CYP7B1-mediated metabolism of and 5a--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 CYP7B1, a cytochrome P450 enzyme, metabolizes several involved cytochrome P450; estrogen receptor; in hormonal signaling including 5a-androstane-3b,17b-diol (3b-Adiol), an hydroxylase; ; estrogen receptor agonist, and dehydroepiandrosterone, a precursor for sex metabolism hormones. Previous studies have suggested that CYP7B1-dependent metab- Correspondence olism involving dehydroepiandrosterone or 3b-Adiol may play an impor- M. Norlin, Department of Pharmaceutical tant role for estrogen receptor b-mediated signaling. However, conflicting Biosciences, Division of Biochemistry, data are reported regarding the influence of different CYP7B1-related University of Uppsala, Box 578, steroids on estrogen receptor b activation. In the present study, we investi- S-751 23 Uppsala, Sweden gated CYP7B1-mediated conversions of dehydroepiandrosterone and Fax: +46 18 558 778 3b-Adiol in porcine microsomes and human kidney cells. As part of these Tel: +46 18 471 4331 studies, we compared the effects of 3b-Adiol (a CYP7B1 substrate) and E-mail: [email protected] 7a-hydroxy-dehydroepiandrosterone (a CYP7B1 product) on estrogen (Received 19 December 2007, revised 13 receptor b activation. The data obtained indicated that 3b-Adiol is a more February 2008, accepted 14 February 2008) efficient activator, thus lending support to the notion that CYP7B1 cataly- sis may decrease estrogen receptor b activation. Our data on metabolism doi:10.1111/j.1742-4658.2008.06336.x 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.

The steroid hydroxylase CYP7B1, a member of the cyto- and metabolizes several steroids involved in hormonal chrome P450 enzyme family, has attracted increasing signaling and other processes. Substrates for CYP7B1 interest in recent years due to its multiple reported roles include 5a-androstane-3b,17b-diol (3b-Adiol), an estro- for key events in cellular physiology [1–9]. CYP7B1 is gen receptor (ER) agonist, and dehydroepiandrosterone widely expressed in tissues of human and other species (DHEA), an essential precursor for and

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

1778 FEBS Journal 275 (2008) 1778–1789 ª 2008 The Authors Journal compilation ª 2008 FEBS H. Pettersson et al. CYP7B1-mediated metabolism of DHEA and 3b-Adiol estrogens. In addition to its role as sex hormone precur- mediated formation of hydroxymetabolites from sor, DHEA is reported to affect a number of processes DHEA and 3b-Adiol was analyzed in microsomes in various tissues, including central nervous system func- prepared from various tissues obtained from pigs of dif- tion, immune system, lipid profiles and cellular growth ferent ages. Because this is the first study on CYP7B1- [8–13]. mediated 3b-Adiol metabolism in the pig, GC ⁄ MS The action of CYP7B1 in various tissues results in analysis was carried out to determine the structure of the formation of 7- and ⁄ or 6-hydroxymetabolites, the 3b-Adiol hydroxymetabolite formed. Previous stud- which can be eliminated from the cell, thereby decreas- ies report the formation of both 6- and 7-hydroxyme- ing intracellular levels of CYP7B1 substrates. Some tabolites from 3b-Adiol [17–19]. The GC ⁄ MS analysis reports, however, suggest that CYP7B1-mediated catal- carried out in the present study showed that the main ysis might lead to formation of active hormones with product formed from 3b-Adiol in pig liver is 5a-andro- impact on several processes, including cellular growth, stane-3b,7a,17b-triol (for GC ⁄ MS chromatogram, see immune system and brain function [7–9]. In view of its Supplementary material). Only minor amounts of a high catalytic activity towards sex hormone precursors, 6-hydroxy derivative were observed. Also, trace amounts as well as towards certain estrogens, the action of of 5a-androstane-3b,7b,17b-diol were detected by CYP7B1 may affect hormonal signaling in several GC ⁄ MS. From present and previous findings, it ways [5,7,14]. appears that CYP7B1 is capable of carrying out both Recent studies have indicated that CYP7B1-depen- 6- and 7-hydroxylation of 3b-Adiol. It is possible that dent metabolism may play an important role for ERb- the main product formed from 3b-Adiol may vary in mediated signaling. The manner in which CYP7B1 different species and different cellular environments. affects this, however, remains unclear. The results of The results of the analyses of porcine DHEA and some studies indicate that CYP7B1-mediated catalysis 3b-Adiol hydroxylation in liver, kidney and lung are leads to formation of an ERb ligand, whereas other shown in Table 1. The data indicate marked tissue- studies have proposed that CYP7B1 catalysis instead specific differences between younger and older ani- counteracts ERb ligand activation [5,7]. As ERb is mals. In liver, 7a-hydroxylation of both substrates considered to affect a wide range of biological systems increased with age, whereas the rate of hydroxylation throughout the body, events regulating its function are decreased with age in the kidney. These findings of of considerable interest [5,15]. age-dependent differences are in agreement with previ- In the present study, we used porcine tissues and ous data on DHEA metabolism [6] and indicate a human kidney cells to investigate and compare similar pattern for 7a-hydroxylation of 3b-Adiol in CYP7B1-mediated conversions of DHEA and 3b-Adi- pig tissues. ol, both of which are reported to affect ERb activa- In a separate set of experiments, microsomes were tion. The pig is a useful animal model for studies of prepared from tissues of a 2.5-year-old boar to exam- CYP7B1-mediated catalytic reactions due to the high ine male reproductive tissues in an older individual. CYP7B1 content in pig tissues and the closer similarity In this animal, hepatic hydroxylase activities towards of porcine and human cytochrome P450 enzymes com- DHEA and 3b-Adiol were 591 and 659 pmolÆmg)1 pared with rodent isoforms [16]. Our findings indicate microsomal protein · min, respectively. Hydroxylase that CYP7B1 action is subject to age- and tissue- activities in testicle and prostate were approximately specific differences. Furthermore, the data indicate that 5% of that in liver. In liver, kidney, testicle and tissue-specific steroid concentrations may have a large prostate, the catalytic activities towards DHEA and impact on CYP7B1-dependent catalysis and thus on 3b-Adiol were of the same order of magnitude (data the levels of different CYP7B1-related steroids that not shown). can influence ERb signaling. CYP7B1-mediated activities towards DHEA and Results 3b-Adiol in different sexes We also compared CYP7B1-mediated hydroxylation of Tissue- and age-specific differences in porcine DHEA and 3b-Adiol in tissues from male and female CYP7B1-mediated hydroxylase activities towards pigs. As the analysis of metabolism in pig tissues is DHEA and 3b-Adiol often carried out with organs from castrated pigs, In previous studies, we described CYP7B1-mediated available from slaughterhouses, we also included cas- 7a-hydroxylation of DHEA in microsomes from pig trated male pigs in these studies. The results from liver and kidney [3,6]. In the current study, CYP7B1- incubations with microsomes from kidneys, livers and

FEBS Journal 275 (2008) 1778–1789 ª 2008 The Authors Journal compilation ª 2008 FEBS 1779 CYP7B1-mediated metabolism of DHEA and 3b-Adiol H. Pettersson et al.

Table 1. Difference in CYP7B1-mediated hydroxylase activity Table 2. CYP7B1-mediated hydroxylase activity towards DHEA and between adult male pig and male piglet. The animals were approxi- 3b-Adiol in different sexes. The animals were approximately mately 10 months (adult) and 5 days (piglet) of age. Microsome 10 months of age. Microsome fractions were prepared from tis- fractions were prepared from tissues and the catalytic activity was sues and the catalytic activity was measured by incubation with measured by incubation with radiolabeled substrates and analysis radiolabeled substrates and analysis by RP-HPLC, as described in by RP-HPLC, as described in the Experimental procedures. Hydrox- the Experimental procedures. Experiments were carried out in ylase activity is displayed as pmolÆmg)1 of microsomal pro- three sets of triplicate incubations based on material from three dif- tein · min ± SD (n = 7 samples). Incubations without NADPH were ferent individuals per group. Hydroxylase activity is displayed as used as negative controls. Product formation in negative controls pmolÆmg)1 of microsomal protein · min ± SD (n = 9 samples). ) corresponded to an activity of £ 10 pmolÆmg 1 protein · min. A Incubations without NADPH were used as negative controls. Prod- comparison of hydroxyproduct formation in tissues obtained from uct formation in negative controls corresponded to an activity of ) piglets and sexually mature animals indicates marked tissue-spe- £ 10 pmolÆmg 1 protein · min. There is no significant difference cific differences between younger and older animals. There is a sig- between the different groups for either of the two substrates. In nificant difference in hydroxyproduct formation between older and lung tissue, there is a significant difference between the hydroxy- younger animals in the kidney and liver for both substrates. In the lase activity towards DHEA and 3b-Adiol. There is no significant liver, the activity is higher in older animals than in piglets whereas, different between the hydroxylase activity towards the two sub- in the kidney, the activity decreased with age (P < 0.05, one-way strates in liver tissue (P < 0.05, two-way ANOVA). BLD, below limit ANOVA). BLD, below limit of detection. of detection.

) ) pmolÆmg 1 protein · min pmolÆmg 1 protein · min

Substrate Liver Kidney Lung Substrate Liver Kidney Lung

Adult male DHEA 441 ± 127 BLD 82 ± 10 Male castrated DHEA 290 ± 73 BLD 80 ± 15 Male piglet DHEA 244 ± 158 130 ± 64 104 ± 46 Female DHEA 254 ± 24 BLD 78 ± 37 Adult male 3b-Adiol 443 ± 66 15 ± 10 33 ± 19 Male DHEA 441 ± 127 BLD 82 ± 10 Male piglet 3b-Adiol 76 ± 40 62 ± 31 43 ± 22 Male castrated 3b-Adiol 448 ± 111 12 ± 10 39 ± 8 Female 3b-Adiol 309 ± 30 16 ± 5 31 ± 11 Male 3b-Adiol 443 ± 66 15 ± 10 33 ± 19 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- DHEA 7a-hydroxylation in the present study is in ylation of DHEA or 3b-Adiol in any of the tissues agreement with our previously reported data on this analyzed. Also, formation of 7a-hydroxyproducts from reaction in purified protein fractions [3]. From the DHEA and 3b-Adiol was found to be of a similar present study, it may be concluded that the efficien- magnitude in normal and castrated males. 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 To study which of these two CYP7B1 substrates is most efficiently metabolized and to obtain more HEK293 cells are known to have a high endogenous information regarding which reaction might be of the CYP7B1 expression, although, to our knowledge, no most physiological importance, we determined kinetic data have been reported on 3b-Adiol metabolism in parameters for 7a-hydroxylation of DHEA and 3b- these cells. In the present study, we examined the Adiol using pig liver microsomes. Kinetic parameters, endogenous hydroxylase activities towards 3b-Adiol including Kcat and apparent Km values for these reac- and DHEA in HEK293 cells. The endogenous 7a- tions, are summarized in Table 3. Data were hydroxylase activity towards DHEA in HEK293 cells obtained by incubation with various amounts of in these experiments was approximately 300 pmolÆmg)1 DHEA or 3b-Adiol as described in the Experimental cell protein · 24 h. Analysis of 3b-Adiol metabolism procedures and the parameters were determined by indicated the formation of more than one hydroxy- nonlinear regression fitting of the data to the metabolite in HEK293 cells. GC ⁄ MS analysis of meta- Michaelis–Menten equation or calculated from dou- bolites demonstrated the formation of both 6- and ble reciprocal plots. The catalytic efficiencies for 7-hydroxy derivatives under these conditions. The mass hydroxylation of DHEA and 3b-Adiol as described spectrum of the main metabolite formed from 3b-Adi- by the Kcat ⁄ Km values were in same order of ol in HEK293 cell cultures was consistent with a magnitude (Table 3). The Km value for porcine 6-hydroxy derivative, i.e. 5a-androstane-3b,6n,17b-triol

1780 FEBS Journal 275 (2008) 1778–1789 ª 2008 The Authors Journal compilation ª 2008 FEBS H. Pettersson et al. CYP7B1-mediated metabolism of DHEA and 3b-Adiol

Table 3. Kinetic parameters for CYP7B1-mediated 7a-hydroxylation of DHEA and 3b-Adiol in pig liver. Experiments to determine kinetic 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.

)1 )1 Km (lM) Vmax (pmolÆmg protein · min) Kcat (nmolÆnmol P450 · min) Kcat ⁄ Km

7a-hydroxylation of DHEA 5 1000 2 0.4 7a-hydroxylation of 3b-Adiol 3 500 1 0.33

(for GC ⁄ MS chromatogram, see Supplementary mate- the presence of increasing amounts of unlabeled 3b- rial). We were unable to distinguish between a or b Adiol. The results of these experiments are shown in orientation of the 6-hydroxygroup, as indicated by the Fig. 1. 3b-Adiol inhibited DHEA hydroxylation by letter n (Greek letter ‘xi’, which corresponds to ‘x’ in approximately 60–70% when both steroids were pres- our alphabet, representing an unknown configuration). ent at equimolar concentrations. Further, a 10-fold Small amounts of 5a-androstane-3b,7a,17b-triol were higher concentration of 3b-Adiol than of DHEA in also formed. Endogenous hydroxylase activity towards the incubation mixture resulted in the suppression of 3b-Adiol in HEK293 cells was approximately 450 DHEA hydroxylation by 80%. In HEK293 cells, the ) pmolÆmg 1 cell protein · 24 h. suppressive effect of 3b-Adiol of DHEA metabolism We also examined the activities towards 3b-Adiol was statistically significant also at a 10-fold lower con- and DHEA in HEK293 cells transfected with an centration of 3b-Adiol than of DHEA in the incuba- expression vector containing human CYP7B1 cDNA. tion mixture (Fig. 1). As expected, overexpression of human CYP7B1 sig- nificantly increased the hydroxylation of both 3b-Adi- 3β-Adiol as inhibitor of ol and DHEA in HEK293 cultures. HEK293 cells DHEA hydroxylation in pig transfected with the CYP7B1 expression vector dis- liver microsomes played three- to six-fold higher hydroxylase activity 3β-Adiol as inhibitor of than cells transfected with empty vector (data not 120 DHEA hydroxylation in HEK293 cells shown). GC ⁄ MS analysis of 3b-Adiol metabolites 100 formed in CYP7B1-transfected cell cultures also * 80 showed formation of both 6- and 7-hydroxy deriva- tives with 5a-androstane-3b,6n,17b-triol as the main 60 * metabolite. * 40 * * Rate of DHEA 20 Effects of 3b-Adiol on CYP7B1-mediated 0 hydroxylation of DHEA (% of control) hydroxylation Control [0.1/1] [1/1] [10/1] [0/1] As the studies indicated that the rate of hydroxylation Concentration ratio [inhibitor/substrate] and affinity of CYP7B1 for DHEA and 3b-Adiol are similar, we conducted experiments to study how the Fig. 1. Effects of 3b-Adiol on DHEA hydroxylation in pig liver micro- concentration of 3b-Adiol would affect the CYP7B1- somes (black bars) and HEK293 cells (grey bars). Experiments were mediated metabolism of DHEA and vice versa. These carried out with a constant concentration (52 lM) of radiolabeled DHEA to study the inhibitory effect by varying levels of 3b-Adiol on experiments were carried out with both porcine micro- DHEA hydroxylation. Concentrations are shown as ratio between somes and human HEK293 cells. The main reason for added inhibitor (3b-Adiol) and substrate (DHEA). Analysis of the our interest in the effects of steroid concentrations on catalytic activity was carried out as described in the Experimental the rate of metabolic reactions is that the concentra- procedures. Catalytic activity is shown as percent of the rate of tions of these steroids vary considerably in different DHEA hydroxylation in controls (± SD) (n = 4–9). Controls consisted tissues and species. For example, the concentration of of incubations without added inhibitor [0 ⁄ 1]. 3b-Adiol inhibited DHEA in human prostate is reported to be approxi- DHEA hydroxylation by approximately 60–75% when both steroids were present at equimolar concentrations, and when the concen- mately 10-fold higher than that of 3b-Adiol [20]. tration of 3b-Adiol was increased to 10-fold, compared with the In the first set of experiments, we studied the effect concentration of DHEA, the hydroxylation of DHEA decreased by of 3b-Adiol on DHEA hydroxylation. Hydroxylation approximately 80%. *Statistically significant suppression compared of DHEA was measured with radiolabeled DHEA in with control (P < 0.05, one-way ANOVA).

FEBS Journal 275 (2008) 1778–1789 ª 2008 The Authors Journal compilation ª 2008 FEBS 1781 CYP7B1-mediated metabolism of DHEA and 3b-Adiol H. Pettersson et al.

Analysis of the type of inhibition by 3b-Adiol on DHEA as inhibitor of DHEA hydroxylation was carried out by a series of 3β-Adiol hydroxylation in incubations with pig liver microsomes containing 1– pig liver microsomes 5 lm of DHEA in the presence of 0, 5, 10, or 20 lm DHEA as inhibitor of unlabeled 3b-Adiol (added as inhibitor). Lineweaver– 3β-Adiol hydroxylation in 140 Burk and Dixon plots were constructed using linear HEK293 cells regression fitting of the data. The data obtained indi- 120 * cated that 3b-Adiol is a mixed inhibitor of DHEA 100 -Adiol hydroxylation (for a Lineweaver–Burk plot showing β 80 * inhibition by 3b-Adiol on DHEA hydroxylation, see 60 Supplementary material). Thus, the inhibition includes 40 *

Rate of 3 * competition at the active site but involves both com- 20 petitive and uncompetitive components. The patterns 0 hydroxylation (% of control) (% of control) hydroxylation of Lineweaver–Burk and Dixon plots were not consis- Control [0/1] [0.1/1] [1/1] [10/1] tent with a pure noncompetitive or uncompetitive type Concentration ratio [inhibitor/substrate] of inhibition (data not shown). The K value for the i Fig. 2. Effects of DHEA on 3b-Adiol hydroxylation in pig liver micro- inhibition of 3b-Adiol on DHEA hydroxylation, calcu- somes (black bars) and HEK293 cells (grey bars). Experiments were m lated from the Dixon plot, was 6 l . 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 Effects of DHEA on CYP7B1-mediated 3b-Adiol added inhibitor (DHEA) and substrate (3b-Adiol). Analysis of the hydroxylation catalytic activity was carried out as described in the Experimental procedures. Catalytic activity is shown as percent of the rate of We also carried out corresponding studies on the effect 3b-Adiol hydroxylation in controls (± SD) (n = 3–9). Controls of different concentrations of DHEA on the 6- and consisted of incubations without added inhibitor [0 ⁄ 1]. DHEA 7-hydroxylation of 3b-Adiol. In these experiments, we inhibited 3b-Adiol hydroxylation by approximately 70–90% when measured 3b-Adiol hydroxylation in pig liver micro- the concentration of DHEA was increased to 10-fold compared with somes and human HEK293 cells with radiolabeled the concentration of 3b-Adiol. *Statistically significant suppression 3b-adiol in the presence of increasing amounts of unla- compared with control (P < 0.05, one-way ANOVA). beled DHEA (Fig. 2). The most efficient inhibition of 3b-Adiol metabolism was obtained at high amounts of DHEA hydroxylation by 3b-Adiol (6 lm; see above). DHEA, although, in experiments with HEK293 cells, Thus, it appears that 3b-Adiol may be a more efficient statistically significant effects were observed also at inhibitor of the hydroxylation of DHEA than vice lower concentrations. A 10-fold higher concentration versa. of DHEA than of 3b-Adiol in the incubation mixture Some inhibition experiments were also carried out decreased the rate of 3b-Adiol hydroxylation by with DHEA-sulfate, the sulfated ester of DHEA, 70–90% (Fig. 2). which is present in even higher amounts than DHEA Similarly, as for the inhibition of DHEA hydroxyl- in human plasma. However, DHEA-sulfate had no ation, we carried out experiments to analyze the type significant effect on the rate of CYP7B1-mediated of inhibition of 3b-Adiol hydroxylation by DHEA. hydroxylation of either DHEA or 3b-Adiol, even at Incubations were performed with pig liver microsomes 10-fold higher concentrations of DHEA-sulfate in the and radiolabeled 3b-Adiol (1–5 lm) in the presence of incubation mixture (data not shown). , unlabeled DHEA (0, 5, 10, or 20 lm, added as inhibi- which is formed from DHEA and is present in high tor). Construction of Lineweaver–Burk and Dixon levels in the same cell types, also did not affect plots from the data indicated that DHEA is a mixed CYP7B1-mediated hydroxylase activity. It may there- inhibitor of 3b-Adiol hydroxylation, similar to that fore be concluded that the steroid inhibition described found for the inhibition of DHEA hydroxylation by in the present study is specific and not a generalized 3b-Adiol (for a Lineweaver–Burk plot showing inhibi- effect by related steroids of a similar structure. tion by DHEA on 3b-Adiol hydroxylation, see Supple- mentary material). The patterns of Lineweaver–Burk Analysis of ERb activation by 3b-Adiol and and Dixon plots were not consistent with a pure non- 7a-hydroxy-DHEA competitive or uncompetitive type of inhibition. The

Ki for inhibition of 3b-Adiol hydroxylation by DHEA Previous studies indicate that ERb is activated by was 24 lm, a higher value than the Ki for inhibition of 3b-Adiol, whereas others report that this receptor is

1782 FEBS Journal 275 (2008) 1778–1789 ª 2008 The Authors Journal compilation ª 2008 FEBS H. Pettersson et al. CYP7B1-mediated metabolism of DHEA and 3b-Adiol

3.5 * *

3 * 2.5 *

Fig. 3. Effects of (E2), 7a-OH- 2

DHEA (7HD) or 3b-Adiol on an ER-respon- -gal) – fold stimulation sive ERE luciferase reporter vector. HEK293 β 1.5 cells were transiently transfected with ERb (1 lgÆwell)1) and treated with steroids as 1 described in the Experimental procedures. Data are presented as the fold stimulation 0.5 compared with controls (treated with vehi- cle). *Statistically significant stimulation 0 compared with control (P < 0.05, one-way Control 0.1 μM 1 μM 0.1 μM 1 μM 0.1 μM 1 μM β β ANOVA). Reporter activity (RLU/ EtOH E2 E2 7HD 7HD 3 -Adiol 3 -Adiol activated by 7a-hydroxy-DHEA [5,7]. In the present The concentrations of DHEA and 3b-Adiol and study, we compared the effects of these two steroids related steroids vary considerably between different tis- on ERb activation in the same experiment, using the sues [10,20,22]. For example, concentrations of DHEA same methodology. Receptor activation was studied by in tissue samples are reported to be 20-fold higher reporter assay with an ER-responsive luciferase repor- in human prostate (100 pmolÆg)1) than in muscle ter vector transfected in HEK293 cells overexpressed (5 pmolÆg)1). In general, physiological levels of 3b-Adi- with ERb, in a similar fashion to that previously ol are lower than those of DHEA in both humans and described [14,21]. This ER-responsive vector contains a pigs, particularly in human adults where plasma con- strong estrogen response element (ERE) coupled to centrations are reported to be approximately 1.5 nm luciferase. Thus, luciferase expression levels are deter- for 3b-Adiol and approximately 10- to 30-fold higher mined by activation of ER and its subsequent binding for DHEA [20,22,23]. In human plasma, DHEA-sul- to the ERE. ERb-transfected cells were treated with fate is present in higher concentrations than DHEA 3b-Adiol or 7a-hydroxy-DHEA in different concentra- [23,24]. The plasma levels of these steroids are subject tions and the levels of luciferase in steroid-treated cells to developmental variation, depending on the stage of were compared with the luciferase levels in cells treated sexual maturation [10,24]. From the results of the pres- with the same volume of vehicle (ethanol). Treatment ent study, it is clear that high concentrations of with 17b-estradiol, a known ER agonist, was used as DHEA, but not of DHEA-sulfate, strongly suppress positive control. The results of these experiments CYP7B1-mediated metabolism of 3b-Adiol. The cur- (Fig. 3) indicate that 3b-Adiol is a more efficient ERb rent data imply that human CYP7B1-mediated metab- activator than 7a-hydroxy-DHEA. 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 Discussion most likely would suppress 3b-Adiol hydroxylation In the present study, we examined CYP7B1-mediated during physiological conditions. metabolism of 3b-Adiol and DHEA in porcine micro- CYP7B1-mediated metabolism of DHEA and 3b- somal fractions and human kidney cells. The current Adiol are of interest in connection with several physio- data indicate that the efficiencies of these two reac- logical processes. One of these concerns the proposed tions are very similar. As the kcat ⁄ Km values are role of this enzyme for ER-mediated signaling and the within the same order of magnitude for both sub- potential ER-mediated control of cellular growth, par- strates, we propose that relative formation of differ- ticularly of the prostate [5,25]. However, the available ent CYP7B1-related steroids may be dependent on data regarding the effect of CYP7B1 on ERb activa- the concentration of substrate(s) present in each tis- tion are conflicting. Data obtained by Weihua et al. sue. Consequently, cellular steroid levels may have a [5,26] in rodents indicate that 3b-Adiol binds strongly strong impact on the resulting physiological levels of to ERb and that CYP7B1-mediated metabolism of CYP7B1 substrates as well as of CYP7B1-formed 3b-Adiol therefore should lead to induced growth by . abolishing ERb action. By contrast, Martin et al. [7],

FEBS Journal 275 (2008) 1778–1789 ª 2008 The Authors Journal compilation ª 2008 FEBS 1783 CYP7B1-mediated metabolism of DHEA and 3b-Adiol H. Pettersson et al. 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. 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. DHEA concentrations are generally higher in humans than in rodents and exceed, by at least 10-fold, the concentration of 3b-Adiol in human pros- Fig. 4. Suggested effects of a high DHEA ⁄ 3b-Adiol ratio (such as tate [22]. Even though DHEA circulates in micromolar in prostate tissue) on 3b-Adiol-mediated ERb activation. High DHEA concentrations in plasma, DHEA levels measured in levels strongly suppresses CYP7B1-mediated 3b-Adiol metabolism, human prostate tissue are reported to be approxi- resulting in higher 3b-Adiol levels and increased ERb activation. The ) CYP7B1-mediated 7 -OH-DHEA metabolite (not shown) is most mately 100 pmolÆg 1 [20,22]. Thus, the amount of a likely not formed in sufficient amounts to compete with 3b-Adiol 7a-hydroxy-DHEA needed for activation of ERb for ERb binding. appears to be approximately 50- to 100-fold higher than the reported content of DHEA in human prostate lar DHEA ⁄ 3b-Adiol ratios, such as in human prostate, tissue. The concentration of DHEA-sulfate, which can on ERb activation is outlined in Fig. 4. At high be converted to DHEA by a sulfatase, is in the same DHEA levels, CYP7B1-mediated 3b-Adiol hydroxyl- order of magnitude in prostate tissue as that of DHEA ation is strongly suppressed, resulting in higher 3b- despite the abundance of DHEA-sulfate in blood Adiol levels and increased ERb activation (Fig. 4). The [10,22]. It is therefore unlikely that the concentration steroid suppression of CYP7B1-mediated metabolism of a DHEA-metabolite such as 7a-hydroxy-DHEA observed in the present study is apparently not an should reach a high enough concentration to be of unspecific steroid effect because our present and previ- physiological relevance for ERb activation, at least in ous data have shown that neither DHEA-sulfate, nor this tissue. However, Weihua et al. [26] reported that testosterone are able to inhibit CYP7B1-mediated very low concentrations of 3b-Adiol are able to bind catalysis [3]. ERb. Ligand-competition experiments with [125I]estra- In conclusion, the results obtained in the present diol indicated a Ki of 2 nm for 3b-Adiol, a value closer study indicate that the efficiencies of DHEA and 3b- to the reported physiological levels of this steroid, Adiol hydroxylation by CYP7B1 are similar, but that which are in the nanomolar range [22,26,27]. a high DHEA ⁄ 3b-Adiol ratio (similar to the ratio of From the data on the catalytic properties of these steroids in many human tissues) strongly sup- CYP7B1 obtained in the present study, it is clear that presses CYP7B1-mediated 3b-Adiol metabolism. Our high concentrations of DHEA strongly suppress data indicate that tissue-specific steroid concentrations CYP7B1-mediated 3b-Adiol metabolism. Although 3b- may have a large impact on CYP7B1-dependent catal- Adiol is able to compete for the active site of CYP7B1 ysis and thus on the levels of different CYP7B1-related to some degree even at a 10-fold higher DHEA con- steroids that can influence ERb-signaling. centrations, as the present data indicate, the rate of its hydroxylation was decreased by 70–90% under these Experimental procedures conditions. It is therefore likely that the comparatively higher DHEA content in prostate and many other Materials tissues may increase the level of 3b-Adiol, and thus of 3b-Adiol-mediated ERb activation, by suppression of Human embryonic kidney cells (HEK293) (ATCC CRL 3b-Adiol metabolism. A proposed effect of high cellu- 1573) were purchased from ATCC (Rockville, MD, USA).

1784 FEBS Journal 275 (2008) 1778–1789 ª 2008 The Authors Journal compilation ª 2008 FEBS H. Pettersson et al. CYP7B1-mediated metabolism of DHEA and 3b-Adiol

DMEM (containing 1000 mgÆL)1 glucose), fetal bovine total volume of 1 mL of 0.1 m potassium phosphate buffer serum, antibiotics ⁄ antimycotics, non-essential amino acids containing 0.1 mm EDTA (pH 7.4) for 10 min at 37 Cin and trypsin were obtained from Invitrogen (Carlsbad, CA, a water bath. The reaction was quenched and extracted USA). The pCMV6 vector containing cDNA encoding for with 5 mL of ethyl acetate. The organic phase was col- human CYP7B1 was kindly provided by D. W. Russell lected, evaporated under nitrogen gas, dissolved in a small (University of Texas, Dallas, TX, USA). The ERb expres- amount of acetone and applied on a silica gel TLC plate. sion vector and the ERE luciferase reporter vector were The TLC plate was developed three times in toluene ⁄ meth- generous gifts from P. Chambon (Institut de ge´ne´tique et de anol (90 : 10, v ⁄ v). TLC plates with unlabeled DHT, biologie mole´culaire et cellulaire, Strasbourg, France) and 3b-Adiol and 5a-androstane-3a,17b-diol were used as refer- K. Arcaro (University of Massachusetts, MA, USA), respec- ences and developed together with the sample plate. The tively. 3b-Adiol, 5a-androstane-3a,17b-diol, DHEA, DHEA- sample TLC plate was scanned for localization of the sulfate, dihydrotestosterone (DHT) and radioactive products, using a Berthold Tracemaster 20 TLC dehydrogenase (from Pseudomonas testosteroni) were from scanner (Berthold ⁄ Frieske GmbH, Karlsruhe-Durlach, Ger- Sigma Chemical Co. (St Louis, MO, USA). 3H-Labeled many). The reference TLC plates were exposed to iodine DHEA and DHT were from Perkin Elmer Life Sciences vapours (o ⁄ n) to visualize the steroids and the retention (Waltham, MA, USA). All remaining chemicals were of times of reference compounds were compared with those of analytical grade and purchased from commercial sources. 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 Animals and tissue sample collection of the 3a-hydroxyderivative, 5a-androstane-3a,17b-diol, Liver, kidney and lung tissues from adult female, male and were also formed in the reaction. The formed radioactive castrated male pigs (aged 10 months) and liver, kidney, tes- 3b-Adiol was extracted from the silica gel with ethyl ace- ticle and prostate tissues from an adult domestic boar (aged tate. The extraction procedure was repeated twice. The 3 2.5 years) were obtained from the Funbo-Lo¨ vsta Research obtained solution of H-labeled 3b-Adiol was evaporated Center [Department of Animal Breeding and Genetics, under nitrogen gas and dissolved in 100 lL of ethyl acetate. )1 Swedish University of Agricultural Sciences (SLU), Ultuna, Radioactivity (c.p.m.ÆlL ) was determined by injection of Sweden]. Liver, kidney and lung tissues from male uncas- an aliquot on a RP-HPLC (125 · 4 mm LiChrosphere RP trated piglets (aged 5 days) were a generous gift from 18 column, 5 lm; Merck, Darmstadt, Germany). Elution P. Wallgren [Department of Ruminant and Porcine Dis- was monitored by a Radiomatic 150TR Flow Scintillation eases, National Veterinary Institute (SVA), Uppsala, Analyzer (Hewlett-Packard, Palo Alto, CA, USA). The 3 Sweden]. All the animals were healthy and untreated at the solution of H-labeled 3b-Adiol was diluted to a working )1 time of euthanasia. The domestic pig is considered to reach concentration of 50 000 c.p.m.ÆlL . sexual maturity at approximately 6 months of age. All organ tissue samples were stored at )80 C until micro- Incubations with microsomes somal preparation was performed. Incubations with microsomes (0.5–1.0 mg of microsomal protein) were carried out at 37 C for 20 or 30 min. The Microsomal preparation substrates DHEA (52 lm,1lCi) or 3b-Adiol (52 lm, The tissue samples were weighed and minced, respectively, in 200 000 c.p.m.) dissolved in 25 lL of acetone, were incu- sucrose buffer containing 0.25 m sucrose, 10 mm Tris–Cl bated with 1 lmol of NADPH in a total volume of 1 mL (pH 7.4) and 1 mm EDTA to a 20% suspension. Microsomes of 50 mm Tris-acetate buffer (pH 7.4) containing 20% glyc- were prepared from the tissues according to standard meth- erol and 0.1 mm EDTA. Incubations were performed under ods [28], and were suspended in 50 mm Tris-acetate buffer conditions where the enzyme was saturated with substrate. (pH 7.4) containing 20% glycerol and 0.1 mm EDTA and The incubations were quenched and extracted with 5 mL of stored at )80 C until incubation. Protein contents of the mi- ethyl acetate. The organic phase was collected and stored at crosomes were assayed by the method of Lowry et al. [29]. )20 C until analysis. Incubations without NADPH were performed at the same time and used as negative controls. For incubations where steroids were added as potential Preparation of 3b-Adiol inhibitors, incubations with labeled substrates were carried 3H-Labeled 3b-Adiol was prepared from 3H-labeled DHT out as described above, except that various amounts of (Perkin Elmer) by bioconversion using a commercially unlabeled 3b-Adiol, DHEA, DHEA-sulfate or testosterone available hydroxysteroid dehydrogenase from P. testoste- were added to the incubation mixtures. For kinetic analysis, roni (Sigma H8879) [30]. A mixture of labeled (100 lCi) pig liver microsomes were incubated with 1, 2, 3, 4 or 5 lm and unlabeled (10 lg) DHT was incubated with 0.32 U of of radiolabeled substrate (DHEA or 3b-Adiol) in the pres- hydroxysteroid dehydrogenase and 1 lmol of NADH in a ence of 0, 5, 10 or 20 lm of unlabeled DHEA or 3b-Adiol

FEBS Journal 275 (2008) 1778–1789 ª 2008 The Authors Journal compilation ª 2008 FEBS 1785 CYP7B1-mediated metabolism of DHEA and 3b-Adiol H. Pettersson et al. added as inhibitors. Experiments were carried out in tripli- Analysis of incubations with DHEA cates or quadruplicates and kinetic parameters were deter- mined by nonlinear regression fitting of the data to the Incubations with 3H-labeled DHEA were analyzed as previ- Michaelis–Menten equation or calculated from Line- ously described [3,32]. The organic phase of the incubations weaver–Burk and Dixon plots. 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- Cultures of HEK293 cells HPLC using a 125 · 4 mm LiChrosphere RP 18 column HEK293 cells were seeded at approximately 7.5 · 105 cells (5 lm; Merck). Elution of labeled steroids was monitored per 60 mm tissue culture dish in DMEM supplemented by a Radiomatic 150TR Flow Scintillation Analyzer (Hew- with 10% fetal bovine serum and antibiotics ⁄ antimycotics. lett-Packard). The RP-HPLC mobile phase system con- Endogenous enzymatic activity towards DHEA or 3b-Adiol sisted of methanol ⁄ water (50 : 50, v ⁄ v) for 10 min, a linear in these cells was examined by addition of 15 lg of sub- gradient from 50–100% methanol for the next 10 min and strate dissolved in dimethyl sulfoxide to the medium and then 100% methanol for the remaining 10 min [3,32]. The incubation for 3, 6, 12 or 24 h at 37 C with 5% CO2. retention times were 8–9 min for 7a-hydroxy-DHEA and Most experiments were carried out with an incubation time 19–20 min for DHEA. of 24 h. Following incubations with substrate, the medium was collected and extracted and the organic phase was ana- HPLC analysis of incubations with 3b-Adiol lyzed for hydroxylated metabolites, as described below. Incubations terminated immediately after addition of sub- Incubations with 3b-Adiol were analyzed either by radio strate (corresponding to an incubation time of 0 h) were RP-HPLC using 3H-labeled 3b-Adiol, prepared as described used as negative controls. Protein contents of the HEK293 above, or by GC ⁄ MS analysis (see below) using unlabeled cells were assayed by the method of Lowry et al. [29]. 3b-Adiol (Sigma). Incubations where steroids were added as potential inhib- Incubations with 3H-labeled 3b-Adiol were analyzed itors were carried out as described above, except that 1.5– using a similar system as for incubations with 3H-labeled 150 lg of unlabeled 3b-Adiol, DHEA or DHEA-sulfate DHEA. The organic phase of the incubations was evapo- were added to the cell media together with the labeled sub- rated, dissolved in 50% methanol and subjected to RP- strates. Inhibition experiments in HEK293 cell cultures HPLC using a 125 · 4 mm LiChrosphere RP 18 column were generally carried out using incubation times of 24 h. (5 lm; Merck). The RP-HPLC mobile phase system was In a separate set of experiments, cells were incubated with the same as for incubations with DHEA. The retention inhibitors for 12 h instead of 24 h to examine whether a times were 7–8 min for 5a-androstane-3b,7a,17b-triol and difference in incubation time might influence the results. 5a-androstane-3b,6n,17b-triol and 19–20 min for 3b-Adiol. Effects of steroid inhibitors, however, were found to be An additional polar metabolite with a retention time of similar with 12 h of incubation as with 24 h of incubation. 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 HEK293 cells were cultured as described above and trans- fected with the pCMV6 vector containing cDNA encoding Metabolites of 3b-Adiol present in incubation mixtures or for human CYP7B1 [31]. In control experiments, cells were fractions collected from the (radio) HPLC system were transfected with the same amount of empty pCMV vector identified by GC ⁄ MS. Prior to GC ⁄ MS analysis, the incu- without the CYP7B1 insert. Transfection was carried out bation mixtures or HPLC fractions were extracted using by electroporation in 0.4 cm cuvettes (Gene Pulser II; Bio- ethyl acetate. The ethyl acetate phase was collected and Rad, Hercules, CA, USA), using a single pulse of 0.4 kV dried under a gentle stream of nitrogen. The residue was and 100 lF. In each experiment, 20 · 106 cells were trans- then dissolved in 3 mL of methanol followed by addition of fected with 20 lg of DNA in a volume of 0.8 mL of phos- 2 mL of water. The solution was passed through a Sep-Pak phate-buffered saline containing chloride and C18 cartridge containing octadecylsilane bonded silica chloride (Dulbecco’s, Life Technologies, Inc., (Waters Associates Inc, Milford, MA, USA) followed by Grand Island, NY, USA). After transfection, the cells were 5 mL of water. The total effluent was collected and the cultured for 24 h on 60 mm plates in medium containing organic solvent was removed in vacuo. The remaining aque- 3b-Adiol or DHEA (15 lg) dissolved in dimethylsulfoxide. ous phase was then passed through the same unwashed Following incubations with substrate, the medium was Sep-Pak C18 cartridge again, before washing with 5 mL of collected and extracted and the organic phase was analyzed water. Steroids were then eluted with 10 mL of 75% aque- for hydroxylated metabolites, as described below. ous methanol. This solution was passed through a column

1786 FEBS Journal 275 (2008) 1778–1789 ª 2008 The Authors Journal compilation ª 2008 FEBS H. Pettersson et al. CYP7B1-mediated metabolism of DHEA and 3b-Adiol

(40 · 4 mm) of the strong lipophilic anion exchanger, expression vector and a pCMV b-galactosidase plasmid (to TEAP-LH-20, in bicarbonate form [33]. After elution with control for transfection efficiency) using calcium co-precipi- an additional 5 mL of methanol, the total effluent from the tation, as previously described [14]. The ER-responsive vec- column was taken to dryness in vacuo. The residue was tor contains a strong ERE coupled to luciferase [21]. then transferred with methanol to a stoppered tube, dried Transfected cells were treated with 3b-Adiol, 7a-hydroxy- under nitrogen, and steroids were trimethylsilylated in DHEA or estradiol (0.1–1 lm), dissolved in ethanol, and 0.2 mL of pyridine ⁄ hexamethyldisilazane ⁄ trimethylchloro- the levels of luciferase in steroid-treated cells were com- silane (3 : 2 : 1, v ⁄ v ⁄ v), by heating at 60 C for 30 min. pared with the luciferase levels in cells treated with the The reagents were removed under nitrogen, and the deriva- same volume of vehicle. Luciferase and b-galactosidase tives were redissolved in hexane. GC ⁄ MS was performed activities were assayed as previously described [14]. ERE- using a Finnigan (San Jose, CA, USA) SSQ 710 instrument reporter luciferase activity is expressed as relative light units housing a fused silica column (25 · 0.32 mm) coated with divided by b-galactosidase activity (A420). a 0.17-lm layer of cross-linked methyl silicone (Ultra 1; Hewlett-Packard) ending in the source. An on-column Statistical analysis injection device was used. The oven temperature was 50 C during the injection and, after 3 min, it was rapidly Data are expressed as mean ± SD. Statistical analysis was increased to 185 C, and then programmed to 280 Cata performed using one- and two-way analysis of variance )1 rate of 5 C min . The electron energy was 50 eV, and followed by Dunnet’s post-hoc test, comparing hydroxyl- )1 repetitive scanning (30 scans min ) over the m ⁄ z range 50– ation of different substrates and ⁄ or groups of sexes. 800 was started after a suitable delay. The identification of P < 0.05 was considered statistically significant. The soft- a steroid was based on the retention time and ⁄ or complete ware used was minitab, release 14 (Minitab Ltd, Coven- mass spectrum, which were compared with those of refer- try, UK). ence steroids. The retention indices (Kovats) for 3b-Adiol (5a-androstane-3b,17b-diol) and its characterized major metabolites 5a-androstane-3b,7a,17b-triol (formed in incu- Acknowledgements bations with pig liver microsomes) and 5a-androstane- The present study was supported by grants from the 3b,6n,17b-triol (formed in incubations with HEK293) were Swedish Research Council Medicine and the A˚ke 2595, 2640 and 2755, respectively. In addition, trace Wiberg foundation. amounts of 5a-androstane-3b,7b,17b-triol (Kovats 2785) were also detected in some incubations. The mass spectrum of the isolated 5a-androstane-3b,7a,17b-triol showed the References following intense ⁄ significant : m ⁄ z 524 (molecular ion), 1 Wu Z, Martin KO, Javitt NB & Chiang JY (1999) 509, 434, 393 (base peak), 344, 254, 239 and 129. The same Structure and functions of human oxysterol 7a-hydroxy- significant ions were seen in the spectrum of isolated 5a- lase cDNAs and gene CYP7B1. J Lipid Res 40, androstane-3b,7b,17b-triol, but the base peak was then m ⁄ z 2195–2203. 434. These mass spectra were essentially identical to those 2 Sulcova J & Starka L (1968) Characterisation of of the corresponding reference compounds. Major signifi- microsomal dehydroepiandrosterone 7-hydroxylase from cant ions in the mass spectrum of isolated 5a-androstane- rat liver. Steroids 12, 113–126. 3b,6n,17b-triol were m ⁄ z 524 (molecular ion), 509, 434, 419, 3 Norlin M & Wikvall K (1998) Biochemical characteriza- 344, 329, 318, 254, 239, 228 and 129 (base peak). This mass tion of the 7a-hydroxylase activities towards 27-hy- spectrum was very similar to that of authentic 5a-andro- droxycholesterol and dehydroepiandrosterone in pig stane-3b,6b,17b-triol and differed from those of 3b-Adiol liver microsomes. Biochim Biophys Acta 1390, 269–281. with an extra hydroxy group in another position (i.e. 1, 2, 4 Shoda J, Toll A, Axelson M, Pieper F, Wikvall K & 7, 11, 15, 16, 18 or 19 position). However, because we did Sjo¨ vall J (1993) Formation of 7a- and 7b-hydroxylated not have a mass spectrum of 5a-androstane-3b,6a,17b-triol precursors from 27-hydroxycholesterol in for comparison, a 6a-hydroxy group of the isolated metab- human liver microsomes and mitochondria. Hepatology olite could not be excluded. Thus, the absolute configura- 17, 395–403. tion of the 6-hydroxy group of this metabolite was not 5 Weihua Z, Lathe R, Warner M & Gustafsson JA˚ established, as indicated by the letter n. 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FEBS Journal 275 (2008) 1778–1789 ª 2008 The Authors Journal compilation ª 2008 FEBS 1787 CYP7B1-mediated metabolism of DHEA and 3b-Adiol H. Pettersson et al.

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32 Norlin M, Andersson U, Bjo¨ rkhem I & Wikvall K cells. (B) GC ⁄ MS analysis of trimethylsilylated steroids (2000) Oxysterol 7a-hydroxylase activity by cholesterol isolated after incubating 3b-Adiol with pig liver micro- 7a-hydroxylase (CYP7A). J Biol Chem 275, 34046– somes. 34053. Fig. S2. Effects of various concentrations of 3b-Adiol 33 Axelson M, Ellis E, Mo¨ rk B, Garmark K, Abrahamsson on DHEA hydroxylation, shown as a Lineweaver– A, Bjo¨ rkhem I, Ericzon BG & Einarsson C (2000) Bile Burk plot. acid synthesis in cultured human hepatocytes: support Fig. S3. Effects of various concentrations of DHEA for an alternative biosynthetic pathway to cholic acid. on 3b-Adiol hydroxylation, shown as a Lineweaver– Hepatology 31, 1305–1312. Burk plot. This material is available as part of the online article Supplementary material from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible The following supplementary material is available for the content or functionality of any supplementary online: materials supplied by the authors. Any queries (other Fig. S1. (A) Gas chromatographic-mass spectrometric than missing material) should be directed to the corre- analysis of trimethylsilylated steroids isolated from the sponding author for the article. cell medium after incubating 3b-Adiol with HEK293

FEBS Journal 275 (2008) 1778–1789 ª 2008 The Authors Journal compilation ª 2008 FEBS 1789