Báo cáo khoa học: Assessment of porcine and human 16-ene-synthase, a third activity of P450c17, in the formation of an androstenol precursor doc

Assessment of porcine and human 16-ene-synthase, a third activity
of P450c17, in the formation of an androstenol precursor
Role of recombinant cytochrome
b
5
and P450 reductase
Penny Soucy, Lucille Lacoste and Van Luu-The
Molecular Endocrinology and Oncology Research Center, Laval University Medical Center (CHUL) and Laval University,
Que
´
bec, Canada
Recently, we have shown that the biosynthesis of androste-
nol, a potential endogenous ligand for the orphan receptors
constitutive androstane receptor and pregnane-X-receptor,
requires the presence of enzymes of the steroidogenic path-
way, such as 3b-hydroxysteroid dehydrogenase, 5a-reduc-
tase and 3a-hydroxysteroid dehydrogenase. In this report,
we examine at the molecular level whether the enzyme 17a-
hydroxylase/17,20-lyase (P450c17), which possesses dual
17a-hydroxylase and 17,20-lyase activities and catalyzes the
production of precursors for glucocorticoids and sex ster-
oids, is also able to catalyze the formation of a third class of
active steroids, 16-ene steroids (including androstenol). The
role of components of the P450 complex is also assessed. We
transfected human embryonic kidney (HEK-293) cells with
various amounts of vectors expressing P450c17, NADPH-
cytochrome P450 reductase, and cytochrome b
5
. Our results
showed that P450c17 possesses a 16-ene-synthase activity
able to transform pregnenolone into 5,16-androstadien-

3b-ol, without the formation of the precursor 17-hydroxy-
pregnenolone. Cytochrome b
5
has a much stronger effect on
the 16-ene-synthase activity than on the 17a-hydroxylase/
17,20-lyase activities. On the other hand, P450reductase has
a drastic effect on the latter, but a negligible one on 5,16-
androstadien-3b-ol synthesis. Our results therefore demon-
strate that human P450c17, as other enzymes of the classical
steroidogenic pathway, is involved in the biosynthetic
pathway leading to the formation of androstenol.
Keywords: 16-ene-synthase; 17a-hydroxylase/17,20-lyase;
cytochrome b
5
; 5,16-androstadien-3b-ol; pregnenolone.
It has been established that human cytochrome P450c17
(product of the CYP17 gene) has two distinct activities
responsible for the synthesis of glucocorticoid and sex
steroid precursors from pregnenolone (preg). A 17a-
hydroxylase activity, which converts preg into 17a-OHpreg,
is necessary for cortisol synthesis, and a 17,20-lyase activity
further transforms 17a-OHpreg into dehydroepiandroster-
one (DHEA), the precursor of sex steroids (Fig. 1). Several
different studies have revealed that these two activities are
differentially modulated by many factors, two of the most
important being the abundance of the redox partner
cytochrome P450reductase (P450red) [1,2] and the inter-
action with cytochrome b
5
(cyt b

5
), an allosteric effector
[3–6].
As for 16-androstenes, the precise mechanism by which
they are biosynthesized has been until now subject for
debate. Early studies reported testosterone to be a precursor
for these steroids [7–12]. Later, however, testosterone and
a large number of other compounds, including epitestos-
terone, DHEA, 16a-hydroxypregnenolone and 16a-
hydroxyprogesterone, were excluded as precursors for
16-androstenes, whereas preg and progesterone were found
to be putative precursors. Multiple pathways have been
suggested for the transformation of C21-steroids into
16-unsaturated C19-steroids in porcine testicular homogen-
ates. These included 20b-reduction (preg fi pregnenediol fi
5,16-androstadien-3b-ol) [12], 21-hydroxylation (preg fi
21-OHpreg fi 5,16-androstadien-3b-ol) [10,13] and 16–
17-dehydrogenation (preg fi 17a-OHpreg fi 16-dehydro-
preg fi 5,16-androstadien-3b-ol) [7,14]. A concerted
process (preg fi 5,16-androstadien-3b-ol) has also been
suggested. Finally, results published by Weusten et al.
provided evidence that androstadienol was synthesized from
preg in a single step by a 16-ene-synthase enzyme system in
human testicular homogenates [15]. However, the molecular
mechanism responsible for this biosynthesis remains to be
elucidated.
It is well recognized that 16-androstenes are produced by
Leydig cells of porcine testis [8] and that these steroids have
Correspondence to V. Luu-The, Oncology and Molecular Endocrino-
logy Research Center, Laval University Medical Center (CHUL),

2705 Laurier Boulevard, Que
´
bec (QC) G1V 4G2, Canada.
Fax: + 1 418 654 2761, Tel.: + 1 418 654 2296,
E-mail:
Abbreviations: P450c17, 17a-hydroxylase/17,20-lyase; P450red,
NADPH cytochrome P450 reductase; cyt b
5
,cytochromeb
5
;3b-HSD,
3b-hydroxysteroid dehydrogenase/D5fiD4 isomerase; 3a-HSD,
3a-hydroxysteroid dehydrogenase; preg, pregnenolone; DHEA,
dehydroepiandrosterone; 17a-OHpreg, 17a-hydroxypregnenolone;
androstadienol, 5,16-androstadien-3b-ol; androstenol, 5a-16-
androsten-3a-ol; CAR, constitutive androstane receptor; PXR,
pregnane-X-receptor; RXR, retinoid-X-receptor; HEK-293, trans-
formed human embryonic kidney 293 cells.
Enzyme: NADPH-cytochrome P450reductase (P450red, EC 1.6.2.4).
(Received 4 November 2002, revised 23 January 2003,
accepted 10 February 2003)
Eur. J. Biochem. 270, 1349–1355 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03508.x
pheromonal activity in pigs. In humans, the physiological
role of 16-androstene steroids is still ill-defined. It has been
proposed that these compounds may have significant effects
on behavior, namely reducing nervousness, tension and
other negative emotional states in women [16]. Another
study demonstrated a positive relationship between
menstrual synchrony and the ability to smell certain
16-androstene steroids [17]. Recent reports in the literature

show that androstenol (5a-16-androsten-3a-ol) could
modulate the activity of two orphan receptors, the recently
renamed CAR (constitutive androstane receptor) [18],
previously known as constitutively active receptor [19],
and PXR (pregnane-X-receptor) [20]. It has been suggested
that androstenol is an endogenous ligand for these receptors
[20], which share a common hetero-dimerization partner,
RXR (retinoid-X-receptor), and are subject to cross talk
interactions with other nuclear receptors and with a broad
range of other intracellular signaling pathways [21,22].
The purpose of this research is to examine, at the
molecular level, whether the human P450c17 overexpressed
in HEK-293 cells possesses 16-ene-synthase activity and
how it differs from the 17a-hydroxylase and 17,20-lyase
activities. We also compare the human P450c17 with its
porcine counterpart.
Experimental procedures
Construction of P450red, cyt
b
5
and P450c17
expression vectors
The cDNA fragments containing the entire coding regions
of human NADPH-cytochrome P450reductase (P450red,
EC 1.6.2.4) [23,24] and cyt b
5
were isolated as previously
described [25]. The cDNAs were then subcloned into a
pCMV expression vector. Porcine P450c17 cDNA was
amplified by PCR using Taq DNA polymerase (Perkin-

Elmer Cetus, Emerville, CA, USA) [26] and an oligo-primer
pair (5¢-GGGGTCGACATGTGGGTGCTCTTGGTTT
TCTTCTTG-3¢ and 5¢-GGGGTCGACTCAGGAGGT
ACTCCCCTCAGTGTGGGC-3¢) and poly(A)+ RNA
isolated from pig testis. The cDNA was then subcloned
into a pCMV expression vector. The cDNA coding for
human P450c17 (EC 1.14.99.9) was kindly provided by Y.
Tremblay (CHUL Research Center, Quebec, Canada).
Transient expression in transformed human embryonic
kidney (HEK-293) cells
Vectors expressing P450c17 (pCMV-P450c17), P450red
(pCMV-P450red) and cyt b
5
(pCMV-cyt b
5
)weretrans-
fected into HEK-293 cells using the Ex-gene kit according to
the manufacturer’s instructions (MBI Fermentas, Amherst,
NY, USA). Cells were initially plated at 5 · 10
5
cells per
well in six-well falcon flasks and grown in Dulbecco’s
modified Eagle’s medium (Gibco, Grand Island, NY, USA)
supplemented with 10% (v/v) fetal bovine serum (Hyclone,
Logan, UT, USA) at 37 °C under a 95% air, 5% CO
2
humidified atmosphere.
Assay of enzymatic activity
Determination of the activities was performed in intact cells
transiently transfected with P450c17 and/or P450red and/

or cyt b
5
as previously described [25]. Briefly, [
3
H]preg,
[
3
H]17a-OHpreg or [
3
H]DHEA was added to freshly
changed culture medium in six-well culture plates. For
Fig. 1. Central role of cytochrome P450c17 in the biosynthetic pathways leading to the formation of 16-ene steroids, sex steroids and glucocorticoids.
1350 P. Soucy et al. (Eur. J. Biochem. 270) Ó FEBS 2003
enzymatic assays performed with intact cells in culture, we
have previously established 16 h to be an appropriate
incubation time period, as the activity vs. time graph still
shows linearity. After 16 h of incubation, the steroids were
extracted twice with 2 mL of ether. The organic phases
were pooled and evaporated to dryness. The steroids were
Fig. 2. Identification by HPLC of pregnenolone metabolites from HEK-293 cells transfected with P450c17 and cyt b
5
. (A) [
3
H]standard preg (left
panel), 17a-OHpreg (middle panel) and DHEA (right panel), (B) nonlabeled preg (left panel) and androstadienol (right panel). Products extracted
from cells transfected with 1 lg of pCMV-cyt b
5
and 0.1 lg of (C) human or (D) porcine pCMV-P450c17. Separation and identification of
metabolites were performed as described in Experimental procedures.
Ó FEBS 2003 Biosynthesis of 5,16-androstadien-3b-ol by human P450c17 (Eur. J. Biochem. 270) 1351

solubilized in 50 lL of dichloromethane, applied to a Silica
Gel 60 TLC plate (Merck, Darmstadt, Germany) before
separation by migration in the toluene/acetone (4 : 1, v/v)
solvent system. Substrates and metabolites were identified
by comparison with reference steroids, revealed and quan-
tified using phosphoimaging, Storm 860 (Molecular
Dynamics Inc., Sunnyvale, CA, USA). Nonlabeled refer-
ence steroids were revealed with a solution of molybdate/
sulfuric acid (10 : 10, v/v).
Analysis by HPLC
3
H-Labelled steroids were analyzed using Waters Nova-Pak
reverse-phase C18 HPLC column (3.9 · 150 mm, 4 lm).
The mobile phase was MeOH/H
2
O (80 : 20, v/v) with 2 m
M
ammonium acetate and 0.1% acetic acid, with a flow rate of
1mLÆmin
)1
. Radioactivity was monitored in the eluent
using Beckman 171 HPLC Radioactivity Monitoring
System. Nonlabeled androstadienol and preg standards
were monitored using UV at 216 nm.
Results
Identification of metabolites by HPLC analysis
and corecrystallization
To verify the nature of the metabolites obtained from the
transformation of preg by human and porcine P450c17, we
identified by HPLC analysis, extracts of HEK-293 cells

transfected with P450c17 and cyt b
5
.
3
H-Labeled preg,
17a-OHpreg, and DHEA, used as standards, showed
elution peaks at 4.70, 2.30 and 2.50 min, respectively
(Fig. 2). In both porcine and human assays using preg as
a substrate, an additional peak of elution appeared at
15 min (panels C and D, respectively). This additional peak
coincides with the elution time of nonlabeled commercial
androstadienol monitored using UV at 216 nm. This data
shows that one of the metabolites obtained in assays using
human and porcine P450c17 is androstadienol. In addition
to comigratory behavior on both HPLC and TLC analyses,
the identity of the radiolabeled androstadienol product was
confirmed by cocrystallization with commercial steroid
(data not shown).
Assessment of the 16-ene-synthase, 17a-hydroxylase
and 17,20-lyase activities of human
and porcine P450c17
In order to produce DHEA from preg, P450c17 first
transforms preg into 17a-OHpreg through its 17a-hydroxy-
lase activity and then transforms this intermediate into
DHEA through its 17,20-lyase activity. In order to deter-
mine whether the transformation of preg into androstadie-
nol requires prior synthesis of 17a-OHpreg or DHEA, we
performed enzymatic assays using human and porcine
P450c17 in the presence of various substrates – preg, 17a-
OHpreg and DHEA – and analyzed androstadienol

formation from each substrate. As observed in Fig. 3, the
biosynthesis of androstadienol in humans (A) and pigs (B)
does not require prior formation of 17a-OH-preg and
DHEA. The lack of androstadienol synthesis in the presence
of 1 l
M
of ketoconazole (C), an inhibitor of cytochrome
P450, further demonstrates the specific implication of
P450c17 in the formation of this metabolite.
Formation of androstadienol by human and porcine
P450c17, with and without cyt
b
5
Using porcine and human P450c17 expressed in HEK-
293 cells in culture, we compared the formation of
Fig. 3. Thin layer chromatography showing the transformation of
pregnenolone, 17a-OHpregnenolone and DHEA by human and porcine
P450c17. HEK-293 cells transfected with 1 lg pCMV-cyt b
5
and
0.1 lg (A) human, or (B) porcine pCMV-P450c17 were treated with
5n
M
of the indicated
3
H-labeled substrates and analyzed for their
ability to produce androstadienol after overnight incubation (16 h).
(C) HEK-293 cells transfected with 1 lgpCMV-cytb
5
and 0.1 lg

human P450c17 were incubated with 5 n
M
of [
3
H]preg in the absence/
presence of 1 l
M
ketoconazole. Metabolites were analyzed after
overnight incubation (16 h). (D) Nonlabeled standards revealed with
molybdate/sulfuric acid (10 : 10, v/v).
1352 P. Soucy et al. (Eur. J. Biochem. 270) Ó FEBS 2003
androstadienol from preg in the pig and the human. As
illustrated in Fig. 4, both human and porcine enzymes have
the ability to produce androstadienol in presence of cyt b
5
.
When exogenous cyt b
5
is omitted from the transfection
assays, both human and porcine P450c17 poorly catalyze
the formation of androstadienol from preg (less than 2% of
preg transformation). Porcine P450c17 shows a slightly
stronger stimulation by cyt b
5
, its activity increasing to 15%
of preg transformation while the activity of human P450c17
increases to 12%. These results show that human and
porcine P450c17 have similar catalytic activities and that, in
both species, P450c17 is involved in the biosynthesis of
androstadienol.

Effect of cyt
b
5
on DHEA and androstadienol
biosynthesis
In order to determine the effect of cyt b
5
in P450c17
16-ene-synthase and 17a-hydroxylase/17,20-lyase activities,
we performed transfection assays with increasing amounts
of DNA fragments encoding cyt b
5
and monitored the
formation of androstadienol and DHEA. As shown in
Fig. 5 the stimulation of DHEA and androstadienol
production from preg increases with increasing amounts
of cyt b
5
in presence of endogenous levels of P450red. In
presence of these low levels of P450red, cyt b
5
shows a
slight stimulatory effect on DHEA formation. However,
we observe a more profound effect on the synthesis of
androstadienol. More precisely, an increase of androsta-
dienol formation was observed at a cyt b
5
/P450c17 ratio
of 5 : 1 (Fig. 5). The activity reached a maximum at a
ratio of 12 : 1. Thus, the influence of human cyt b

5
changes dramatically as the cyt b
5
/P450c17 ratio varies.
Effect of P450red on DHEA and androstadienol synthesis
To further investigate the modulation of human P450c17
16-ene-synthase activity, we proceeded to analyze the
relative effect of P450red on 17a-hydroxylase/17,20-lyase
and 16-ene-synthase activities. To do so, we cotransfected
P450c17 and cyt b
5
in amounts determined to be optimal for
androstadienol formation along with increasing amounts of
P450red. It can clearly be seen in Fig. 6 that the addition of
P450red, even in small amounts, has a profound effect on
17a-hydroxylase/17,20-lyase activities. In presence of only
endogenous P450red levels, DHEA formation from preg is
below 10%. Increasing P450red up to 0.25 lgcausesa
drastic increase of DHEA formation, reaching levels up to
50% of preg transformation. On the other hand, increasing
amounts of P450red do not significantly stimulate 16-ene-
synthase activity. For this activity, endogenous levels of
P450red seem to be sufficient for optimal cyt b
5
stimulation
as increasing amounts of P450red do not further stimulate
androstadienol production. These results clearly show a
differential modulation of 17a-hydroxylase/17,20-lyase and
16-ene-synthase activities by P450red and cyt b
5

.
Discussion
It is already known that human cytochrome P450c17
possesses two distinct activities, a 17a-hydroxylase and a
17,20-lyase activity, responsible for the biosynthesis of
glucocorticoid and sex steroid precursors. In this report we
show that human and porcine P450c17 also possess a 16-ene-
synthase activity that catalyzes the transformation of preg
into androstadienol, a precursor in the biosynthesis of
Fig. 5. Influence of increasing concentrations of cyt b
5
on the relative
formation of androstadienol and DHEA by human P450c17. HEK-293
cells were transfected with 0.1 lg pCMV-P450c17 and the indicated
amounts of pCMV-cyt b
5
. The transfected cells were analyzed for their
ability to catalyze the transformation of 5 n
M
of [
3
H]preg to andro-
stadienol. An increase in 16-ene-synthase activity is observed at a cytb
5
/
P450c17 ratio of 5 : 1 (0.25 lgcytb
5
/0.1 lg P450c17) while the optimal
stimulationisobservedataratioof12:1(1lgofcytb
5

/0.1 lgof
P450c17). Transfections and enzymatic assays were performed as
described in Experimental procedures. The results are the mean ±
SEM of three independent experiments.
Fig. 4. Role of cyt b
5
in the formation of androstadienol from preg-
nenolone by human and porcine P450c17. HEK-293 cells were trans-
fected with 0.1 lg human or porcine pCMV-P450c17 in the presence
or absence of 1 lgpCMV-cytb
5
. Their ability to catalyze the trans-
formation of 5 n
M
of [
3
H]preg into androstadienol after overnight
incubation (16 h) was determined. Transfections and enzymatic assays
were performed as described in Experimental procedures. The results
are the mean ± SEM of three independent experiments.
Ó FEBS 2003 Biosynthesis of 5,16-androstadien-3b-ol by human P450c17 (Eur. J. Biochem. 270) 1353
androstenol (Fig. 1). This reaction differs from the produc-
tion of DHEA through the 17a-hydroxylase/17,20-lyase
activity, in that it does not require the 17a-OH-preg
formation step and it is strongly stimulated by optimal
amounts of cyt b
5
. Indeed, results obtained from assays
using 17a-hydroxypregnenolone as a substrate demonstrate
that the synthesis of androstadienol diverges from the

biosynthetic pathway of sex steroids at the level of preg
transformation and that it does not involve the 17a-hydroxy-
lase activity. Furthermore, time course experiments (data not
shown) using preg as a substrate do not show the production
of any intermediates in the formation of androstadienol
which suggests that it is synthesized from pregnenolone in a
single step. Therefore, although the synthesis of glucocorti-
coid/sex steroid precursors and 16-ene-steroids result
from the same enzyme, the activities responsible for their
formation and the regulation of these activities are distinct.
Gower et al. had previously demonstrated, using porcine
testis microsomes, the formation of 16-androstenes from
17a-hydroxypregnenolone [8]. However, this situation is not
observed in the intact transfected cell system using porcine or
human P450c17. In combination, these results suggest the
presence, in pig testis, of another system that can use
17a-hydroxypregnenolone to form a different product.
P450c17 is therefore a crucial enzyme, not only in the
formation of sex steroid precursors, but also in the
production of androstadienol which is considered to be an
intermediate in the 16-androstene pathway leading to the
biosynthesis of androstenol and of 5a(16)androsten-3-one, a
pheromonally active steroid in the pig [8,14]. A recent report
by our group [27] shows that further transformation of
androstadienol into androstenol involves the classic
enzymes of the steroidogenic pathway, namely 3b-HSD,
5a-reductase and 3a-HSD. Interestingly, because of the lack
of either a 17-keto or a 17-hydroxy group, this pathway
does not require the enzyme 17b-HSD which is specific to
sex steroid biosynthesis.

Although it is well known that androstenol is a phero-
mone in the pig, its role in the human is still ill-defined.
Recent findings show that androstenol is able to modulate
the expression of certain cytochrome P450s and alcohol
dehydrogenases through interactions with the orphan
receptors CAR and PXR [18,20]. These receptors belong
to the P450-regulatory nuclear receptors, in the subfamily
NR1 (nuclear binding site 1) [21]. Other members of this
NR1 orphan nuclear receptor gene subfamily are PPAR
(peroxisome proliferator-activated receptor), LXR (liver X
receptor) and FXR (farnesol-X-receptor). They share a
common hetero-dimerization partner, the RXR, and are
subject to cross-talk interactions with other nuclear recep-
tors and with a broad range of other intracellular signaling
pathways, including those activated by certain cytokines
and growth factors [21,22]. It has been shown that the
steroids androstenol [16(5a)-androsten-3a-ol] and 5b-preg-
nanedione (5b-pregnane-3,20-dione) modulate the action of
these receptors and thus are putative endogenous ligands for
these receptors [20]. Upon binding to the ligands, PXR and
CAR bind DNA as a heterodimer with the RXR and
modulate the expression of cytochromes P450, especially
CYP2B and CYP3A families. Because P450s play an
essential role in the detoxification of drugs and of a large
series of exogenous compounds from the environment,
androstenol and 5b-pregnanedione that modulate cyto-
chrome P450 levels could have a profound effect on the
detoxification process.
Because of the low affinity of these orphan nuclear
receptors (in the range of 1–10 · 10

)6
M
) and their relatively
broad spectrum of ligand specificity, many researchers that
are familiar with classic steroid receptors, namely androgen,
estrogen, progesterone, glucocorticoid and mineralocorti-
coid receptors, that bind to their corresponding specific
ligand with a very high affinity (10
)10
)10
)9
M
), are skeptical
about the idea of androstenol and 5b-pregnanedione being
ligands for these orphan receptors. However, it is notewor-
thy that, as classic active steroids are diluted in the blood,
their concentration is very low and thus they require high
affinity receptors to pick them up. On the other hand, the
orphan receptor ligands are most probably produced locally
in the various tissues. Because of the small volume of the
cell, the production of a little amount of ligand will give a
relatively high concentration (up to 1–10 · 10
)6
M
). We
hypothesize that this low affinity combined with the local
biosynthesis of ligands represents a mechanism allowing the
selective regulation of the action of the receptor: ligands that
enter the cell or tissue by chance will not have a high enough
concentration to turn on the receptor. Only for ligands that

are produced locally or accumulated in the tissue (probably
through active transport or hydrophobicity) is the concen-
tration high enough to modulate the receptor activity.
As suggested above, local biosynthesis in various tissues
such as the liver, could constitute a way to selectively
regulate the activity of nuclear orphan receptors such as
CAR and PXR. Although there is no evidence of P450c17
expression in the human liver, many other enzymes such as
3b-HSD, 5a-reductase and 3a-HSD, whose activities lead to
androstenol synthesis from androstadienol, are present in
Fig. 6. Influence of increasing amounts of P450red on the relative for-
mation of DHEA and androstadienol by P450c17. HEK-293 cells were
transfected with 0.1 lg pCMV-P450c17, 1 lg pCMV-cyt b
5
and the
indicated amounts of pCMV-P450red. The transfected cells were
analyzed for their ability to catalyze the transformation of 5 n
M
of
[
3
H]preg into DHEA (j) and androstadienol (n). Transfections
and enzymatic assays were performed as described in Experimental
procedures. The results are the mean ± SEM of three independent
experiments.
1354 P. Soucy et al. (Eur. J. Biochem. 270) Ó FEBS 2003
the liver and many other peripheral tissues. Furthermore,
androstadienol is found in circulation suggesting that it is
synthesized at the sites of expression of P450c17 and
eventually converted to androstenol by different enzymes in

peripheral tissues such as the liver and adipose tissue. Our
study aimed at elucidating the nature and the mechanism of
the reactions involved in the local formation of androstenol
is thus of major importance.
Acknowledgements
This work has been supported by a grant from the Canadian Institutes
of Health Research. The authors would like to thank Guy Reimnitz,
Nathalie Paquet and Mei Wang for their technical assistance and Sylvie
Me
´
thot for careful reading of the manuscript.
References
1. Lin, D., Black, S.M., Nagahama, Y. & Miller, W.L. (1993) Steroid
17 alpha-hydroxylase and 17,20-lyase activities of P450c17: con-
tributions of serine106 and P450 reductase. Endocrinology 132,
2498–2506.
2. Yanagibashi, K. & Hall, P.F. (1986) Role of electron transport in
the regulation of the lyase activity of C21 side-chain cleavage
P-450 from porcine adrenal and testicular microsomes. J. Biol.
Chem. 261, 8429–8433.
3. Auchus, R.J., Lee, T.C. & Miller, W.L. (1998) Cytochrome b5
augments the 17,20-lyase activity of human P450c17 without
direct electron transfer. J. Biol. Chem. 273, 3158–3165.
4. Kominami, S., Ogawa, N., Morimune, R., De-Ying, H. & Take-
mori, S. (1992) The role of cytochrome b5 in adrenal microsomal
steroidogenesis. J. Steroid Biochem. Mol. Biol. 42, 57–64.
5. Onoda, M. & Hall, P.F. (1982) Cytochrome b5 stimulates purified
testicular microsomal cytochrome P-450 (C21 side-chain clea-
vage). Biochem. Biophys. Res. Commun. 108, 454–460.
6. Aoyama, T., Nagata, K., Yamazoe, Y., Kato, R., Matsunaga, E.,

Gelboin, H.V. & Gonzalez, F.J. (1990) Cytochrome b5potentia-
tion of cytochrome P-450 catalytic activity demonstrated by a
vaccinia virus-mediated in situ reconstitution system. Proc. Natl
Acad. Sci. USA 87, 5425–5429.
7. Ahmad, N. & Gower, D.B. (1966) The in vitro metabolism of
pregn-5-en-3-beta-OL-20-one by rat testicular tissue. Formation
of delta-16-steroids. Steroids 7, 273–288.
8. Gower, D.B. (1972) 16-Unsaturated C 19 steroids. A review of
their chemistry, biochemistry and possible physiological role.
J. Steroid Biochem. 3, 45–103.
9. Gower, D.B. (1981) The biosynthesis and occurence of
16-androstenes in man. In Hormones in Normal and Abnormal
Human Tissues (Fotherby, K. & Pal, S.B., eds), pp. 1–27, Walter
de Gruyter, Inc., Berlin, Germany.
10. Knuppen,R.&Breuer,H.(1963)Biogenesevono
¨
strate-traenol
beim menschen. Acta Endocr. Copenh. 42, 129–134.
11. Stylianou, M., Forchielli, E., Tummillo, M. & Dorfman, R.I.
(1961) Metabolism in vitro of 4-C-14 testosterone by human liver
homogenate. J. Biol. Chem. 236, 692–694.
12. Stylianou, M., Forchielli, E. & Dorfman, R.I. (1961) The meta-
bolism of 4-C14-testosterone in rat testis homogenates. J. Biol.
Chem. 236, 1318–1320.
13. Brooksbank, B.W. & Haslewood, G.A.D. (1950) The nature of
pregnanediol-like glucuronide. Biochem. J. 47, 36–43.
14. Melrose, D.R., Reed, H.C. & Patterson, R.L. (1971) Androgen
steroids associated with boar odour as an aid to the detection of
oestrus in pig artificial insemination. Br. Vet J. 127, 497–502.
15. Weusten, J.J., Legemaat, G., van der Wouw, M.P., Smals, A.G.,

Kloppenborg, P.W. & Benraad, T. (1989) The mechanism of the
synthesis of 16-androstenes in human testicular homogenates.
J. Steroid Biochem. 32, 689–694.
16. Grosser, B.I., Monti-Bloch, L., Jennings-White, C. & Berliner,
D.L. (2000) Behavioral and electrophysiological effects of andro-
stadienone, a human pheromone. Psychoneuroendocrinology 25,
289–299.
17. Morofushi, M., Shinohara, K., Funabashi, T. & Kimura, F.
(2000) Positive relationship between menstrual synchrony and
ability to smell 5alpha-androst-16-en-3alpha-ol. Chem. Senses. 25,
407–411.
18. Forman, B.M., Tzameli, I., Choi, H.S., Chen, J., Simha, D., Seol,
W., Evans, R.M. & Moore, D.D. (1998) Androstane metabolites
bind to and deactivate the nuclear receptor CAR-beta. Nature 395,
612–615.
19. Choi, H.S., Chung, M., Tzameli, I., Simha, D., Lee, Y.K., Seol,
W. & Moore, D.D. (1997) Differential transactivation by two
isoforms of the orphan nuclear hormone receptor CAR. J. Biol.
Chem. 272, 23565–23571.
20. Moore, L.B., Parks, D.J., Jones, S.A., Bledsoe, R.K., Consler,
T.G., Stimmel, J.B., Goodwin, B., Liddle, C., Blanchard, S.G.,
Willson, T.M., Collins, J.L. & Kliewer, S.A. (2000) Orphan
nuclear receptors constitutive androstane receptor and pregnane
X receptor share xenobiotic and steroid ligands. J. Biol. Chem.
275, 15122–15127.
21. Waxman, D.J. (1999) P450 gene induction by structurally diverse
xenochemicals: central role of nuclear receptors CAR, PXR, and
PPAR. Arch. Biochem. Biophys. 369, 11–23.
22. Savas, U., Griffin, K.J. & Johnson, E.F. (1999) Molecular
mechanisms of cytochrome P-450 induction by xenobiotics: An

expanded role for nuclear hormone receptors. Mol. Pharmacol. 56,
851–857.
23. Baron, J., Taylor, W.E. & Masters, B.S. (1972) Immuno-
chemical studies on electron transport chains involving cyto-
chrome P-450. The role of the iron-sulfur protein, adrenodoxin, in
mixed-function oxidation reactions. Arch. Biochem. Biophys. 150,
105–115.
24. Kominami, S., Ochi, H., Kobayashi, Y. & Takemori, S. (1980)
Studiesonthesteroidhydroxylationsysteminadrenalcortex
microsomes. Purification and characterization of cytochrome
P-450 specific for steroid C-21 hydroxylation. J. Biol. Chem. 255,
3386–3394.
25. Soucy, P. & Luu-The, V. (2000) Conversion of pregnenolone to
DHEA by human 17alpha-hydroxylase/17, 20-lyase (P450c17).
Evidence that DHEA is produced from the released inter-
mediate, 17alpha-hydroxypregnenolone. Eur. J. Biochem. 267,
3243–3247.
26. Huang, X.F. & Luu-The, V. (2000) Molecular characterization of
a first human 3 (alpha–beta)-hydroxysteroid epimerase. J. Biol.
Chem. 275, 29452–29457.
27. Dufort, I., Soucy, P., Lacoste, L. & Luu-The, V. (2001) Com-
parative biosynthetic pathway of androstenol and androgens.
J. Steroid Biochem. Mol. Biol. 77, 223–227.
Ó FEBS 2003 Biosynthesis of 5,16-androstadien-3b-ol by human P450c17 (Eur. J. Biochem. 270) 1355