ChlorodehydromethyltestosteroneBy contrast, microsomal steroid hydroxylases did not convert OT. Spectroscopic binding assays revealed dissociation constants of Products of OT metabolism by the CYP11B subfamily members were produced at a milligram scale with a recombinant Sarm anabolic minds coli —based whole-cell turabolin que es. All three enzymes are shown to be inhibited by OT in their natural function. The extent of inhibition thereby depends on the affinity of the enzyme for OT and turabolin que es strongest effect was demonstrated for CYP11B2.
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By contrast, microsomal steroid hydroxylases did not convert OT. Spectroscopic binding assays revealed dissociation constants of Products of OT metabolism by the CYP11B subfamily members were produced at a milligram scale with a recombinant Escherichia coli —based whole-cell system. All three enzymes are shown to be inhibited by OT in their natural function. The extent of inhibition thereby depends on the affinity of the enzyme for OT and the strongest effect was demonstrated for CYP11B2.
These findings suggest that steroidogenic cytochrome P enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies. In humans, most steps of steroid biosynthesis are catalyzed by monooxygenases from the cytochrome P P superfamily, which synthesize glucocorticoids, mineralocorticoids, and sex hormones. Steroidogenesis is initiated by CYP11A1, which cleaves the side chain of cholesterol, thereby producing pregnenolone, the common precursor for all steroid hormones.
CYP21A2 generates the substrates for gluco- and mineralocorticoid biosynthesis by its hydroxylase activity. P catalysis requires the presence of a suitable electron transport system, which delivers the electrons necessary for the activation of molecular oxygen from the external electron donor NADPH. Several studies have recently hinted at an involvement of steroidogenic P enzymes in the biotransformation of xenobiotic compounds, which is, according to traditional classifications of human P enzymes, believed to be solely conducted by microsomal isoforms from the liver Guengerich, Anabolic agents, particularly AASs, are widely misused for doping purposes in all sports.
In antidoping controls, AASs represent the most frequently detected class of substances prohibited by the World Anti-Doping Agency Numerous side effects—including physical phenomena such as cardiovascular risks Angell et al.
Although several hypothetical models exist to describe the mechanisms behind the cardiovascular issues Melchert and Welder, ; Deligiannis et al. Increases in blood pressure up to hypertension are described to be secondary to increases in blood volume, which can result from a disruption of mineralocorticoid signaling Rockhold, Mineralocorticoids, the most important of which are DOC and aldosterone in humans, regulate water and electrolyte homeostasis by controlling renal water and sodium retention as well as potassium secretion via the mineralocorticoid receptor MR signaling pathway Funder, Ligand-induced activation of the cytosolic MR leads to the release of bound chaperones and nuclear localization, followed by DNA binding and the recruitment of specific coactivators, which subsequently initiates the transcription of specific target genes Galigniana et al.
Here, we analyzed the metabolism of the xenobiotic steroid OT by human steroidogenic P enzymes on the molecular level to explore their drug-metabolizing capabilities.
Therefore, we took advantage of the recombinant, high-yield expression of these enzymes in the bacterial host Escherichia coli , which only recently became feasible for all of these P enzymes. Dissociation constants were determined by UV-visible spectroscopy to enable a comparison with affinities toward endogenous substrates, and kinetic studies on OT metabolism were carried out using a reconstituted in vitro system with purified enzymes combined with high-performance liquid chromatography HPLC analysis.
In addition, potential physiologic consequences of OT metabolism were investigated by demonstrating the influence of OT on the natural P function in a reconstituted system and by studying the effect of the metabolites on MR transactivation in a reporter gene assay.
All reagents were obtained from standard sources with the highest purity available. Other steroids were purchased from Sigma-Aldrich St. The column was washed with buffer A, followed by buffer A containing 30 mM potassium phosphate pH 6. AdR Sugiyama and Yamano, ; Sagara et al. The truncation of 27 amino acids at the N terminus enables high-yield expression of a soluble, catalytically active CPR. Slight modifications were made during the purification: The protein was finally dialyzed against buffer C described above for the removal of imidazole.
Bovine cytochrome b 5 was purified as reported previously by others Neunzig et al. Prior to use, the buffer was sonicated in a sonication water bath for 5 minutes for the reconstitution of 1,2-dilauroyl- sn -glycerophosphocholine vesicles. The system contained 0. For mitochondrial P enzymes, 0. The final ethanol concentration was adjusted within each set of reactions. Steroids were extracted twice with chloroform, evaporated, and suspended in acetonitrile for HPLC analysis.
For product quantification, progesterone was added as an internal standard prior to extraction and quantification was performed by HPLC using a calibration curve. For OT turnover, product formation was calculated from the applied OT concentration deducting OT consumption, which was determined with a calibration curve.
Cortisol was used for quantification of product formation in this case. For the determination of kinetic parameters of substrate conversion, enzyme concentrations were scaled down to 0.
For CYP11A1, the conditions described above were maintained. Reactions were stopped under steady-state conditions by freezing in liquid nitrogen. The determination of dissociation constants was performed by difference spectroscopy using tandem cuvettes as described Schenkman, with a Jasco V spectrophotometer. Titrations were performed three times. Plots were fitted with Origin 8. Terrific broth media ml; 24 g yeast extract technical, 12 g peptone, 4 ml glycerol, 4.
Steroids were extracted twice with one culture volume of ethyl acetate and the organic phase was evaporated to dryness. The eluents were 0. All assignments were based on extensive NMR spectral evidence.
Aldosterone, which was used as positive control, was supplied with the assay. Conversion of OT by the human steroid hormone—synthesizing P enzymes was assayed with recombinant proteins purified from E. Whereas CYP11B1 forms one main metabolite 1 as well as two side products 2 and 3 in minor amounts, CYP11B2 produces three main products 1, 4, and 5 and three intermediate 2 or side products 3 and 6.
For the CYP11B2-catalyzed reaction, time dependence of the product pattern could be observed. Peak area portions of metabolites 1 and 2 are reduced over the time, whereas those of metabolites 4 and 5 increase Fig.
AdR and Adx; microsomal: By means of the accurate masses of mono- and dihydroxylated analogs to OT as well as the consideration of diagnostic product ions generated from protonated molecules of the observed analytes, several different metabolic products were identified. The extracted ion chromatograms of mono- and dihydroxylated OT are illustrated in Fig. The product ion mass spectrum of metabolite 7 is depicted in Fig. However, in the absence of further information, a structural assignment of this analyte was not possible.
Proposed structures of the respective OT metabolites are shown. Fragmentation and the resulting fragment ion masses are indicated. In addition to the aforementioned monohydroxylated metabolites of OT, dihydroxylated species were also observed by means of the accurate mass of the protonated molecules Fig.
To assess the efficiency of OT metabolism by the human CYP11 family members, in vitro characterization of substrate affinity and reaction kinetics was performed. This feature was used to determine dissociation constants K d from the type I difference spectra recorded during the titration of P with increasing concentrations of OT Fig. CYP11B2 showed a K d value of 5. CYP11A1, however, showed a putative type II—like difference spectrum with a minimum around nm and a maximum between and nm, which was not quantifiable even under high P concentrations and in the presence of Adx Fig.
The observation of a type II difference spectrum with OT is unexpected because as all other type II ligands described in the literature, to our knowledge, possess a nitrogen atom, whose association with the heme iron induces a low spin shift. However, the spin-state equilibrium can also be influenced by distortions of the porphyrin molecule Groenhof, Because of the small size of OT compared with cholesterol, two molecules of OT might bind in the active site, resulting in a very close position of one OT molecule above the heme.
Putative emerging substrate-induced heme deformation could then lead to a spin-state crossover toward the low-spin state. Measurements were performed in triplicate. Moreover, we determined kinetic parameters of OT conversion by performing in vitro reactions under steady-state conditions and quantification of OT consumption Fig.
Molar ratios of P and its redox partners were 1: The system was reconstituted with the respective P, Adx, and AdR at a ratio of 1: All values represent the mean of triplicates with the standard deviation.
The binding and metabolism of OT at relevant catalytic rates by the three enzymes suggests possible interference with the conversion of their natural substrates. The regulation of steroid biosynthesis is also the result of signaling on multiple levels hypothalamic-pituitary-adrenal axis, renin-angiotensin system, etc.
A deeper characterization of the inhibitory effects of OT was thus not considered as conducive for the interpretation of their relevance. Relative product formation from the natural substrates in the presence of OT in a reconstituted in vitro system.
The system consisted of 0. DOC consumption or pregnenolone formation was determined by HPLC using progesterone or cortisol, respectively, as an internal standard. Each bar represents the mean of three reactions with the respective standard deviation.
The complete redox chain was transferred into E. All three products were purified in milligram amounts by preparative HPLC. Purified products were structurally characterized by NMR spectroscopy. Elucidated structures are illustrated in Fig. Two-dimensional NMR measurements supported the structure and led to the full assignments.
CYP11B-derived OT metabolites carry the same oxy-functionalizations as are introduced into the steran scaffold during the biosynthesis of natural mineralocorticoids. Therefore, we evaluated their potential to activate the human MR to investigate putative new or altered functions of the OT metabolites.
The assay was performed with a commercially available cell culture—based system, which gives a luminescence signal upon activation of the MR. Purity of the test compounds was verified by LC-MS prior to the assay. Test concentrations ranged from 2 to 20, pM, which resulted in full dose response for the natural MR ligand aldosterone. The luminescence signal of MR-responsive luciferase reporter gene expression was normalized to the signal of the vehicle control and is plotted against the test compound concentration in a logarithmic scale.
The assay was performed in triplicate for each concentration. Human P enzymes are traditionally classified into a group of drug-metabolizing P enzymes expressed in the liver and those that carry out the biosynthesis of endogenous compounds such as steroid hormones. In this study, we examined whether the second group might additionally contribute to the metabolism of xenobiotics by investigating the synthetic steroidal drug OT, which is a common doping agent.
All six steroidogenic P enzymes were tested for their activity toward OT. It was postulated that the extended functional spectrum of CYP11B2 is enabled by an increase in retention time of the intermediates in the active site due to higher intrinsic flexibility compared with CYP11B1 Strushkevich et al. A reduced flexibility of CYP11B1 might impede the access of the non-natural substrate to the active site and thus matches the lower binding affinity and catalytic efficiency of CYP11B1 toward OT.
To the best of our knowledge, the respective products have not been described in the literature thus far.