Oxidation of dihydrotestosterone by human cytochromes P450 19A1 and 3A4

Article abstract of DOI:10.1074/jbc.M112.390047

Dihydrotestosterone is a more potent androgen than testosterone and plays an important role in endocrine function. We demonstrated that, like testosterone, dihydrotestosterone can be oxidized by human cytochrome P450 (P450) 19A1, the steroid aromatase. The products identified include the 19-hydroxy-and 19-oxo derivatives and the resulting Δ^1,10-, Δ^5,10-, and Δ^9,10-dehydro 19-norsteroid products (loss of 19-methyl group). The overall catalytic efficiency of oxidation was ～10-fold higher than reported for 3α-reduction by 3α-hydroxysteroid dehydrogenase, the major enzyme known to deactivate dihydrotestosterone. These and other studies demonstrate the flexibility of P450 19A1 in removing the 1- and 2-hydrogens from 19-norsteroids, the 2-hydrogen from estrone, and (in this case) the 1-, 5β-, and 9β-hydrogens of dihydrotestosterone. Incubation of dihydrotestosterone with human liver microsomes and NADPH yielded the 18- and 19-hydroxy products plus the Δ^1,10-dehydro 19-nor product identified in the P450 19A1 reaction. The 18- and 19-hydroxylation reactions were attributed to P450 3A4, and 18- and 19-hydroxydihydrotestosterone were identified in human plasma and urine samples. The change in the pucker of the A ring caused by reduction of the Δ^4,5 bond is remarkable in shifting the course of hydroxylation from the 6β-, 2β-, 1β-, and 15β-methylene carbons (testosterone) to the axial methyl groups (18, 19) in dihydrotestosterone and demonstrates the sensitivity of P450 3A4, even with its large active site, to small changes in substrate structure.

Full text of DOI:10.1074/jbc.M112.390047

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 35, pp. 29554–29567, August 24, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Oxidation of Dihydrotestosterone by Human Cytochromes
P450 19A1 and 3A4*
Received for publication, June 11, 2012, and in revised form, July 3, 2012 Published, JBC Papers in Press, July 7, 2012, DOI 10.1074/jbc.M112.390047
Qian Cheng, Christal D. Sohl1, Francis K. Yoshimoto, and F. Peter Guengerich2
From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232-0146
Background: The fate of dihydrotestosterone has been characterized only in terms of reduction of the 3-ketone.
Results: Human P450s 19A1 and 3A4 oxidized dihydrotestosterone to several new products, detected in vivo.
Conclusion: The new P450 19A1 and 3A4 pathways introduce an oxidative dimension to the metabolism of
dihydrotestosterone.
Significance: Small changes in the steroid A ring can produce major changes in P450 3A4 catalytic selectivity.
Dihydrotestosterone is a more potent androgen than testos-
terone and plays an important role in endocrine function. We
demonstrated that, like testosterone, dihydrotestosterone can
be oxidized by human cytochrome P450 (P450) 19A1, the ste-
roid aromatase. The products identified include the 19-hy-
droxy- and 19-oxo derivatives and the resulting ⌬1,10-, ⌬5,10-,
and ⌬9,10-dehydro 19-norsteroid products (loss of 19-methyl
group). The overall catalytic efficiency of oxidation was ϳ10-
fold higher than reported for 3␣-reduction by 3␣-hydroxys-
teroid dehydrogenase, the major enzyme known to deactivate
dihydrotestosterone. These and other studies demonstrate the
flexibility of P450 19A1 in removing the 1- and 2-hydrogens
from 19-norsteroids, the 2-hydrogen from estrone, and (in this
case) the 1-, 5␤-, and 9␤-hydrogens of dihydrotestosterone.
Incubation of dihydrotestosterone with human liver micro-
somes and NADPH yielded the 18- and 19-hydroxy products
plus the ⌬1,10-dehydro 19-nor product identified in the P450
19A1 reaction. The 18- and 19-hydroxylation reactions were
attributed to P450 3A4, and 18- and 19-hydroxydihydrotestos-
terone were identified in human plasma and urine samples. The
change in the pucker of the A ring caused by reduction of the
drugs, carcinogens, and other endogenous and xenobiotic
chemicals (1–3). The importance of P450s in steroid metabo-
lism is documented by the large number of instances of inher-
ited endocrine diseases now linked to defects in P450 (CYP)
genes (4, 5). Roughly one-fourth of the human P450s have
important roles in steroid metabolism (6, 7). The effects of
small chemical changes in steroids (introduced by P450s and
other enzymes) are rather remarkable in terms of the resulting
changes in biological activities.
P450 19A1, the steroid aromatase, is a critical enzyme that
converts testosterone and androstenedione to estrogens (8).
Deficiencies in P450 19A1 present clinical problems, e.g. female
virilization (4). However, P450 19A1 is also a drug target in the
treatment of estrogen-dependent cancers, e.g. breast and ovary
(9). The catalytic mechanism of P450 19A1 involves a three-
step reaction, consisting of 19-hydroxylation, oxidation of the
19-hydroxy product to an aldehyde, and the unusual loss of the
19-formyl group as HCO2H accompanying the aromatization
of the steroid A ring (10, 11). P450 3A4 is best known as a
hepatic/intestinal enzyme and is involved in the metabolism of
roughly one-half of drugs (that are metabolized) (12). However,
it is also active in the oxidations of many steroids, including
cholesterol (13), estradiol (14), progesterone (15), cortisol (16),
and testosterone and androstenedione (14, 15). The major reac-
tion observed with testosterone is 6␤-hydroxylation (14, 15,
17); hydroxylation at the 2␤, 1␤, and 15␤ sites also occurs (17,
18). However, no physiological relevance of any of these
hydroxylations has been established. Crystal structures of both
P450 19A1 (19) and P450 3A4 (20–23) have been reported,
some with bound steroids (19, 21), but the structures have
yielded limited rationalization of catalytic selectivity.
⌬
4,5 bond is remarkable in shifting the course of hydroxylation
from the 6␤-, 2␤-, 1␤-, and 15␤-methylene carbons (testoster-
one) to the axial methyl groups (18, 19) in dihydrotestosterone
and demonstrates the sensitivity of P450 3A4, even with its large
active site, to small changes in substrate structure.
P4503 enzymes have long been studied because of their roles
in the metabolism of steroids, fat-soluble vitamins, fatty acids,
Dihydrotestosterone is a more potent androgen than testos-
terone and is important, particularly in virilization (24). Men
with genetic deficiency of steroid 5␣-reductase Type 2 show a
syndrome termed pseudohermaphroditism (25). The enzymes
that reduce testosterone to dihydrotestosterone (5␣-steroid
reductase, isozymes 1 and 2 (24)) are targets for drugs used to
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grants R37 CA090426, R01 GM069970, T32 ES007028, and P30 ES000267.
1 Present address: Dept. of Pharmacology, Yale University School of Medicine,
333 Cedar St., SHM B-350, New Haven, CT 06520.
2 To whom correspondence should be addressed: Dept. of Biochemistry, Van-
derbilt University School of Medicine, 638 Robinson Research Bldg., 2200
Pierce Ave., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-
4349; E-mail: f.guengerich@vanderbilt.edu.
3 The abbreviations used are: P450, cytochrome P450; 2,4-DNPH, 2,4-dinitro-
phenylhydrazine; ESI, electrospray ionization; HMBC, heteronuclear multi-
ple bond correlated (spectroscopy); HRMS, high-resolution mass spec-
trometry;HSQC, heteronuclearsinglequantumcorrelation(spectroscopy);
THF, tetrahydrofuran; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl;
dihydrotestosterone, refers to testosterone reduced at the ⌬4,5 double
bond.
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P450 Oxidation of Dihydrotestosterone
FIGURE 1. Chemical synthesis of 19-hydroxydihydrotestosterone. PPTS, pyridinium p-toluenesulfonate; PTSA, p-toluenesulfonic acid. Yields are shown in
the scheme. 19-Hydroxydihydrotestosterone exists in equilibrium with its hemiketal form (31). See “Experimental Procedures” for details.
treat prostate hypertrophy, a common problem in older men, as performed either on Thermo LTQ linear ion-trap or TSQ triple
well as some other androgen-dependent conditions (26). The
only enzyme reported to be involved in the metabolism of
dihydrotestosterone is a 3␣-hydroxysteroid dehydrogenase
(AKR1C2) that reduces the 3-keto group to an alcohol, which is
devoid of androgen activity (27).
quadrapole instruments. All spectrometers were operated in
the positive ESI mode. NMR spectra of reaction products were
recorded on a Bruker AV-II-600 instrument, equipped with a
cryoprobe, in the Vanderbilt facility or on other Bruker instru-
ments as noted. Spectra were referenced to the residual solvent
signals (see above). Two-dimensional spectra (HSQC, HMBC)
were acquired using standard Bruker programs.
We became interested in the possibility of dihydrotestoster-
one oxidation following a series of mechanistic studies on
androgen oxidation by recombinant P450 19A1 (11). Some
alternate substrates of P450 19A1 were shown to be oxidized
(estrone and 19-nortestosterone), confirming earlier reports.
The 2-electron difference in the oxidation states of testosterone
and dihydrotestosterone suggested that aromatic (estrogenic)
products might not be formed from oxidations of the latter; we
identified three unsaturated 19-norsteroids as products, plus a
further hydroxylation product of one of these. Human liver
microsomes (in the presence of NADPH) also formed oxidation
products from dihydrotestosterone, and P450 3A4 was impli-
cated in the production of 18- and 19-hydroxydihydrotestos-
terone. The latter two hydroxylated products were identified in
vivo in human plasma and urine samples.
Chemicals
Steroids were obtained from either Sigma or Steraloids (Wil-
ton, NH). 2,4-DNPH-HCl (Sigma) was recrystallized from
C2H5OH before use.
Synthesis of 19-Hydroxydihydrotestosterone (Fig. 1)
General—19-Hydroxyandrostenedione was purchased from
Waterstone Technologies Ltd. (Carmel, IN). Compounds were
monitored by TLC under UV light or staining with CAM rea-
gent (Ce(NH4)2(NO3)6 (0.5%, w/v), ammonium molybdate
(12%, w/v), and H2SO4 (20%, v/v) in H2O) for non-UV active
compounds (compounds 2, 2a, 3, 4, 5, and 6). Synthetic com-
pounds were characterized by NMR (300 or 400 MHz, CDCl3,
or CD3OD solvent). The CDCl3 peak was referenced to ␦ 7.26
ppm in the 1H NMR spectra and the triplet corresponding to
CDCl3 was referenced to 77.16 ppm in the 13C NMR spectra.
The methyl peak of CD3OD was referenced to ␦ 3.49 ppm in the
1H NMR spectra (32).
EXPERIMENTAL PROCEDURES
Enzymes
Human P450 19A1 was heterologously expressed in Esche-
richia coli and purified as described earlier (11). Recombinant
rat NADPH-P450 reductase was also expressed in E. coli and
purified as described (28). E. coli membranes expressing human
P450 3A4 and human NADPH-P450 reductase were prepared
as described previously (29). Microsomes were prepared from
human livers as described (30) (samples were originally
obtained through the Nashville Regional Organ Procurement
Agency, according to Vanderbilt Institutional Review Board
procedures).
19-(Tetrahydropyran-2Ј-yl-oxy)-androst-4-ene-3,17-dione
(1 in Fig. 1)—3,4-Dihydro-2H-pyran (0.30 ml, 0.28 g, 2.3 mmol,
8 mol eq) was added to a mixture of 19-hydroxyandrostenedi-
one (95 mg, 0.3 mmol, 1.0 mol eq) and pyridinium p-toluene-
sulfonate (2.6 mg, 0.1 mmol, 0.3 mol eq) in THF (5 ml). The
reaction was stirred at room temperature under an argon atmo-
sphere for 20 h. The reaction was concentrated and purified via
preparative TLC (silica gel G, ethyl acetate:hexanes, 1:1, v/v) to
furnish a diastereomeric mixture of 19-(tetrahydropyran-2Ј-yl-
oxy)-androst-4-ene-3,17-dione (1, 72 mg, 0.2 mmol, 67%). Rf
0.50 (1:1, ethyl acetate:hexanes, v/v). 1H NMR (400 MHz,
CDCl3): ␦ 5.91 (br s, 1H, H-4), 5.89 (br s, 1H, H-4), 4.63–4.56
Plasma and urine samples were obtained from healthy indi-
viduals in accord with the Vanderbilt Institutional Review
Board policies.
MS and NMR Spectroscopy
HRMS analyses were done with an LTQ-Orbitrap (Thermo)
spectrometer in the Vanderbilt facility. Other MS analyses were (m, 2H), 0.92 (s, 3H, 19-H), 0.91 (s, 3H, 19-H).
AUGUST 24, 2012•VOLUME 287•NUMBER 35
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P450 Oxidation of Dihydrotestosterone
19-(Tetrahydropyran-2Ј-yl-oxy)-5␣-androstan-3-one (2)—A the organic extracts were concentrated in vacuo. The crude
modified Birch reduction procedure was based on the work of material was purified by flash column chromatography (100%
Oh and Robinson (33). Lithium (0.5 g) was pre-rinsed in
hexanes and added to a 3-necked oven-baked round bottom
flask equipped with a cold finger, oil bubbler outlet, and stirrer.
Ammonia (150 ml) was used to fill the flask (Ϫ78 °C). The mix-
ture was stirred for 45 min at Ϫ78 °C. A solution of 19-(tetra-
hydropyran-2Ј-yl-oxy)-androst-4-ene-3,17-dione (1, 71 mg,
0.18 mmol) in THF (3 ml) was added dropwise. After 10 min,
NH4Cl (0.5 g) was added. The reaction was warmed to 23 °C
and diluted with ethyl acetate (50 ml) and H2O (20 ml). The
organic extract was concentrated in vacuo and purified by flash
column chromatography on silica gel (100% hexanes to 50%
hexanes/ethyl acetate, v/v) to afford 19-(tetrahydropyran-2Ј-yl-
oxy)-5␣-androstan-3,17-diol (3, 21 mg, 53 ␮mol, 29%) and
17␤-hydroxy-19-(tetrahydropyran-2Ј-yl-oxy)-5␣-androstan-
3-one (2, 17 mg, 44 ␮mol, 24%). (Note: if not enough NH4Cl was
added to quench the reaction, the 17␤-hydroxy group became
acetylated upon addition of ethyl acetate to yield 3-oxo-19-
(tetrahydropyran-2Ј-yl-oxy)-5␣-androstan-17␤-yl acetate 2a,
which could be converted back to 2 with stoichiometric potas-
sium carbonate in CH3OH.) Rf of diol 3, 0.22 (1:1, ethyl acetate:
hexanes, v/v). 1H NMR of diol 3 (300 MHz, CDCl3): ␦ 4.63–4.55
(m, 1H), 3.91–3.81 (m, 1H), 2.31–2.19 (m, 1H), 2.10–1.99 (m,
1H), 0.76 (s, 3H), 0.73 (s, 3H). Rf of 3-ketosteroid 2, 0.36 (1:1,
hexanes to 50% hexanes in ethyl acetate to 10% CH3OH:
CH2Cl2, v/v) to afford 19-hydroxydihydrotestosterone (10 mg,
33 ␮mol, 45%), which was further purified on a second column
to yield material for 1H NMR spectroscopy (1 mg).
19-Hydroxydihydrotestosterone (4), from 3—p-Toluenesul-
fonic acid monohydrate (17 mg, 89 ␮mol) was added to a solu-
tion of tetrahydropyran ether 3 (16 mg, 41 ␮mol) in 4 ml of
CH3OH:CH2Cl2 (1:1, v/v) and the reaction was stirred for 3 h.
The reaction mixture was purified directly by flash column
chromatography (silica gel G, 100% hexanes to 50% ethyl ace-
tate: hexanes, v/v) to yield the triol 5 (10 mg, 32 ␮mol, 78%). 1H
NMR of triol 5 (300 MHz, CD3OD): ␦ 4.04 and 3.93 (ABq, 2H,
J ϭ 8.7 Hz, 19-H), 3.79–3.66 (m, 2H, 3-H and 17-H), 2.51–2.44
(m, 1H), 2.20–2.09 (m, 1H), 2.03–1.72 (m, 8H), 1.69–1.37 (m,
7H), 1.18–1.05 (m, 3H), 0.96 (s, 3H, 18-H), 0.93–0.82 (m, 1H).
The NMR spectrum showed that 4 exists in equilibrium with its
3,19-hemiketal form (Fig. 1), consonant with previous litera-
ture (31).
Conversion of Triol 5 to 19-Hydroxydihydrotestosterone (4)
using Cholesterol Oxidase—Triol 5 (5 mg), dissolved in CH3OH
(50 ␮l), was incubated with Brevibacterium sp. cholesterol oxi-
dase (Sigma, 25 units) in 50 mM potassium phosphate buffer
(pH 7.4) in a total volume of 1 ml (30 °C, shaking at 100 rpm,
3 h). The reaction was terminated by the addition of CH2Cl2 (1
ml) and the organic layer was dried under a stream of N2.
1
ethyl acetate:hexanes, v/v). H NMR of 3-ketosteroid 2 (300
MHz, CDCl3): ␦ 4.64–4.56 (m, 1H), 4.15 (dd, J ϭ 14.4, 10.6 Hz),
0.77 (s, 3H, 19-H), 0.76 (s, 3H, 19-H). Rf of acetate 2a, 0.8 (10:1,
CH2Cl2:CH3OH, v/v).
Enzyme Reactions
19-Hydroxydihydrotestosterone (19-Hydroxy-5␣-androstan-
3-one) (4), from 2—p-Toluenesulfonic acid monohydrate (123
mg, 646 ␮mol) was added to a stirring solution of 17␤-hydroxy-
19-(tetrahydropyran-2Ј-yl-oxy)-5␣-androstan-3-one (2, 65
mg, 167 ␮mol) in 4 ml of CH3OH:CH2Cl2 (1:1, v/v), and the
reaction was stirred for 10 h at 23 °C. The reaction mixture was
washed with saturated NaHCO3 solution and purified by flash
column chromatography (silica gel G, 100% hexanes to 50%
ethyl acetate:hexanes, v/v) to afford a mixture of 19-hydroxy-
dihydrotestosterone 4 (4 mg, 13 ␮mol, 8%) and 3,19-epoxy-3-
methoxy-5␣-androstan-17␤-ol 6 (30 mg, 89 ␮mol, 53%). (Note:
formation of 6 from 4 increased with reaction time, and the use
of HCl instead of p-toluenesulfonic acid monohydrate formed 6
at a faster rate.) Rf of 19-hydroxydihydrotestosterone 4, 0.45
(3:1, ethyl acetate:hexanes, v/v). Rf of 6, 0.76 (3:1, ethyl acetate:
hexanes, v/v).
Dihydrotestosterone-P450 19A1 Titrations—UV-visible
spectra were recorded using an Aminco DW2/OLIS spectro-
photometer (On-Line Instrument Systems, Bogart, GA). Dihy-
drotestosterone (final organic solvent concentration Յ3%, v/v)
was added to P450 19A1 (2 ␮M), and duplicate scans were made
from 350 to 500 nm and averaged using the manufacturer’s
software.
Conversion of Estrone to 2-Hydroxyestrone—The reconsti-
tuted enzyme system contained 0.4 ␮M P450 19A1, 0.8 ␮M
NADPH-P450 reductase, and 36 ␮M L-␣-1,2-dilauroyl-sn-
glycero-3-phosphocholine (dispersed into vesicles by sonica-
Ϫ1
tion prior to use, as a 1 mg ml stock solution). Potassium
phosphate buffer (100 mM, pH 7.4) and estrone (in CH3OH,
final organic solvent content Յ1% (v/v)) were added, and the
final incubation volume was 1.0 ml. An NADPH-generating
system (30) was added to initiate incubations (in a shaking
water bath, 37 °C, 50 rpm shaking, Amerex Instruments Model
903, Lafayette, CA). Reactions were run in duplicate and, after
20 min, quenched with 1.0 ml of CH2Cl2. The layers in the
samples were separated by centrifugation (3 ϫ 103 ϫ g, 10 min),
and 0.8 ml of each organic layer was removed and dried under
N2. HPLC (20-␮l injections) was used to separate the com-
pounds (octadecylsilane (C18) column, 6.2 mm ϫ 80 mm, 3 ␮m,
Agilent Technologies, Palo Alto, CA). A Spectra Series UV3000
rapid-scanning UV detector (Thermo Fisher Scientific) (285
nm) was used to quantitate the product 2-hydroxyestrone. The
3,19-Epoxy-3-methoxy-5␣-androstan-17␤-ol (6) from 2a—
HCl (12 M, 50 ␮l) was added to a solution of 2a (80 mg, 190
␮mol) in CH3OH (2 ml) and the reaction was stirred for 4 h.
The reaction mixture was purified directly by flash column
chromatography to afford 3,19-epoxy-3-methoxy-5␣-andro-
stan-17␤-ol 6 (35 mg, 100 ␮mol, 53%).
19-Hydroxydihydrotestosterone (4), from 6—A solution of
BBr3 (0.1 M solution in CH2Cl2, 500 ␮l, 50 ␮mol) was added to
a stirring solution of 3,19-epoxy-3-methoxy-5␣-androstan-
17␤-ol (6, 25 mg, 74 ␮mol) in CH2Cl2 (5 ml) at Ϫ78 °C under an
argon atmosphere. After stirring for 5 min, the reaction mix-
ture was poured into a flask containing H2O (20 ml). The reac- following gradient was used (1.0 ml minϪ1, solvent A, 90% H2O,
tion mixture was extracted with ethyl acetate (3 ϫ 20 ml), and 10% CH3OH (v/v); solvent B, 100% CH3OH (v/v)): initial com-
29556 JOURNAL OF BIOLOGICAL CHEMISTRY
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P450 Oxidation of Dihydrotestosterone
position was 50% B (v/v) from 0 to 2 min, linear gradient to was either a linear ion-trap (LTQ) or an Orbitrap (Thermo),
operating in the positive ESI/full-scan mode.
100% B from 2 to 4 min, held at 100% B from 4 to 9 min.
Conversion of 19-Norandrostenedione to Estrone—The same
incubation conditions were used as for the estrone 2-hydroxyl-
ation experiment (see above); 0.20 ␮M P450 19A1, 0.40 ␮M
NADPH-P450 reductase, and 18 ␮M L-␣-1,2-dilauroyl-sn-
glycero-3-phosphocholine were used. Separation was achieved
by HPLC (octadecylsilane (C18), 150 mm ϫ 4.6 mm, 5 ␮m,
YMC, Kyoto, Japan), using an isocratic mixture of 40% B (v/v)
(1.0 ml minϪ1, solvent A, 90% H2O, 10% CH3CN (v/v); solvent
B, 100% CH3CN (v/v)), with UV detection (A280).
P450 19A1 Incubations with Dihydrotestesterone—The LC-MS
conditions of the human liver microsomal study were used for the
incubations of P450 19A1 with dihydrotestosterone, except that
1.0 ␮M P450 19A1, 2.0 ␮M NADPH-P450 reductase, and 90 ␮M
L-␣-1,2-dilauroyl-sn-glycero-3-phosphocholine were mixed prior
to the addition of dihydrotestosterone.
Analysis of Human Plasma and Urine Samples
Steroid Extraction from Plasma—Pooled plasma samples
(600 ␮l, 200 ␮l from each of three healthy individuals) were
extracted twice with 5 ml of a mixture of ethyl acetate and
hexane (4:1, v/v). The organic phases were combined and dried
under a stream of N2.
Time Course of the Reaction of Dihydrotestosterone with P450
19A1—The reconstituted enzyme system contained 5 nM P450
19A1, 150 nM NADPH-P450 reductase, and 4.5 ␮M L-␣-1,2-
dilauroyl-sn-glycero-3-phosphocholine (dispersed into vesicles
Ϫ1
Steroid Extraction from Urine—Urine (20 ml collected from
each male volunteer) was diluted with 80 ml of 0.5 M sodium
acetate buffer (pH 5.0). ␤-Glucuronidase type HP-2 (Helix
pomatia, Sigma, 105 units) was added and samples were incu-
bated at 55 °C for 24 h. The urinary steroids were recovered
either by liquid extraction (using equal volumes of ethyl ace-
tate) or solid phase extraction (Oasis HLB columns, Waters). In
the latter case the columns were loaded, washed with H2O, and
eluted with CH3OH. The organic phase from liquid extraction
was dried using a rotary evaporator, and the eluates from solid
phase extraction columns were dried under a stream of N2. The
resulting urinary steroids were derivatized with dansyl chloride
or 2,4-DNPH (see below) prior to LC-MS analyses.
by sonication prior to use, as a 1 mg ml stock solution).
[1,2,4,5,6,7-3H]Dihydrotestosterone (PerkinElmer Life Sci-
ences) (in CH3OH, Յ1%, v/v) was added (1.0 ␮M, 0.27 ␮Ci
nmolϪ1), and the sample was incubated at 37 °C, with 50 rpm
shaking for 0.5 to 40 min. After quenching reactions with 1.0 ml
of CH2Cl2, each sample was mixed with a vortex device and
separated by centrifugation (3 ϫ 103 ϫ g, 10 min). The organic
layer (0.75 ml) was removed, dried under an N2 stream, and
dissolved in CH3OH prior to injection. Radio-HPLC (20-␮l
injections) was used to separate the products using an octade-
cylsilane (C18) column (6.2 ϫ 80 mm, 3 ␮m, Agilent Technol-
ogies). The following gradient was used (1 ml minϪ1; solvent A,
83% H2O, 17% THF (v/v); solvent B, 83% H2O, 17% THF (v/v)):
isocratic 20% B from 0 to 11 min, linear gradient to 60% B (v/v)
from 11 to 13 min, hold at 60% B (v/v) from 13 to 22 min.
The separations of the P450 19A1 dihydrotestosterone oxi-
dation products were not successful on octadecylsilane HPLC
columns without the inclusion of THF. However, THF has
undesirable properties in terms of storage and its compatibility
with some LC-MS fittings. Accordingly we substituted a phe-
nyl-hexyl column and obtained good resolution with only H2O,
CH3CN, and trace HCO2H (see below).
Dansylation—The dansylation reactions were performed as
previously described (34). Briefly, samples were dissolved in 200
␮l of CH2Cl2 containing 2 mg of dansyl chloride, 1 mg of 4,4-
dimethylaminopyridine, and 20 ␮l of triethylamine and incu-
bated at 65 °C for 1 h. The samples were dried under an N2
stream and then dissolved in 50 ␮l of CH3CN for analysis.
Derivatization with 2,4-DNPH—Each sample was dissolved
in 100 ␮l of 2,4-DNPH-saturated CH3OH containing 0.10 M
HCl and incubated at room temperature for 16 h. Samples were
then directly injected in LC-MS analysis.
Human Liver Microsomal Incubations with Dihydrotes-
tosterone—Human liver microsomes (mixture of samples from
10 individuals, equal weights combined) (0.5 ␮M total P450)
were incubated with 100 ␮M dihydrotestosterone (dissolved in
CH3OH, Յ1% (v/v)) in 100 mM potassium phosphate buffer
(pH 7.4), with an NADPH-generating system (30). After a
20-min incubation at 37 °C, 50 rpm shaking, each reaction was
quenched with 1.0 ml of CH2Cl2, mixed with a vortex device,
and separated by centrifugation (3 ϫ 103 ϫ g, 10 min). The
(lower) organic layer (0.8 ml) was removed and dried under N2.
Aliquots were loaded onto a Luna phenyl-hexyl HPLC column
(250 ϫ 4.6 mm, 5 ␮m, Phenomenex) equilibrated with solvent
A (95% H2O, 5% CH3CN, 0.1% HCO2H, v/v) and solvent B (5%
H2O, 95% CH3CN, 0.1% HCO2H, v/v), using a flow rate of 1.0
ml minϪ1). The column was maintained at the initial condition
of 40% (v/v) solvent B from 0 to 14 min, followed by a linear
gradient increasing to 50% (v/v) solvent B over 1 min. This
condition was maintained for 10 min and then returned to the
initial condition. About 20% of the flow was diverted into a mass
Analysis of Dansylated Plasma and Urinary Steroids—Dan-
sylated products were analyzed by LC-MS with an HPLC sys-
tem connected to a TSQ Quantum mass spectrometer
(Thermo) using a Pursuit UPS octadecylsilane (C18) column
(2.1 ϫ 100 mm, 3 ␮m, Varian, Palo Alto, CA). HPLC conditions
were as follows. Solvent A contained 95% H2O, 5% CH3CN, and
0.1% HCO2H (v/v), and solvent B contained 5% H2O, 95%
CH3CN, and 0.1% HCO2H(v/v). The column was maintained at
an initial condition of 78% (v/v) solvent B for 4 min at a flow rate
of 300 ␮l minϪ1, followed by a linear gradient increasing to 95%
(v/v) solvent B over 1 min. This mixture was maintained for 2
min and then returned to the initial condition over 1 min and
maintained until the end of a 10-min run. The column temper-
ature was maintained at 25 °C. The injection volume onto the
column was 15 ␮l. MS analyses were performed in a multiple-
reaction monitoring mode (dansylated dihydrotestosterone,
m/z 524 3 252, collision energy of 35 V; dansylated hydroxy-
dihydrotestosterone, m/z 540 3 252, collision energy of 35 V).
The transitions correspond to the release of the dansyl (sulfonic
spectrometer using a T-shaped sleeve. The mass spectrometer acid) from the derivative. Standard curves were prepared from
AUGUST 24, 2012•VOLUME 287•NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 29557

P450 Oxidation of Dihydrotestosterone
FIGURE2. Steady-statekineticsofalternateoxidationscatalyzedbyP450
19A1. A, 2-hydroxylation of estrone; B, oxidation of 19-norandrostenedione
to estrone.
FIGURE 3. Binding and oxidation of dihydrotestosterone by P450 19A1.
A, binding as observed by perturbation of the heme Soret spectra. B, radio-
HPLC of oxidation products of dihydrotestosterone formed by P450 19A1.
The m/z 275 products, including the ⌬1,10 compound whose structure is
shown, were identified by MS and NMR (Figs. 4, 5, and 7).
synthetic material and utilized for quantitation of 19-hydroxy-
dihydrotestosterone in urine.
Quantitation of Urinary Steroids (2,4-DNPH Derivatives)—
2,4-DNPH-derivatized products were analyzed by LC-MS
using similar LC conditions as in the analysis of dansylated ste-
roids, except that the initial condition was 72% solvent B (v/v).
The volume injected onto the column was 10 ␮l. MS analyses
were performed in a multiple-reaction monitoring mode (2,4-
DNPH-dihydrotestosterone, m/z 471 3 242, collision energy
of 35 V; 2,4-DNPH-19-hydroxydihydrotestosterone, m/z
487 3 268, collision energy of 35 V). The transitions corre-
spond to the loss of a 2,4-dinitroaniline moiety plus H2O (or
two molecules of H2O in the case of hydroxydihydrotestoster-
one). Standard curves were prepared from synthetic material
and utilized for quantitation of dihydrotestosterone in urine.
ciency (kcat/Km) was ϳ105 Ϫ1 minϪ1, or 1/750 of that of con-
M
version of testosterone to estradiol (11).
Binding of Dihydrotestosterone to P450 19A1 and Oxidation—
Dihydrotestosterone was found to bind to P450 19A1, as judged
by the observed spectral shifts (increase of A392, decrease of
A
421) indicative of a change in the iron spin-state from low to
high spin (11, 37). The apparent Kd was 6.5 (Ϯ 0.7) ␮M (Fig. 3A).
Preliminary incubation experiments with P450 19A1,
NADPH-P450 reductase, and NADPH showed the appearance
of several products of dihydrotestosterone (Fig. 3B). Subse-
quent studies (e.g. Fig. 9, see below) showed Ͼ95% recovery of
the radiolabel from dihydrotestosterone. With a substrate con-
RESULTS
Oxidation of Alternative Substrates by P450 19A1—A P450 centration of 1.0 ␮M and P450 19A1 concentration of 5 nM,
19A1 preparation purified from human placental microsomes dihydrotestosterone disappearance occurred at a rate of ϳ7.5
Ϫ1
has been reported to catalyze estrone 2-hydroxylation (35). We pmol (pmol P450) minϪ1, suggesting a catalytic efficiency
also observed this activity with recombinant human P450 19A1, Ն7.5 ϫ 106 Ϫ1 minϪ1, which is ϳ10% of the observed catalytic
M
with a kcat of 0.046 Ϯ 0.003 minϪ1 and Km of 2.7 Ϯ 0.7 ␮M (Fig. efficiency of a similar preparation of P450 19A1 with andro-
Ϫ1
2A). The catalytic efficiency (kcat/Km) was ϳ2 ϫ 104
M
Ϫ1 min
,
stenedione as the substrate (11).
Ϫ1
Ϫ1
compared with ϳ7.5 ϫ 107
M
min for the conversion of
Identification of Dihydrotestosterone Oxidation Products
Formed by P450 19A1—Five products were isolated and iden-
androstenedione to estrone (11).
19-Nortestosterone lacks the 19-CH3 group (of testoster- tified by MS and NMR. The structures are indicated in Fig. 4
one), and ⌬1,2-desaturation would yield a product that should and the NMR spectra are shown in Figs. 5–8.
rearrange nonenzymatically to estradiol. The oxidation of
Initial LC-MS analysis indicated the presence of products
ϩ
19-nortestosterone to estradiol occurred with human recombi- with MH ions at m/z 307 and 305 and several at m/z 275.
nant P450 19A1, as reported earlier by Harada (36) with an Subsequent HRMS yielded molecular formula consistent with
enzyme purified from placenta, with a kcat of 0.15 (Ϯ0.01) the structures indicated in Fig. 4 (all within 5 ppm of calcu-
minϪ1 and a Km of 1.1 (Ϯ 0.1) ␮M (Fig. 2B). The catalytic effi- lated). None of the radiolabeled products co-chromatographed
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P450 Oxidation of Dihydrotestosterone
FIGURE 4. HRMS of oxidation products of dihydrotestosterone formed by P450 19A1. HRMS parent ions (m/z values) are those determined, with the
calculated values (calcd.) are listed below each. All measured values were within 5 ppm of the calculated values.
with authentic 19-nortestosterone, the ⌬4,5-19-norsteroid
(Steraloids) (absorbance monitored at 240 nm). We found no
evidence for rearrangement of the products following initial
isolation, ruling out migration of the olefinic bonds.
19-Hydroxydihydrotestosterone was verified by its 1H
NMR spectrum, with a shift of the singlet at ␦ 1.00 to a
doublet (3.92 and 4.21 ppm, data not shown). The peak co-
eluted with and had an identical mass spectrum with syn-
thetic 19-hydroxydihydrotestosterone.
The 19-aldehyde product was characterized by HRMS (Fig.
4) and the NMR spectra (Fig. 5). The CHO proton appeared as
a singlet at ␦ 10.2 and, in the HSQC spectrum, was coupled to a
carbon peak at 210 ppm, indicative of a carbonyl.
ϩ
The major peak with MH at m/z 275 was assigned the struc-
ture ⌬1,10-dehydro 19-nordihydrotestosterone (Fig. 4) based on
HRMS and the one-dimensional, HSQC, and HMBC NMR
spectra (Fig. 6). In particular, the H-1 proton (singlet) appeared
at ␦ 5.3, indicative of a change to an olefin (Fig. 6A). In the
HSQC spectrum, that proton is bonded to a carbon at 112 ppm
(Fig. 6B). The H-1 proton is linked to the C3 carbonyl at 210
ppm in the HMBC spectrum (Fig. 6C).
Two smaller m/z 275 peaks in the radiochromatogram (Fig.
3B) were characterized, although with less material than the
others. Both had HRMS molecular formula of 19-nor products
with one degree of unsaturation (Fig. 4). The 1H NMR spectra
were used to assign the compounds as the ⌬5,10- and ⌬9,10
-
dehydro 19-nor steroids (Fig. 7). Both spectra are devoid of
vinyl protons, ruling out several possibilities. In Fig. 7A the H-4
protons are shifted downfield, as would be expected if they were
moved into a conjugated system (⌬5,10). In Fig. 7B there is no
proton shift characteristic of an olefin, and we assigned this as
the ⌬9,10-dehydro structure.
2-Hydroxy-⌬5,10-dehydro 19-nordihydrotestosterone (tR ϳ
7 min using the conditions described in Fig. 3B, following pro-
longed incubation, data not shown for such a chromatogram)
had the HRMS formula of a hydroxylated product of one of the
dehydro 19-nor products (Fig. 4). The structure was assigned
FIGURE 5. NMR spectra of oxidation products of dihydrotestosterone
formed by P450 19A1: dihydrotestosterone 19-aldehyde (see Fig. 3).
A, 1H NMR spectrum; B, HSQC spectrum (CDCl3). Key assignments are shown.
based on the NMR spectra (Fig. 8). Particularly characteristic spectrum, and the C-2 carbinol appears at 70 ppm, both linked
are the H-1 and H-2 protons (␦ 5.9 and 4.2) (Fig. 8A). The H-1 to the assigned protons (Fig. 8B). The HMBC spectrum (Fig.
proton is linked to the C-1 carbon (117 ppm) in the HSQC 8C) shows the coupling to the C-3 carbonyl.
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P450 Oxidation of Dihydrotestosterone
(for the formation of the various products, using dihydrotestos-
terone as substrate): 19-hydroxydihydrotestosterone, kcat 0.27
minϪ1, Km 3.8 ␮M; dihydrotestosterone 19-aldehyde, kcat 0.32
minϪ1, Km 3.2 ␮M; ⌬1,10-dehydro 19-nordihydrotestosterone,
kcat 0.77 minϪ1, Km 7.6 ␮M. Thus, the apparent catalytic effi-
ciencies were similar.
Oxidation of Dihydrotestosterone by Human Liver Micro-
somes—Incubation of [3H]dihydrotestosterone with (a pooled
sample of 10) human liver microsomes and NADPH yielded
several products (Fig. 10A). The production of the major peaks
(except ⌬1,10-dehydro 19-nordihydrotestosterone) was strongly
inhibited in the presence of 2 ␮M ketoconazole, a selective
inhibitor of P450 3A4 (6, 38). Quinidine (2 ␮M), sulfaphenazole
(2 ␮M), 4-methylpyrazole (2 ␮M), nor ␣-naphthoflavone (2 ␮M)
inhibited product formation (these are, respectively, inhibitors
of P450s 2D6, 2C9, 2E1, and 1A2 (6, 38)). Furthermore, E. coli
membranes containing recombinant P450 3A4 and NADPH-
P450 reductase (29) formed the same major products, except
for ⌬1,10-dehydro 19-nordihydrotestosterone, in different
ratios (Fig. 10B). These results lead to the conclusion that the
major catalyst of oxidation is P450 3A4.
Identification of P450 3A4 Oxidation Products—The two
major hydroxylated products (Figs. 10B) were isolated from
incubations with P450 3A4-containing E. coli membranes and
the structures were determined. The ⌬1,10-dehydro 19-nordi-
hydrotestosterone product (see above) was also present in liver
microsomes (identified by co-chromatography and LC-MS,
Figs. 4 and 7) but not the P450 3A4 incubations.
19-Hydroxydihydrotestosterone was identified by co-elu-
tion, HRMS, and fragmentation (Figs. 11 and 12) with the prod-
uct formed by P450 19A1 and the synthetic material (see
1
above). The H NMR spectrum was also consistent with the
assigned structure (Fig. 13A).
18-Hydroxydihydrotestosterone also showed HRMS data con-
sistent with the assigned structure (Fig. 12). The 1H NMR spec-
trum (Fig. 13B) showed the total loss of the 18-CH3 signal (␦ 0.75
ppm) and the appearance of carbinol protons (␦ 3.9 ppm) over-
lapped with the C17 carbinol proton, which had also moved.
Catalytic Efficiency of Oxidation of Dihydrotestosterone by
P450 3A4 and Human Liver Microsomes—Rates of formation
of the major oxidation products were measured at varying con-
centrations of dihydrotestosterone (Fig. 14). With both a
recombinant human P450 3A4 system and human liver micro-
somes, plots of velocity versus substrate concentrations were
not very saturable (up to 300 ␮M dihydrotestosterone). The
FIGURE 6. NMR spectra of oxidation products of dihydrotestosterone
formed by P450 19A1: ⌬1,10-dehydro 19-nordihydrotestosterone (see
Fig. 3). A, 1H NMR spectrum; B, HSQC spectrum; C, HMBC spectrum (CDCl3).
Key assignments are shown.
Time Course and Catalytic Efficiencies of Formation of P450 slopes of these plots provide estimates of catalytic efficiency, i.e.
19A1 Dihydrotestosterone Products—P450 19A1 was incubated 1.1 ϫ 104 and 7.5 ϫ 103 Ϫ1 minϪ1 for the formation of 18- and
M
with a slight excess (2-fold) of dihydrotestosterone, and prod- 19-hydroxydihydrotestosterone, respectively, by P450 3A4 and
ucts were analyzed as a function of time (Fig. 9). The substrate, 2400 and 880 MϪ1 minϪ1, respectively, for the formation of 18-
dihydrotestosterone, was depleted within 30 min. 19-Hydroxy- and 19-hydroxydihydrotestosterone by human liver micro-
dihydrotestosterone increased and did not decrease. The 19-al- somes (based on total P450). These values are ϳ30-fold lower
dehyde (19 ϭ O) product increased and then slowly decreased. than the catalytic efficiencies of testosterone 6␤-hydroxylation
The major desaturation product (⌬1,10-dehydro 19-nordihy- reported previously with similar enzyme preparations (3 ϫ 105
drotestosterone, Fig. 3B) increased without a noticeable lag.
cat and Km values were estimated (using a P450 19A1 con-
M
Ϫ1 minϪ1) (17).
k
Identification of Dihydrotestosterone Oxidation Products in
centration of 30 nM and a reaction time of 15 min). The exact Vivo—Human plasma and urine samples were analyzed for the
meaning of these parameters is not clear because of the flux of presence of the dihydrotestosterone oxidation products. Dan-
products (Fig. 9). The following parameters were determined syl (34) and 2,4-DNPH derivatives were analyzed by LC-MS
29560 JOURNAL OF BIOLOGICAL CHEMISTRY
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P450 Oxidation of Dihydrotestosterone
FIGURE 7. 1H NMR spectra of oxidation products of dihydrotestosterone formed by P450 19A1 (see Fig. 3). A, ⌬5,10-dehydro 19-nordihydrotestosterone;
B, ⌬9,10-dehydro 19-nordihydrotestosterone. The solvent was CDCl3. Key assignments are shown.
using multiple reaction monitoring mode to achieve appropri- major products obtained with liver microsomes were 18- and
ate sensitivity.
19-hydroxy dihydrotestosterone, involving the action of P450
3A4. These latter two compounds were also found in vivo.
Estrone 2-hydroxylation and the conversion of 19-norandro-
stenedione to estrone had previously been reported as reactions
of horse (39, 40) and human P450 19A1 preparations (35, 36,
41). We confirmed these reactions with our purified recombi-
nant P450 19A1 (Fig. 2). The apparent catalytic efficiencies
were ϳ3,000- and 750-fold lower than for the conversion of
androstenedione or testosterone to the respective estrogens
(11).
LC-MS profiles of a sample of urine (from a single individual)
and a pooled sample of sera (3 individuals) are shown (Figs. 15 and
16). The identities were established by co-elution with derivatives
of the oxidation products identified from the P450 3A4 incuba-
tions (18-hydroxy) or synthetic material (19-hydroxy).
Concentrations of the oxidation product 19-hydroxydihy-
drotestosterone could be quantitated, based on the synthetic
material and fragmentation of the dansyl group (34), and the
values of multiple analyses with several samples of these prod-
ucts were ϳ40-fold less than dihydrotestosterone (determined
as 2,4-DNPH derivative) in both plasma and urine (Table 1).
The range of concentrations was expected to be variable due to
variations in individual urinary volumes; the concentrations
varied 2-fold. Normalization based on the concentrations of
dihydrotestosterone indicated an ϳ3-fold variation among the
five healthy individuals (Table 1). The concentration of urinary
18-hydroxydihydrotestoterone was much less, but we did not
attempt to quantify this in the lack of a standard (other than the
material recovered from P450 3A4 incubations, Fig. 13).
Characterization of two dihydrotestosterone oxidation
products formed by P450 19A1, the 19-alcohol and 19-aldehyde
(Fig. 4), was straightforward in that the mass spectra were
expected from the known pathways for testosterone and andro-
stenedione (3, 8), and the NMR data (19-aldehyde, Fig. 5) are
supportive. Three desaturated products were characterized
(Fig. 4). The ⌬1,10-dehydro product was the major one, and the
HRMS and two-dimensional NMR data are quite clear regard-
ing its identity. The ⌬5,10-dehydro product, obtained in lower
yield and not amenable to two-dimensional analysis, was iden-
tified by the change in the C-4 protons in the 1H NMR spectrum
(Fig. 5), as well as the absence of any vinyl protons (cf. ⌬1,10, Fig.
5). Assigning the ⌬9,10 structure was more difficult; this product
had the same molecular ion/formula (Fig. 4) and was devoid of
vinyl protons (Fig. 7B). A ⌬8,9-dehydro structure is conceivable
(in the absence of more NMR data) but is considered less likely
in any mechanistic context.
DISCUSSION
Recombinant human P450 19A1 was demonstrated to use
dihydrotestosterone as a substrate, with a 3- to 4-step reaction
pathway similar to that demonstrated for testosterone and
androstenedione (Fig. 4). An interesting feature is the abstrac-
tion of any of several hydrogens, 1, 5, and 9, to yield the resulting
dehydro 19-norsteroids. Dihydrotestosterone was also oxi-
dized by human liver microsomes to yield one of the dehydro
A time course with a 2-fold molar excess of dihydrotestoster-
19-norsteroid products (⌬1,10) observed with P450 19A1. The one relative to P450 19A1 showed disappearance of the sub-
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P450 Oxidation of Dihydrotestosterone
FIGURE 10. Radio-HPLC traces of [3H]dihydrotestosterone oxidation
products. A, human liver microsomes; B, P450 3A4 bicistronic membranes.
The reaction time was 60 min in both cases.
FIGURE 8. NMR spectra of oxidation products of dihydrotestosterone
formed by P450 19A1: 2-hydroxy-⌬1,10-dehydro 19-nordihydrotestos-
terone (see Fig. 3). A, 1H NMR spectrum; B, HSQC spectrum; C, HMBC spec-
trum (CDCl3). Key assignments are shown.
FIGURE 11. LC-MS of metabolites of dihydrotestosterone formed by P450
3A4. Fourhydroxymetabolites(m/z307)weredetected. Thetwomajorprod-
ucts were identified as 19-OH dihydrotestosterone (DHT) by co-chromatog-
raphy and 18-OH DHT by 1H NMR (see Fig. 13), respectively. A, m/z 291; B, m/z
307.
strate and immediate formation of the 19-hydroxy, 19-alde-
hyde, and ⌬1,10-dehydro products (Fig. 9). Levels of the
19-hydroxy and ⌬1,10-dehydro products did not decrease with
time but the 19-aldehyde did. Apparent kcat and Km values were
FIGURE 9. Time course of conversion of dihydrotestosterone to products
by P450 19A1. The initial concentration of [3H]dihydrotestosterone was 1.0
␮M and the P450 19A1 concentration was 0.5 ␮M (the NADPH-P450 reductase
concentration was 1.0 ␮M).
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P450 Oxidation of Dihydrotestosterone
FIGURE12. HRMSofoxidationproductsofdihydrotestosteroneformedinhumanlivermicrosomes. HRMSparentions(m/zvalues)arethosedetermined,
with the calculated values (calcd.) listed below each.
FIGURE 13. One-dimensional 1H NMR spectra of dihydrotestosterone and 18-OH and 19-OH dihydrotestosterone recovered from P450 3A4 incuba-
tions. A, 19-hydroxydihydrotestosterone; B, 18-hydroxydihydrotestosterone; C, dihydrotestosterone. The 18-methyl signal of dihydrotestosterone is missing
in the 18-OH dihydrotestosterone spectrum and appears (as -CH2OH) at ␦ 3.82.
FIGURE 14. Steady-state kinetics of P450 3A4-catalyzed 18- and 19-hydroxylations of dihydrotestosterone. A, 19-hydroxylation by P450 3A4;
B, 18-hydroxylation by P450 3A4. The fits shown were done using a hyperbolic fit (A) and linear regression (B) (only slope estimates are considered in the
text).
estimated for the conversion of dihydrotestosterone to each of initial concentration) in the presence of a limiting amount of
the observed products. However, these values can be mislead- P450 19A1 (5 nM), yielding a rate of ϳ7.5 minϪ1. A lower limit
Ϫ1 Ϫ1
Ϫ1
ing for a multistep reaction. A more useful parameter was of 1.2 ϫ 105
M
s
(i.e. 7.5 ϫ 106
M
minϪ1) can be esti-
obtained based on disappearance of dihydrotestosterone (1 ␮M mated, which is ϳ10-fold greater than the kcat/Km of 104
M
Ϫ1
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P450 Oxidation of Dihydrotestosterone
Ϫ1
s
reported for reduction of dihydrotestosterone by 3␣-ste-
roid dehydrogenase AKR1C2 (27).
Incubation of dihydrotestosterone with P450 19A1 yielded
several additional minor products with molecular ions (m/z 275
ϩ
for MH ) corresponding to the elemental formula of the
dehydro products (⌬1,10-, ⌬5,10-, ⌬9,10-), observed in LC-MS,
but the amounts were too small for further identification (con-
trol experiments showed lack of isomerization of the isolated
products under these conditions). Also, the ⌬1,10-dehydro
product was shown to be subsequently hydroxylated at C-2, but
the stereochemistry was not established (Figs. 4 and 8).
Incubation of dihydrotestosterone with human liver micro-
somes yielded the 18- and 19-hydroxy products and the ⌬1,10
-
dehydro product (Fig. 10). The characterization of the two
hydroxylated products was straightforward with NMR and,
with 19-hydroxydihydrotestosterone, comparison with the
synthetic and other biosynthetic material (Figs. 11 and 13). The
formation of the 18- and 19-hydroxy products was catalyzed by
P450 3A4, as shown by inhibition studies and work with the
recombinant enzyme (Fig. 10B). At least two other putative
hydroxylated products were formed from dihydrotestosterone
by P450 3A4 (Fig. 11B). Formation of the ⌬1,10-dehydro prod-
uct, which presumably proceeds from the 19-hydroxy product,
was not attributed to a specific P450. Although we did not
address this issue further, our tentative view is that the ⌬1,10
-
FIGURE 15. LC-MS of dansylated 19-hydroxydihydrotestosterone in
human urine and plasma. The transition m/z 540 [rarrrow] 252 was moni-
tored. A, derivatization of synthetic 19-hydroxydihydrotestosterone led to
one major product (identified as a mono-dansyl derivative) and two minor
products (unidentified); B, dansylated 19-hydroxydihydrotestosterone in a
human urine sample; C, dansylated 19-hydroxydihydrotestosterone in
human plasma.
dehydro 19-nordihydrotestosterone product (Fig. 10A) may
result from P450 19A1. The literature indicates some ambiguity
about the expression of P450 19A1, with some studies reporting
the absence of P450 19A1 in adult human liver (42). However,
in other work the aromatization of androstenedione (43) and
the presence of P450 19A1 mRNA (44, 45) have been reported
in (noncancerous) adult human liver. However, the role of
another, unidentified P450 in the liver cannot be ruled out at
this time. The presence of 19-hydroxydihydrotestosterone
and the ⌬1,10-dehydro 19-norsteroid product are consistent
with the involvement of P450 19A1 (Fig. 9). 19-Hydroxydihy-
drotestosterone was clearly shown to be formed by P450 3A4,
however (Figs. 10B and 11B).
Initial in vivo experiments were done with human plasma,
and 18- and 19-hydroxydihydrotestosterone were identified in
a pooled sample (Fig. 15C). These two hydroxylated steroids
were also identified in individual urine samples (Figs. 15B and
16B, Table 1). The extent of variation (normalized for dihy-
drotestosterone) was 3-fold (Table 1). At this time we cannot
define the origin of the 19-hydroxydihydrotestosterone in the
urine or plasma. P450 3A4 was much less efficient than P450
19A1 in forming 19-hydroxydihydrotesotosterone (ϳ103-fold)
but the amount of P450 3A4 in human liver (46) is probably
much larger than the total amount of P450 19A1 in the body.
Two other issues are (i) the tendency of P450 19A1 to further
oxidize 19-hydroxydihydrotestosterone (Fig. 9) and (ii) the dif-
ferential locations of P450 19A1 and P450 3A4 in tissues and
the excretion of 19-hydroxydihydrotestosterone from these
into plasma and urine. Although the concentration of 19-hy-
droxydihydrotestosterone in urine was much lower than that of
dihydrotestosterone, we do not know if this reflects the ratios in
any tissues. To expand on this latter point, P450 19A1 is pref-
erentially expressed in female steroidogenic tissues (e.g. ovaries,
FIGURE 16. LC-MS of 2,4-DNPH derivatives of 19-hydroxydihydrotestos-
terone in human urine. The transition m/z 487 3 268 was monitored. A, syn-
thetic 19-hydroxydihydrotestosterone derivatized with 2,4-DNPH; B, a
human urine sample extracted and derivatized with 2,4-DNPH.
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P450 Oxidation of Dihydrotestosterone
TABLE 1
Concentrations of steroids in human urine samples
Dihydrotestosterone
19-Hydroxy-dihydrotestosterone
19-Hydroxy-dihydrotestosterone/dihydroestosterone
ng/ml
0.13
0.23
0.20
0.22
0.29
%
Subject 1
Subject 2
Subject 3
Subject 4
Subject 5
2.2
5.9
2.0
1.8
2.8
2.6
11.2
11.2
7.6
10.9
FIGURE 17. Some oxidations of steroids catalyzed by P450 19A1. Site of oxidation by P450 FeO complexes are shown. The losses of individual protons of
dihydrotestosterone 19-aldehyde are shown with arrows.
ready conversion to estradiol in vivo is a complicating factor (in
some models 19-hydroxytestosterone has been used as an
estrogen precursor) (47, 48).
The catalytic mechanisms of the oxidations are considered
here for P450s 19A1 and 3A4 (Figs. 17 and 18). P450 19A1 can
catalyze oxidations at carbons 1, 2, and 19 of these steroids. The
oxidation of the 19-formyl group could proceed via an iron
Ϫ
peroxy complex (FeOO ) (49, 50) or the “Compound I” FeO3ϩ
species invoked for other P450 reactions (10, 51). Regardless of
the oxidizing species, the formation of the characterized
dehydro products requires the removal of protons at the 1, 5␤,
or 9␤ sites. We presume that the 1␤-hydrogen is removed, in
FIGURE 18. Oxidations of testosterone and dihydrotestosterone cata-
the absence of direct evidence, in that H-1␤ is known to be
lyzed by P450 3A4. Sites of hydroxylation are shown with arrows. All testos-
removed in the usual P450 19A1 aromatase reaction with 3-ke-
terone hydroxylations occur at the ␤ face (18).
to-⌬4,5-dehydro steroids (52). It is not clear if the iron-oxygen
complex assists in the proton removal, as shown for P450
placenta) and adipose tissue, and expression in tissues of most
N-dealkylation reactions (53). Alternatively base catalysis by
amino acid residues might be involved in catalysis (19), or even
water-bonded networks to amino acids. The ability of P450
19A1 to remove a variety of ␤-hydrogens is of interest (Fig. 17).
The catalytic selectivity of P450 3A4 in dihydrotestosterone
oxidation (18-, 19-hydroxylation) was unexpected, in that the
well characterized 6␤, 1␤, 2␤, and 15␤ hydroxylations of testos-
terone (14, 18) were not observed (Fig. 18). The only difference
relevance to dihydrotestosterone action (e.g. prostate) is very
low (44). We do not know the levels of expression of P450 19A1
protein relative to the 3␣-reductase AKR1C2, the other enzyme
that acts on dihydrotestosterone. Finally (regarding relevance
to physiology), the biological activity of the products we have
identified here is unknown. We might presume that any of the
oxidations would decrease the androgenic activity of dihy-
drotestosterone. 19-Hydroxylation of testosterone appears to
decrease its androgenic activity in rat models, although the between the structures of testosterone and dihydrotestosterone
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P450 Oxidation of Dihydrotestosterone
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is in the electronics and the pucker of the A ring (Fig. 18).
Reduction of the ⌬4,5 bond remarkably shifts the sites of oxida-
tion to the attached axial (␤) methyl groups (18 and 19), which
are chemically more difficult to oxidize than methylenes due to
C-H bond strength (54, 55). This specificity was unexpected
and argues for caution in relying on predictions of P450 reac-
tion sites.
15. Waxman, D. J., Attisano, C., Guengerich, F. P., and Lapenson, D. P. (1988)
Cytochrome P-450 steroid hormone metabolism catalyzed by human liver
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In conclusion, we report several oxidations of the important
androgen dihydrotestosterone. Several interesting reactions
occur with the steroid aromatase, P450 19A1. One of these
products (⌬1,10-dehydro 19-nordihydrotestosterone, Fig. 4)
was also formed in liver microsomes. Two major liver micro-
somal products, the 18- and 19-alcohols, were also detectable in
vivo, in plasma and urine. Because of the variability of P450 3A4
activity among humans (56, 57), variation in the rates of degra-
dation may occur (Table 1) and influence the homeostasis of
dihydrotestosterone.
19. Ghosh, D., Griswold, J., Erman, M., and Pangborn, W. (2009) Structural
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20. Yano, J. K., Wester, M. R., Schoch, G. A., Griffin, K. J., Stout, C. D., and
Johnson, E. F. (2004) The structure of human microsomal cytochrome
P450 3A4 determined by X-ray crystallography to 2.05-Å resolution.
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Acknowledgments—We thank M. V. Martin and L. M. Folkmann for
preparing NADPH-P450 reductase, D. Stec for assistance with some of
the NMR spectroscopy, M. W. Calcutt for assistance with MS, and K.
Trisler for assistance in preparation of the manuscript.
21. Williams, P. A., Cosme, J., Vinkovic, D. M., Ward, A., Angove, H. C., Day,
P. J., Vonrhein, C., Tickle, I. J., and Jhoti, H. (2004) Crystal structures of
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