Suppression of 5 alpha-androstane-3 beta,17 beta-diol hydroxylase activity in rat pituitary by cobalt protoporphyrin: implications for testosterone homeostasis.

Cobalt protoporphyrin (CoPP) administered subcutaneously to adult male rats caused a marked reduction in the conversion of 5 alpha-androstane-3 beta-17 beta-diol (3 beta-adiol) to its main triol derivative (6 alpha-atriol) by homogenates of the pituitary but not of the prostate or brain (ventromedial hypothalamus and cortex). No effect in the brain was observed when this heme analogue was infused intracerebroventricularly. 3 beta-adiol hydroxylase, the enzyme responsible for the reaction and whose main function is thought to be the elimination of dihydrotestosterone and its metabolites from target tissues, was also inhibited by CoPP and SKF-525A added in vitro. The reaction was microsomal and dependent on NADPH. It is proposed that the lack of reciprocal elevation of luteinizing hormone in the face of the low testosterone levels observed following treatment with CoPP may be due, in part, to increased levels of androstanediols. These metabolites accumulate because of increased production from testosterone and decreased conversion to their triol derivatives in the pituitary.     

3β-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5α-androstane-3β,17β-diol and dehydroepiandrosterone as substrates

3β-Hydroxysteroid dehydrogenase (3β-HSD)/Δ^5→4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3β-HSD/Δ^5→4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3β-HSD activity. For this purpose, we compared the efficiencies of a 3β-hydroxy-5-ene steroid (DHEA) and a 3β-hydroxy-5α-reduced steroid (5α-androstane-3β,17β-diol, 5α-A-diol) as substrates for the enzyme. The apparent Michaelis constant (K_m) for 5α-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent K_m of placental 3β-HSD for 5α-A-diol was in the range of 18 to 40 μmol/l (n = 3) vs 0.45 to 4 μmol/l for DHEA (n = 3); for the liver enzyme, 17 μmol/l for 5α-A-diol and 0.60 μmol/l for DHEA, and for the skin enzyme 14 and 0.18 μmol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5α-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to K_ms and rates of product formation was obtained by use of purified placental 3β-HSD with DHEA, pregnenolone, and 3β-hydroxy-5α-androstan-17-one (epiandrosterone) as substrates: the K_m of 3β-HSD for DHEA was 2.8 μmol/l, for pregnenolone 1.9 μmol/l, and for epiandrosterone 25 μmol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein·min and, with epiandrosterone, 127 nmol/mg protein·min. With placental homogenate as the source of 3β-HSD, DHEA at a constant level of 5 μmol/l behaved as a competitive inhibitor when the radiolabeled substrate, [³H]5α-A-diol, was present in concentrations of 20 to 60 μmol/l, but a lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [³H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5α-A-diol (40 μmol/l). These findings are indicative that both steroids bind to a common site on the enzyme, however, the binding affinity for these steroids appear to differ markedly as suggested by the respective K_ms. Studies of inactivation of purified placental 3β-HSD/Δ^5→4-isomerase by an irreversible inhibitor, viz 5,10-secoestr-4-yne-3,10,17-trione, were suggestive that the placental protein adopts different conformations depending on whether the steroidal substrate has a 5α-configuration, e.g. epiandrosterone, or a C-5,C-6-double bond e.g. DHEA or pregnenolone. The lower rates of product formation obtained with placenta and fetal tissues by use of 3β-hydroxy-5-ene steroids as substrates when compared with those obtained with 3β-hydroxy-5α-reduced steroids may be explained by a combination of factors, including: (i) inhibition of 3β-HSD activity by end products of metabolism of 3β-hydroxy-5-ene steroids, e.g. 4-androstene-3,17-dione formed with DHEA as substrate; (ii) higher binding affinity of the enzyme for 3β-hydroxy-5-ene steroids—and possibly for their 3-oxo-5-ene metabolites; (iii) lack of a requirement for the isomerization step with 5α-reduced steroids as substrates, and (iv) the possible presence in fetal tissues of an enzyme with 3β-HSD activity only (i.e. no Δ^5→4-isomerase).     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

An alternate pathway for androgen regulation of brain function: Activation of estrogen receptor beta by the metabolite of dihydrotestosterone, 5α-androstane-3β,17β-diol

The complexity of gonadal steroid hormone actions is reflected in their broad and diverse effects on a host of integrated systems including reproductive physiology, sexual behavior, stress responses, immune function, cognition, and neural protection. Understanding the specific contributions of androgens and estrogens in neurons that mediate these important biological processes is central to the study of neuroendocrinology. Of particular interest in recent years has been the biological role of androgen metabolites. The goal of this review is to highlight recent data delineating the specific brain targets for the dihydrotestosterone metabolite, 5α-androstane, 3β,17β-diol (3β-Diol). Studies using both in vitro and in vivo approaches provide compelling evidence that 3β-Diol is an important modulator of the stress response mediated by the hypothalmo–pituitary–adrenal axis. Furthermore, the actions of 3β-Diol are mediated by estrogen receptors, and not androgen receptors, often through a canonical estrogen response element in the promoter of a given target gene. These novel findings compel us to re-evaluate the interpretation of past studies and the design of future experiments aimed at elucidating the specific effects of androgen receptor signaling pathways.     

A chemiluminescent immunoassay for the direct measurement of urinary 5α-androstane-3α, 17β-diol-glucuronide in urine

We described a chemiluminescent immunoassay (CIA) for 5α-androstane-3α, 17β-diol-glucuronide (3α-diol-G) in human diluted urine. This method allowed the direct measurement in 1μl of urine avoiding the hydrolysis and extraction steps for sample pretreatment commonly used in routine methods. The hapten 3α-diol-G was synthesized by a Koenigs–Knorr reaction. The immunogenic complex, 3α-diol-G conjugated to bovine serum albumin (BSA), was employed to induce the formation of specific antibodies in New Zealand rabbits. In addition, the required chemiluminescent (CL) tracer was prepared. The characteristics of the antibody was determined as regard to specificity and sensitivity and the precision of the assay methods established. In 22 hirsute women affected by policystic ovarian syndrome we found 3α-diol-G values significantly (p < 0.01) higher (146.28 ± 73.77μg/g of creatinine; mean ± SD) than those observed in normal women (72.1 ± 32.58 μg/g of creatinine; mean ± SD).     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

A Potential Role for 5‐Androstene‐3β,7β,17β‐triol in Obesity and Metabolic Syndrome

Metabolic syndrome is marked by perturbed glucocorticoid (GC) signaling, systemic inflammation, and altered immune status. Dehydroepiandrosterone (DHEA), a major circulating adrenal steroid and dietary supplement, demonstrates antiobesity, anti‐inflammatory, GC‐opposing and immune‐modulating activity when administered to rodents. However, plasma DHEA levels failed to correlate with metabolic syndrome and oral replacement therapy provided only mild benefits to patients. Androstene‐3β,7β,17β‐triol (β‐AET) an anti‐inflammatory metabolite of DHEA, also exhibits GC‐opposing and immune‐modulating activity when administered to rodents. We hypothesized a role for β‐AET in obesity. We now report that plasma levels of β‐AET positively correlate with BMI in healthy men and women. Together with previous studies, the observations reported here may suggest a compensatory role for β‐AET in preventing the development of metabolic syndrome. The β‐AET structural core may provide the basis for novel pharmaceuticals to treat this disease.     

Marine sterols. XIII. Isolation and synthesis of 1β,3β,5,6β-tetrahydroxy-5α-androstan-17-one from the soft coral

A minor C₂₇ sterol, glaucasterol, was isolated from the soft coral Based on the spectroscopic evidence and the correlation to cholestanol and 26-nor-27-homocholestanol, its structure was proposed to be 24ξ, 25ξ-24,26-cyclocholesta-5,22E-dien-3β-ol ( ), the first example of a natural C₂₇ sterol having a cyclopropane ring in the side chain.     

17β-acylurea derivatives of 4-azasteroids as inhibitors of testosterone 5α-reductase

A series of 17 beta-acylurea-4-aza-5 alpha-androstan-3-one derivatives has been assayed in vitro as inhibitors of testosterone 5 alpha-reductase, using the particulate fraction of human hyperplastic prostate and rat prostate as enzyme sources. The most active derivatives were 1-[4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carbonyl]- 1,3-dicyclohexylurea (compound 1) and 1-[4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carbonyl]- 1,3-diisopropylurea (compound 3) which demonstrated IC50 values of 41 and 55 nM for the human enzyme and of 83 and 53 nM for the rat enzyme, respectively. Neither compound showed any relevant binding affinity to the rat prostate androgen receptor (IC50 of approximately 100 and 84 microM). When given orally in immature castrated rats together with subcutaneous testosterone propionate (TP) for 7 consecutive days, compound 3 (laboratory code FCE 26073), at 3 mg/kg/day, significantly decreased the ventral prostate growth promoting effect of TP by 40-50%, whereas compound 1 was ineffective up to the dose of 10 mg/kg/day.     

Biotransformation of 5α-androstane-3,17-dione by microalgal cultures.

Twenty-four different strains of unicellular algae have been used in the bioconversion of 5α-androstane-3,17-dione.5_α-androstane-3,17-dione has been bioconverted by twenty-four microalgae The activity of algae seems to be species-specific. Reducted, hydroxylated or more unsaturated steroids have been isolated from the cultures.     

5α,6α-Epoxy-cholestan-3β-ol (cholesterol α-oxide): A specific substrate for rat liver glutathione transferase B

A semi-micro assay was developed for the conjugation of 5α,6α-epoxy-cholestan-3β-ol (cholesterol α-oxide) with glutathione. The soluble supernatant of rat liver homogenate catalysed the reaction at a rate of 0.2–0.5 pmol.min⁻¹ .mg protein⁻¹ with 4μM cholesterol α-oxide, while the reaction in the presence of GSH alone was barely detectable. Enzymic activity in the soluble supernatant was due equally to the two forms of glutathione transferase B (100 pmol.min⁻.mg protein⁻¹), glutathione transferases AA, A, C and E being unreactive. The activity of purified glutathione transferase B was about 5-times that expected from the activity of the soluble supernatant. Complex enzyme kinetics were obtained suggestive of substrate inhibition.     

Parallel solid-phase synthesis of 3β-peptido-3α-hydroxy-5α-androstan-17-one derivatives for inhibition of type 3 17β-hydroxysteroid dehydrogenase

Type 3 17β-hydroxysteroid dehydrogenase (17β-HSD), a key steroidogenic enzyme, transforms 4-androstene-3,17-dione (Δ⁴-dione) into testosterone. In order to produce potential inhibitors, we performed solid-phase synthesis of model libraries of 3β-peptido-3α-hydroxy-5α-androstan-17-ones with 1, 2, or 3 levels of molecular diversity, obtaining good overall yields (23–58%) and a high average purity (86%, without any purification steps) using the Leznoff's acetal linker. The libraries were rapidly synthesized in a parallel format and the generated compounds were tested as inhibitors of type 3 17β-HSD. Potent inhibitors were identified from these model libraries, especially six members of the level 3 library having at least one phenyl group. One of them, the 3β-(N-heptanoyl- -phenylalanine- -leucine-aminomethyl)-3α-hydroxy-5α-androstan-17-one (42) inhibited the enzyme with an IC₅₀ value of 227 nM, which is twice as potent as the natural substrate Δ⁴-dione when used itself as an inhibitor. Using the proliferation of androgen-sensitive (AR⁺) Shionogi cells as model of androgenicity, the compound 42 induced only a slight proliferation at 1 μM (less than previously reported type 3 17β-HSD inhibitors) and, interestingly, no proliferation at 0.1 μM.     

β-Turn conformations in crystal structures of model peptides containing α,α-Di-n-propylglycine and α,α-Di-n-butylglycine

The crystal state conformations of three peptides containing the α,α-dialkylated residues. α,α-di-n-propylglycine (Dpg) and α,α-di-n-butylglycine (Dbg), have been established by x-ray diffraction. Boc-Ala-Dpg-Alu-OMe (I) and Boc-Ala-Dbg-Ala-OMe (III) adopt distorted type II β-turn conformations with Ala (1) and Dpg/Dbg (2) as the corner residues. In both peptides the conformational angles at the Dxg residue (I: ? = 66.2°, ψ = 19.3°; III: ? = 66.5°. ψ = 21.1°) deviate appreciably from ideal values for the i + 2 residue in a type II β-turn. In both peptides the observed (N…O) distances between the Boc CO and Ala (3) NH groups are far too long (1: 3.44 Å: III: 3.63 Å) for an intramolecular 4 → 1 hydrogen bond. Boc-Ala-Dpg-Ata-NHMe (II) crystallizes with two independent molecules in the asymmetric unit. Both molecules HA and HB adopt consecutive β-turn (type III-III in HA and type III-I in IIB) or incipient 3₁₀-helical structures, stabilized by two intramolecular 4 → 1 hydrogen bonds. In all four molecules the bond angle N-C^α-C′ (τ) at the Dxg residues are ≥ 110°. The observation of conformational angles in the helical region of ?,ψ space at these residues is consistent with theoretical predictions. © 1995 John Wiley & Sons, Inc.     

Ecosystem stability in space: α, β and γ variability

The past two decades have seen great progress in understanding the mechanisms of ecosystem stability in local ecological systems. There is, however, an urgent need to extend existing knowledge to larger spatial scales to match the scale of management and conservation. Here, we develop a general theoretical framework to study the stability and variability of ecosystems at multiple scales. Analogously to the partitioning of biodiversity, we propose the concepts of alpha, beta and gamma variability. Gamma variability at regional (metacommunity) scale can be partitioned into local alpha variability and spatial beta variability, either multiplicatively or additively. On average, variability decreases from local to regional scales, which creates a negative variability–area relationship. Our partitioning framework suggests that mechanisms of regional ecosystem stability can be understood by investigating the influence of ecological factors on alpha and beta variability. Diversity can provide insurance effects at the various levels of variability, thus generating alpha, beta and gamma diversity–stability relationships. As a consequence, the loss of biodiversity and habitat impairs ecosystem stability at the regional scale. Overall, our framework enables a synthetic understanding of ecosystem stability at multiple scales and has practical implications for landscape management.     

Synthesis of 1-hydroxylated bile acids: Methyl 1α, 3α-dihydroxy 5β-cholan-24-oate

Methyl 1α, 3α-dihydroxy 5β-cholan-24-oate was synthesized to provide a model compound for the mass spectrometric identification of 1α-hydroxylated bile acids.     

Inhibition of adipose differentiation by 9α, 11β-prostaglandin F2α

Prostaglandin F2α (PGF2α) is a potent adipose differentiation inhibitor for the adipogenic cell line 1246 and for adipocyte precursors in primary culture with an ED50 of 3×10⁻⁸ M. In this paper, we examined the effect of several prostaglandins which have structural similarities with PGF2α on the differentiation of 1246 cells and of adipocyte precursors in primary culture. The results show that only 9α,11β-PGF2α is as potent as PGF2α to inhibit differentiation of adipocyte precursors in primary culture and of the adipogenic cell line 1246. In the presence of 9α,11β-PGF2α, the cells remained fibroblast-like, typical of undifferentiated adipocyte precursors. Triglyceride accumulation and increase of specific activity for glycerol-3-phosphate dehydrogenase were inhibited. In addition, mRNA expression of early markers of differentiation such as lipoprotein lipase (LPL) and fatty acid binding protein (FAB) was decreased. The isomer 9β,11α-PGF2α and other PGF2α derivatives were inactive. These results provide new information on the biological activity of 9α,11β-PGF2α as an inhibitor of adipose differentiation and about the structural characteristics of prostaglandins required for maintenance of a high adipose differentiation inhibitory effect.     

Biotransformations of α,β-Epoxyalcohols Catalyzed by Epoxide Hydrolases

Microsomal and cytosolic fractions of mammalian livers were screened for their capacity to resolve racemic mixtures of trans -2,3-epoxy-l-alkanols. The epoxide hydrolase activities showed some specificity for the 2S, 3S enantiomers which were attacked at the proximate carbon atom. The best resolutions were observed with guinea pig liver microsomal enzymes.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

Studies on (β,1→5) and (β,1→6) linked octyl Galf disaccharides as substrates for mycobacterial galactosyltransferase activity

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pandemic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl Galf(β,1→5)Galf and octyl Galf(β,1→6)Galf derivatives. A cell-free assay system has been utilized for galactosyltransferase activity using UDP[¹⁴C]Galf as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that more closely resemble the natural substrates in cell wall biosynthesis.