Microbial Modifications of Androstane and Androstene Steroids by Penicillium vinaceum.

The biotransformation of steroid compounds is a promising, environmentally friendly route to new pharmaceuticals and hormones. One of the reaction types common in the metabolic fate of steroids is Baeyer-Villiger oxidation, which in the case of cyclic ketones, such as steroids, leads to lactones. Fungal enzymes catalyzing this reaction, Baeyer-Villiger monooxygenases (BVMOs), have been shown to possess broad substrate scope, selectivity, and catalytic performance competitive to chemical oxidation, being far more environmentally green. This study covers the biotransformation of a series of androstane steroids (epiandrosterone and androsterone) and androstene steroids (progesterone, pregnenolone, dehydroepiandrosterone, androstenedione, 19-OH-androstenedione, testosterone, and 19-nortestosterone) by the cultures of filamentous fungus Penicillium vinaceum AM110. The transformation was monitored by GC and the resulting products were identified on the basis of chromatographic and spectral data. The investigated fungus carries out effective Baeyer-Villiger oxidation of the substrates. Interestingly, introduction of the 19-OH group into androstenedione skeleton has significant inhibitory effect on the BVMO activity, as the 10-day transformation leaves half of the 19-OH-androstenedione unreacted. The metabolic fate of epiandrosterone and androsterone, the only 5α-saturated substrates among the investigated compounds, is more complicated. The transformation of these two substrates combined with time course monitoring revealed that each substrate is converted into three products, corresponding to oxidation at C-3 and C-17, with different time profiles and yields.

Antiandrogens act as selective androgen receptor modulators at the proteome level in prostate cancer cells.

Current therapies for prostate cancer include antiandrogens, inhibitory ligands of the androgen receptor, which repress androgen-stimulated growth. These include the selective androgen receptor modulators cyproterone acetate and hydroxyflutamide and the complete antagonist bicalutamide. Their activity is partly dictated by the presence of androgen receptor mutations, which are commonly detected in patients who relapse while receiving antiandrogens, i.e. in castrate-resistant prostate cancer. To characterize the early proteomic response to these antiandrogens we used the LNCaP prostate cancer cell line, which harbors the androgen receptor mutation most commonly detected in castrate-resistant tumors (T877A), analyzing alterations in the proteome, and comparing these to the effect of these therapeutics upon androgen receptor activity and cell proliferation. The majority are regulated post-transcriptionally, possibly via nongenomic androgen receptor signaling. Differences detected between the exposure groups demonstrate subtle changes in the biological response to each specific ligand, suggesting a spectrum of agonistic and antagonistic effects dependent on the ligand used. Analysis of the crystal structures of the AR in the presence of cyproterone acetate, hydroxyflutamide, and DHT identified important differences in the orientation of key residues located in the AF-2 and BF-3 protein interaction surfaces. This further implies that although there is commonality in the growth responses between androgens and those antiandrogens that stimulate growth in the presence of a mutation, there may also be influential differences in the growth pathways stimulated by the different ligands. This therefore has implications for prostate cancer treatment because tumors may respond differently dependent upon which mutation is present and which ligand is activating growth, also for the design of selective androgen receptor modulators, which aim to elicit differential proteomic responses dependent upon cellular context.

The isomerization of Δ5-androstene-3,17-dione by the human glutathione transferase A3-3 proceeds via a conjugated heteroannular diene intermediate.

The seemingly simple proton abstraction reactions underpin many chemical transformations, including isomerization reactions, and are thus of immense biological significance. Despite the energetic cost, enzyme-catalyzed proton abstraction reactions show remarkable rate enhancements. The pathways leading to these accelerated rates are numerous and on occasion partly enigmatic. The isomerization of the steroid Δ(5)-androstene-3,17-dione by the glutathione transferase A3-3 in mammals was investigated to gain insight into the mechanism. Particular emphasis was placed on the nature of the transition state, the intermediate suspected of aiding this process, and the hydrogen bonds postulated to be the stabilizing forces of these transient species. The UV-visible detection of the intermediate places this species in the catalytic pathway, whereas fluorescence spectroscopy is used to obtain the binding constant of the analog intermediate, equilenin. Solvent isotope exchange reveals that proton abstraction from the substrate to form the intermediate is rate-limiting. Analysis of the data in terms of the Marcus formalism indicates that the human glutathione transferase A3-3 lowers the intrinsic kinetic barrier by 3 kcal/mol. The results lead to the conclusion that this reaction proceeds through an enforced concerted mechanism in which the barrier to product formation is kinetically insignificant.

A new derivative for oxosteroid analysis by mass spectrometry.

Here we report a new method for oxosteroid identification utilizing "tandem mass tag hydrazine" (TMTH) carbonyl-reactive derivatisation reagent. TMTH is a reagent with a chargeable tertiary amino group attached through a linker to a carbonyl-reactive hydrazine group. Thirty oxosteroids were analysed after derivatisation with TMTH by electrospray ionization mass spectrometry (ESI-MS) and were found to give high ion-currents compared to underivatised molecules. ESI-tandem mass spectrometry (MS/MS) analysis of the derivatives yielded characteristic fragmentation patterns with specific mass reporter ions derived from the TMT group. A shotgun ESI-MS method incorporating TMTH derivatisation was applied to a urine sample.

The use of symmetry in enantioselective synthesis: four pairs of chrysene enantiomers prepared from 19-nortestosterone.

Expansion of the D-ring of 19-norsteroids with incorporation of the steroid C-18 methyl group into a newly formed six-membered ring provides easy access to the chrysene ring system. By taking advantage of the symmetry of the chrysene ring system and avoiding meso chrysene intermediates, four optically pure 2,8-difunctionalized (C-2 hydroxyl group and C-8 oxo group) hexadecahydrochrysene diastereomers, and their corresponding optically pure enantiomers were prepared from 19-nortestosterone. The eight chrysene stereoisomers are of interest as starting materials for preparing chrysene analogues of physiologically important neurosteroids.

Production and characterisation of monoclonal antibodies against 19-Nortestosterone.

OBJECTIVE: To produce anti-19-Nortestosterone (NT) monoclonal antibodies and identify their immunological characteristics. METHODS: Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cell clones. A caprylic acid ammonium sulphate (CAAP) method was used to purify NT mAb, and the Batty saturation method was used to determine the affinity constant (Kaff). RESULTS: Five hybridoma cell lines, named NT-1, NT-2, NT-3, NT-4, and NT-5, were identified and their corresponding mAbs were of the IgG(1) isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7 × 10(9) L/mol. The titers and IC(50) values of purified ascite fluids were in the range of (0.64-2.56) × 10(5) and (0.55-1.0) ng/mL, respectively. Of all the cross-reacting steroids, (-NT was the most reactive with the mAbs at 62% with NT-1 mAb and 64% with NT-2 mAb. Negligible cross-reactivity (<0.01%) with other steroids was observed. CONCLUSION: The establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals.

Structurally different anabolic androgenic steroids reduce neurite outgrowth and neuronal viability in primary rat cortical cell cultures.

The illicit use of anabolic androgenic steroids (AAS) among adolescents and young adults is a major concern due to the unknown and unpredictable impact of AAS on the developing brain and the consequences of this on mental health, cognitive function and behaviour. The present study aimed to investigate the effects of supra-physiological doses of four structurally different AAS (testosterone, nandrolone, stanozolol and trenbolone) on neurite development and cell viability using an in vitro model of immature primary rat cortical cell cultures. A high-throughput screening image-based approach, measuring the neurite length and number of neurons, was used for the analysis of neurite outgrowth. In addition, cell viability and expression of the Tubb3 gene (encoding the protein beta-III tubulin) were investigated. Testosterone, nandrolone, and trenbolone elicited adverse effects on neurite outgrowth as deduced from an observed reduced neurite length per neuron. Trenbolone was the only AAS that reduced the cell viability as indicated by a decreased number of neurons and declined mitochondrial function. Moreover, trenbolone downregulated the Tubb3 mRNA expression. The adverse impact on neurite development was neither inhibited nor supressed by the selective androgen receptor (AR) antagonist, flutamide, suggesting that the observed effects result from another mechanism or mechanisms of action that are operating apart from AR activation. The results demonstrate a possible AAS-induced detrimental effect on neuronal development and regenerative functions. An impact on these events, that are essential mechanisms for maintaining normal brain function, could possibly contribute to behavioural alterations seen in AAS users.

Determination of nandrolone metabolites in human urine: comparison between liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry.

Nandrolone (19-nortestosterone) is an androgenic anabolic steroid illegally used as a growth-promoting agent in animal breeding and as a performance enhancer in athletics. Therefore, its use was officially banned in 1974 by the Medical Commission of the International Olympic Committee (IOC). Following nandrolone administration, the main metabolites in humans are 19-norandrosterone, 19-norethiocolanolone and 19-norepiandrosterone, and their presence in urine is the basis of detecting its abuse. The present work was undertaken to determine, in human urine, nandrolone metabolites (phase I and phase II) by developing and comparing multiresidue liquid chromatography/tandem mass spectrometry (LC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) methods. A double extraction by solid-phase extraction (SPE) was necessary for the complete elimination of the interfering compounds. The proposed methods were also tested on a real positive sample, and they allow us to determine the conjugated/free fractions ratio reducing the risk of false positive or misleading results and they should allow laboratories involved in doping control analysis to monitor the illegal use of steroids. The advantages of LC/MS/MS over GC/MS (which is the technique mainly used) include the elimination of the hydrolysis and derivatization steps: it is known that during enzymatic hydrolysis several steroids can be converted into related compounds and deconjugation is not always 100% effective. The validation parameters for the two methods were similar (limit of quantification (LOQ) <1 ng/mL and percentage coefficient of variance (CV%) <16.4), and both were able to confirm unambiguously all the analytes, thus confirming the validity of both techniques.

Why use of dienogest for the first contraceptive pill with estradiol?

Dienogest (DNG) has the essential properties of an effective progestogen for use in a new contraceptive pill using estradiol valerate as estrogenic component -- it inhibits ovulation and protects against endometrial proliferation. DNG is a derivative of norethisterone (NET), but has a cyanomethyl- instead of an ethinyl-group in C17 position which may offer a variety of benefits regarding hepatic effects. The similarity to NET is reflected in the high endometriotropy and in similar pharmacokinetics like short plasma half-live and high bioavailability. However, DNG also elicits properties of progesterone derivatives like neutrality in metabolic and cardiovascular system and considerable antiandrogenic activity, the latter increased by lack of binding to SHBG as specific property of DNG. It has no glucocorticoid and antimineralocorticoid activity and has no antiestrogenic activity with the consequence that possible beneficial estradiol effects should not be antagonized. This may be of special importance for the tolerability and safety of the first pill with estradiol valerate instead of ethinylestradiol, although well-designed postmarketing studies are still ongoing to demonstrate what can be expected on the basis of pharmacology.

A progestin isn't a progestin: dienogest for endometriosis as a blueprint for future research - Review as a contribution for discussion.

The different etiopathogenetic mechanisms and the diversity of clinical features of endometriosis has not yet allowed to identify a causal pharmacological monotherapy satisfying the unresolved medical needs in this important female disease. Therefore, despite the search for new therapeutic principles for the indication, the strategy of gradual optimization of established therapeutic principles should not be disregarded.In the case of progestins, the fact that each compound has its own, specific profile may allow to study the therapeutic relevance of the various signal cascades influenced by their receptors.Using the example of the progestin dienogest, the different genomic and non-genomic mechanisms of action are discussed. It is pharmacodynamic profile is unique compared to other progestins.In light of the emerging multitude of pathomechanisms in endometriosis, a monotherapy may not be possible, and then the search for broad spectrum compounds or combination therapies with dual or multiple mode of action in a clinically relevant dose range might be considered. The progestogenic action may greatly benefit from, by way of example, additional anti-inflammatory and/or anti-fibrotic and/or pro-apoptotic activities. Such a strategy could lead to new drug classes.

Enzymatic Synthesis of Anabolic Steroid Glycosides by Glucosyltransferase from Terribacillus sp. PAMC 23288.

The application of steroids has steadily increased thanks to their therapeutic effects. However, alternatives are required due their severe side effects; thus, studies on the activities of steroid derivatives are underway. Sugar derivatives of nandrolone, which is used to treat breast cancer, as well as cortisone and prednisone, which reduce inflammation, pain, and edema, are unknown. We linked O-glucose to nandrolone and testosterone using UDP-glucosyltransferase (UGT-1) and, then, tested their bioactivities in vitro. Analysis by NMR showed that the derivatives were 17ß-nandrolone ß-D-glucose and 17ß-testosterone ß-D-glucose, respectively. The viability was higher and cytotoxicity was evident in PC12 cells incubated with rotenone and, testosterone derivatives, compared to the controls. SH-SY5Y cells incubated with H2O2 and nandrolone derivatives remained viable and cytotoxicity was attenuated. Both derivatives enhanced neuronal protective effects and increased the amounts of cellular ATP.

Bioactive Rearrangement Products from Aqueous Photolysis of Pharmaceutical Steroids.

In an ongoing effort to study the environmental fate of endocrine-active steroid hormones, we report the formation of phenolic rearrangement products (3 and 4) with a novel 6,5,8,5-ring system following aqueous photolysis of dienogest (1) and methyldienolone (2). The structures were established by analysis of 2D NMR and HRMS data, and that of 3 was confirmed by X-ray diffraction analysis. These photoproducts exhibit progestogenic and androgenic activity, albeit with less potency than their parent compounds.

15N NMR relaxation studies of Y14F mutant of ketosteroid isomerase: the influence of mutation on backbone mobility.

The backbone dynamics of Y14F mutant of Delta(5)-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by 15N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S2) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.

In vivo and in vitro effect of novel 4,16-pregnadiene-6,20-dione derivatives, as 5alpha-reductase inhibitors.

In this study, we report the synthesis and biological evaluation of several new 3-substituted pregna-4,16-diene-6,20-dione derivatives (11a-11d). These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological effect of these steroids was demonstrated in in vivo and in vitro experiments. In the in vivo experiments, we measured the activity of the 11a-11d on the weight of the prostate gland of gonadectomized hamsters treated with testosterone plus finasteride or with the new steroids. For the studies in vitro, we determined the IC50 values by measuring the steroid concentration that inhibits 50% of the activity of 5alpha-reductase present in human prostate. In order to study the mechanism of action of 11a-11d, we also determined the capacity of these steroids to bind to the androgen receptor (AR) present in the rat prostate cytosol using labeled mibolerone as a tracer. The results from this work indicated that compounds 11a-11d significantly decreased the weight of the prostate as compared to testosterone treated animals and this reduction of the weight of the prostate was comparable to that produced by the finasteride. On the other hand 11a-11d exhibited a high inhibitory activity for the human 5alpha-reductase enzyme with IC50 values of 1.4 x 10(-8), 1.8 x 10(-9), 1.0 x 10(-8) and 4 x 10(-5) respectively. However the IC50 value of 11a (1.8 x 10(-9)) was the only one lower than that of finasteride (8.5 x 10(-9)). Nevertheless this compound did not show a higher potency in vivo as compared to that of compounds 11b-11d. The competition analysis for the androgen receptor indicated that the IC50 value of non-labeled mibolerone used in this experiment was 1nM, whereas steroids 10, 11a-11d did not inhibit the labeled mibolerone binding to the androgen receptor. On the other hand, steroid 10 did not show any activities in vitro or in vivo, and for this reason these steroidal derivatives (11a-11d) cannot be considered as prodrugs of compound 10. In conclusion, the compounds containing chlorine 11a, bromine 11b, iodine 11c atoms, and 11d (without any substituent in the ester moiety) at C-3 produce a significant decrease of the prostate weight in castrated animals treated with T and inhibits the activity of the 5alpha-reductase. Apparently the presence of the halogen atoms in compounds 11a-11c enhances the inhibitory activity for the 5alpha-reductase enzyme.

Quantitative Monitoring the Anti-Solvent Crystallization and Storage Process for Nandrolone by Near-Infrared Spectroscopy.

A novel hydrate (SH2O) of nandrolone was prepared by anti-solvent methods. The crystallization processes with 2 schemes (A and B) were monitored by in-line near-infrared (NIR) spectroscopy. The amounts of SH2O in powder samples obtained by the anti-solvent crystallization and storage process were quantified by NIR combined with chemometrics methods. In-line NIR spectra from 4500 to 8000 cm-1 were chosen to capture physicochemical changes during the whole crystallization process. The combination of the principal component results with offline characterization (scanning electron microscopy, powder X-ray diffraction, NIR) data showed that both schemes yielded high purity SH2O products, but the crystallization speed of scheme B was significantly accelerated. It was demonstrated that in-line NIR spectroscopy combined with principal component analysis can be very useful to monitor in real time and control the anti-solvent crystallization process. Moreover, the solubility and the solid-state transformation of nandrolone under different storage conditions were investigated. The apparent solubility of SH2O was 2.19-2.44 times of Form I, and SH2O was relatively stable when stored at a high relative humidity and temperature below 25°C.

Synthesis and spectral properties of estrogen- and androgen-BODIPY conjugates.

To develop receptor based fluorescence ligands for imaging breast and prostate cancer, a series of estrogen-, testosterone- and 19-nortestosterone conjugates linked to BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) or aza-BODIPY, were prepared. Their synthesis involves attachment of iodo derivatives of differently substituted BODIPY and aza-BODIPY analogs to the C17α-position of the steroid moieties using either the Sonogashira coupling or Click reaction. The UV-Vis absorption spectra of the conjugates range from 500 to 710nm with fluorescence emission properties ranging from 520 to 700nm, facilitating observations in living cells and tissues. Selection of the site of substitution, as well as the type of substituents on the steroidal moiety and the use of different linkers, provides a library of fluorescing conjugates to explore the effect of structural modifications on biological properties.

Effects and detection of Nandrosol and ractopamine administration in veal calves.

The present study describes different effects of the selective androgen receptor modulator (SARM) nandrolone phenylpropionate (Nandrosol) and the ß-agonist ractopamine administration in veal calves, and it investigates different strategies applied to trace these molecules. Morphological changes of gonads and accessory glands attributed to androgen effects, such as testicular atrophy, seminiferous tubule diameter reduction and hyperplasia of prostate epithelium, were detected, although SARMs are not described to cause these lesions. The gene expression analysis showed an anabolic activity of Nandrosol in Longissimus dorsi muscle, where myosin heavy chain (MYH) was significantly up-regulated. An IGF1 increase was weakly significant only in Vastus lateralis muscle. In conclusion, the anatomo-histopathological observations and the MYH mRNA up-regulation in Longissimus dorsi muscle confirm the androgenic treatment in experimental animals. The biosensor assay was not enough sensitive to detect residues in urines and only the direct chemical analysis of urine samples confirmed both ß-agonist and SARM treatment.

Environmental photochemistry of dienogest: phototransformation to estrogenic products and increased environmental persistence via reversible photohydration.

Potent trienone and dienone steroid hormones undergo a coupled photohydration (in light)-thermal dehydration (in dark) cycle that ultimately increases their environmental persistence. Here, we studied the photolysis of dienogest, a dienone progestin prescribed as a next-generation oral contraceptive, and used high resolution mass spectrometry and both 1D and 2D nuclear magnetic resonance spectroscopy to identify its phototransformation products. Dienogest undergoes rapid direct photolysis (t1/2 ∼ 1-10 min), forming complex photoproduct mixtures across the pH range examined (pH 2 to 7). Identified products include three photohydrates that account for ∼80% of the converted mass at pH 7 and revert back to parent dienogest in the absence of light. Notably, we also identified two estrogenic compounds produced via the A-ring aromatization of dienogest, evidence for a photochemically-induced increase in estrogenic activity in product mixtures. These results imply that dienogest will undergo complete and facile photolytic transformation in sunlit surface water, yet exhibit greater environmental persistence than might be anticipated by inspection of kinetic rates. Photoproduct mixtures also include transformation products with different nuclear receptor binding capabilities than the parent compound dienogest. These outcomes reveal a dynamic fate and biological risk profile for dienogest that must also take into account the composition and endocrine activity of its transformation products. Collectively, this study further illustrates the need for more holistic regulatory, risk assessment, and monitoring approaches for high potency synthetic pharmaceuticals and their bioactive transformation products.

Evidence of boldenone, nandrolone, 5(10)-estrene-3ß-17α-diol and 4-estrene-3,17-dione as minor metabolites of testosterone in equine.

The detection of boldenone, nandrolone, 5(10)-estrene-3ß,17α-diol, and 4-estrene-3,17-dione in a urine sample collected from a gelding having been treated with testosterone (500 mg 'Testosterone Suspension 100', single dose, injected intramuscularly) in 2009 led the authors' laboratory to suspect that these 'testicular' steroids could be minor metabolites of testosterone in geldings. Administration trials on six castrated horses with Testosterone Suspension 100 confirmed that low levels of boldenone, nandrolone, 5(10)-estrene-3ß,17α-diol, and 4-estrene-3,17-dione could indeed be detected and confirmed in the early post-administration urine samples from all six geldings. Although boldenone has been reported to be present in urine after testosterone administration, there has been no direct evidence reported that boldenone, nandrolone, 5(10)-estrene-3ß,17α-diol, and 4-estrene-3,17-dione are metabolites of testosterone in geldings. Subsequent in vitro experiments involving the incubation of testosterone with horse liver microsomes, liver, adipose and muscle tissues, and adrenal cortex homogenates failed to provide evidence that these four substances are minor metabolites of testosterone. An administration trial using 'Testosterone Suspension 100' supplemented with 13 C-labelled testosterone (500 mg, 1:1 ratio, injected intramuscularly) was performed. The similarities of the excretion curves of 12 C-testosterone and 13 C-testosterone in urine suggest that there was minimal kinetic isotope effect. 13 C-Labelled boldenone, nandrolone and 4-estrene-3,17-dione were detected but not 5(10)-estrene-3ß,17α-diol and its 13 C-counterpart. This is the first unequivocal evidence of boldenone, nandrolone and 4-estrene-3,17-dione being metabolites of testosterone in geldings. In view of these results, caution should be exercised when interpreting findings of boldenone, nandrolone and/or 4-estrene-3,17-dione together with a relatively high level of testosterone in gelding urine. Copyright © 2017 John Wiley & Sons, Ltd.

Regio- and stereoselective reduction of 17-oxosteroids to 17ß-hydroxysteroids by a yeast strain Zygowilliopsis sp. WY7905.

The reduction of 17-oxosteroids to 17ß-hydroxysteroids is one of the important transformations for the preparation of many steroidal drugs and intermediates. The strain Zygowilliopsis sp. WY7905 was found to catalyze the reduction of C-17 carbonyl group of androst-4-ene-3,17-dione (AD) to give testosterone (TS) as the sole product by the constitutive 17ß-hydroxysteroid dehydrogenase (17ß-HSD). The optimal conditions for the reduction were pH 8.0 and 30°C with supplementing 10g/l glucose and 1% Tween 80 (w/v). Under the optimized transformation conditions, 0.75g/l AD was reduced to a single product TS with >90% yield and >99% diastereomeric excess (de) within 24h. This strain also reduced other 17-oxosteroids such as estrone, 3ß-hydroxyandrost-5-en-17-one and norandrostenedione, to give the corresponding 17ß-hydroxysteroids, while the C-3 and C-20 carbonyl groups were intact. The absence of by-products in this microbial 17ß-reduction would facilitate the product purification. As such, the strain might serve as a useful biocatalyst for this important transformation.