Impact of Continuous Estroprogestin Treatment on Circulating Microparticle Levels in Deep Endometriosis Patients.

There has been increasing interest in the study of new pathogenic mechanisms in endometriosis (END), including the coagulation/fibrinolysis system and its link with inflammation and tissue remodeling. It has been suggested that END patients, especially with deep-infiltrating (DE) forms, could present a hypercoagulable state revealing higher levels of proinflammatory and procoagulant markers, such as total circulating microparticles (cMPs) and cMP-TF (tissue factor), released by cells in response to damage, activation, or apoptosis. However, no previous study has assessed the effect of END hormonal treatments on cMP and cMP-TF levels. Therefore, the aim of this study was to evaluate the impact of these treatments on cMP and cMP-TF levels in DE patients. Three groups were compared: DE patients receiving a continuous combined oral contraceptive regimen (CCOCR) (n = 41), DE patients without CCOCR (n = 45), and a control group (n = 43). cMP and cMP-TF levels were evaluated in platelet-free plasma. A significant decrease in the total cMP levels was found in the DE group with CCOCR versus the group without CCOCR, reflecting a higher chronic inflammatory status in DE patients that decreased with the treatment. cMP-TF levels were higher in DE patients receiving CCOCR versus those not receiving CCOCR, suggesting that treatments containing estrogens play a predominant role in suppressing the inhibitory pathway of TF.

The potent contraceptive gestodene exerts insulinotropic effects through its a-ring reduced metabolites with intrinsic estrogen-like activity in pancreatic ß-cells.

PURPOSE: The contraceptive gestodene is a potent synthetic progestin used in several low-dose contraceptive formulations. Clinical studies reported a relationship between long-term use of combined oral contraceptives containing gestodene (GDN) and profound alterations in glucose metabolism in women. The observation that contraceptive synthetic progestins exert hormone-like effects other than their progestational activities, prompted us to investigate whether GDN may induce estrogen-like effects, even though GDN does not interact with estrogen receptors. The aim of this study was to investigate whether GDN affect pancreatic ß-cell activity, directly or through its conversion to other bioactive metabolites. METHODS: The effects of GDN and its two derivatives 3ß,5α-tetrahydro-GDN and 3α,5α-tetrahydro-GDN on insulin 2 (Ins II) and glucokinase (Gk) expression and glucose-stimulated insulin secretion were determined in pancreatic islets from female rats. RESULTS: Gestodene did exert significant effects on islet ß-cells activity. The most striking finding was that 3ß,5α-tetrahydro-GDN and 3α,5α-tetrahydro-GDN had greater stimulatory effects on Ins II and Gk expression than that observed with GDN, consistent with their effects on glucose-stimulated insulin secretion. The effects on gene expression induced by GDN-derivatives were abolished by ICI 182,780 and MPP. In addition, the presence of inhibitors of androgen and progestin-metabolizing enzymes eliminated gene expression induced by GDN. These results indicated that GDN is metabolized to A-ring reduced metabolites with estrogen-like activities and through this mechanism, GDN may affect ß-cell activity. CONCLUSIONS: Altogether, the data suggest that 19-nortestosterone-derived contraceptives such as GDN, possess insulinotropic effects through their conversion into metabolites with intrinsic estrogen-like activity in pancreatic ß-cells.

Effects of Human Sulfotransferase 2A1 Genetic Polymorphisms 3 on the Sulfation of Tibolone.

BACKGROUND AND OBJECTIVES: Previous studies have demonstrated the metabolism of tibolone through sulfation, with the cytosolic sulfotransferase (SULT) SULT2A1 as the major responsible enzyme. The current study aimed to investigate how SULT2A1 genetic polymorphisms may affect the dehydroepiandrosterone (DHEA)- and tibolone-sulfating activity of SULT2A1. METHODS: Site-directed mutagenesis was employed to generate cDNAs encoding ten different SULT2A1 allozymes. Recombinant SULT2A1 allozymes were expressed in BL21 E. coli cells, and purified using glutathione-sepharose affinity chromatography. An established sulfotransferase assay was used to analyze DHEA- and tibolone-sulfating activity of the purified SULT2A1 allozymes. RESULTS: The nine human SULT2A1 allozymes plus the wild-type SULT2A1 were found to display differential sulfating activity toward DHEA and tibolone. Kinetic analysis revealed that different SULT2A1 allozymes exhibited differential substrate affinity and catalytic efficiency toward the two substrates tested. CONCLUSION: The results obtained provided useful information concerning the differential metabolism of tibolone through sulfation in individuals with different SULT2A1 genotypes.

Identification and characterization of the steroid 15α-hydroxylase gene from Penicillium raistrickii.

Penicillium raistrickii ATCC 10490 is used for the commercial preparation of 15α-13-methy-estr-4-ene-3,17-dione, a key intermediate in the synthesis of gestodene, which is a major component of third-generation contraceptive pills. Although it was previously shown that a cytochrome P450 enzyme in P. raistrickii is involved in steroid 15α-hydroxylation, the gene encoding the steroid 15α-hydroxylase remained unknown. In this study, we report the cloning and characterization of the 15α-hydroxylase gene from P. raistrickii ATCC 10490 by combining transcriptomic profiling with functional heterologous expression in Saccharomyces cerevisiae. The full-length open reading frame (ORF) of the 15α-hydroxylase gene P450pra is 1563 bp and predicted to encode a cytochrome P450 protein of 520 amino acids. Targeted gene deletion revealed that P450pra is solely responsible for 15α-hydroxylation activity on 13-methy-estr-4-ene-3,17-dione in P. raistrickii ATCC 10490. The identification of the 15α-hydroxylase gene from P. raistrickii should help elucidate the molecular basis of regio- and stereo-specificity of steroid 15α-hydroxylation and aid in the engineering of more efficient industrial strains for useful steroid 15α-hydroxylation reactions.

The synthetic progestin, gestodene, affects functional biomarkers in neonatal rat osteoblasts through an estrogen receptor-related mechanism of action.

BACKGROUND: Clinical studies have shown that gestodene (GDN), a potent third-generation synthetic progestin, affects bone resorption. However, its mode of action in bone cells is not fully understood. The aim of this study was to establish whether GDN affects bone directly or through its bioconversion to other metabolites with different biological activities. METHODS: In this study, we investigated the effects of GDN and its A-ring reduced metabolites on proliferation, differentiation, and mineralization of calvarial osteoblasts isolated from neonatal rat and their capacity to displace [3H]-E2 at ER binding sites. RESULTS: In contrast to progesterone, gestodene did exert significant effects on osteoblast activities. The most striking finding was the observation that the A-ring reduced derivatives 3ß,5α-tetrahydro-GDN and 3α,5α-tetrahydro-GDN, though to a lesser extent, had greater stimulatory effects on the osteoblast activity than those observed with GDN. The effects on osteoblast proliferation and differentiation induced by GDN-reduced derivatives were abolished by the antiestrogen ICI 182780, consistent with their binding affinities for the estrogen receptor. In addition, the presence of a 5α-reductase inhibitor or inhibitors of aldo-keto hydroxysteroid dehydrogenases abolished the GDN-induced enhancement of osteoblast differentiation. These results indicated that GDN is metabolized to the A-ring reduced metabolites with estrogen-like activities and through this mechanism, GDN may affect the osteoblast activity. CONCLUSION: Together, the data suggest that synthetic progestins derived from 19-nortestosterone such as GDN, have beneficial effects on bone due to their biotransformation into metabolites with intrinsic estrogenic activity.

Antioxidant effect of the active metabolites of tibolone.

Certain steroidal compounds have an antioxidant effect in humans. Our aim was to test whether the synthetic steroid tibolone and its metabolites are also able to display such a property. For this, granulocytes from healthy men and women were incubated for two hours with different concentrations (10(-7), 10(-8), 10(-9 )M) of either estradiol, tibolone, 3α-hydroxytibolone, 3ß-hydroxytibolone, Δ(4)-tibolone, 3α-sulfated-tibolone, 3α-17ß-disulfated-tibolone, 3ß-sulfated-tibolone or 3ß-17ß-disulfated-tibolone. Superoxide anion generation of neutrophils was measured by photometry. Results of different steroids were given as percentages of their controls. A more simple superoxide generating system, the xanthine-xanthine oxidase reaction was also tested. We found that granulocyte superoxide production did not differ from the control using 10(-9 )M of steroids. Using 10(-8 )M concentration: estradiol (80.9 ± 2.5%); 3ß-sulfated-tibolone (83.3 ± 4.7%); 3ß-17ß-disulfated-tibolone (81.0 ± 4.2%) caused a significant decrease in superoxide production, compared to the control. In addition at 10(-7 )M, 3ß-hydroxytibolone and 3α-sulfated-tibolone also showed antioxidant effects. In the xanthine-xanthine oxidase system estradiol (67.4 ± 1.0%), 3α-sulfated-tibolone (85.8 ± 5.3%), 3α-17ß-disulfated-tibolone (71.9 ± 2.5%), 3ß-sulfated-tibolone (73.9 ± 5.0%), and 3ß-17ß-disulfated-tibolone (65.8 ± 3.4%) caused a significant decrease in superoxide production. Conclusively, although tibolone itself did not show significant antioxidant capacity, most of its active metabolites have antioxidant effects.

Molecular and structural basis of androgen receptor responses to dihydrotestosterone, medroxyprogesterone acetate and Δ(4)-tibolone.

Medroxyprogesterone acetate (MPA) has widely been used in hormone replacement therapy (HRT), and is associated with an increased risk of breast cancer, possibly due to disruption of androgen receptor (AR) signaling. In contrast, the synthetic HRT Tibolone does not increase breast density, and is rapidly metabolized to estrogenic 3α-OH-tibolone and 3ß-OH-tibolone, and a delta-4 isomer (Δ(4)-TIB) that has both androgenic and progestagenic properties. Here, we show that 5α-dihydrotestosterone (DHT) and Δ(4)-TIB, but not MPA, stabilize AR protein levels, initiate specific AR intramolecular interactions critical for AR transcriptional regulation, and increase proliferation of AR positive MDA-MB-453 breast cancer cells. Structural modeling and molecular dynamic simulation indicate that Δ(4)-TIB induces a more stable AR structure than does DHT, and MPA a less stable one. Microarray expression analyses confirms that the molecular actions of Δ(4)-TIB more closely resembles DHT in breast cancer cells than either ligand does to MPA.

Biotransformation of 17α-ethynyl substituted steroidal drugs with microbial and plant cell cultures: a review.

Structural modification of steroids through whole-cell biocatalysis is an invaluable procedure for the production of active pharmaceutical ingredients (APIs) and key intermediates. Modifications could be carried out with regio- and stereospecificity at positions hardly available for chemical agents. Much attention has been focused recently on the biotransformation of 17α-ethynyl substituted steroidal drugs using fungi, bacteria and plant cell cultures in order to obtained novel biologically active compounds with diverse structure features. Present article includes studies on biotransformation on 17α-ethynyl substituted steroidal drugs using microorganisms and plant cell cultures. Various experimental and structural elucidation methods used in biotransformational processes are also highlighted.

Alpha-glucosidase and tyrosinase inhibitors from fungal hydroxylation of tibolone and hydroxytibolones.

Sixteen new and one known metabolites 4-20 were obtained by incubation of tibolone (1) and hydroxytibolones (2 and 3) with various fungi. Their structures were elucidated by means of a homo and heteronuclear 2D NMR and by HREI-MS techniques. The relative stereochemistry was deduced by 2D NOESY experiment. Metabolites of tibolone (1) exhibited significant inhibitory activities against alpha-glucosidase and tyrosinase enzymes. Hydroxylations at C-6, C-10, C-11, C-15 positions and alpha,beta-unsaturation at C-1/C-2, C-4/C-5 showed potent inhibitory activities against these enzymes.

Regulation of activities of steroid hormone receptors by tibolone and its primary metabolites.

This work was undertaken (i) to study deeply the estrogen, androgen and progestative activities of tibolone and its metabolites (ii) to determine whether tibolone and its metabolites present glucocorticoid or mineralocorticoid activity. For this purpose, we used human cell lines bearing a luciferase gene with a responsive element under the control of human estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) or androgen receptor (AR) or chimeric Gal4 fusion with progesterone receptor (PR), glucocorticoid receptor (GR) or mineralocorticoid receptor (MR). The major tibolone metabolites, the two hydroxymetabolites, bind and activate ER with a preference for ERalpha. Tibolone and the Delta(4)-tibolone are agonists for AR and PR and surprisingly 3alpha- and 3beta-OH-tibolone are antagonists for them. Moreover we showed for the first time that tibolone and its primary metabolites are GR and MR antagonists with a stronger affinity for MR than for GR. In conclusion, tibolone by these actions on different receptors and by this capacity to transform in different metabolites, has more complex effects than initially supposed.

Progestins induce catalase activities in breast cancer cells through PRB isoform: correlation with cell growth inhibition.

Reactive oxygen species (ROS) have been suggested to participate in tumor emergence due to their mitogenic and apoptotic signaling, and as contributors to DNA structural damage. Here we report that progesterone and various synthetic steroids with progestin potencies (norethisterone acetate, MPA, and Tibolone) counteract cell growth induced by hydrogen peroxide (H(2)O(2)), through a potent induction of catalase activities, in breast cancer cells and normal human epithelial breast cells. At physiological concentrations, progesterone and the pure progestin, Org2058, displayed the most potent H(2)O(2) detoxification ability suggesting its effect was characteristic of its progestin potency. We also report on the enhancement of catalase activities by progesterone receptor isoform B (PRB), as determined from experiments using antiprogestins and MDA-MB-231, cells engineered for the selective expression of progesterone receptor isoform A or B. The potent action of progesterone on catalase activities indicates its contribution to a beneficial role in breast cell homeostasis.

Effects of hormone replacement therapy on insulin resistance and platelet function tests.

OBJECTIVES: The aim of this study was to evaluate measures of insulin resistance and platelet function in postmenopausal women with oral or transdermal hormone replacement therapy (HRT). SUBJECTS AND METHODS: Eighty women divided into four groups of 20 each were enrolled in the study. Group 1: postmenopausal hysterectomized women who received only transdermal estradiol (13.9 mg/12.5 cm(2)); group 2: women with intact uterus who were treated with estrogen-progestin combination (HRT); group 3: postmenopausal women who were treated with the selective estrogen receptor modulator tibolone, and group 4: women who were not taking any drugs for HRT were chosen as a control group (group 4). RESULTS: In group 2, homeostasis model assessment of insulin resistance and fasting insulin levels were 2.90 +/- 0.37 and 9.3 +/- 3.0 microU/ml, respectively, prior to administration of HRT. These levels were reduced to 1.91 +/- 0.41 (p = 0.001) and 7.1 +/- 2.7 microU/ml (p = 0.002), respectively, after drug therapy. Mean levels of high-sensitivity C-reactive protein (hsCRP) were decreased with HRT only in group 2 (p = 0.002). No changes for biochemical and hematological parameters were observed in the other groups. Platelet function tests showed no differences after HRT in any group. CONCLUSIONS: Estrogen-progestin combination HRT decreased measures of insulin resistance and hsCRP levels, but had no effect on platelet function tests in postmenopausal women.

Tibolone vaginal versus per os administration in the management of post-menopausal symptoms.

UNLABELLED: Tibolone is a selective tissue estrogenic activity regulator effective in the management of climacteric symptoms, without stimulating endometrial and breast tissue proliferation. It is a well-tolerated treatment regimen with minor adverse effects. The purpose of the current trial was to investigate the efficacy of tibolone administration per os versus per vaginal, concerning the treatment of climacteric symptoms. MATERIAL AND METHOD: A total of 64 post-menopausal healthy women aged 44-55 years (mean 52,5) were enrolled in the study. The patients were divided into three groups: Group A with 24 patients receiving per os Tibolone 2.5 mg x 1 daily for 6 months, Group B with 21 patients receiving vaginally Tibolone 2.5 mg x 1 for 6 months and Group C--with 19 patients receiving only hygienodietetics advices and psychological support for 6 months. All subjects underwent a physical examination, including a transvaginal ultrasonography for the measurement of endometrial thickness and a mammography for the assessment of breast density. RESULTS: In Group A and B, climacteric symptoms were similarly reduced with non significant differences between groups: 79% (19 patients) vs 76% (16 patients) reduction for hot flushes and 83% (20 patients) vs 76% (16 patients) for sweating episodes respectively (p > 0.05). Reduction of hot flushes and sweating episodes was observed in only 2 of 19 patients (10.5%) in group C (p < 0.01). Vaginal route had better results on vaginal dryness-dyspareunia in group B (in 14 patients taken tibolone orally-58% vs 16 patients taken tibolone vaginally-76%). No statistically significant difference was found related to urine symptoms between A and B groups. Tibolone by vaginal route did not cause any severe side effects (apart of a slight discharge) and it was equally (or better) tolerated than oral route. No significant alterations in breast density and BIRADS and no increase in endometrial thickness were observed in all study groups. CONCLUSION: Tibolone is effective in relieving climacteric symptoms indifferent from applications-modus: orally or vaginally. However, further and larger studies are required to confirm results.

Tibolone rapidly attenuates the GABAB response in hypothalamic neurones.

Tibolone is primarily used for the treatment of climacteric symptoms. Tibolone is rapidly converted into three major metabolites: 3 alpha- and 3beta-hydroxy (OH)-tibolone, which have oestrogenic effects, and the Delta 4-isomer (Delta 4-tibolone), which has progestogenic and androgenic effects. Because tibolone is effective in treating climacteric symptoms, the effects on the brain may be explained by the oestrogenic activity of tibolone. Using whole-cell patch clamp recording, we found previously that 17beta-oestradiol (E(2)) rapidly altered gamma-aminobutyric acid (GABA) neurotransmission in hypothalamic neurones through a membrane oestrogen receptor (mER). E(2) reduced the potency of the GABA(B) receptor agonist baclofen to activate G-protein-coupled, inwardly rectifying K(+) (GIRK) channels in hypothalamic neurones. Therefore, we hypothesised that tibolone may have some rapid effects through the mER and sought to elucidate the signalling pathway of tibolone's action using selective inhibitors and whole cell recording in ovariectomised female guinea pigs and mice. A sub-population of neurones was identified post hoc as pro-opiomelanocortin (POMC) neurones by immunocytochemical staining. Similar to E(2), we have found that tibolone and its active metabolite 3 beta OH-tibolone rapidly reduced the potency of the GABA(B) receptor agonist baclofen to activate GIRK channels in POMC neurones. The effects were blocked by the ER antagonist ICI 182 780. Other metabolites of tibolone (3 alpha OH-tibolone and Delta 4-tibolone) had no effect. Furthermore, tibolone (and 3 beta OH-tibolone) was fully efficacious in ER alpha knockout (KO) and ER beta KO mice to attenuate GABA(B) responses. The effects of tibolone were blocked by phospholipase C inhibitor U73122. However, in contrast to E(2), the effects of tibolone were not blocked by protein kinase C inhibitors or protein kinase A inhibitors. It appears that tibolone (and 3 beta OH-tibolone) activates phospholipase C leading to phosphatidylinositol bisphosphate metabolism and direct alteration of GIRK channel function. Therefore, tibolone may enhance synaptic efficacy through the G(q) signalling pathways of mER in brain circuits that are critical for maintaining homeostatic functions.

Bioequivalence studies of tibolone in premenopausal women and effects on expression of the tibolone-metabolizing enzyme AKR1C (aldo-keto reductase) family caused by estradiol.

This study aimed to investigate the bioequivalence of a test formulation of tibolone with the marketed reference formulation in 24 young healthy female volunteers. Tibolone is a synthetic steroid hormone for menopausal women. Volunteers were treated with the 2 formulations of tibolone (total dose of active ingredient 2.5 mg) according to a 2 x 2 crossover design with a 1-week washout period. Plasma concentrations of 3alpha- and 3beta-hydroxytibolone, which are major metabolites of tibolone, were assayed in timed samples over a 24-hour period with a validated gas chromatography/mass spectrometry (GC/MS) method that had a lower limit of quantification of 0.5 ng/mL. The reference and test formulations gave a mean 3alpha-hydroxytibolone C(max) of 5.0 and 5.2 ng/mL, respectively, and a mean 3beta-hydroxytibolone C(max) of 16.4 and 16.5 ng/mL, respectively. The mean AUC(t) of 3alpha-hydroxytibolone was 24.7 and 24.3 ng h/mL, whereas the mean AUC(t) of 3beta-hydroxytibolone was 57.6 and 54.8 ng h/mL for the test and reference formulations, respectively. The authors did not find significant differences in pharmacokinetic parameters between the 2 formulations, but metabolite formation was different from reports in postmenopausal women. The authors therefore measured the effects of estradiol on the expression of the tibolone-metabolizing enzymes, from the aldo-keto reductase (AKR1C) family, using HepG2 cell (human hepatoma cells) and MCF-7 cell (human breast cancer cells). Estradiol increased mRNA levels of AKR1C1, AKR1C2, and AKR1C3 and protein levels of total AKR1C in HepG2 cells. Estradiol selectively enhanced levels of AKR1C2 mRNA in MCF-7 cells. Thus, changes in the major metabolites of tibolone might result from changes in AKR1C family expression by patient estrogen status.

Detection of anabolic steroid abuse using a yeast transactivation system.

The classical analytical method for detection of anabolic steroid abuse is gas chromatography followed by mass spectrometry (GC/MS). However, even molecules with a chemical structure typical for this class of substances, are sometimes not identified in routine screening by GC/MS when their precise chemical structure is still unknown. A supplementary approach to identify anabolic steroid abuse could be a structure-independent identification of anabolic steroids based on their biological activity. To test the suitability of such a system, we have analyzed the yeast androgen receptor (AR) reporter gene system to identify anabolic steroids in human urine samples. Analysis of different anabolic steroids dissolved in buffer demonstrated that the yeast reporter gene system is able to detect a variety of different anabolic steroids and their metabolites with high specificity, including the so-called 'designer steroid' tetrahydrogestrinone. In contrast, other non-androgenic steroids, like glucocordicoids, progestins, mineralocordicoids and estrogens had a low potency to stimulate transactivation. To test whether the system would also allow the detection of androgens in urine, experiments with spiked urine samples were performed. The androgen reporter gene in yeast responds very sensitive to 5alpha-dihydrotestosterone (DHT), even at high urine concentrations. To examine whether the test system would also be able to detect anabolic steroids in the urine of anabolic steroid abusers, anonymous urine samples previously characterized by GCMS were analyzed with the reporter gene assay. Even when the concentration of the anabolic metabolites was comparatively low in some positive samples it was possible to identify the majority of positive samples by their biological activity. In conclusion, our results demonstrate that the yeast reporter gene system detects anabolic steroids and corresponding metabolites with high sensitivity even in urine of anabolic steroid abusing athletes. Therefore we believe that this system can be developed towards a powerful (pre) screening tool for the established doping tests. The system is easy to handle, robust, cost-efficient and needs no high-tech equipment. But most importantly, a biological test system does not require knowledge of the chemical structure of androgenic substances and therefore suitable to detect previously unidentified substances, especially those of the class of so-called designer steroids.

Structure-activity relationships of synthetic progestins in a yeast-based in vitro androgen bioassay.

The recent identification of tetrahydrogestrinone (THG), a non-marketed designer androgen used for sports doping but previously undetectable by established mass spectrometry-based urine drug screens, and its production by a facile chemical modification of gestrinone has raised concerns about the risks of developing designer androgens from numerous marketed progestins. We therefore have used yeast-based in vitro androgen and progesterone bioassays to conduct a structure-activity study assessing the intrinsic androgenic potential of commercially available progestins and their derivatives, to identify those compounds or structures with the highest risk of forming a basis for such misapplication. Progestins had a wide range of androgenic bioactivity that was not reliably predicted for individual steroids by their progestin bioactivity. 17alpha-Hydroxyprogesterone and 19-norprogesterone derivatives with their bulky 17beta-substituents were strong progestins but generally weak androgens. 17alpha-Ethynylated derivatives of testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone such as gestrinone, ethisterone, norethisterone and norgestrel had the most significant intrinsic androgenicity of all the commercially marketed progestins. Facile chemical modification of the 17alpha-ethynyl group of each of these progestins produces 17alpha-methyl, ethyl and allyl derivatives, including THG and norbolethone, which further enhanced androgenic bioactivity. Thus by using the rapid and sensitive yeast bioassay we have screened a comprehensive set of progestins and associated structures and identified the ethynylated testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone derivatives as possessing the highest risk for abuse and potential for conversion to still more potent androgens. By contrast, modern progestins such as progesterone, 17alpha-hydroxyprogesterone and 19-norprogesterone derivatives had minimal androgenic bioactivity and pose low risk.

Enantioselective production of 3-hydroxy metabolites of tibolone by yeast reduction.

The enantioselective reduction of tibolone into the corresponding 3alpha-hydroxy or 3beta-hydroxy metabolite can be controlled by choosing suited strains of yeasts and biotransformation conditions. A restricted screening performed among 52 yeasts showed that the 3alpha-epimer was preferentially obtained with high epimeric purity with various strains (i.e. with Kluyveromyces lactis CBS 2359), while only Saccharomyces cerevisiae CBS 3093 gave the 3beta-epimer as major product. The reduction of tibolone with K. lactis CBS 2359 and S. cerevisiae CBS 3093 was optimised. S. cerevisiae CBS 3093 furnished a 96:4 ratio of 3beta/3alpha with complete molar conversion within 72h when the initial concentration of substrate was below 2.5g/L. K. lactis CBS 2359 gave a 99:1 ratio of 3alpha/3beta with complete conversion in 64h.

3-ketosteroid reductase activity and expression by fetal rat osteoblasts.

In addition to reproductive tissue, sex hormones induce transcriptional events in many connective tissue cells, including osteoblasts. Some sex hormone receptor modulators with bone sparing effects selectively target estrogen or androgen receptors, whereas others appear more promiscuous, in part through enzymatic metabolism. Rat osteoblasts express significant oxidative 3alpha-hydroxysteroid dehydrogenase activity, which can convert precursor substrates to potent androgen receptor agonists. Here we show that they also express 3-ketosteroid reductase activity, exemplified by 7-methyl-17-ethynyl-19-norandrostan-5 (10)en-3-one (tibolone) conversion to potent estrogen receptor alpha agonists. Conversion was rapid and quantitative, with 3alpha-hydroxytibolone as the primary metabolite. Consistently, tibolone induced estrogen receptor alpha-dependent gene promoter activity through cis-acting estrogen response elements, increased the stimulatory effect of TGF-beta on Smad-dependent gene promoter activity, and enhanced prostaglandin E2-induced activity of transcription factor Runx2. Rat osteoblasts express the 3-ketosteroid reductase AKR1C9, an aldo-keto reductase gene family member. Exposure to prostaglandin E2 increased AKR1C9 gene promoter activity and mRNA expression. AKR1C9 promoter activity was also enhanced by overexpression of protein kinase A catalytic subunit or transcription factor C/EBPdelta, and the effect of PGE2 was reduced by dominant negative C/EBPdelta competition or C/EBPdelta antisense expression. Moreover, prostaglandin E2 increased the amount of functional endogenous nuclear C/EBPdelta that could bind specifically to a distinct domain approximately 1.8-kb upstream from the start site of AKR1C9 transcription. In summary, in addition to 3alpha-hydroxysteroid dehydrogenase, rat osteoblasts express significant and regulatable 3-ketosteroid reductase activity. Through these enzymes, they may selectively metabolize precursor compounds into potent steroid receptor agonists locally within bone.