Báo cáo khoa học: CYP7B1-mediated metabolism of dehydroepiandrosterone and 5a-androstane-3b,17b-diol – potential role(s) for estrogen signaling

CYP7B1-mediated metabolism of dehydroepiandrosterone and 5a-androstane-3b,17b-diol – potential role(s) for estrogen signaling Hanna Pettersson1, Lisa Holmberg1, Magnus Axelson2 and Maria Norlin1

1 Department of Pharmaceutical Biosciences, Division of Biochemistry, University of Uppsala, Sweden 2 Department of Clinical Chemistry, Karolinska Hospital, Stockholm, Sweden

Keywords cytochrome P450; estrogen receptor; hydroxylase; sex hormone; steroid metabolism

Correspondence M. Norlin, Department of Pharmaceutical Biosciences, Division of Biochemistry, University of Uppsala, Box 578, S-751 23 Uppsala, Sweden Fax: +46 18 558 778 Tel: +46 18 471 4331 E-mail: maria.norlin@farmbio.uu.se

(Received 19 December 2007, revised 13 February 2008, accepted 14 February 2008)

doi:10.1111/j.1742-4658.2008.06336.x

CYP7B1, a cytochrome P450 enzyme, metabolizes several steroids involved in hormonal signaling including 5a-androstane-3b,17b-diol (3b-Adiol), an estrogen receptor agonist, and dehydroepiandrosterone, a precursor for sex hormones. Previous studies have suggested that CYP7B1-dependent metab- olism involving dehydroepiandrosterone or 3b-Adiol may play an impor- tant role for estrogen receptor b-mediated signaling. However, conflicting data are reported regarding the influence of different CYP7B1-related steroids on estrogen receptor b activation. In the present study, we investi- gated CYP7B1-mediated conversions of dehydroepiandrosterone and 3b-Adiol in porcine microsomes and human kidney cells. As part of these studies, we compared the effects of 3b-Adiol (a CYP7B1 substrate) and 7a-hydroxy-dehydroepiandrosterone (a CYP7B1 product) on estrogen receptor b activation. The data obtained indicated that 3b-Adiol is a more efficient activator, thus lending support to the notion that CYP7B1 cataly- sis may decrease estrogen receptor b activation. Our data on metabolism indicate that the efficiencies of CYP7B1-mediated hydroxylations of dehy- droepiandrosterone and 3b-Adiol are very similar. The enzyme catalyzed both reactions at a similar rate and the Kcat ⁄ Km values were in the same order of magnitude. A high dehydroepiandrosterone ⁄ 3b-Adiol ratio in the incubation mixtures, similar to the ratio of these steroids in many human tissues, strongly suppressed CYP7B1-mediated 3b-Adiol metabolism. As the efficiencies of CYP7B1-mediated hydroxylation of dehydroepiandrosterone and 3b-Adiol are similar, we propose that varying steroid concentrations may be the most important factor determining the rate of CYP7B1-medi- ated metabolism of dehydroepiandrosterone or 3b-Adiol. Consequently, tissue-specific steroid concentrations may have a strong impact on CYP7B1-dependent catalysis and thus on the levels of different CYP7B1- related steroids that can influence estrogen receptor b signaling.

and metabolizes several steroids involved in hormonal signaling and other processes. Substrates for CYP7B1 include 5a-androstane-3b,17b-diol (3b-Adiol), an estro- gen receptor (ER) agonist, and dehydroepiandrosterone (DHEA), an essential precursor for androgens and

The steroid hydroxylase CYP7B1, a member of the cyto- chrome P450 enzyme family, has attracted increasing interest in recent years due to its multiple reported roles for key events in cellular physiology [1–9]. CYP7B1 is widely expressed in tissues of human and other species

Abbreviations 3b-Adiol, 5a-androstane-3b,17b-diol; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; ER, estrogen receptor; ERE, estrogen response element; HEK, human embryonic kidney.

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estrogens. In addition to its role as sex hormone precur- sor, DHEA is reported to affect a number of processes in various tissues, including central nervous system func- tion, immune system, lipid profiles and cellular growth [8–13].

The action of CYP7B1 in various tissues results in the formation of 7- and ⁄ or 6-hydroxymetabolites, which can be eliminated from the cell, thereby decreas- ing intracellular levels of CYP7B1 substrates. Some reports, however, suggest that CYP7B1-mediated catal- ysis might lead to formation of active hormones with impact on several processes, including cellular growth, immune system and brain function [7–9]. In view of its high catalytic activity towards sex hormone precursors, as well as towards certain estrogens, the action of CYP7B1 may affect hormonal signaling in several ways [5,7,14].

from mediated formation of hydroxymetabolites DHEA and 3b-Adiol was analyzed in microsomes prepared from various tissues obtained from pigs of dif- ferent ages. Because this is the first study on CYP7B1- mediated 3b-Adiol metabolism in the pig, GC ⁄ MS analysis was carried out to determine the structure of the 3b-Adiol hydroxymetabolite formed. Previous stud- ies report the formation of both 6- and 7-hydroxyme- tabolites from 3b-Adiol [17–19]. The GC ⁄ MS analysis carried out in the present study showed that the main product formed from 3b-Adiol in pig liver is 5a-andro- stane-3b,7a,17b-triol (for GC ⁄ MS chromatogram, see Supplementary material). Only minor amounts of a 6-hydroxy derivative were observed. Also, trace amounts of 5a-androstane-3b,7b,17b-diol were detected by GC ⁄ MS. From present and previous findings, it appears that CYP7B1 is capable of carrying out both 6- and 7-hydroxylation of 3b-Adiol. It is possible that the main product formed from 3b-Adiol may vary in different species and different cellular environments.

Recent studies have indicated that CYP7B1-depen- dent metabolism may play an important role for ERb- mediated signaling. The manner in which CYP7B1 affects this, however, remains unclear. The results of some studies indicate that CYP7B1-mediated catalysis leads to formation of an ERb ligand, whereas other studies have proposed that CYP7B1 catalysis instead counteracts ERb ligand activation [5,7]. As ERb is considered to affect a wide range of biological systems throughout the body, events regulating its function are of considerable interest [5,15].

The results of the analyses of porcine DHEA and 3b-Adiol hydroxylation in liver, kidney and lung are shown in Table 1. The data indicate marked tissue- specific differences between younger and older ani- mals. In liver, 7a-hydroxylation of both substrates increased with age, whereas the rate of hydroxylation decreased with age in the kidney. These findings of age-dependent differences are in agreement with previ- ous data on DHEA metabolism [6] and indicate a in similar pattern for 7a-hydroxylation of 3b-Adiol pig tissues.

that

In a separate set of experiments, microsomes were prepared from tissues of a 2.5-year-old boar to exam- ine male reproductive tissues in an older individual. In this animal, hepatic hydroxylase activities towards DHEA and 3b-Adiol were 591 and 659 pmolÆmg)1 microsomal protein · min, respectively. Hydroxylase activities in testicle and prostate were approximately 5% of testicle and in liver. In liver, kidney, prostate, the catalytic activities towards DHEA and 3b-Adiol were of the same order of magnitude (data not shown).

In the present study, we used porcine tissues and to investigate and compare human kidney cells CYP7B1-mediated conversions of DHEA and 3b-Adi- ol, both of which are reported to affect ERb activa- tion. The pig is a useful animal model for studies of CYP7B1-mediated catalytic reactions due to the high CYP7B1 content in pig tissues and the closer similarity of porcine and human cytochrome P450 enzymes com- pared with rodent isoforms [16]. Our findings indicate that CYP7B1 action is subject to age- and tissue- specific differences. Furthermore, the data indicate that tissue-specific steroid concentrations may have a large impact on CYP7B1-dependent catalysis and thus on the levels of different CYP7B1-related steroids that can influence ERb signaling.

CYP7B1-mediated activities towards DHEA and 3b-Adiol in different sexes

Results

Tissue- and age-specific differences in porcine CYP7B1-mediated hydroxylase activities towards DHEA and 3b-Adiol

In previous studies, we described CYP7B1-mediated 7a-hydroxylation of DHEA in microsomes from pig liver and kidney [3,6]. In the current study, CYP7B1-

We also compared CYP7B1-mediated hydroxylation of DHEA and 3b-Adiol in tissues from male and female pigs. As the analysis of metabolism in pig tissues is often carried out with organs from castrated pigs, available from slaughterhouses, we also included cas- trated male pigs in these studies. The results from incubations with microsomes from kidneys, livers and

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Table 1. Difference in CYP7B1-mediated hydroxylase activity between adult male pig and male piglet. The animals were approxi- mately 10 months (adult) and 5 days (piglet) of age. Microsome fractions were prepared from tissues and the catalytic activity was measured by incubation with radiolabeled substrates and analysis by RP-HPLC, as described in the Experimental procedures. Hydrox- ylase activity is displayed as pmolÆmg)1 of microsomal pro- tein · min ± SD (n = 7 samples). Incubations without NADPH were used as negative controls. Product formation in negative controls corresponded to an activity of £ 10 pmolÆmg)1 protein · min. A comparison of hydroxyproduct formation in tissues obtained from piglets and sexually mature animals indicates marked tissue-spe- cific differences between younger and older animals. There is a sig- nificant difference in hydroxyproduct formation between older and younger animals in the kidney and liver for both substrates. In the liver, the activity is higher in older animals than in piglets whereas, in the kidney, the activity decreased with age (P < 0.05, one-way ANOVA). BLD, below limit of detection.

Table 2. CYP7B1-mediated hydroxylase activity towards DHEA and 3b-Adiol in different sexes. The animals were approximately 10 months of age. Microsome fractions were prepared from tis- sues and the catalytic activity was measured by incubation with radiolabeled substrates and analysis by RP-HPLC, as described in in the Experimental procedures. Experiments were carried out three sets of triplicate incubations based on material from three dif- ferent individuals per group. Hydroxylase activity is displayed as pmolÆmg)1 of microsomal protein · min ± SD (n = 9 samples). Incubations without NADPH were used as negative controls. Prod- uct formation in negative controls corresponded to an activity of £ 10 pmolÆmg)1 protein · min. There is no significant difference between the different groups for either of the two substrates. In lung tissue, there is a significant difference between the hydroxy- lase activity towards DHEA and 3b-Adiol. There is no significant different between the hydroxylase activity towards the two sub- strates in liver tissue (P < 0.05, two-way ANOVA). BLD, below limit of detection.

pmolÆmg)1 protein · min

pmolÆmg)1 protein · min

Liver

Kidney

Lung

Liver

Kidney

Lung

Substrate

Substrate

Adult male Male piglet Adult male Male piglet

441 ± 127 244 ± 158 443 ± 66 76 ± 40

BLD 130 ± 64 15 ± 10 62 ± 31

82 ± 10 104 ± 46 33 ± 19 43 ± 22

DHEA DHEA 3b-Adiol 3b-Adiol

Male castrated Female Male Male castrated Female Male

290 ± 73 254 ± 24 441 ± 127 448 ± 111 309 ± 30 443 ± 66

BLD BLD BLD 12 ± 10 16 ± 5 15 ± 10

80 ± 15 78 ± 37 82 ± 10 39 ± 8 31 ± 11 33 ± 19

DHEA DHEA DHEA 3b-Adiol 3b-Adiol 3b-Adiol

lungs obtained from normal males, females (gilts) and castrated males are shown in Table 2. No significant differences between sexes were observed for 7a-hydrox- ylation of DHEA or 3b-Adiol in any of the tissues analyzed. Also, formation of 7a-hydroxyproducts from DHEA and 3b-Adiol was found to be of a similar magnitude in normal and castrated males.

DHEA 7a-hydroxylation in the present study is in agreement with our previously reported data on this reaction in purified protein fractions [3]. From the present study, it may be concluded that the efficien- cies of CYP7B1-mediated 7a-hydroxylation of DHEA and 3b-Adiol appear to be very similar.

Kinetic analysis of CYP7B1-mediated metabolism of DHEA and 3b-Adiol

CYP7B1-mediated activities towards DHEA and 3b-Adiol in human kidney (HEK293) cells

are

summarized

regression fitting of

HEK293 cells are known to have a high endogenous CYP7B1 expression, although, to our knowledge, no data have been reported on 3b-Adiol metabolism in these cells. In the present study, we examined the endogenous hydroxylase activities towards 3b-Adiol and DHEA in HEK293 cells. The endogenous 7a- hydroxylase activity towards DHEA in HEK293 cells in these experiments was approximately 300 pmolÆmg)1 cell protein · 24 h. Analysis of 3b-Adiol metabolism indicated the formation of more than one hydroxy- metabolite in HEK293 cells. GC ⁄ MS analysis of meta- bolites demonstrated the formation of both 6- and 7-hydroxy derivatives under these conditions. The mass spectrum of the main metabolite formed from 3b-Adi- ol in HEK293 cell cultures was consistent with a 6-hydroxy derivative, i.e. 5a-androstane-3b,6n,17b-triol

To study which of these two CYP7B1 substrates is most efficiently metabolized and to obtain more information regarding which reaction might be of the most physiological importance, we determined kinetic parameters for 7a-hydroxylation of DHEA and 3b- Adiol using pig liver microsomes. Kinetic parameters, including Kcat and apparent Km values for these reac- tions, in Table 3. Data were obtained by incubation with various amounts of DHEA or 3b-Adiol as described in the Experimental procedures and the parameters were determined by nonlinear the data to the Michaelis–Menten equation or calculated from dou- ble reciprocal plots. The catalytic efficiencies for hydroxylation of DHEA and 3b-Adiol as described by the Kcat ⁄ Km values were in same order of for porcine magnitude

(Table 3). The Km value

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in pig liver. Experiments to determine kinetic Table 3. Kinetic parameters for CYP7B1-mediated 7a-hydroxylation of DHEA and 3b-Adiol parameters were carried out by assay of catalytic activities in adult pig liver microsomes with 1, 2, 3, 4 or 5 lM of substrate (DHEA or 3b- Adiol). Parameters were determined by nonlinear regression fitting of the data to the Michaelis–Menten equation or calculated from double reciprocal plots. Experiments were performed in triplicates or quadruplicates. Catalytic activity was measured by RP-HPLC as described in the Experimental procedures.

Km (lM)

Vmax (pmolÆmg)1 protein · min)

Kcat (nmolÆnmol)1 P450 · min)

Kcat ⁄ Km

5 3

1000 500

2 1

0.4 0.33

7a-hydroxylation of DHEA 7a-hydroxylation of 3b-Adiol

(for GC ⁄ MS chromatogram, see Supplementary mate- rial). We were unable to distinguish between a or b orientation of the 6-hydroxygroup, as indicated by the letter n (Greek letter ‘xi’, which corresponds to ‘x’ in our alphabet, representing an unknown configuration). Small amounts of 5a-androstane-3b,7a,17b-triol were also formed. Endogenous hydroxylase activity towards 3b-Adiol in HEK293 cells was approximately 450 pmolÆmg)1 cell protein · 24 h.

the presence of increasing amounts of unlabeled 3b- Adiol. The results of these experiments are shown in inhibited DHEA hydroxylation by Fig. 1. 3b-Adiol approximately 60–70% when both steroids were pres- ent at equimolar concentrations. Further, a 10-fold higher concentration of 3b-Adiol than of DHEA in the incubation mixture resulted in the suppression of DHEA hydroxylation by 80%. In HEK293 cells, the suppressive effect of 3b-Adiol of DHEA metabolism was statistically significant also at a 10-fold lower con- centration of 3b-Adiol than of DHEA in the incuba- tion mixture (Fig. 1).

120

3β-Adiol as inhibitor of DHEA hydroxylation in pig liver microsomes 3β-Adiol as inhibitor of DHEA hydroxylation in HEK293 cells

100

*

80

) l o r t n o c f o %

A E H D

60

*

We also examined the activities towards 3b-Adiol and DHEA in HEK293 cells transfected with an expression vector containing human CYP7B1 cDNA. As expected, overexpression of human CYP7B1 sig- nificantly increased the hydroxylation of both 3b-Adi- ol and DHEA in HEK293 cultures. HEK293 cells transfected with the CYP7B1 expression vector dis- played three- to six-fold higher hydroxylase activity than cells transfected with empty vector (data not shown). GC ⁄ MS analysis of 3b-Adiol metabolites cultures also formed in CYP7B1-transfected cell showed formation of both 6- and 7-hydroxy deriva- tives with 5a-androstane-3b,6n,17b-triol as the main metabolite.

*

40

* *

f o e t a R

20

0

( n o i t a l y x o r d y h

[0.1/1]

[1/1]

[10/1]

Effects of 3b-Adiol on CYP7B1-mediated hydroxylation of DHEA

Control [0/1]

Concentration ratio [inhibitor/substrate]

incubations without added inhibitor

[0 ⁄ 1]. 3b-Adiol

As the studies indicated that the rate of hydroxylation and affinity of CYP7B1 for DHEA and 3b-Adiol are similar, we conducted experiments to study how the concentration of 3b-Adiol would affect the CYP7B1- mediated metabolism of DHEA and vice versa. These experiments were carried out with both porcine micro- somes and human HEK293 cells. The main reason for our interest in the effects of steroid concentrations on the rate of metabolic reactions is that the concentra- tions of these steroids vary considerably in different tissues and species. For example, the concentration of DHEA in human prostate is reported to be approxi- mately 10-fold higher than that of 3b-Adiol [20].

Fig. 1. Effects of 3b-Adiol on DHEA hydroxylation in pig liver micro- somes (black bars) and HEK293 cells (grey bars). Experiments were carried out with a constant concentration (52 lM) of radiolabeled DHEA to study the inhibitory effect by varying levels of 3b-Adiol on DHEA hydroxylation. Concentrations are shown as ratio between added inhibitor (3b-Adiol) and substrate (DHEA). Analysis of the catalytic activity was carried out as described in the Experimental procedures. Catalytic activity is shown as percent of the rate of DHEA hydroxylation in controls (± SD) (n = 4–9). Controls consisted of inhibited DHEA hydroxylation by approximately 60–75% when both steroids were present at equimolar concentrations, and when the concen- tration of 3b-Adiol was increased to 10-fold, compared with the concentration of DHEA, the hydroxylation of DHEA decreased by approximately 80%. *Statistically significant suppression compared with control (P < 0.05, one-way ANOVA).

In the first set of experiments, we studied the effect of 3b-Adiol on DHEA hydroxylation. Hydroxylation of DHEA was measured with radiolabeled DHEA in

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DHEA as inhibitor of 3β-Adiol hydroxylation in pig liver microsomes

140

DHEA as inhibitor of 3β-Adiol hydroxylation in HEK293 cells

120

l

i

*

100

80

) l o r t n o c f o %

*

60

*

40

o d A - β 3 f o e t a R

*

20

0

( n o i t a l y x o r d y h

Control [0/1]

[0.1/1]

[1/1]

[10/1]

Concentration ratio [inhibitor/substrate]

Analysis of the type of inhibition by 3b-Adiol on DHEA hydroxylation was carried out by a series of incubations with pig liver microsomes containing 1– 5 lm of DHEA in the presence of 0, 5, 10, or 20 lm unlabeled 3b-Adiol (added as inhibitor). Lineweaver– Burk and Dixon plots were constructed using linear regression fitting of the data. The data obtained indi- cated that 3b-Adiol is a mixed inhibitor of DHEA hydroxylation (for a Lineweaver–Burk plot showing inhibition by 3b-Adiol on DHEA hydroxylation, see Supplementary material). Thus, the inhibition includes competition at the active site but involves both com- petitive and uncompetitive components. The patterns of Lineweaver–Burk and Dixon plots were not consis- tent with a pure noncompetitive or uncompetitive type of inhibition (data not shown). The Ki value for the inhibition of 3b-Adiol on DHEA hydroxylation, calcu- lated from the Dixon plot, was 6 lm.

Effects of DHEA on CYP7B1-mediated 3b-Adiol hydroxylation

Fig. 2. Effects of DHEA on 3b-Adiol hydroxylation in pig liver micro- somes (black bars) and HEK293 cells (grey bars). Experiments were carried out with a constant concentration (52 lM) of radiolabeled 3b-Adiol to study the inhibitory effect by varying levels of DHEA on 3b-Adiol hydroxylation. Concentrations are shown as ratio between added inhibitor (DHEA) and substrate (3b-Adiol). Analysis of the catalytic activity was carried out as described in the Experimental procedures. Catalytic activity is shown as percent of the rate of (n = 3–9). Controls 3b-Adiol hydroxylation in controls (± SD) consisted of [0 ⁄ 1]. DHEA incubations without added inhibitor inhibited 3b-Adiol hydroxylation by approximately 70–90% when the concentration of DHEA was increased to 10-fold compared with the concentration of 3b-Adiol. *Statistically significant suppression compared with control (P < 0.05, one-way ANOVA).

DHEA hydroxylation by 3b-Adiol (6 lm; see above). Thus, it appears that 3b-Adiol may be a more efficient inhibitor of the hydroxylation of DHEA than vice versa.

We also carried out corresponding studies on the effect of different concentrations of DHEA on the 6- and 7-hydroxylation of 3b-Adiol. In these experiments, we measured 3b-Adiol hydroxylation in pig liver micro- somes and human HEK293 cells with radiolabeled 3b-adiol in the presence of increasing amounts of unla- beled DHEA (Fig. 2). The most efficient inhibition of 3b-Adiol metabolism was obtained at high amounts of DHEA, although, in experiments with HEK293 cells, statistically significant effects were observed also at lower concentrations. A 10-fold higher concentration of DHEA than of 3b-Adiol in the incubation mixture decreased the rate of 3b-Adiol hydroxylation by 70–90% (Fig. 2).

Some inhibition experiments were also carried out with DHEA-sulfate, the sulfated ester of DHEA, which is present in even higher amounts than DHEA in human plasma. However, DHEA-sulfate had no significant effect on the rate of CYP7B1-mediated hydroxylation of either DHEA or 3b-Adiol, even at 10-fold higher concentrations of DHEA-sulfate in the incubation mixture (data not shown). Testosterone, which is formed from DHEA and is present in high types, also did not affect levels in the same cell CYP7B1-mediated hydroxylase activity. It may there- fore be concluded that the steroid inhibition described in the present study is specific and not a generalized effect by related steroids of a similar structure.

Analysis of ERb activation by 3b-Adiol and 7a-hydroxy-DHEA

Previous studies indicate that ERb is activated by 3b-Adiol, whereas others report that this receptor is

Similarly, as for the inhibition of DHEA hydroxyl- ation, we carried out experiments to analyze the type of inhibition of 3b-Adiol hydroxylation by DHEA. Incubations were performed with pig liver microsomes and radiolabeled 3b-Adiol (1–5 lm) in the presence of unlabeled DHEA (0, 5, 10, or 20 lm, added as inhibi- tor). Construction of Lineweaver–Burk and Dixon plots from the data indicated that DHEA is a mixed inhibitor of 3b-Adiol hydroxylation, similar to that found for the inhibition of DHEA hydroxylation by 3b-Adiol (for a Lineweaver–Burk plot showing inhibi- tion by DHEA on 3b-Adiol hydroxylation, see Supple- mentary material). The patterns of Lineweaver–Burk and Dixon plots were not consistent with a pure non- competitive or uncompetitive type of inhibition. The Ki for inhibition of 3b-Adiol hydroxylation by DHEA was 24 lm, a higher value than the Ki for inhibition of

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*

3.5

*

n o i t a l u m

3

*

l

*

2.5

2

1.5

i t s d o f – ) l a g - β / U L R

1

0.5

0

( y t i v i t c a r e t r o p e R

Control EtOH

0.1 μM E2

1 μM E2

0.1 μM 7HD

1 μM 7HD

0.1 μM 3β-Adiol

1 μM 3β-Adiol

Fig. 3. Effects of estradiol (E2), 7a-OH- DHEA (7HD) or 3b-Adiol on an ER-respon- sive ERE luciferase reporter vector. HEK293 cells were transiently transfected with ERb (1 lgÆwell)1) and treated with steroids as described in the Experimental procedures. Data are presented as the fold stimulation compared with controls (treated with vehi- cle). *Statistically significant stimulation compared with control (P < 0.05, one-way ANOVA).

it

activated by 7a-hydroxy-DHEA [5,7]. In the present study, we compared the effects of these two steroids on ERb activation in the same experiment, using the same methodology. Receptor activation was studied by reporter assay with an ER-responsive luciferase repor- ter vector transfected in HEK293 cells overexpressed with ERb, in a similar fashion to that previously described [14,21]. This ER-responsive vector contains a strong estrogen response element (ERE) coupled to luciferase. Thus, luciferase expression levels are deter- mined by activation of ER and its subsequent binding to the ERE. ERb-transfected cells were treated with 3b-Adiol or 7a-hydroxy-DHEA in different concentra- tions and the levels of luciferase in steroid-treated cells were compared with the luciferase levels in cells treated with the same volume of vehicle (ethanol). Treatment with 17b-estradiol, a known ER agonist, was used as positive control. The results of these experiments (Fig. 3) indicate that 3b-Adiol is a more efficient ERb activator than 7a-hydroxy-DHEA.

Discussion

The concentrations of DHEA and 3b-Adiol and related steroids vary considerably between different tis- sues [10,20,22]. For example, concentrations of DHEA in tissue samples are reported to be 20-fold higher in human prostate (100 pmolÆg)1) than in muscle (5 pmolÆg)1). In general, physiological levels of 3b-Adi- ol are lower than those of DHEA in both humans and pigs, particularly in human adults where plasma con- centrations are reported to be approximately 1.5 nm for 3b-Adiol and approximately 10- to 30-fold higher for DHEA [20,22,23]. In human plasma, DHEA-sul- fate is present in higher concentrations than DHEA [23,24]. The plasma levels of these steroids are subject to developmental variation, depending on the stage of sexual maturation [10,24]. From the results of the pres- ent study, is clear that high concentrations of DHEA, but not of DHEA-sulfate, strongly suppress CYP7B1-mediated metabolism of 3b-Adiol. The cur- rent data imply that human CYP7B1-mediated metab- olism of 3b-Adiol should be comparatively lower than that of DHEA in many human tissues due to the gen- erally higher tissue concentrations of DHEA, which most likely would suppress 3b-Adiol hydroxylation during physiological conditions.

In the present study, we examined CYP7B1-mediated metabolism of 3b-Adiol and DHEA in porcine micro- somal fractions and human kidney cells. The current data indicate that the efficiencies of these two reac- tions are very similar. As the kcat ⁄ Km values are within the same order of magnitude for both sub- strates, we propose that relative formation of differ- ent CYP7B1-related steroids may be dependent on the concentration of substrate(s) present in each tis- sue. Consequently, cellular steroid levels may have a strong impact on the resulting physiological levels of CYP7B1 substrates as well as of CYP7B1-formed hydroxysteroids.

CYP7B1-mediated metabolism of DHEA and 3b- Adiol are of interest in connection with several physio- logical processes. One of these concerns the proposed role of this enzyme for ER-mediated signaling and the potential ER-mediated control of cellular growth, par- ticularly of the prostate [5,25]. However, the available data regarding the effect of CYP7B1 on ERb activa- tion are conflicting. Data obtained by Weihua et al. [5,26] in rodents indicate that 3b-Adiol binds strongly to ERb and that CYP7B1-mediated metabolism of 3b-Adiol therefore should lead to induced growth by abolishing ERb action. By contrast, Martin et al. [7],

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who studied DHEA 7a-hydroxylation in human prostate cells, report that the CYP7B1-formed product 7a-hydroxy-DHEA is a ligand of ERb and conclude that CYP7B1 metabolism therefore should activate this receptor. The contrasting findings on the role of CYP7B1 in connection with ERb action were obtained in different species using different CYP7B1 substrates and methodologies.

than in rodents and exceed, by at

Fig. 4. Suggested effects of a high DHEA ⁄ 3b-Adiol ratio (such as in prostate tissue) on 3b-Adiol-mediated ERb activation. High DHEA levels strongly suppresses CYP7B1-mediated 3b-Adiol metabolism, resulting in higher 3b-Adiol levels and increased ERb activation. The CYP7B1-mediated 7a-OH-DHEA metabolite (not shown) is most likely not formed in sufficient amounts to compete with 3b-Adiol for ERb binding.

lar DHEA ⁄ 3b-Adiol ratios, such as in human prostate, on ERb activation is outlined in Fig. 4. At high DHEA levels, CYP7B1-mediated 3b-Adiol hydroxyl- ation is strongly suppressed, resulting in higher 3b- Adiol levels and increased ERb activation (Fig. 4). The steroid suppression of CYP7B1-mediated metabolism observed in the present study is apparently not an unspecific steroid effect because our present and previ- ous data have shown that neither DHEA-sulfate, nor testosterone are able to inhibit CYP7B1-mediated catalysis [3].

In the present study, we carried out experiments to compare effects of 3b-Adiol and 7a-hydroxy-DHEA on activation of ERb in the same assay and cell type. Our experiments indicate that 3b-Adiol is a more efficient activator than 7a-hydroxy-DHEA. These data are in agreement with the findings obtained by Martin et al. [7] who observed activation of ERb by 7a-hydroxy-DHEA only at 5 lm or higher concentrations of this metabolite. in DHEA concentrations are generally higher humans least 10-fold, the concentration of 3b-Adiol in human pros- tate [22]. Even though DHEA circulates in micromolar concentrations in plasma, DHEA levels measured in human prostate tissue are reported to be approxi- mately 100 pmolÆg)1 [20,22]. Thus, the amount of 7a-hydroxy-DHEA needed for activation of ERb appears to be approximately 50- to 100-fold higher than the reported content of DHEA in human prostate tissue. The concentration of DHEA-sulfate, which can be converted to DHEA by a sulfatase, is in the same order of magnitude in prostate tissue as that of DHEA despite the abundance of DHEA-sulfate in blood [10,22]. It is therefore unlikely that the concentration of a DHEA-metabolite such as 7a-hydroxy-DHEA should reach a high enough concentration to be of physiological relevance for ERb activation, at least in this tissue. However, Weihua et al. [26] reported that very low concentrations of 3b-Adiol are able to bind ERb. Ligand-competition experiments with [125I]estra- diol indicated a Ki of 2 nm for 3b-Adiol, a value closer to the reported physiological levels of this steroid, which are in the nanomolar range [22,26,27].

From the data on the

In conclusion, the results obtained in the present study indicate that the efficiencies of DHEA and 3b- Adiol hydroxylation by CYP7B1 are similar, but that a high DHEA ⁄ 3b-Adiol ratio (similar to the ratio of these steroids in many human tissues) strongly sup- presses CYP7B1-mediated 3b-Adiol metabolism. Our data indicate that tissue-specific steroid concentrations may have a large impact on CYP7B1-dependent catal- ysis and thus on the levels of different CYP7B1-related steroids that can influence ERb-signaling.

Experimental procedures

Materials

Human embryonic kidney cells (HEK293) (ATCC CRL 1573) were purchased from ATCC (Rockville, MD, USA).

catalytic properties of CYP7B1 obtained in the present study, it is clear that high concentrations of DHEA strongly suppress CYP7B1-mediated 3b-Adiol metabolism. Although 3b- Adiol is able to compete for the active site of CYP7B1 to some degree even at a 10-fold higher DHEA con- centrations, as the present data indicate, the rate of its hydroxylation was decreased by 70–90% under these conditions. It is therefore likely that the comparatively higher DHEA content in prostate and many other tissues may increase the level of 3b-Adiol, and thus of 3b-Adiol-mediated ERb activation, by suppression of 3b-Adiol metabolism. A proposed effect of high cellu-

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DMEM (containing 1000 mgÆL)1 glucose), fetal bovine serum, antibiotics ⁄ antimycotics, non-essential amino acids and trypsin were obtained from Invitrogen (Carlsbad, CA, USA). The pCMV6 vector containing cDNA encoding for human CYP7B1 was kindly provided by D. W. Russell (University of Texas, Dallas, TX, USA). The ERb expres- sion vector and the ERE luciferase reporter vector were generous gifts from P. Chambon (Institut de ge´ ne´ tique et de biologie mole´ culaire et cellulaire, Strasbourg, France) and K. Arcaro (University of Massachusetts, MA, USA), respec- tively. 3b-Adiol, 5a-androstane-3a,17b-diol, DHEA, DHEA- sulfate, dihydrotestosterone (DHT) and hydroxysteroid dehydrogenase (from Pseudomonas testosteroni) were from Sigma Chemical Co. (St Louis, MO, USA). 3H-Labeled DHEA and DHT were from Perkin Elmer Life Sciences (Waltham, MA, USA). All remaining chemicals were of analytical grade and purchased from commercial sources.

Animals and tissue sample collection

total volume of 1 mL of 0.1 m potassium phosphate buffer containing 0.1 mm EDTA (pH 7.4) for 10 min at 37 (cid:2)C in a water bath. The reaction was quenched and extracted with 5 mL of ethyl acetate. The organic phase was col- lected, evaporated under nitrogen gas, dissolved in a small amount of acetone and applied on a silica gel TLC plate. The TLC plate was developed three times in toluene ⁄ meth- anol (90 : 10, v ⁄ v). TLC plates with unlabeled DHT, 3b-Adiol and 5a-androstane-3a,17b-diol were used as refer- ences and developed together with the sample plate. The sample TLC plate was scanned for localization of the radioactive products, using a Berthold Tracemaster 20 TLC scanner (Berthold ⁄ Frieske GmbH, Karlsruhe-Durlach, Ger- many). The reference TLC plates were exposed to iodine vapours (o ⁄ n) to visualize the steroids and the retention times of reference compounds were compared with those of the sample plate. Under these conditions, the main product formed was 3b-Adiol. The yield of 3b-Adiol under the con- ditions used was approximately 70%. Very small amounts of the 3a-hydroxyderivative, 5a-androstane-3a,17b-diol, were also formed in the reaction. The formed radioactive 3b-Adiol was extracted from the silica gel with ethyl ace- tate. The extraction procedure was repeated twice. The obtained solution of 3H-labeled 3b-Adiol was evaporated under nitrogen gas and dissolved in 100 lL of ethyl acetate. Radioactivity (c.p.m.ÆlL)1) was determined by injection of an aliquot on a RP-HPLC (125 · 4 mm LiChrosphere RP 18 column, 5 lm; Merck, Darmstadt, Germany). Elution was monitored by a Radiomatic 150TR Flow Scintillation Analyzer (Hewlett-Packard, Palo Alto, CA, USA). The solution of 3H-labeled 3b-Adiol was diluted to a working concentration of 50 000 c.p.m.ÆlL)1.

Incubations with microsomes

Liver, kidney and lung tissues from adult female, male and castrated male pigs (aged 10 months) and liver, kidney, tes- ticle and prostate tissues from an adult domestic boar (aged 2.5 years) were obtained from the Funbo-Lo¨ vsta Research Center [Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences (SLU), Ultuna, Sweden]. Liver, kidney and lung tissues from male uncas- trated piglets (aged 5 days) were a generous gift from P. Wallgren [Department of Ruminant and Porcine Dis- eases, National Veterinary Institute (SVA), Uppsala, Sweden]. All the animals were healthy and untreated at the time of euthanasia. The domestic pig is considered to reach sexual maturity at approximately 6 months of age. All organ tissue samples were stored at )80 (cid:2)C until micro- somal preparation was performed.

Microsomal preparation

The tissue samples were weighed and minced, respectively, in sucrose buffer containing 0.25 m sucrose, 10 mm Tris–Cl (pH 7.4) and 1 mm EDTA to a 20% suspension. Microsomes were prepared from the tissues according to standard meth- ods [28], and were suspended in 50 mm Tris-acetate buffer (pH 7.4) containing 20% glycerol and 0.1 mm EDTA and stored at )80 (cid:2)C until incubation. Protein contents of the mi- crosomes were assayed by the method of Lowry et al. [29]. Incubations with microsomes (0.5–1.0 mg of microsomal protein) were carried out at 37 (cid:2)C for 20 or 30 min. The substrates DHEA (52 lm, 1 lCi) or 3b-Adiol (52 lm, 200 000 c.p.m.) dissolved in 25 lL of acetone, were incu- bated with 1 lmol of NADPH in a total volume of 1 mL of 50 mm Tris-acetate buffer (pH 7.4) containing 20% glyc- erol and 0.1 mm EDTA. Incubations were performed under conditions where the enzyme was saturated with substrate. The incubations were quenched and extracted with 5 mL of ethyl acetate. The organic phase was collected and stored at )20 (cid:2)C until analysis. Incubations without NADPH were performed at the same time and used as negative controls.

Preparation of 3b-Adiol

3H-Labeled 3b-Adiol was prepared from 3H-labeled DHT (Perkin Elmer) by bioconversion using a commercially available hydroxysteroid dehydrogenase from P. testoste- roni (Sigma H8879) [30]. A mixture of labeled (100 lCi) and unlabeled (10 lg) DHT was incubated with 0.32 U of hydroxysteroid dehydrogenase and 1 lmol of NADH in a

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For incubations where steroids were added as potential inhibitors, incubations with labeled substrates were carried out as described above, except that various amounts of unlabeled 3b-Adiol, DHEA, DHEA-sulfate or testosterone were added to the incubation mixtures. For kinetic analysis, pig liver microsomes were incubated with 1, 2, 3, 4 or 5 lm of radiolabeled substrate (DHEA or 3b-Adiol) in the pres- ence of 0, 5, 10 or 20 lm of unlabeled DHEA or 3b-Adiol

H. Pettersson et al.

CYP7B1-mediated metabolism of DHEA and 3b-Adiol

Analysis of incubations with DHEA

added as inhibitors. Experiments were carried out in tripli- cates or quadruplicates and kinetic parameters were deter- mined by nonlinear regression fitting of the data to the Michaelis–Menten equation or calculated from Line- weaver–Burk and Dixon plots.

Cultures of HEK293 cells

Incubations with 3H-labeled DHEA were analyzed as previ- ously described [3,32]. The organic phase of the incubations with microsomes or HEK293 cell cultures was evaporated under nitrogen gas, dissolved in 100 lL of mobile phase methanol ⁄ water (50 : 50, v ⁄ v) and subjected to radio RP- HPLC using a 125 · 4 mm LiChrosphere RP 18 column (5 lm; Merck). Elution of labeled steroids was monitored by a Radiomatic 150TR Flow Scintillation Analyzer (Hew- lett-Packard). The RP-HPLC mobile phase system con- sisted of methanol ⁄ water (50 : 50, v ⁄ v) for 10 min, a linear gradient from 50–100% methanol for the next 10 min and then 100% methanol for the remaining 10 min [3,32]. The retention times were 8–9 min for 7a-hydroxy-DHEA and 19–20 min for DHEA.

HPLC analysis of incubations with 3b-Adiol

Incubations with 3b-Adiol were analyzed either by radio RP-HPLC using 3H-labeled 3b-Adiol, prepared as described above, or by GC ⁄ MS analysis (see below) using unlabeled 3b-Adiol (Sigma). HEK293 cells were seeded at approximately 7.5 · 105 cells per 60 mm tissue culture dish in DMEM supplemented with 10% fetal bovine serum and antibiotics ⁄ antimycotics. Endogenous enzymatic activity towards DHEA or 3b-Adiol in these cells was examined by addition of 15 lg of sub- strate dissolved in dimethyl sulfoxide to the medium and incubation for 3, 6, 12 or 24 h at 37 (cid:2)C with 5% CO2. Most experiments were carried out with an incubation time of 24 h. Following incubations with substrate, the medium was collected and extracted and the organic phase was ana- lyzed for hydroxylated metabolites, as described below. Incubations terminated immediately after addition of sub- strate (corresponding to an incubation time of 0 h) were used as negative controls. Protein contents of the HEK293 cells were assayed by the method of Lowry et al. [29].

Incubations where steroids were added as potential inhib- itors were carried out as described above, except that 1.5– 150 lg of unlabeled 3b-Adiol, DHEA or DHEA-sulfate were added to the cell media together with the labeled sub- strates. Inhibition experiments in HEK293 cell cultures were generally carried out using incubation times of 24 h. In a separate set of experiments, cells were incubated with inhibitors for 12 h instead of 24 h to examine whether a difference in incubation time might influence the results. Effects of steroid inhibitors, however, were found to be similar with 12 h of incubation as with 24 h of incubation.

Incubations with 3H-labeled 3b-Adiol were analyzed using a similar system as for incubations with 3H-labeled DHEA. The organic phase of the incubations was evapo- rated, dissolved in 50% methanol and subjected to RP- HPLC using a 125 · 4 mm LiChrosphere RP 18 column (5 lm; Merck). The RP-HPLC mobile phase system was the same as for incubations with DHEA. The retention times were 7–8 min for 5a-androstane-3b,7a,17b-triol and 5a-androstane-3b,6n,17b-triol and 19–20 min for 3b-Adiol. An additional polar metabolite with a retention time of 4–5 min was also detected in the incubations with porcine liver microsomes, but we were unable to characterize this compound further.

Overexpression of recombinant human CYP7B1 in HEK293 cells

GC ⁄ MS: identification of 3b-Adiol metabolites

cartridge

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Metabolites of 3b-Adiol present in incubation mixtures or fractions collected from the (radio) HPLC system were identified by GC ⁄ MS. Prior to GC ⁄ MS analysis, the incu- bation mixtures or HPLC fractions were extracted using ethyl acetate. The ethyl acetate phase was collected and dried under a gentle stream of nitrogen. The residue was then dissolved in 3 mL of methanol followed by addition of 2 mL of water. The solution was passed through a Sep-Pak containing octadecylsilane bonded silica C18 (Waters Associates Inc, Milford, MA, USA) followed by 5 mL of water. The total effluent was collected and the organic solvent was removed in vacuo. The remaining aque- ous phase was then passed through the same unwashed Sep-Pak C18 cartridge again, before washing with 5 mL of water. Steroids were then eluted with 10 mL of 75% aque- ous methanol. This solution was passed through a column HEK293 cells were cultured as described above and trans- fected with the pCMV6 vector containing cDNA encoding for human CYP7B1 [31]. In control experiments, cells were transfected with the same amount of empty pCMV vector without the CYP7B1 insert. Transfection was carried out by electroporation in 0.4 cm cuvettes (Gene Pulser II; Bio- Rad, Hercules, CA, USA), using a single pulse of 0.4 kV and 100 lF. In each experiment, 20 · 106 cells were trans- fected with 20 lg of DNA in a volume of 0.8 mL of phos- containing calcium chloride and phate-buffered saline magnesium chloride (Dulbecco’s, Life Technologies, Inc., Grand Island, NY, USA). After transfection, the cells were cultured for 24 h on 60 mm plates in medium containing 3b-Adiol or DHEA (15 lg) dissolved in dimethylsulfoxide. the medium was Following incubations with substrate, collected and extracted and the organic phase was analyzed for hydroxylated metabolites, as described below.

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CYP7B1-mediated metabolism of DHEA and 3b-Adiol

expression vector and a pCMV b-galactosidase plasmid (to control for transfection efficiency) using calcium co-precipi- tation, as previously described [14]. The ER-responsive vec- tor contains a strong ERE coupled to luciferase [21]. Transfected cells were treated with 3b-Adiol, 7a-hydroxy- DHEA or estradiol (0.1–1 lm), dissolved in ethanol, and the levels of luciferase in steroid-treated cells were com- pared with the luciferase levels in cells treated with the same volume of vehicle. Luciferase and b-galactosidase activities were assayed as previously described [14]. ERE- reporter luciferase activity is expressed as relative light units divided by b-galactosidase activity (A420).

Statistical analysis

it was

substrates and ⁄ or groups of

Data are expressed as mean ± SD. Statistical analysis was performed using one- and two-way analysis of variance followed by Dunnet’s post-hoc test, comparing hydroxyl- ation of different sexes. P < 0.05 was considered statistically significant. The soft- ware used was minitab(cid:3), release 14 (Minitab Ltd, Coven- try, UK).

Acknowledgements

In addition, respectively.

The present study was supported by grants from the Swedish Research Council Medicine and the A˚ ke Wiberg foundation.

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cells. (B) GC ⁄ MS analysis of trimethylsilylated steroids isolated after incubating 3b-Adiol with pig liver micro- somes. Fig. S2. Effects of various concentrations of 3b-Adiol on DHEA hydroxylation, shown as a Lineweaver– Burk plot. Fig. S3. Effects of various concentrations of DHEA on 3b-Adiol hydroxylation, shown as a Lineweaver– Burk plot.

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Supplementary material

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The following supplementary material online: Fig. S1. (A) Gas chromatographic-mass spectrometric analysis of trimethylsilylated steroids isolated from the cell medium after incubating 3b-Adiol with HEK293

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