Báo cáo khoa học: Testosterone 1b-hydroxylation by human cytochrome P450 3A4 pdf

Peter Guengerich1 1 Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine and 2 Department of Chemistry and Center in Molecular Toxicolo

Testosterone 1b-hydroxylation by human cytochrome P450 3A4

Joel A Krauser1, Markus Voehler2, Li-Hong Tseng3, Alexandre B Schefer3, Markus Godejohann3

and F Peter Guengerich1

1 Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine and 2 Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, TN, USA; 3 Bruker Bio-Spin GmbH, Rheinstetten, Germany

Human cytochrome P450 3A4 forms a series of minor

testosterone hydroxylation products in addition to

6b-hy-droxytestosterone, the major product One of these, formed

at the next highest rate after the 6b- and 2b-hydroxy

prod-ucts, was identified as 1b-hydroxytestosterone This product

was characterized from a mixture of testosterone oxidation

products using an HPLC-solid phase extraction-cryoprobe

NMR/time-of-flight mass spectrometry system, with an

estimated total of
6 lg of this product Mass spectrometry

established the formula as C19H29O3(MH+305.2080) The

1-position of the added hydroxyl group was established by

correlated spectroscopy and heteronuclear spin quantum correlation experiments, and the b-stereochemistry of the added hydroxyl group was assigned with a nuclear Over-hauser correlated spectroscopy experiment (1a-H) Of several human P450s examined, only P450 3A4 formed this product The product was also formed in human liver microsomes

Keywords: cytochrome P450; NMR spectroscopy; HPLC-NMR combinations; testosterone

Cytochrome P450 (P450; also termed heme-thiolate P450

[1]) enzymes have long been of interest because of their roles

in steroid metabolism [2,3] These oxidations are most

critical in steroidogenic tissues, and a set of
12 P450s are

most important [4,5] The hepatic P450s have also been

studied extensively in the context of their abilities to

hydroxylate steroids, even though few of the oxidations

involve the generation of products with distinctive biological

activities Seminal in this area is the work of Conney and his

associates, who studied the hydroxylation of testosterone in

rat liver systems and developed the hypothesis that different

hydroxylations are catalyzed by individual P450 enzymes

[6] Subsequently testosterone hydroxylation patterns have

been utilized extensively as probes of the presence and

function of individual rat liver P450s [7–11]

Testosterone hydroxylation has also been studied

exten-sively with human liver microsomal P450s Early work with

liver microsomes resulted in reports of hydroxylation at the

2b, 6a, 6b, 7a, 15b, 16a, and 17 positions (17-hydroxylation

yields the ketone androstenedione) [12–17] A human liver

P450 was isolated that was shown to be the major

6b-hydroxylase [18]; this P450 was originally termed nifedi-pine oxidase (P450NF) and subsequently named P450 3A4 Other work confirmed the role of P450 3A4 as the major enzyme involved in testosterone 6b-hydroxylation [19] Other P450 3A subfamily enzymes (3A5, 3A7, 3A43) can also catalyze this reaction [20,21]

Testosterone hydroxylation is in general use today as one

of the characteristic assays of P450 3A4, which was subsequently shown to be the most abundant P450 in human liver and small intestine [22] and involved in the oxidation of approximately one-half the drugs used today [23] The major product is 6b-hydroxytestosterone [18,19] but several other hydroxylations occur, including those at the 2b and 15b positions [18,19] (Fig 1) Not all of the products have been identified, however In the course of our investigations we noted that a peak (X) formed at a rate in the order 6b > 2b > X
15b (hydroxylation) had not been characterized and did not correspond to any of our standards available in our set, including 2a-, 2b-, 6a-, 6b-, 11b-, 15b-, 16a-, or 16b-hydroxytestosterone or androsten-edione We utilized an HPLC-solid phase extraction (SPE)-cryoprobe NMR/MS system and now provide a full spectral characterization of this product from
6 lg of the product injected The product is 1b-hydroxytestosterone (Scheme 1) and is formed only by P450 3A4, of the set of human P450s examined

Experimental procedures

Chemicals GC-grade acetonitrile (CH3CN) and HPLC-grade H2O for the separation (combined HPLC-MS-NMR) was from Merck (Darmstadt, Germany) CD3CN and CD3OD (both 99.8% deuterium-enriched) were from Deutero GmbH

Correspondence to F P Guengerich, Department of Biochemistry and

Center in Molecular Toxicology Vanderbilt University School of

Medicine Nashville, Tennessee 37232–0146, USA.

Fax: +1 615 3223141, Tel.: +1 615 3222261,

E-mail: f.guengerich@vanderbilt.edu

Abbreviations: P450, cytochrome P450 (also termed heme-thiolate

P450, substrate, reduced flavoprotein: oxygen oxidoreductase);

HSQC, heteronuclear spin quantum correlation; SPE, solid phase

extraction.

Enzymes: P450, substrate, reduced flavoprotein:oxygen

oxidoreductase (EC 1.14.14.1).

(Received 16 July 2004, accepted 19 August 2004)

(Kastellaun, Germany) Testosterone (Sigma-Aldrich, St.

Louis, MO, USA) was used without further purification

Hydroxytestosterone standards were purchased from

Stearoaloids (Newport, RI, USA)

Enzymes

Microsomes were prepared [24] from a human liver sample

(denoted HL 97), which had been used in some previous

investigations in this laboratory [25] Recombinant

P450 3A4 was used either in the form of Escherichia coli

membranes in which both P450 3A4 and NADPH-P450

reductase were coexpressed [26] (termed
bicistronic

mem-brane system) or microsomes prepared from insect cells

infected with a baculovirus vector and expressing

NADPH-P450 reductase in excess of NADPH-P450 3A4 (PanVera, Madison,

WI, USA) Other human P450s were expressed together

with NADPH-P450 reductase in the bicistronic membrane

systems (E coli membranes) for use [26]

Testosterone hydroxylation assays

Incubations (0.5 mL total volume) were carried out with

bacterial membranes (from E coli, bicistronic expression

vectors, see above) containing P450 (100 pmol of P450 1A1,

1A2, 1B1, 2C9, or 2D6, or 40 pmol of P450 3A4) human

liver microsomes containing 100 pmol total P450, or

microsomes from baculovirus-infected insect cells

contain-ing 4 pmol of P450 3A4 (Varycontain-ing amounts of P450 were

used because of differences in rates of the systems, in order

to maintain linearity of product formation vs time.) A

typical system contained the NADPH-P450 reductase (see

above), an NADPH-generating system [24], and varying

concentrations of testosterone, and was incubated for

8–10 min at 37C [27]

HPLC-UV HPLC-UV assays were used to quantify the rates of formation of individual testosterone hydroxylation prod-ucts The dichloromethane extract from each incubation was taken to dryness under a stream of N2 Aliquots were dissolved in 30 lL of methanol, injected into a 20-lL loop, and separated on a 4.6· 150 mm Phenomenex Prodigy ODS octadecylsilane HPLC column (C18, 3-lm particle size, Phenomenex, Torrence, CA, USA) with a gradient formed from solvent A (95% CH3CN, 5% H2O, v/v) and solvent B (H2O), using the schedule as follows: 0–5.5 min, 75% (v/v) solvent B; 5.5–12 min, 75% to 64% solvent B; 12–24 min, 64% (v/v) solvent B; 25–26 min, 64% to 75% (v/v) solvent B; and 26–30 min, hold at 75% solvent B The pumping system was a Hitachi-L-7100 single pump ternary apparatus (Hitachi High Technologies America, San Jose,

CA, USA) A244measurements were used, with a UV3000 rapid scanning detector (ThermoSeparations, Piscataway,

NJ, USA), and integration was done using the software supplied by the manufacturer

HPLC-MS-NMR Sample preparation A preparative incubation was done with the P450 3A4 bicistronic membrane preparation (200 pmol P450 3A4, total volume 5 mL) containing

500 lM testosterone and an NADPH-generating system [24] for 12 min at 37C The reaction was extracted with dichloromethane (15 mL) and the organic phase was washed with brine, dried over magnesium sulfate, filtered, and concentrated to dryness The resulting solid was dissolved in

300 lL of CD3OD and filtered prior to injection

HPLC (including UV) The HPLC system consisted of an Agilent 1100 System including a vacuum degasser, quaternary HPLC pump, an autosampler, and a diode array detector

Chromatographic separation was carried out on a Phenomenex Prodigy ODS3 (5-lm particle size, 4.6· 250 mm, Phenomenex, Torrence, CA, USA) The chromatographic conditions were as follows: solvent A,

CH3CN; solvent B, H2O; initial conditions 5% A/95% B (v/v), followed by a linear gradient to 95% A/5% B (v/v) over 30 min; 10-min linear gradient to 100% A and held for

5 min; back to initial conditions in 0.1 min; re-equilibration for 10 min at a flow rate of 0.8 mLÆmin)1 The peaks were detected at a wavelength of 244 nm using a diode array detector

MS (time-of-flight)

An aliquot (5%, v/v) of the eluent from the HPLC column was split to the mass spectrometer using a splitter from LCPackings (Amsterdam, the Netherlands) The split ratio was guided to a MicroTOF mass spectrometer (Bruker Daltonic, Bremen, Germany) equipped with an orthogonal electrospray ion source Measurements were carried out in the positive mode with a scan range from 20 to 800 mass-to-charge ratio (m/z) The capillary was set to 4500 V with

an end-plate offset of)400 V The nebulizer was operated

Scheme 1 1b-Hydroxytestosterone.

Fig 1 HPLC of testosterone oxidation products.

at 1.3 bar and the dry gas was set to 4.3 LÆh)1 at a

temperature of 200C The capillary exit was to 120 V

with a skimmer voltage of 40 V The hexapole RF was set

to 50 Vpp (volts peak to peak) to enable the detection of

smaller masses

Solid phase extraction of peaks

After detection of peaks with the diode array detector, H2O

was added using a Knauer K120 pump operated at a flow

rate of 1.6 mLÆmin)1 The flow was guided to a modified

Prospekt 2 solid phase extraction unit from Bruker/Spark

(Bruker Biospin, Rheinstetten, Germany/Spark Holland,

Emmen, the Netherlands) Peaks were automatically

trapped on 2· 10 mm SPE cartridges filled with Hysphere

GP, a cross-linked polystyrene-divinylbenzyl copolymer

(Spark Holland)

After the trapping step the cartridges were automatically

dried for 30 min under a stream of N2gas and eluted into

the NMR flow cell with CD3OD

Cryo-NMR

An Avance spectrometer equipped with a Dual Inverse

1H/13C 30-lL Cryofit Probe operated at 600.13 MHz from

Bruker BioSpin (Rheinstetten, Germany) was used for

NMR investigation The data was obtained after threefold

trapping of the peak on GP cartridges and elution with

subsequent on-line NMR analysis The analyses were

performed with three 20-lL injections, each containing

420 lg of total material (substrate plus other products) The

chromatographic separation is shown below (Fig 1) The

spectra of testosterone and 6b-hydroxytestosterone were

recorded but are not presented here Spectra of the

previously unidentified oxidation product were recorded

eluting at 12.1 min, which was subsequently identified as

1b-hydroxytestosterone using the LC-NMR data discussed

below

The trapped product was eluted in a mixture of

d6-methanol and D2O with small amounts of residual

CH3CN present The temperature was controlled at

25 ± 0.1C Chemical shifts were referenced to the water

resonance at 4.88 p.p.m at 25C The 1D spectrum utilized

double presaturation to minimize any residual water and

methanol signals A total of 65 536 complex data points

were recorded with a sweep width of 5531 Hz and 32 scans

The data was processed with a line broadening of 0.3 Hz

Two-dimensional techniques (1H-1H COSY, 1H-1H

NOESY, and 1H-13C HSQC) were also used for the

structure elucidation of the trapped compounds The

parameters for the phase sensitive (States-TPPI mode)

1H-1H COSY spectra with water suppression were: spectral

width, 5531 Hz, 4096 complex data points, relaxation

delay 2 s, and eight scans for each of the 512 increments

The same parameters were used for phase sensitive1H-1H

NOESY with H2O suppression on the water signal except

for the number of scans (32), the number of data points

(2 k) and number of increments (256) The mixing time

was 500 ms The1H-13C HSQC experiment was acquired

in the phase sensitive mode with sensitivity enhancement,

echo/anti-echo-TPPI gradient selection and adiabatic

car-bon decoupling during evolution and acquisition [28–30]

Further parameters were: spectral width of 5531 Hz, 4096 data points in the1H dimension, 25 000 Hz with 256 data points in the13C dimension and a relaxation delay of 2 s The data was processed using BrukerXWINNMR software

on an SGI workstation (Silicon Graphics, Mountain View,

CA, USA) The data was zero-filled in the acquisition dimension and linear prediction was applied in the indirect dimension

Results

HPLC of testosterone oxidation products The chromatogram acquired at 244 nm (Fig 1) showed a number of UV absorbing peaks eluting at shorter retention times when compared with the major peak (tR¼ 23 min), which can be easily assigned to the substrate testosterone This indicates the presence of more polar components at much lower concentrations Several of these were known because of their coelution with standards in this and previous work However, the peak eluted at 12.1 min did not correspond to any of the available standards in our collection (2a-, 2b-, 6a-, 6b-, 11b-, 15b-, 16a- or 16b-hydroxytestosterone or androstenedione), and the sample was submitted for HPLC-solid phase extraction-cryoprobe NMR/time-of-flight MS analysis

Mass spectrometry Preliminary HPLC-electrospray MS experiments indicated

an [M + H]+ion at m/z 305, corresponding to a mono-hydroxylated testosterone product The result was con-firmed in the HPLC-MS-NMR work with the MicroTOF instrument, yielding MH+ at m/z 305.2080 (theoretical m/z for C19H29O3305.2111) (Fig 2)

NMR The total amount of the product estimated to have been collected for the analysis is
6 lg The 1D1H spectrum was devoid of impurities (Fig 3) The carbinol peak of interest was noted at d 3.95 p.p.m., observed as a multiplet The COSY spectrum (Fig 4) was very informative The carbinol proton of interest (d 3.95 p.p.m.) was coupled to two protons in the d 2.5 p.p.m region, indicating that the hydroxylation was at either C-1 or C-7, i.e the carbinol is coupled to either an H-2 or H-6 proton The lack of coupling to the H-8 proton (d 1.69 p.p.m.) indicates that the proton can only be at C-1

The HSQC spectrum (Fig 5) allowed complete assign-ment of all proton-attached13C signals, confirming the basic structure The resulting information is presented in Table 1 The NOESY spectrum (Fig 6) clearly indicates that the added hydroxyl group at C-1 must be b The H-1 carbinol proton clearly shows correlation peaks with protons established as C-2 (d 2.53, 2.46 p.p.m.), C-9 (d 1.15 p.p.m.) and the equatorial positioned proton C-11 (d 2.07 p.p.m.) (but not with the C-19 methyl group) Thus, the carbinol proton must be in the a-position If the proton were b, it would be expected

to show a strong interaction with the C-19 methyl, as indicated in Figs 5 and 6

One synthesis of 1b-hydroxytestosterone was found in the

literature (seven steps from dihydrotestosterone benzoate)

[31] The chemical shifts presented in that paper (1, 2, 4, 17,

18 and 19 protons assigned) are similar to ours However,

the J1a,2aand J1a,2bvalues differ Our assignments are also

consistent with those reported for 1a-hydroxytestosterone,

except for the differences at and near C-1 (http://www

unibas.ch/mdpi/ecsoc-4/a0099/a0099.htm)

Formation of 1b-hydroxytestosterone by recombinant

P450 3A4 systems

The 1b-hyroxylation of testosterone was observed in both

bacterial- and baculovirus-based P450 3A4 expression

sys-tems (Table 2) Rates of formation of the products were

similar The 1b-hydroxy product accounts for
5% of all testosterone products formed in both systems

The formation of 1b-hydroxytestosterone was also observed in human liver microsomes The ratio of 1b- to 6b-hydroxylation was less than that measured with the recombinant P450 3A4 systems due to contribution of some other P450s to 6b-hydroxylation (Table 3; see below also) The liver sample used (HL 97) had previously been shown

to have a concentration of P450 3A4 intermediate between that of high and low individuals [25]

Testosterone hydroxylation by other human P450s Several human P450s were examined for the ability to form the individual hydroxylated testosterone products, at a

Fig 2 MS of previously unidentified

testo-sterone oxidation product (A) Experimental

spectrum (B) Theoretical The molecular ion

(MH+305.2080) corresponds to the formula

C 19 H 29 O 3 (theoretical 305.2111).

Fig 3 COSY ( 1 H) NMR spectrum of

testo-sterone oxidation product Eight scans,

4096 · 512 acquisition matrix, 2-s relaxation

delay, with water suppression See Table 1 for

assignments.

single substrate concentration of 100 lM (Table 3) All of

the P450s examined produced some products, but only

P450 3A4 formed 1b-hydroxytestosterone

Discussion

The use of a combined HPLC-MS-NMR system facilitated

the characterization of one of the minor hydroxylation

products of testosterone, with an estimated total amount of

6 lg Spectroscopy alone yielded an unequivocal assign-ment of the product Traces of a product designated 1a,b-hydroxytestosterone had been reported previously in rat and mouse liver systems but only on the basis of the expected tR[11,32,33]

The 1(b)-hydroxylation of androgens has been reported previously Dodson et al [34] reported that microbial

ppm

20

30

40

50

60

70

80

ppm

ppm

125

130 6.0 5.8 2.0

Fig 4 HSQC NMR spectrum of testo-sterone oxidation product Sixty-four scans,

4096 · 256 acquisition matrix, 2-s relaxation delay, with PFG coherence selection The inset shows a cross-peak out of the range of the rest

of the scale.

Fig 5 NOESY ( 1 H)NMR spectrum of tes-tosterone oxidation product Thirty-two scans,

2048 · 256 acquisition matrix, 2-s relaxation delay, with water suppression H-1 a cross-peaks are boxed.

(Xylaria sp.) oxidation of androstenedione yielded a product

identified as the 1b-hydroxy derivative The assignment was

based largely on chemical conversion to D1,2

-dehydrotesto-serone and the optical rotation [34] This compound was

reduced to 1b-hydroxytestosterone, which has been used as

a standard or substrate in most subsequent work, either directly or by indirect comparisons Gustafsson’s group reported 1b-hydroxylation of testosterone by human fetal liver micorosomes, using the Xylaria-derived product as a standard [35,36] On the basis of our own study, it may be speculated that the enzyme responsible is P450 3A7, an enzyme closely related to P450 3A4 and fetal-specific (P450 3A4 is not expressed until after birth) [37]

Other work with the 1b-hydroxytestosterone derived from Xyleria oxidations [34] has yielded reports that it is a weak inhibitor of human placental aromatase (P450 19A1), the enzyme that oxidizes testosterone to 17b-estradiol, with

an IC50value of
1 mM[38] Another report indicated that human placental microsomes used 1b-hydroxytestosterone

30% as efficiently as testosterone or antrostenedione [35], but apparently has not been confirmed

Very recent work on possible functions of 1b-hydroxy-testosterone has appeared in a paper published after our own work was submitted [39] Porcine gonadal P450 19A1 (aromatase) converted testosterone to significant amounts

of 1b-hydroxytestosterone, as well as 19-hydroxy- and 19-oxotestosterone and 17b-estradiol [39] The assignment

of the structure was based on (a) comparison of an MS fragmentation pattern with an earlier literature spectrum [35] (going back to the original Xylaria product [34]), and (b) labilization of3H from [1b-3H]-testosterone [39] Corbin

Table 1 NMR shifts (see Figs 3–5) N/A, no protons attached;
– indicates that the shift was not identified 1b-OH, 1b-hydroxytestosterone; 2b-OH, 2b-hydroxytestosterone.

Atom

1

H

d (p.p.m.)

Multiplicity and coupling, J (Hz)

13

C

d (p.p.m.)

1

H

d (p.p.m.)

13

C

d (p.p.m.)

1

H

d (p.p.m.)

13

C

d (p.p.m.) 1a 3.95 dd 1H, J ¼ 10.2 Hz, H 2a , 4.6 Hz, H 2b 74.3 2.31 40.46 1.64 36.22

2a 2.46 dd 1H, J ¼ 4.6 Hz, H 1 , 16.1 Hz, H 2b 44.05 4.11 69.06 2.18 34.33 2b 2.53 dd 1H, J ¼ 10.2 Hz, H 1 , 16.1 Hz, H 2b 44.1 N/A N/A 2.41 34.33

9 1.15 ddd 1H, J ¼ 4.2 Hz, 10.3Hz, 16.2 Hz 55.65 1.39 50.90 0.90 54.76

12 1.09 dd 1H, J ¼ 3.9 Hz, H 11 , 13.1 Hz, H 12 37.8 0.94 34.96 0.94 32.32

Fig 6 Space-filling model of 1b-hydroxytestosterone The model was

produced with the program CHEM 3 D PRO v.5, CambridgeSoft Corp.

(Cambridge, MA, USA) Black denotes oxygen, medium gray denotes

carbon, and light gray denotes hydrogen atoms.

et al [39] postulated physiological activity of

1b-hydroxy-testosterone and showed activation of the androgen

recep-tor in two different cell lines 1b-Hydroxytestosterone was

not (enzymatically) reduced to the D4,5derivative

Interest-ingly, the 1b-hydroxylation reaction was not catalyzed by

human P450 19A1 or by any other (tissue-specific) form of

porcine P450 19A1 Although some biological activity has

been demonstrated, the relevance of 1b-hydroxytestosterone

to human physiology is not clear at this point

The biological properties of 1b-hydroxytestosterone,

although speculated (see above), are currently unknown

It is of interest to note that almost all of the P450

3A4-catalyzed hydroxylations of testosterone are on the b face

This information is of interest in considerations of the

steroselectivity of P450 3A4 and general considerations

about the juxtaposition of the substrate in the active site,

particularly in predicting sites and rates of P450 3A4

reactions deals with models based on chemical reactivity

The concept has often been proposed that P450 3A4 has a

relatively open active site and that reactions are influenced

largely by the chemical lability of C-H bonds [40,41]

However, the striking stereochemical selectivity at each of

the several hydroxylation positions would appear to argue

against this and in favor of a relatively large but organized

active site

Acknowledgements

The authors thank M V Martin and C G Turvy for preparing

bacterial membranes This work was supported in part by United States

Public Health Service grants R01 CA90426, P30 ES00267, and T32

ES07028.

References

1 Palmer, G & Reedijk, J (1992) Nomenclature of electron-transfer proteins: recommendations 1989 J Biol Chem 267, 665–677.

2 Dorfman, R., Cook, J.W & Hamilton, J.B (1939) Conversion by the human of the testis hormone, testosterone, into the urinary androgen, androsterone J Biol Chem 130, 285–295.

3 Ryan, K.J (1958) Conversion of androstenedione to estrone by placental microsomes Biochim Biophys Acta 27, 658–662.

4 Kagawa, N & Waterman, M.R (1995) Regulation of steroido-genic and related P450s In Cytochrome P450: Structure, Mechanism, and Biochemistry, 2nd edn (Ortiz de Montellano, P.R., ed.), pp 419–442 Plenum Press, New York.

5 Guengerich, F.P (2003) Cytochrome P450s, drugs, and diseases Mol Interventions 3, 8–18.

6 Conney, A.H., Levin, W., Jacobsson, M & Kuntzman, R (1969) Specificity in the regulation of the 6b, 7a, and 16a-hydroxylation

of testosterone by rat liver microsomes In Microsomes and Drug Oxidations (Gillette, J.R., ed.), pp 279–301 Academic Press, New York.

7 Wood, A.W., Ryan, D.E., Thomas, P.E & Levin, W (1983) Regio- and stereoselective metabolism of two C19 steroids by five highly purified and reconstituted rat hepatic cytochrome P-450 isozymes J Biol Chem 258, 8839–8847.

8 van der Hoeven, T (1984) Assay of hepatic microsomal testo-sterone hydroxylases by high-performance liquid chromatogra-phy Anal Biochem 138, 57–65.

9 Dutton, D.R., McMillen, S.K., Sonderfan, A.J., Thomas, P.E & Parkinson, A (1987) Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme: isozyme interaction Arch Biochem Biophys 255, 316–328.

10 Sonderfan, A.J., Arlotto, M.P & Parkinson, A (1989) Identifi-cation of the cytochrome P-450 isozymes responsible for testo-sterone oxidation in rat lung, kidney, and testis: evidence that cytochrome P-450a (P450IIA1) is the physiologically important testosterone 7a-hydroxylase in rat testis Endocrinology 125, 857–866.

11 Purdon, M.P & Lehman-McKeeman, L.D (1997) Improved high-performance liquid chromatographic procedure for the separation and quantification of hydroxytestosterone metabolites.

J Pharmacol Toxicol Methods 37, 67–73.

12 Gustafsson, J.A & Lisboa, B.P (1968) Biosynthesis of 6-beta-hydroxytestosterone from testosterone by human fetal liver microsomes Steroids 11, 555–563.

13 Lisboa, B.P & Gustafsson, J.A (1969) Studies on the metabolism

of steroids in the foetus: biosynthesis of 6a-hydroxytestosterone in the human foetal liver Biochem J 115, 583–586.

14 Yaffe, S.J., Rane, A., Sjoqvist, F., Boreus, L.O & Orrenius, S (1970) The presence of a monooxygenase system in human fetal liver microsomes Life Sci II, 1189–1200.

Table 2 Hydroxylation of testosterone by recombinant human P450 3A4 systems and human liver microsomes The range of substrate concentrations used in most cases was 25–400 l M

Product

E coli membranes Baculovirus microsomes Human liver microsomes

k cat (min)1) K m (l M ) k cat /K m k cat (min)1) K m (l M ) k cat /K m k cat (min)1)a K m (l M ) k cat /K m

1b-OH 4.1 ± 0.1 10 ± 2 0.40 7.1 ± 0.3 17 ± 2 0.41 1.9 ± 0.1 55 ± 9 0.035 2b-OH 11 ± 1 49 ± 6 0.23 14 ± 4 44 ± 4 0.30 12 ± 1 170 ± 40 0.072 6b-OH 78 ± 2 26 ± 3 3.0 78 ± 3 23 ± 2 3.4 88 ± 5 90 ± 10 0.98 15b-OH 3.0 ± 0.2 41 ± 12 0.072 7.1 ± 0.2 32 ± 3 0.22 8.4 ± 0.8 81 ± 20 0.10

a Based on total P450.

Table 3 Testosterone hydroxylation by various human P450 enzymes.

Assays were done (in triplicate) with bacterial membranes

(
bicistro-nic) containing P450 and NADPH-P450 reductase [26] using a single

testosterone concentration of 100 l M
– Indicates rate < 0.1 min)1.

P450

Rate (min)1)

1b-OH 2b-OH 6b-OH 15b-OH

2D6 – 0.31 ± 0.01 0.91 ± 0.01 –

3A4 4.8 ± 0.1 11 ± 0.1 83 ± 1 5.0 ± 0.1

15 Kremers, P., Beaune, P., Cresteil, T., de Graeve, J., Columelli, S.,

Leroux, J.P & Gielen, J.E (1981) Cytochrome P-450

mono-oxygenase activities in human and rat liver microsomes Eur J.

Biochem 118, 599–606.

16 Cresteil, T., Beaune, P., Kremers, P., Flinois, J.P & Leroux, J.P.

(1982) Drug-metabolizing enzymes in human foetal liver: partial

resolution of multiple cytochromes P450 Pediatr Pharmacol.

(New York) 2, 199–207.

17 Distlerath, L.M & Guengerich, F.P (1987) Enzymology of

human liver cytochromes P-450 In Mammalian Cytochromes

P-450, Vol 1 (Guengerich, F.P., ed.), pp 133–198 CRC Press,

Boca Raton, Florida.

18 Guengerich, F.P., Martin, M.V., Beaune, P.H., Kremers, P.,

Wolff, T & Waxman, D.J (1986) Characterization of rat and

human liver microsomal cytochrome P-450 forms involved in

nifedipine oxidation, a prototype for genetic polymorphism in

oxidative drug metabolism J Biol Chem 261, 5051–5060.

19 Waxman, D.J., Attisano, C., Guengerich, F.P & Lapenson, D.P.

(1988) Cytochrome P-450 steroid hormone metabolism catalyzed

by human liver microsomes Arch Biochem Biophys 263, 424–

436.

20 Kitada, M., Kamataki, T., Itahashi, K., Rikihisa, T & Kanakubo,

Y (1987) Significance of cytochrome P-450 (P-450 HFLa) of

human fetal livers in the steroid and drug oxidations Biochem.

Pharmacol 36, 453–456.

21 Wrighton, S.A., Brian, W.R., Sari, M.A., Iwasaki, M.,

Guenge-rich, F.P., Raucy, J.L., Molowa, D.T & VandenBranden, M.

(1990) Studies on the expression and metabolic capabilities of

human liver cytochrome P450IIIA5 (HLp3) Mol Pharmacol 38,

207–213.

22 Guengerich, F.P (1995) Human cytochrome P450 enzymes In

Cytochrome P450: Structure, Mechanism, and Biochemsitry (Ortiz

de Montellano, P.R., ed.), pp 473–535 Plenum Press, New York.

23 Evans, W.E & Relling, M.V (1999) Pharmacogenomics:

trans-lating function genomics into rational therapeutics Science 286,

487–491.

24 Guengerich, F.P (2001) Analysis and characterization of enzymes

and nucleic acids In Principles and Methods of Toxicology (Hayes,

A.W., ed.), pp 1625–1687 Taylor & Francis, Philadelphia.

25 Guengerich, F.P (1988) Oxidation of 17a-ethynylestradiol by

human liver cytochrome P-450 Mol Pharmacol 33, 500–508.

26 Parikh, A., Gillam, E.M.J & Guengerich, F.P (1997) Drug

metabolism by Escherichia coli expressing human cytochromes

P450 Nat Biotechnol 15, 784–788.

27 Yamazaki, H., Nakano, M., Imai, Y., Ueng, Y.-F., Guengerich,

F.P & Shimada, T (1996) Roles of cytochrome b 5 in the oxidation

of testosterone and nifedipine by recombinant cytochrome P450

3A4 and by human liver microsomes Arch Biochem Biophys.

325, 174–182.

28 Palmer, A.G., 3rd, Cavanagh, J., Wright, P.E & Rance, M (1991)

Sensitivity improvement in proton-detected two-dimensional

heteronuclear correlation NMR spectroscopy J Mag Res 93, 151–170.

29 Kay, L.E., Keifer, P & Saarinen, T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity J Am Chem Soc 114, 10663–10665.

30 Schleucher, J., Schwendinger, M., Sattler, M., Schmidt, P., Schedletzky, O., Glaser, S.J., Sorensen, O.W & Griesinger, C (1994) A general enhancement scheme in heteronuclear multi-dimensional NMR employing pulsed field gradients J Biomol NMR 4, 301–306.

31 Sharma, P.K & Akhila, A (1991) A facile synthesis of 1b-hy-droxytestosterone Indian J Chem., Sect B 30B, 554–556.

32 Ford, H.C., Wheeler, R & Engel, L.L (1975) Hydroxylation of testosterone at carbons 1, 2, 6, 7, 15 and 16 by the hepatic microsomal fraction from adult female C57BL/6J mice Eur J Biochem 57, 9–14.

33 Halvorson, M., Greenway, D., Eberhart, D., Fitzgerald, K & Parkinson, A (1990) Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1) Arch Biochem Biophys.

277, 166–180.

34 Dodson, R.M., Kraychy, S., Nicholson, R.T & Mizuba, S (1962) Microbiological transformations IX The 1b-hydroxylation of androstenedione J Org Chem 27, 3159–3164.

35 Lisboa, B.P & Gustafsson, J.A (1968) Biosynthesis of two new steroids in the human foetal liver, 1b- and 2b-hydroxytestosterone Eur J Biochem 6, 419–424.

36 Ingelman-Sundberg, M., Rane, A & Gustafasson, J.A (1975) Properties of hydroxylase systems in the human fetal liver active

on free and sulfoconjugated steroids Biochemistry 14, 429–437.

37 Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K & Kamataki, T (1990) Fetus-specific expression of a form of cytochrome P-450 in human livers Biochemistry 29, 4430–4433.

38 Schwarzel, W.C., Kruggel, W.G & Brodie, H.J (1973) Studies on the mechanism of estrogen biosynthesis 8 The development of inhibitors of the enzyme system in human placenta Endocrinology

92, 866–880.

39 Corbin, C.J., Mapes, S.M., Marcos, J., Shackleton, C.H., Mor-row, D., Safe, S., Wise, T., Ford, J.J & Conley, A.J (2004) Paralogues of porcine aromatase cytochrome P450: a novel hydroxylase activity is associated with the survival of a duplicated gene Endocrinology 145, 2157–2164.

40 Smith, D.A & Jones, B.C (1992) Speculations on the substrate structure–activity relationship (SSAR) of cytochrome P450 enzymes Biochem Pharmacol 44, 2089–2098.

41 Singh, S.B., Shen, L.Q., Walker, M.J & Sheridan, R.P (2003) A model for predicting likely sites of CYP3A4-mediated metabolism

on drug-like molecules J Med Chem 46, 1330–1336.