doi:10.1111/j.1432-1033.2004.04339.x

Eur. J. Biochem. 271, 3962–3969 (2004) (cid:1) FEBS 2004

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

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.

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 (cid:1) 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

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].

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 (cid:1) 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

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 (cid:1) 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 (cid:1) 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)

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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). A244 measurements 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

Fig. 1. HPLC of testosterone oxidation products.

(Kastellaun, Germany). Testosterone (Sigma-Aldrich, St. Louis, MO, USA) was used without further purification. Hydroxytestosterone standards were purchased from Stearoaloids (Newport, RI, USA).

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 37 (cid:2)C. 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.

Enzymes

HPLC (including UV)

in this

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

(5-lm particle

Microsomes were prepared [24] from a human liver sample (denoted HL 97), which had been used in some previous investigations 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 (cid:1)bicistronic mem- brane(cid:2) system) or microsomes prepared from insect cells infected with a baculovirus vector and expressing NADPH- P450 reductase in excess of 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

Chromatographic separation was carried out on a Phenomenex Prodigy ODS3 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

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. (Varying 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 37 (cid:2)C [27].

Scheme 1. 1b-Hydroxytestosterone.

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at 1.3 bar and the dry gas was set to 4.3 LÆh)1 at a temperature of 200 (cid:2)C. 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

Further parameters were: spectral width of 5531 Hz, 4096 data points in the 1H dimension, 25 000 Hz with 256 data points in the 13C dimension and a relaxation delay of 2 s. The data was processed using Bruker XWINNMR 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

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 N2 gas and eluted into the NMR flow cell with CD3OD.

Cryo-NMR

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 (tR ¼ 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 C19H29O3 305.2111) (Fig. 2).

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 the recorded but are not presented here. Spectra of 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 total amount of the product estimated to have been collected for the analysis is (cid:1) 6 lg. The 1D 1H 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.

techniques

Two-dimensional

(1H-1H COSY,

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-attached 13C 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 (but not with the C-19 methyl C-11 (d 2.07 p.p.m.) 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.

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 ± 0.1 (cid:2)C. Chemical shifts were referenced to the water resonance at 4.88 p.p.m. at 25 (cid:2)C. 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. 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 sensitive 1H-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. The 1H-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].

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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 C19H29O3 (theoretical 305.2111).

similar. The 1b-hydroxy product accounts for (cid:1) 5% of all testosterone products formed in both systems.

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,2a and J1a,2b values 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).

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].

Formation of 1b-hydroxytestosterone by recombinant P450 3A4 systems

Testosterone hydroxylation by other human P450s

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

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

Fig. 3. COSY (1H) 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.

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ppm

20

30

40

50

ppm

60

70

125

80

130

6.0

5.8

ppm

2.0

4.0

3.5

3.0

2.5

1.5

1.0 ppm

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.

single substrate concentration of 100 lM (Table 3). All of the P450s examined produced some products, but only P450 3A4 formed 1b-hydroxytestosterone.

Fig. 5. NOESY (1H)NMR spectrum of tes- tosterone oxidation product. Thirty-two scans, 2048 · 256 acquisition matrix, 2-s relaxation delay, with water suppression. H-1a cross- peaks are boxed.

Discussion

products of testosterone, with an estimated total amount of (cid:1) 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 [34] reported that microbial

previously. Dodson et al.

The use of a combined HPLC-MS-NMR system facilitated the characterization of one of the minor hydroxylation

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

1b-OH 2b-OH Testosterone

1H d (p.p.m.)

13C d (p.p.m.)

1H d (p.p.m.)

13C d (p.p.m.)

1H d (p.p.m.)

13C d (p.p.m.)

Atom Multiplicity and coupling, J (Hz)

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 IC50 value of (cid:1) 1 mM [38]. Another report indicated that human placental microsomes used 1b-hydroxytestosterone (cid:1) 30% as efficiently as testosterone or antrostenedione [35], but apparently has not been confirmed.

1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12 12b 13 14 15a 15b 16a 16b 17 18 19 3.95 – 2.46 2.53 N/A 5.75 N/A 2.31 2.54 1.00 1.90 1.69 1.15 N/A 2.07 1.55 1.09 1.84 N/A 0.98 1.60 1.31 1.98 1.47 3.56 0.78 1.24 dd 1H, J ¼ 10.2 Hz, H2a, 4.6 Hz, H2b – dd 1H, J ¼ 4.6 Hz, H1, 16.1 Hz, H2b dd 1H, J ¼ 10.2 Hz, H1, 16.1 Hz, H2b N/A – N/A m 1H m 1H m 1H m 1H m 1H ddd 1H, J ¼ 4.2 Hz, 10.3Hz, 16.2 Hz N/A m 1H m 1H dd 1H, J ¼ 3.9 Hz, H11, 13.1 Hz, H12 m 1H N/A m 1H m 1H m 1H m 1H m 1H dd 1H, J ¼ 8.5 Hz s 3H s 3H 74.3 – 44.05 44.1 – 123.55 – 34.3 34.3 33.7 33.7 37.35 55.65 – 24.20 24.20 37.8 37.8 – 51.65 24.1 24.1 30.2 30.2 82.2 11.4 13.25 2.31 1.48 4.11 N/A N/A 5.70 N/A 2.20 2.51 1.09 1.77 1.90 1.39 N/A 1.71 1.15 0.94 1.92 N/A 0.96 1.51 1.27 1.88 1.36 3.49 0.71 1.15 40.46 40.46 69.06 N/A – 119.33 – 33.18 33.18 37.08 37.08 30.55 50.90 – 22.89 22.89 34.96 34.96 – 51.03 23.57 23.57 30.60 30.60 81.45 11.36 22.88 1.64 2.02 2.18 2.41 N/A 5.64 N/A 2.23 2.40 1.01 1.77 1.59 0.90 N/A 1.56 1.43 0.94 1.81 N/A 0.93 1.56 1.26 1.91 1.38 3.50 0.72 1.18 36.22 36.22 34.33 34.33 – 123.84 – 33.12 33.12 37.17 37.17 36.19 54.76 – 21.30 21.30 32.32 32.32 – 51.09 23.78 23.78 30.48 30.48 81.54 11.39 17.48

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 of 3H from [1b-3H]-testosterone [39]. Corbin

(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

Fig. 6. Space-filling model of 1b-hydroxytestosterone. The model was produced with the program CHEM3D PRO v.5, CambridgeSoft Corp. (Cambridge, MA, USA). Black denotes oxygen, medium gray denotes carbon, and light gray denotes hydrogen atoms.

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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 lM.

E. coli membranes Baculovirus microsomes Human liver microsomes

Product kcat (min)1) Km (lM) kcat/Km kcat (min)1) Km (lM) kcat/Km kcat (min)1)a Km (lM) kcat/Km

a Based on total P450.

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

References

Table 3. Testosterone hydroxylation by various human P450 enzymes. Assays were done (in triplicate) with bacterial membranes ((cid:1)bicistro- nic(cid:2)) containing P450 and NADPH-P450 reductase [26] using a single testosterone concentration of 100 lM. (cid:1)–(cid:2) Indicates rate < 0.1 min)1.

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4. Kagawa, N. & Waterman, M.R. (1995) Regulation of steroido- In Cytochrome P450: Structure, genic and related P450s. 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. 1A1 1A2 1B1 2C9 2D6 3A4 – – – – – 4.8 ± 0.1 – – – – 0.31 ± 0.01 11 ± 0.1 1.1 ± 0.1 6.7 ± 0.3 7.7 ± 0.6 0.93 ± 0.03 0.91 ± 0.01 83 ± 1 – – – – – 5.0 ± 0.1

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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 D4,5 derivative. 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.

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.

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.

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.

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