel, C.R. Janssen, H.F. De Brabander, Formation of boldenone and
boldenone-analogues by maggots of Lucilia sericata, Anal. Chim. Acta 586 (2007)
163–170.
[11] F. Buiarelli, G.P. Cartoni, F. Coccioli, L. Giannetti, M. Merolle, B. Neri, A.
Terracciano, Detection of boldenone and its major metabolites by liquid
chromatography–tandem mass spectrometry in urine samples, Anal. Chim.
Acta 552 (2005) 116–126.
[33] K. De Wasch, S. Poelmans, T. Verslycke, C.R. Janssen, N. Van Hoof, H.F. De Braban-
der, Alternative to vertebrate animal experiments in the study of metabolism
of illegal growth promotors and veterinary drugs, Anal. Chim. Acta 473 (2002)
59–69.
[12] H.F. De Brabander, B. Le Bizec, G. Pinel, J.-P. Antignac, K. Verheyden, V. Mortier,
D. Courtheyn, H. Noppe, Past, present and future of mass spectrometry in the
analysis of residues of banned substances in meat-producing animals, J. Mass
Spectrom. 42 (2007) 983–998.
[13] S. Impens, K. De Wasch, M. Cornelis, H.F. De Brabander, Analysis on residues
of estrogens, gestagens and androgens in kidney fat and meat with gas
chromatography–tandem mass spectrometry, J. Chromatogr. A 970 (2002)
235–247.
[14] S. Impens, D. Courtheyn, K. De Wasch, H.F. De Brabander, Faster analysis of
anabolic steroids in kidney fat by downscaling the sample size and using
gas chromatography–tandem mass spectrometry, Anal. Chim. Acta 483 (2003)
269–280.
[15] S. Impens, J. Van Loco, M. Degroodt, H.F. De Brabander, A downscaled multi-
residue strategy for detection of anabolic steroids in bovine urine using gas
chromatography tandem mass spectrometry (GC–MS3), Anal. Chim. Acta 586
(2007) 43–48.
[34] H. Hamamoto, A. Tonoike, K. Narushima, R. Horie, K. Sekimizu, Silkworm as
a model animal to evaluate drug candidate toxicity and metabolism, Comp.
Biochem. Physiol. C: Toxicol. Pharmacol. 149 (3) (2009) 334–339.
[35] P. Labadie, M. Peck, C. Minier, E.M. Hill, Identification of the steroid fatty acid
ester conjugates formed in vivo in Mytilus edulis as a result of exposure to
estrogens, Steroids 72 (2007) 41–49.
[36] T. Verslycke, K. De Wasch, H.F. De Brabander, C.R. Janssen, Testosterone
metabolism in the estuarine mysid Neomysis integer (Crustacea; Mysidacea):
identification of testosterone metabolites and endogenous vertebrate-type
steroids, Gen. Comp. Endocrinol. 126 (2) (2002) 190–199.
[37] C.P. Campobasso, M. Gherardi, M. Caligara, L. Sironi, F. Introna, Drug analysis
in blowfly larvae and in human tissues: a comparative study, Int. J. Legal Med.
118 (4) (2004) 210–214.
[38] J. Roberts, P. Brown, J. Fox, M. Lushnikova, M. Dumasia, E. Houghton, In vitro
production of metabolites by the fungus Cunninghamella elegans, in: Proceed-
ings of the 15th International Conference of Racing Analysts and Veterinarians,
2004, pp. 1–8.
[16] M.W.F. Nielen, J.J.P. Lasaroms, P.P.J. Mulder, J. Van Hende, J.H.A. van Rhijn, M.J.
Groot, Multi residue screening of intact testosterone esters and boldenone
undecylenate in bovine hair using liquid chromatography electrospray tandem
mass spectrometry, J. Chromatogr. B 830 (2006) 126–134.
[39] Z. Wang, F. Zhao, D. Chen, D. Li, Biotransformation of phytosterol to produce
androsta-diene-dione by resting cells of Mycobacterium in cloud point system,
Process. Biochem. 41 (2006) 557–561.
[17] J. Scarth, C. Akre, L. van Ginkel, B. Le Bizec, H.F. De Brabander, W. Korth, J. Points,
P. Teale, J. Kay, Presence and metabolism of endogenous androgenic–anabolic
steroid hormones in meat-producing animals: a review, Food Addit. Contam.
Part A 26 (5) (2009) 640–671.
[40] M. Van Puymbroeck, E. Royackers, R.F. Witkamp, L. Leyssens, A.S. van Miert,
J. Gelan, D. Vanderzande, J. Raus, Microsomal preparations as an aid for the
characterisation of metabolites of illegal growth promoters, in: Proceedings of
the Euroresidue, vol. III, 1996, pp. 808–813.
[18] C. Van Poucke, Van Peteghem, Development and validation of a multi-analyte
method for the detection of anabolic steroids in bovine urine with liquid
chromatography–tandem mass spectrometry, J. Chromatogr. B 772 (2002)
211–217.
[41] M. Van Puymbroeck, M.E.M. Kuilman, R.F.M. Maas, R.F. Witkamp, L. Leyssens,
D. Vanderzande, J. Gelan, J. Raus, Identification of some important metabo-
lites of boldenone in urine and feces of cattle by gas chromatography–mass
spectrometry, Analyst 123 (1998) 2681–2686.
[19] M. Van Puymbroeck, L. Leyssens, D. Vanderzande, J. Gelan, J. Raus, Metabolites
in feces can be important markers for the abuse of anabolic steroids in cattle,
Analyst 123 (1998) 2449–2452.
[42] Z. Lou, J.V. Johnson, M.O. James, Intestinal and hepatic microsomal metabolism
of testosterone and progesterone by a 3␣-hydroxysteroid dehydrogenase to
the 3␣-hydroxy derivatives in the channel catfish, Ictalurus punctatus, J. Steroid
Biochem. Mol. Biol. 82 (2002) 413–424.
[43] H. Zorn, D.E. Breithaupt, M. Takenberg, W. Schwack, G. Berger, Enzymatic
hydrolysis of carotenoid esters of marigold flowers (Tagetes erecta L.) and red
paprika (Capsicum annuum L.) by commercial lipases and Pleuritus sapidus
extracellular lipase, Enzyme Microb. Technol. 32 (2003) 623–628.
[44] T. Verslycke, S. Poelmans, K. De Wasch, J. Vercauteren, C. Devos, L. Moens, P. San-
dra, H.F. De Brabander, C.R. Janssen, Testosterone metabolism in the estuarine
mysid Neomysis integer (Crustacea; Mysidacea) following tributyltin exposure,
Environ. Toxicol. Chem. 22 (2003) 2030–2036.
[20] M.H. Blokland, D. van Doorn, M.R. Duits, S. Sterk, L. van Ginkel, Phytosterols in
animal feed, Proceedings of the Euroresidue 6 (2008) 593–598.
[21] B. Destrez, E. Bichon, L. Rambaud, F. Courant, F. Monteau, G. Pinel, J.-P. Antignac,
B. Le Bizec, Criteria to distinguish between natural situations and illegal use of
boldenone, boldenone esters and boldione in cattle: 2. Direct measurement
of 17b-boldenone sulpho-conjugate in calf urine by liquid chromatography-
high resolution and tandem mass spectrometry, Steroids 74 (10–11) (2009)
803–808.
[22] B. Le Bizec, F. Courant, I. Gaudin, E. Bichon, B. Destrez, R. Schilt, R. Draisci, F.
Monteau, F. André, Criteria to distinguish between natural situations and illegal
use of boldenone, boldenone esters and boldione in cattle. 1. Metabolite profiles
of boldenone, boldenone esters and boldione in cattle urine, Steroids 71 (2006)
1078–1087.
[45] T. Verslycke, S. Poelmans, K. De Wasch, H.F. De Brabander, C.R. Janssen,
Testosterone and energy metabolism in the estuarine mysid Neomysis integer
(Crustacea: Mysidacea) following exposure to endocrine disruptors, Environ.
Toxicol. Chem. 23 (5) (2004) 1289–1296.
[23] C. Van Poucke, E. Van Vossel, C. Van Peteghem, Fractionation of free and con-
jugated steroids for the detection of boldenone metabolites in calf urine with
ultra-performance liquid chromatography/tandem mass spectrometry, Rapid
Commun. Mass Spectrom. 22 (2008) 2324–2332.
[24] R. Draisci, C. Montesissa, B. Santamaria, C. D’Ambrosio, G. Ferretti, R. Mer-
lanti, C. Ferranti, M. De Liguoro, C. Cartoni, E. Pistarino, L. Ferrara, M. Tiso, A.
Scaloni, M.E. Cosulich, Integrated analytical approach in veal calves admin-
istered the anabolic androgenic steroids boldenone and boldione: urine and
plasma kinetic profile and changes in plasma protein expression, Proteomics 7
(2007) 3184–3193.
[25] M.W.F. Nielen, J.J.P. Lasaroms, M.L. Essers, M.B. Sanders, H.H. Heskamp, T.F.H.
Bovee, J.H.A. van Rhijn, M.J. Groot, The ultimate veal calf reference experiment:
hormone residue analysis data obtained by gas and liquid chromatography
tandem mass spectrometry, Anal. Chim. Acta 586 (2007) 30–34.
[26] K. Verheyden, H. Noppe, J. Vanden Bussche, K. Wille, K. Bekaert, L. De Boever,
J. Van Acker, C.R. Janssen, H.F. De Brabander, L. Vanhaecke, Characterisation
of steroids in wooden crates of veal calves by accelerated solvent extraction
(ASE®) and ultra-high performance liquid chromatography coupled to triple
quadrupole mass spectrometry (U-HPLC–QqQ-MS–MS), Anal. Bioanal. Chem.
(2010), doi:10.1007/s00216-010-3462-9.
[46] S. Poelmans, T. Verslycke, E. Monteyne, H. Noppe, K. Verheyden, C.R. Janssen,
H.F. De Brabander, Testosterone metabolism in Neomysis integer following
exposure to benzo(a)pyrene, Comp. Biochem. Physiol. B 144 (2006) 405–412.
[47] Council Directive 2002/657/EC, Off. J. Eur. Comm. L221 (2002) 8–36.
[48] C.A. Sgoifo Rossi, F. Arioli, A. Bassini, L.M. Chiesa, V. Dell’Orto, M. Montana, G.
Pompa, Evidence for false-positive results for boldenone testing of veal urine
due to faecal cross-contamination during sampling, Food Addit. Contam. 21 (8)
(2004) 756–762.
[49] F. Arioli, L.M. Chiesa, M.L. Fracchiolla, P.A. Biondi, G. Pompa, ADD, AED, a-
boldenone and epitestosterone neo formation in calf faeces: preliminary
results, Vet. Res. Commun. 29 (1) (2005) 355–357.
[50] R. Draisci, R. Merlanti, G. Ferretti, L. Fantozzi, C. Ferranti, F. Capolongo, S. Segato,
C. Montesissa, Excretion profile of boldenone in urine of veal calves fed with
milk replacers, Anal. Chim. Acta 586 (1–2) (2006) 171–176.
[51] G. Pompa, F. Arioli, M.L. Fracchiolla, C.A. Sgoifo Rossi, A.L. Bassini, S. Stella, P.A.
Biondi, Neoformation of boldenone and related steroids in faeces of veal calves,
Food Addit. Contam. 23 (2) (2006) 126–132.
[52] G. Gallina, G. Ferretti, R. Merlanti, C. Civitareale, F. Capolongo, R. Draisci, C. Mon-
tesissa, Boldenone, boldione, and milk replacers in the diet of veal calves: the
effects of phytosterol content on the urinary excretion of boldenone metabo-
lites, J. Agric. Food Chem. 55 (2007) 8275–8283.
[53] A. Tolonen, M. Turpeinen, A. Pelkonen, Liquid chromatography–mass spec-
trometry in in vitro drug metabolite screening, Drug Discov. Today 14 (3–4)
(2009) 120–133.
[27] A. Rocco, S. Fanali, Analysis of phytosterols in extra-virgin olive oil by nano-
liquid chromatography, J. Chromatogr. A 1216 (2009) 7173–7178.
[28] K.B. Hicks, R.A. Moreau, Phytosterols and phytostanols: functional food choles-
terol busters, Food Technol. 55 (2001) 63–67.
[29] V. Piironen, D.G. Lindsay, T.A. Miettinen, J. Toivo, A.-M. Lampi, Plant sterols:
biosynthesis, biological function and their importance to human nutrition, J.
Sci. Food Agric. 80 (2000) 939–966.
[54] S. Casati, R. Ottria, P. Ciuffreda, 17 Alpha- and 17 beta-boldenone 17-
glucuronides: synthesis and complete characterization by H-1 and C-13 NMR,
Steroids 74 (2) (2009) 250–255.
[30] Y.L. Lin, X. Song, J. Fu, J.Q. Lin, Y.B. Qu, Microbial transformation of phytos-
terol in corn flour and soybean flour to 4-androstene-3,17-dione by Fusarium
moniliforme Sheld, Bioresour. Technol. 100 (5) (2009) 1864–1867.
[31] S. Poelmans, K. De Wasch, Y. Martelé, R. Schilt, N. Van Hoof, H. Noppe, T. Versly-
cke, C.R. Janssen, D. Courtheyn, H.F. De Brabander, The possible transformation
[55] M.J. Graham, B.G. Lake, Induction of drug metabolism: species differences
and toxicological relevance, Paper read at Annual Congress of the British-
Toxicological-Society, April 06–09, at Guildford, England, 2008.
[56] T. Verslycke, A. Ghekiere, C.R. Janssen, Seasonal and spatial patterns in cellular
energy allocation in the estuarine mysid Neomysis integer (Crustacea: Mysi-

170
K. Verheyden et al. / Journal of Steroid Biochemistry & Molecular Biology 119 (2010) 161–170
dacea) of the Scheldt estuary (The Netherlands), J. Exp. Mar. Biol. Ecol. 306 (2)
(2004) 245–267.
[57] T. Verslycke, A. Ghekiere, S. Raimondo, C. Janssen, Mysid crustaceans as stan-
dard models for the screening and testing of endocrine-disrupting chemicals,
Ecotoxicology 16 (1) (2007) 205–219.
[58] M. Wood, M. Laloup, K. Pien, N. Samyn, M. Morris, R.A.A. Maes, E.A. de Bruijn, V.
Maes, G. De Boeck, Development of a rapid and sensitive method for the quanti-
tation of benzodiazepines in Calliphora vicina larvae and puparia by LG–MS–MS,
J. Anal. Toxicol. 27 (7) (2003) 505–512.
[64] S. De Saeger, L. Sibanda, C. Van Peteghem, Analysis of zearalenone and ␣-
zearalenol in animal feed using high-performance liquid chromatography,
Anal. Chim. Acta 487 (2003) 137–143.
[65] I.Y. Goryacheva, S. De Saeger, S.A. Eremin, C. Van Peteghem, Immunochemi-
cal methods for rapid mycotoxin detection: evolution from single to multiple
analyte screening: a review, Food Addit. Contam. 24 (10) (2007) 1169–1183.
[66] BEMEFA, Beroepsvereniging voor Mengvoederfabrikanten, Annual Statis-
tics, 2007, Online available: http://www.bemefa.be/AnnualStatistics.aspx
(14/07/2009).
[59] R. Gagliano-Candela, L. Aventaggiato, The detection of toxic substances in ento-
mological specimens, Int. J. Legal Med. 114 (4–5) (2001) 197–203.
[60] G. Brambilla, S. De Filippis, Trends in animal feed composition and the possible
consequences on residue tests, Anal. Chim. Acta 529 (2005) 7–13.
[61] Y.S. Song, C. Jin, E.H. Park, Identification of metabolites of phytosterols in rat
feces using GC/MS, Arch. Pharm. Res. 23 (6) (2000) 599–604.
[62] E. Richard, N. Heutte, V. Bouchart, D. Garon, Evaluation of fungal contamination
and mycotoxin production in maize silage, Anim. Feed Sci. Technol. 148 (2–4)
(2009) 309–320.
[63] B. Delmulle, S. De Saeger, A. Adams, N. De Kimpe, C. Van Peteghem, Develop-
ment of a liquid chromatography/tandem mass spectrometry method for the
simultaneous determination of 16 mycotoxins on cellulose filters and in fungal
cultures, Rapid Commun. Mass Spectrom. 20 (2006) 771–776.
[67] V. Piironen, A.-M. Lampi, Occurrence and levels of phytosterols in food, in: P.C.
Dutta (Ed.), Phytosterols as Functional Food Components and Nutraceuticals,
Marcel Dekker Inc., New York, USA, 2004, pp. 1–32.
[68] F. Ahmed, R.A.D. Williams, K.E. Smith, Microbial transformations of steroids:
X. Cytochromes P-450 11␣-hydroxylase and C17-C20 lyase and a 1-ene dehy-
drogenase transform steroids in Nectria haematococca, J. Steroid Biochem. Mol.
Biol. 58 (1996) 337–349.
[69] A.C. Hunter, K.R. Watts, C. Dedi, H.T. Dodd, An unusual ring-A opening and other
reactions in steroid transformation by the thermophilic fungus Myceliophthora
thermophila, J. Steroid Biochem. Mol. Biol. 116 (2009) 171–177.