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.

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.

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.

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.

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.

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.

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.

Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites.

Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6ß,10ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11ß,17ß-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of ß-glucuronidase enzyme (IC50 = 42.98 ± 1.24 µM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 µM). Its transformed products 3 (IC50 = 79.09 ± 0.06 µM), and 8 (IC50 = 70.09 ± 0.05 µM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 µM), and its metabolite 8 (IC50 = 34.16 ± 5.3 µM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 µM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 µM) and 4 (IC50 = 152.5 ± 2.15 µM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 µM), and its transformed products 2 (IC50 = 43.3 ± 7.7 µM), 3 (IC50 = 65.6 ± 2.5 µM), and 4 (IC50 = 89.4 ± 2.7 µM) were also found to be moderately toxic to 3T3 cell line (mouse fibroblast). Interestingly, metabolite 8 showed no cytotoxicity against 3T3 cell line. Compounds 1-4, and 8 were also evaluated for inhibition of tyrosinase, carbonic anhydrase, and α-glucosidase enzymes, and all were found to be inactive.

Optimised deconjugation of androgenic steroid conjugates in bovine urine.

After administration of steroids to animals the steroids are partially metabolised in the liver and kidney to phase 2 metabolites, i.e., glucuronic acid or sulphate conjugates. During analysis these conjugated metabolites are normally deconjugated enzymatically with aryl sulphatase and glucuronidase resulting in free steroids in the extract. It is well known that some sulphates are not deconjugated using aryl sulphatase; instead, for example, solvolysis can be used for deconjugation of these aliphatic sulphates. The effectiveness of solvolysis on androgenic steroid sulphates was tested with selected aliphatic steroid sulphates (boldenone sulphate, nortestosteron sulphate and testosterone sulphate), and the method was validated for analysis of androgenic steroids in bovine urine using free steroids, steroid sulphates and steroid glucuronides as standards. Glucuronidase and sulphuric acid in ethyl acetate were used for deconjugation and the extract was purified by solid-phase extraction. The final extract was evaporated to dryness, re-dissolved and analysed by LC-MS/MS.

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.

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.

Transferred multipolar atom model for 10ß,17ß-dihydroxy-17α-methylestr-4-en-3-one dihydrate obtained from the biotransformation of methyloestrenolone.

Biotransformation is the structural modification of compounds using enzymes as the catalysts and it plays a key role in the synthesis of pharmaceutically important compounds. 10ß,17ß-Dihydroxy-17α-methylestr-4-en-3-one dihydrate, C19H28O3·2H2O, was obtained from the fungal biotransformation of methyloestrenolone. The structure was refined using the classical independent atom model (IAM) and a transferred multipolar atom model using the ELMAM2 database. The results from the two refinements have been compared. The ELMAM2 refinement has been found to be superior in terms of the refinement statistics. It has been shown that certain electron-density-derived properties can be calculated on the basis of the transferred parameters for crystals which diffract to ordinary resolution.

Fundamental understanding of drug absorption from a parenteral oil depot.

Oil depots are parenteral drug formulations meant for sustained release of lipophilic compounds. Until now, a comprehensive understanding of the mechanism of drug absorption from oil depots is lacking. The aim of this paper was to fill this gap. A clinical study with healthy volunteers was conducted. An oil depot with nandrolone decanoate and benzyl alcohol was subcutaneously administered in the upper arm of female volunteers. Pharmacokinetic profiles of both substances were related to each other and to literature data. Benzyl alcohol absorbs much more rapidly than nandrolone. In detail, it appears that benzyl alcohol enters the central compartment directly, while nandrolone decanoate is recovered in serum after a lag time. This lag time is also seen in literature data, although not reported explicitly. The absorption of nandrolone is enhanced by the presence of benzyl alcohol. This is most likely an effect of altered oil viscosity and partition coefficient between the oil and aqueous phase. The absorption rate constant of compounds is found to be related to the logP of the solubilized prodrug. The absorption rate is however not only determined by the physico-chemical properties of the formulation but also by the tissue properties. Here, it is argued that lymphatic flow must be considered as a relevant parameter.

Stereocontrolled synthesis of the four 16-hydroxymethyl-19-nortestosterone isomers and their antiproliferative activities.

Novel 16-hydroxymethyl-19-nortestosterone diastereomers were prepared by Birch reduction from the corresponding 3-methoxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol isomers with known configurations. The synthesized compounds are 16α- and 16ß-hydroxymethyl-substituted 19-nortestosterone and their 17α-epimers. To prepare 17α-19-nortestosterone, the Mitsunobu inversion reaction of 19-nortestosterone with different alkyl and aryl carboxylic acids was chosen. Deacylation of the new compounds by the Zemplén method yielded the required 17α-19-nortestosterone. The antiproliferative activities of the structurally related compounds were determined in vitro through microculture tetrazolium assays on a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231, MDA-MB-361 and T47D) and ovarian (A2780) cell lines. The 17α epimer of 19-nortestosterone demonstrated considerable activity, selectively for HeLa cells, with a calculated IC50 of 0.65 µM. The reference compound, cisplatin, displayed an order of magnitude higher IC50 (12.4 µM). The cancer selectivity of 17α-19-nortestosterone was tested by MTT assay performed with noncancerous human fibroblast cell line MRC-5. The results indicated that 17α-19-nortestosterone selectively disturbs the viability of HeLa cells without greatly affecting other cancer cell types and intact fibroblasts.

Nortestosterone-derived synthetic progestogens do not activate the progestogen receptor of Murray-Darling rainbowfish (Melanotaenia fluviatilis) but are potent agonists of androgen receptors alpha and beta.

Synthetic progestogens derived from 19-nortestosterone can elicit a number of adverse effects in fish including decreased fecundity, altered hormone levels, disruption of normal breeding cycles, expression in females of male-specific biomarkers, development of male secondary sexual characteristics in females, and changes in the expression of steroidogenic genes. A recent in vitro study showed that a number of representatives from this class of progestins were potent agonists of fathead minnow androgen receptor (AR) and only weak agonists of progesterone receptor (PR) from the same species. This confirms that synthetic progestogens derived from 19-nortestosterone function as AR agonists in otomorphs, which express a single AR subtype. However, numerous perciformes are known to express two AR subtypes. We have recently shown that ARα and ARß from Murray-Darling rainbowfish (Melanotaenia fluviatilis) respond differently to certain androgens and anti-androgens. The goal of the present study was to determine concentration-response profiles for selected progestins in transactivation assays driven by rainbowfish ARα, ARß and PR in order to ascertain the relative potency of progestins against these receptors. As a means of confirming the expected activity of the progestins and reference compounds used in the study against human-derived receptors, we also established concentration-response relationships using transactivation assays driven by human PR and AR. We found that all five 19-nortestosterone-derived progestins tested were highly potent agonists of rainbowfish ARα, but that only four of the five progestins were potent agonists of rainbowfish ARß, with norgestimate exhibiting only weak activity against rainbowfish ARß. The spironolactone-derived progestin, drospirenone, was not an agonist of rainbowfish ARα or ARß but was a weak agonist of rainbowfish PR. None of the 19-nortestosterone-progestins activated rainbowfish PR. These findings confirm that the majority of 19-nortestosterone-derived progestins are likely to elicit strong androgenic activity in teleosts, but that PR-mediated effects would be minimal. In species that express two AR subtypes similar to rainbowfish ARα and ARß, biological processes mediated by a specific subtype may be affected differently by progestins such as norgestimate.

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.

Synthesis and biological evaluation of nandrolone-bodipy conjugates.

Here, we report synthesis and biological evaluation of fluorescent nandrolone-3-carboxymethyloxime derivatives conjugated with green-emitting bodipy dye via PEG linkers. All the newly-synthesized compounds were evaluated for their effect on cell proliferation in vitro in MCF-7, LNCaP, PC-3 and HEK 293T model cell lines using WST-1 assay. By means of live-cell fluorescence microscopy, the intracellular localization of nandrolone-bodipy conjugates was revealed in endoplasmic reticulum. Moreover, we performed competitive localization study with nonfluorescent nandrolone, metandrolone, boldenone, trenbolone, and testosterone.

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.