The source of NADPH-dependent cytosolic 3␤-hydroxysteroid dehydrogenase (3␤-HSD) activity is unknown to date. This important reaction leads e.g. to the reduction of the potent androgen 5␣-dihydrotestosterone (DHT) into inactive 3␤-androstanediol (3␤-Diol). Four human cytosolic aldo-keto reductases (AKR1C1-AKR1C4) are known to act as non-positional-specific 3␣-/ 17␤-/20␣-HSDs. We now demonstrate that AKR1Cs catalyze the reduction of DHT into both 3␣-and 3␤-Diol (established by 1 H NMR spectroscopy). The rates of 3␣-versus 3␤-Diol formation varied significantly among the isoforms, but with each enzyme both activities were equally inhibited by the nonsteroidal anti-inflammatory drug flufenamic acid. In vitro, AKR1Cs also expressed substantial 3␣[17␤]-hydroxysteroid oxidase activity with 3␣-Diol as the substrate. However, in contrast to the 3-ketosteroid reductase activity of the enzymes, their hydroxysteroid oxidase activity was potently inhibited by low micromolar concentrations of the opposing cofactor (NADPH). This indicates that in vivo all AKR1Cs will preferentially work as reductases. Human hepatoma (HepG2) cells (which lack 3␤-HSD/⌬ 5-4 ketosteroid isomerase mRNA expression, but express AKR1C1-AKR1C3) were able to convert DHT into 3␣-and 3␤-Diol. This conversion was inhibited by flufenamic acid establishing the in vivo significance of the 3␣/3␤-HSD activities of the AKR1C enzymes. Molecular docking simulations using available crystal structures of AKR1C1 and AKR1C2 demonstrated how 3␣/3␤-HSD activities are achieved. The observation that AKR1Cs are a source of 3␤-tetrahydrosteroids is of physiological significance because: (i) the formation of 3␤-Diol (in contrast to 3␣-Diol) is virtually irreversible, (ii) 3␤-Diol is a pro-apoptotic ligand for estrogen receptor ␤, and (iii) 3␤-tetrahydrosteroids act as ␥-aminobutyric acid type A receptor antagonists.Two classes of 3␤-hydroxysteroids, i.e. the ⌬ 5 -3␤-hydroxysteroids and the fully saturated 3␤-tetrahydrosteroids, represent pivotal intermediates in steroid hormone metabolism. In steroidogenic glands, ⌬ 5 -3␤-hydroxysteroid precursors are converted into ⌬ 4 -3-ketosteroids to produce active steroid hormones (1, 2), whereas 3-ketosteroid reduction of 5␣/5␤-dihydrosteroids into 3␤-tetrahydrosteroids is an important catabolic step in steroid hormone transformation.Human steroid hormone target tissues like the prostate express membrane attached and/or cytosolic 3␣-HSD 1 and 3␤-HSD activity (3-9). One key example of the catabolic function of these HSDs is the 3-ketosteroid reduction of the potent androgen 5␣-dihydrotestosterone (DHT, 17␤-hydroxy-5␣-androstan-3-one) into the inactive androgens 5␣-androstane-3␣,17␤-diol (3␣-Diol; Fig. 1) and 5␣-androstane-3␤,17␤-diol (3␤-Diol) (10 -12). In vivo, the formation of 3␤-Diol is virtually irreversible, whereas 3␣-Diol can be converted back to DHT via 3␣-hydroxysteroid oxidase activity (13-17). Reformation of DHT from 3␤-Diol is prevented, because 3␤-Diol is either irreversibly hydroxylated at the C-6 and/or C-7 position or ...

Tibolone is used to treat climacteric complaints and prevent osteoporosis. These beneficial effects are exerted via its 3␣-and 3␤-hydroxymetabolites. Undesirable stimulation of the breast and endometrium is not apparent. Endometrial stimulation is prevented by the progestogenic activity of its ⌬ 4 -ene metabolite. The enzymes responsible for the formation of these active metabolites are unknown. Human aldo-keto reductase (AKR)1C isoforms have been shown to act as 3␣/3␤-hydroxysteroid dehydrogenases (HSDs) on 5␣-dihydrotestosterone (5␣-DHT). We show that AKR1Cs also efficiently catalyze the reduction of the ⌬ 5(10) -3-ketosteroid tibolone to yield 3␣-and 3␤-hydroxytibolone. Homogeneous recombinant AKR1C1, AKR1C3, and AKR1C4 gave similar catalytic profiles to those observed with 5␣-DHT. AKR1C1 catalyzed exclusively the formation of 3␤-hydroxytibolone, AKR1C3 showed weak 3␤/3␣-HSD activity, and AKR1C4 acted predominantly as a 3␣-HSD. Whereas AKR1C2 acted as a 3␣-HSD toward 5␣-DHT, it functioned exclusively as a 3␤-HSD on tibolone. Furthermore, strong substrate inhibition was observed for the AKR1C2 catalyzed reduction of tibolone. Using NAD ϩ , the 3-hydroxymetabolites were efficiently oxidized by homogeneous recombinant AKR1C2 and AKR1C4. However, because of potent inhibition of this activity by NADPH, AKR1Cs will probably act only as 3-ketosteroid reductases in vivo. Molecular docking simulations using crystal structures of AKR1C1 and AKR1C2 explained why AKR1C2 inverted its stereospecificity from a 3␣-HSD with 5␣-DHT to a 3␤-HSD with tibolone. The preference for AKR1C1 and AKR1C2 to form 3␤-hydroxytibolone, and the preference of the liver-specific AKR1C4 to form 3␣-hydroxytibolone, may explain why 3␤-hydroxytibolone is the major metabolite in human target tissues and why 3␣-hydroxytibolone is the major circulating metabolite.

Tibolone [[7␣,17␣]-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one] is used to treat climacteric symptoms and prevent osteoporosis. It exerts tissue-selective effects via site-specific metabolism into 3␣-and 3␤-hydroxymetabolites and a ⌬ 4 -isomer. Recombinant human cytosolic aldo-keto reductases 1C1 and 1C2 (AKR1C1 and AKR1C2) produce 3␤-hydroxytibolone, and the liver-specific AKR1C4 produces predominantly 3␣-hydroxytibolone. These observations may account for the appearance of 3␤-hydroxytibolone in target tissues and 3␣-hydroxytibolone in the circulation. Using liver autopsy samples (which express AKR1C1-AKR1C4), tibolone was reduced via 3␣-and 3␤-hydroxysteroid dehydrogenase (HSD) activity. 3␤-Hydroxytibolone was exclusively formed in the cytosol and was inhibited by the AKR1C2-specific inhibitor 5␤-cholanic acid-3␣,7␣-diol. The cytosolic formation of 3␣-hydroxytibolone was inhibited by an AKR1C4-selective inhibitor, phenolphthalein. The ratio of these stereoisomers was 4:1 in favor of 3␤-hydroxytibolone. In HepG2 cell cytosol and intact cells (which do not express AKR1C4), tibolone was exclusively reduced to 3␤-hydroxytibolone and was blocked by the AKR1C1-AKR1C3 inhibitor flufenamic acid. In primary hepatocytes (which express AKR1C1-AKR1C4), time-dependent reduction of tibolone into 3␤-and 3␣-hydroxytibolone was observed again in a 4:1 ratio. 3␤-HSD activity was inhibited by both 5␤-cholanic acid-3␣,7␣-diol and flufenamic acid, implicating a role for AKR1C2 and AKR1C1. By contrast, the formation of 3␣-hydroxytibolone was exclusively inhibited by phenolphthalein implicating AKR1C4 in this reaction. 3␤-and 3␣-Hydroxytibolone were rapidly metabolized into polar metabolites (Ͼ85%). The formation of minor amounts of tibolone was also observed followed by AKR1C-catalyzed epimerization. The low hepatic formation of 3␣-hydroxytibolone suggests that AKR1C4 is not the primary source of this metabolite and instead it maybe formed by an intestinal or enterobacterial 3␣-HSD.Tibolone (Livial) [[7␣,17␣]-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one] is used in the treatment of climacteric symptoms and the prevention of osteoporosis (Albertazzi et al., 1998;Moore, 1999). Its favorable effects on the breast and endometrium (Colacurci et al., 1998;Valdivia and Ortega, 2000;Volker et al., 2001;Gompel et al., 2002;Blok et al., 2003) suggest that it may be an alternative to a selective estrogen receptor modulator (Smith and O'Malley, 2004) and to traditional estrogen and progestogen combined therapy. Tibolone differs from estrogen and progestogen combined therapy, because it exerts tissue selective effects via sitespecific metabolism (Kloosterboer, 2001;Kloosterboer and Ederveen, 2003). The agent has been assigned the acronym selective tissue estrogenic activity regulator to distinguish it from a selective estrogen receptor modulator, since its effects are not solely mediated by the estrogen receptor (Kloosterboer and Ederveen, 2003).After oral administration, tibolone is quickly metabolized into 3␣-and 3␤-h...