17α- and 17β-boldenone 17-glucuronides: Synthesis and complete characterization by H and C NMR

Boldenone is an androgenic anabolic steroid intensively used for growth promoting purposes in animals destined for meat production and as a performance enhancer in athletics. Therefore its use is officially banned either in animals intended for consumption or in humans. Because most anabolic steroids are completely metabolized and usually no parent steroid is excreted, metabolite identification is crucial to detect the illegal use of anabolic steroids either in humans or in livestock. 17α- and 17β-boldenone 17-glucuronides were synthesized, purified and characterized in order to provide suitable standards for the identification and quantification of these metabolites.     

17α-ethinylestradiol cometabolism by bacteria degrading estrone, 17β-estradiol and estriol

17alpha-ethinylestradiol (EE2), the active compound of the contraceptive pill, is a recalcitrant estrogen, which is encountered at ng/l levels in wastewater treatment plant (WWTP) effluents and rivers and can cause feminization of aquatic organisms. The aim of this study was to isolate micro-organisms that could remove such low EE2 concentrations. In this study, six bacterial strains were isolated from compost that cometabolize EE2 when metabolizing estrone (E1), 17beta-estradiol (E2) and estriol (E3). The strains belong to the alpha, beta and gamma-Proteobacteria. All six strains metabolize E2 over E1, at mug/l to ng/l concentrations. In 4 days, initial concentrations of 0.5 mug E2/l and 0.6 mug EE2/l were degraded to 1.8 +/- 0.4 ng E2/l and 85 +/- 16 ng EE2/l, respectively. No other metabolites besides E1, E2, E3 or EE2 were detected, suggesting that total degradation and cleavage of the aromatic ring occurred. This is the first study describing that bacteria able to metabolize E2, can subsequently cometabolize EE2 at low mug/l levels.     

Chemical synthesis of the 17-propanamide derivatives of stereoisomeric Δ14-17α- and 17β-estradiols: potential 17β-hydroxysteroid dehydrogenase inhibitors

The 17-propanamide derivatives of diastereomeric Δ¹⁴-17α- and 17β-estradiols, the potential candidates of a 17β-hydroxysteroid dehydrogenase (17β-HSD) inhibitor, were synthesized in 11 steps from estrone. The principal reactions employed involved in (1) conversion of estrone to the corresponding Δ¹⁴-estrone, (2) Grignard reaction of Δ¹⁴-estrone with allylmagnesium bromide followed by regioselective hydroboration of the resulting stereoisomeric 17ξ-allyl-Δ¹⁴-17ξ-ols with 9-borabicyclo[3.3.1]nonane (9-BBN), and (3) direct amidation of the 17ξ-O-/17ξ-C-spiro-γ-lactones with NH₃ under positive pressure of H₂.     

Occurrence and Fate of Estrone, 17β-estradiol and 17α-ethynylestradiol in STPs for Domestic Wastewater

Estrone (E1), 17β-estradiol (E2) and 17α-ethynylestradiol (EE2) discharged from sewage treatment plants (STPs) into surface waters, are seen as a threat effecting aquatic life by its estrogenic character. Therefore, much research is conducted on the fate and removal of these compounds. Since these compounds are present in influents and effluents in the ng/l range, methods for detection deserve special attention. Most important processes that play a role in the removal of estrogens are: adsorption, aerobic degradation, anaerobic degradation, anoxic biodegradation and photolytic degradation. Halflifes tend to vary and are remarkably shorter when low initial concentrations are applied. In general anaerobic conditions result in longer halflifes then aerobic conditions. EE2 shows far most persistence of the compounds, thereby also the estrogenic effect in vitro is about 2–3-fold higher compared to E2. The three compounds show a higher affinity to sorb to sludge compared to other tested adsorption materials like sediment. Aerobic degradation is far the most efficient in removing these compounds, but adsorption seems to play a significant role in retaining the estrogens inside full-scale STPs. Removal rates in full scale plants depend on the HRT, SRT and loading rates, but lack of information on the exact dependency so far prevents an optimal design able to fully eliminate estrogens from wastewater.     

Ovariectomy and 17β-estradiol replacement in rats and mice: a visual demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17β-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17β-estradiol level/administration method profoundly influences the experimental results. We have in a series of studies characterized the different modes of 17β-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17β-estradiol. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17β-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.     

Inhibition of human and rat 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities by perfluoroalkylated substances

Perfluoroalkylated substances (PFASs) including perfluorooctane acid (PFOA) and perfluorooctane sulfonate (PFOS) have been classified as persistent organic pollutants and are known to cause reduced testosterone production in human males. The objective of the present study was to compare the potencies of five different PFASs including PFOA, PFOS, potassium perfluorooctane sulfonate (PFOSK), potassium perfluorohexane sulfonate (PFHxSK) and potassium perfluorobutane sulfonate (PFBSK) in the inhibition of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) activities in the human and rat testes. Human and rat microsomal enzymes were exposed to various PFASs. PFOS and PFOSK inhibited rat 3β-HSD activity with IC₅₀ of 1.35 ± 0.05 and 1.77 ± 0.04 μM, respectively, whereas PFHxSK and PFBSK had no effect at concentrations up to 250 μM. All chemicals tested weakly inhibited human 3β-HSD activity with IC₅₀s over 250 μM. On the other hand, PFOS, PFOSK and PFOA inhibited human 17β-HSD3 activity with IC₅₀s of 6.02 ± 1.02, 4.39 ± 0.46 and 127.60 ± 28.52 μM, respectively. The potencies for inhibition of 17β-HSD3 activity were determined to be PFOSK > PFOS > PFOA > PFHxSK = PFBSK for human 17β-HSD3 activity. There appears to be a species-dependent sensitivity to PFAS-mediated inhibition of enzyme activity because the IC₅₀s of PFOS(K) for inhibition of rat 17β-HSD3 activity was greater than 250 μM. In conclusion, the present study shows that PFOS and PFOSK are potent inhibitors of rat 3β-HSD and human 17β-HSD3 activity, and implies that inhibition of steroidogenic enzyme activity may be a contributing factor to the effects that PFASs exert on androgen secretion in the testis.     

Molecular mechanisms of estrogen recognition and 17-keto reduction by human 17β-hydroxysteroid dehydrogenase 1

The reduction of inactive estrone (E1) to the active estrogen 17β-estradiol (E2) is catalyzed by type 1 17β-hydroxysteroid dehydrogenase (17HSD1). Crystallographic studies, modeling and activity measurement of mutants and chimeric enzymes have led to the understanding of its mechanism of action and the molecular basis for the estrogenic specificity. An electrophilic attack on the C17-keto oxygen by the Tyr 155 hydroxyl is proposed for initiation of the transition state. The active site is a hydrophobic pocket with catalytic residues at one end and the recognition machinery on the other. Tyr 155, Lys 159 and Ser 142 are essential for the activity. The presence of certain other amino acids near the substrate recognition end of the active site including His 152 and Pro 187 is critical to the shape complementarity of estrogenic ligands. His 221 and Glu 282 form hydrogen bonds with 3-hydroxyl of the aromatic A-ring of the ligand. This mechanism of recognition of E1 by 17HSD1 is similar to that of E2 by estrogen receptor α. In a ternary complex with NADP⁺ and equilin, an equine estrogen with C7=C8 double bond, the orientation of C17=O of equilin relative to the C4-hydride is more acute than the near normal approach of the hydride for the substrate. In the apo-enzyme structure, a substrate-entry loop (residues 186–201) is in an open conformation. The loop is closed in this complex and Phe 192 and Met 193 make contacts with the ligand. Residues of the entry loop could be partially responsible for the estrogenic specificity.     

Syntheses of (14β,17α)-14-Hydroxy- and (14β,17α)-2,14-Dihydroxyestradiols and Their Activities

Structure 1 is proposed for the Inagami-Tamura endogenous digitalis-like factor (EDLF), and (14β,17α)-14-hydroxy- and (14β, 17α)-2,14-dihydroxyestradiols (2 and 3) were synthesized as models for studies on 1. The latter compound was remarkably potent in inducing a contractile response in isolated rat aorta and guinea pig left atrium.     

Regucalcin is expressed in rat mammary gland and prostate and down-regulated by 17β-estradiol

Regucalcin is involved in maintenance of calcium homeostasis due to the activation of Ca²⁺ pumping enzymes in the plasma membrane. It has a suppressive effect in cell proliferation, DNA and RNA synthesis, and may be associated with the abnormal cell division on tumor tissues. On the other hand both estrogens and Ca²⁺ are implicated in breast and prostate cancer but there are no studies focused on the expression of regucalcin in rat mammary gland or prostate. Furthermore, it is known that the expression of regucalcin in rat liver and kidney is regulated by 17β-estradiol (E₂). The aim of this study is to analyze if regucalcin is expressed in rat mammary gland and prostate and if it is regulated by E₂ in these tissues. We demonstrated for the first time that regucalcin mRNA and protein are present in rat mammary gland and prostate by in situ hybridization and immunohistochemistry, respectively. Furthermore, we show by Real-time PCR that E₂ down-regulates regucalcin expression in rat mammary gland and prostate.     

Phenylbenzenesulfonates and -sulfonamides as 17β-hydroxysteroid dehydrogenase type 2 inhibitors: Synthesis and SAR-analysis

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) converts the potent estrogen estradiol into the weakly active keto form estrone. Because of its expression in bone, inhibition of 17β-HSD2 provides an attractive strategy for the treatment of osteoporosis, a condition that is often caused by a decrease of the active sex steroids. Currently, there are no drugs on the market targeting 17β-HSD2, but in multiple studies, synthesis and biological evaluation of promising 17β-HSD2 inhibitors have been reported. Our previous work led to the identification of phenylbenzenesulfonamides and -sulfonates as new 17β-HSD2 inhibitors by ligand-based pharmacophore modeling and virtual screening. In this study, new molecules representing this scaffold were synthesized and tested in vitro for their 17β-HSD2 activity to derive more profound structure-activity relationship rules.     

Effects of phthalates on 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities in human and rat testes

The 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) are involved in the reactions that culminate in androgen biosynthesis in Leydig cells. Human and rat testis microsomes were used to investigate the inhibitory potencies on 3β-HSD and 17β-HSD3 activities of 14 different phthalates with various carbon numbers in the ethanol moiety. The results demonstrated that the half-maximal inhibitory concentrations (IC(50)s) of dipropyl (DPrP), dibutyl (DBP), dipentyl (DPP), bis(2-butoxyethyl) (BBOP) and dicyclohexyl (DCHP) phthalate were 123.0, 24.1, 25.5, 50.3 and 25.5μM for human 3β-HSD activity, and 62.7, 30.3, 33.8, 82.6 and 24.7μM for rat 3β-HSD activity, respectively. However, only BBOP and DCHP potently inhibited human (IC(50)s, 23.3 and 8.2μM) and rat (IC(50)s, 30.24 and 9.1μM) 17β-HSD3 activity. Phthalates with 1-2 or 7-8 carbon atoms in ethanol moieties had no effects on both enzyme activities even at concentrations up to 1mM. The mode of action of DCHP on 3β-HSD activity was competitive with the substrate pregnenolone but noncompetitive with the cofactor NAD+. The mode of action of DCHP on 17β-HSD3 activity was competitive with the substrate androstenedione but noncompetitive with the cofactor NADPH. In summary, our results showed that there are clear structure-activity responses for phthalates in the inhibition of both 3β-HSD and 17β-HSD3 activities. The length of carbon chains in the ethanol moieties of phthalates may determine the potency to inhibit these two enzymes.     

Isolation and characterization of 17β-hydroxy steroid dehydrogenase from human erythrocytes

1. The 17beta-hydroxy steroid dehydrogenase was solubilized during haemolysis of erythrocytes and was isolated from the membrane-free haemolysate. Membrane preparations isolated in different ways did not contain 17beta-hydroxy steroid dehydrogenase activity. The 17beta-hydroxy steroid dehydrogenase activity in the haemolysate was concentrated by repeated ammonium sulphate precipitation and gel filtration on Sephadex G-150. The 17beta-hydroxy steroid dehydrogenase activity of the purified preparation per unit weight of protein was 350-3000 times higher than the activity of the crude erythrocyte haemolysate. The 20alpha-hydroxy steroid dehydrogenase activity was lost during this purification procedure. 2. The 17beta-hydroxy steroid dehydrogenase was NADP-dependent and had a pH optimum for conversion of testosterone between 8.5 and 10. For the molecular weight of the enzyme a value of 64000 was calculated from Sephadex chromatography results. 3. p-Chloromercuribenzoate inhibited the enzymic activity. The oxidative activity of the enzyme for the 17beta-hydroxyl group was only partly inhibited when a large excess of 17-oxo steroids was added. The catalysing activity of the enzyme was influenced by the NADP(+)/NADPH ratio. The oxidation of the 17beta-hydroxyl group in the presence of NADP(+) proceeded faster than the reduction of the 17-oxo group with NADPH. When both reduced and oxidized cofactors were present the oxidation of the 17beta-hydroxyl group was inhibited to a considerable extent. 4. The enzyme had a broad substrate specificity and not only catalysed the conversion of androstanes with a 17beta-hydroxyl group, or 17-oxo group, but also the conversion oestradiolleft arrow over right arrowoestrone. In addition the steroid conjugates dehydroepiandrosterone sulphate and oestrone sulphate were also converted. There were no indications that more than one 17beta-hydroxy steroid dehydrogenase was present in the partially purified preparation.     

Synchronization of estrus in post-partum mares with progesterone and estradiol 17β

Thirty-six mares which foaled over a 10-day period were given 1 to 10 daily intramuscular injections of a combination of 150 mg. progesterone and 10 mg. estradiol 17β. The first injection was given within 18 hours after parturition. Because individual mares foaled on different dates during the 10 day period, commencement of treatment varied, but treatment for all mares ceased on the same day. Teasing and breeding began seven days after the final treatment. The mares were teased daily for 10 days and artifically inseminated every second day until ovulation occurred. The mean interval from the end of treatment to beginning of estrus was 9.4 days (range 7 to 14) and 33 of 26 mares (94.7%) ovulated 10 to 16 days after the final treatment. Both estrus and ovulation were effectively synchronized, resulting in a first estrus pregnancy rate of 80.6% (29 of 36).     

5α-Estrane-3β,17β-diol and 5β-estrane-3α,17β-diol: definitive screening biomarkers to sign nandrolone abuse in cattle?

17β-Nandrolone (17β-NT) is one of the most frequently misused anabolic steroids in meat producing animals. As a result of its extensive metabolism combined with the possibility of interferences with other endogenous compounds, detection of its illegal use often turns out to be a difficult issue. In recent years, proving the illegal administration of 17β-NT became even more challenging since the presence of endogenous presence of 17β-NT or some of its metabolite in different species was demonstrated. In bovines, 17α-NT can occur naturally in the urine of pregnant cows and recent findings reported that both forms can be detected in injured animals. Because efficient control must both take into account metabolic patterns and associated kinetics of elimination, the purpose of the present study was to investigate further some estranediols (5α-estrane-3β,17β-diol (abb), 5β-estrane-3α,17β-diol (bab), 5α-estrane-3β,17α-diol (aba), 5α-estrane-3α,17β-diol (aab) and 5β-estrane-3α,17α-diol (baa)) as particular metabolites of 17β-NT on a large number of injured (n=65) or pregnant (n=40) bovines. Whereas the metabolites abb, bab, aba and baa have previously been detected in urine up to several days after 17β-NT administration, the present study showed that some of the isomers abb (5α-estrane-3β,17β-diol) and bab (5β-estrane-3α,17β-diol) could not be detected in injured or pregnant animals, even at very low levels. This result may open a new way for the screening of anabolic steroid administration considering these 2 estranediols as biomarkers to indicate nandrolone abuse in cattle.     

Solid-state NMR analysis of steroidal conformation of 17α- and 17β-estradiol in the absence and presence of lipid environment

Solid-state {(1)H}(13)C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy has been applied to 17β-estradiol (E2) and 17α-estradiol (E2α), to analyze the steroidal ring conformations of the two isomers in the absence and presence of lipids at the atomic level. In the absence of lipid, the high-resolution (13)C NMR signals of E2 in a powdered form show only singlet patterns, suggesting a single ring conformation. In contrast, the (13)C signals of E2α reveal multiplet patterns with splittings of 20-300Hz, implying multiple ring conformations. In the presence of a mimic of the lipid environment, made by mixing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) in a molar ratio 3:1, E2 and E2α revealed multiplet patterns different from those seen in the absence of lipids, indicating that the two isomers adopt multiple conformations in the lipid environment. In this work, on the basis of chemical shift isotropy and anisotropy analysis, we demonstrated that E2 and E2α prefer to adopt multiple steroidal ring conformations in the presence of a lipid environment, distinct from that observed in solution phase and powdered form.     

Immunoaffinity chromatography and a biotin-streptavidin amplified enzymeimmunoassay for sensitive and specific estimation of estradiol-17β

A sensitive test system has been developed for estimation ofestradiol-17β (E₂) in bovine plasma. Plasma extracts are first purified by a selective immunoaffinity chromatography (IAC) using an antibody raised against estradiol-6-carboxymethyloxime-bovine serum albumin and immobilized to Sepharose. The eluate was analysed by a competitive enzyme immunoassay (EIA) on microtitration plates. For the assay the wells of microtitration plates were coated with affinity purified sheep IgG (antirabbit IgG) that binds the hormone specific antibody raised in rabbits against estradiol-17-hemisuccinate-bovine serum albumin. E₂ is estimated by displacement of biocytinyl-E₂, that was produced by ligation of estradiol-17β, d-glucuronic acid and biocytin. Bound biocytinyl-E₂ is detected after binding of streptavidin-peroxidase and colour production by the enzyme. A very high amplification was possible with this technique and the absolute detection limit amounted to ≈120fg/well at 94% relative binding. By combination of IAC and EIA the following levels of E₂ were found in bovine plasma: male or female calves <2.7pg/ml, cycling cow 0.5–7 pg/ml, cow during last month of pregnancy 9–310 pg/ml, mature bull 5–30 pg/ml. However, up to 1110 pg E₂/ml were found in plasma of a calf after treatment with an illicit hormone preparation used for growth promotion; after 21 days levels declined to 6 pg/ml which is hardly different from controls. In conclusion, the IAC/EIA can be used for sentitive estimation ofestradiol-17β in plasma from all type of cattle and for control of improper use of E₂ after commitment of a threshhold level.     

Mitochondria from distinct tissues are differently affected by 17β-estradiol and tamoxifen

This study was aimed to analyse and compare the bioenergetics and oxidative status of mitochondria isolated from liver, heart and brain of ovariectomized rat females treated with 17β-estradiol (E2) and/or tamoxifen (TAM). E2 and/or TAM did not alter significantly the respiratory chain of the three types of mitochondria. However, TAM significantly decreased the phosphorylation efficiency of liver mitochondria while E2 significantly decreased the phosphorylation efficiency of heart mitochondria. E2 also significantly decreased the capacity of heart and liver mitochondria to accumulate Ca(2+) this effect being attenuated in liver mitochondria isolated from E2+TAM-treated rat females. TAM treatment increased the ratio of glutathione to glutathione disulfide (GSH/GSSG) of liver mitochondria. Brain mitochondria from TAM- and E2+TAM-treated females showed a significantly lower GSH/GSSG ratio. However, heart mitochondria from TAM- and E2+TAM-treated females presented a significant decrease in GSSG and an increase in GSH/GSSG ratio. Thiobarbituric acid reactive substances levels were significantly decreased in liver mitochondria isolated from E2+TAM-treated females. Finally, E2 and/or TAM treatment significantly decreased the levels of hydrogen peroxide produced by brain mitochondria energized with glutamate/malate. These results indicate that E2 and/or TAM have tissue-specific effects suggesting that TAM and hormonal replacement therapies may have some side effects that should be carefully considered.