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 CYP7B1, a cytochrome P450 enzyme, metabolizes several steroids involved cytochrome P450; estrogen receptor; in hormonal signaling including 5a-androstane-3b,17b-diol (3b-Adiol), an hydroxylase; sex hormone; steroid 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 androgens and Abbreviations 3b-Adiol, 5a-androstane-3b,17b-diol; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; 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