Bovine adrenal 3beta-hydroxysteroid dehydrogenase (E.C. 1.1.1. 145)/5-ene-4-ene isomerase (E.C. 5.3.3.1): characterization and its inhibition by isoflavones

The isoflavones daidzein, genistein, biochanin A and formononetin inhibit potently and preferentially the γ-isozymes of mammalian alcohol dehydrogenase (γγ-ADH), the only ADH isozyme that catalyzes the oxidation of 3β-hydroxysteroids. Based on these results, we proposed that these isoflavones might also act on other enzymes involved in 3β-hydroxysteroid metabolism. Recently, we showed that they indeed are potent inhibitors of a bacterial β-hydroxysteroid dehydrogenase (β-HSD). To extend this finding to the mammalian systems, we hereby purified, characterized and studied the effects of isoflavones and structurally related compounds on, a bovine adrenal 3β-hydroxysteroid dehydrogenase (3β-HSD). This enzyme catalyzes the oxidation of 3β-hydroxysteroids but not 3-, 11β- or 17β-hydroxysteroids. The same enzyme also catalyzes 5-ene-4-ene isomerization, converting 5-pregnen 3, 20-dione to progesterone. The K_m values of its dehydrogenase activity determined for a list of 3β-hydroxysteroid substrates are similar (1 to 2 μM) and that of its isomerase activity, determined with 5-pregnen 3, 20-dione as a substrate, is 10 μM. The k_cat value determined for its isomerase activity (18.2 min⁻¹) is also higher than that for its dehydrogenase activity (1.4–2.4 min⁻¹). A survey of more than 30 isoflavones and structurally related compounds revealed that daidzein, genistein, biochanin A and formononetin inhibit both the dehydrogenase and isomerase activity of this enzyme. Inhibition is potent and concentration dependent. IC₅₀ values determined for these compounds range from 0.4 to 11 μM, within the plasma and urine concentration ranges of daidzein and genistein of individuals on vegetarian diet or semi-vegetarian diet. These results suggest that dietary isoflavones may exert their biological effects by inhibiting the action of 3β-HSD, a key enzyme of neurosteroid and/or steroid hormone biosynthesis.     

Structures important in mammalian 11β- and 17β-hydroxysteroid dehydrogenases

We have used the X-ray crystallographic structures of rat and human dihydropteridine reductase and Streptomyces hydrogenans 20β-hydroxysteroid dehydrogenase to model parts of the 3-dimensional structure of human 11β- and 17β-hydroxysteroid dehydrogenases. We use this information along with previous results from studies of Drosophila alcohol dehydrogenase mutants to analyze the structures in binding sites for NAD(H) and NADP(H) in 11β-hydroxysteroid dehydrogenase-types 1 and 2. We also examine the structure of an -helix at catalytic site of 17β-hydroxysteroid dehydrogenase-types 1, 2, 3, and 4. This -helix contains a highly conserved tyrosine and lysine. Adjacent to the carboxyl side of this lysine is a site proposed to be important in subunit association. We find that 11β- and 17β-hydroxysteroid dehydrogenases-type 1 have the same residues at the “anchor site” and conserve other stabilizing features, despite only 20% sequence identity between their entire sequences. Similar conservation of stabilizing structures is found in the 11β- and 17β-hydroxysteroid dehydrogenases-type 2. We suggest that interactions of the dimerization surface of -helix F with proteins or membranes may be important in regulating activity of hydroxysteroid dehydrogenases.     

Metabolism of cortisone-4- 14 C by rat lung tissue

The metabolism of cortisone-4-¹⁴C has been studied in male rat lung tissue preparations. Data indicate the presence of 11β-hydroxysteroid dehydrogenase, Δ⁴-5α-reductase, 3α-hydroxysteroid dehydrogenase and 20α-hydroxysteroid dehydrogenase activity in this tissue. Metabolites identified were hydrocortisone, 17α, 20α, 21-trihydroxy-4-pregnene-3, 11-dione and 3α, 17α, 21-trihydroxy-5α-pregnan-11,20-dione.     

Extraglandular hormonal steroidogenesis in aged rats

We have examined the metabolism in vitro of [4-¹⁴C]pregnenolone by the following organs of 2.4-year-old rats: submandibular gland, stomach, duodenum, liver, lung, heart, spleen, kidney, skin, prostate, testis and adrenal. All tissues converted pregnenolone to progesterone, the highest yields being observed with adrenal, testis and skin. Androgen formation was intense in the testis and absent in the adrenal. Moreover, 17-hydroxylation of pregnenolone occurred moderately in kidney, skin and submandibular gland and markedly in duodenum and stomach, which also produced high amounts of dehydroepiandrosterone and/or 5-androstene-3β,17β-diol. Extratesticular synthesis of androstenedione and testosterone was very low. A significant formation of 20-dihydropregnenolone was observed in all tissues but stomach, duodenum and steroidogenic endocrines. Corticosteroids were not synthesized extraadrenally, except a small amount of 11-deoxycorticosterone in the testis. These results indicate that key steroid-biosynthetic enzymes, such as 3β-hydroxysteroid dehydrogenase/Δ⁵′Δ⁴ isomerase, 17β- and 20-hydroxysteroid dehydrogenases and steroid 17-monooxygenase/17,20-lyase are also expressed extraglandularly in the rat.     

Type 2 11β-hydroxysteroid dehydrogenase in foetal and adult life

Two isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD) catalyse the interconversion of active cortisol to inactive cortisone; 11β-HSD1 is a low affinity, NADP(H)-dependent dehydrogenase/oxo-reductase, and 11β-HSD2 a high affinity, NAD-dependent dehydrogenase. Because of the importance of 11β-HSD in regulating corticosteroid hormone action, we have analysed the distribution of the 11β-HSD isoforms in human adult and foetal tissues (including placenta), and, in addition have performed a series of substrate specificity studies on the novel, kidney 11β-HSD2 isoform. Using an RT-PCR approach, we failed to detect 11β-HSD1 mRNA in any human mid-gestational foetal tissues. In contrast 11β-HSD2 mRNA was present in foetal lung, adrenal, colon and kidney. In adult tissues 11β-HSD2 gene expression was confined to the mineralocorticoid target tissues, kidney and colon, whilst 11β-HSD1 was expressed predominantly in glucocorticoid target tissues, liver, lung, pituitary and cerebellum. In human kidney homogenates, 11-hydroxylated progesterone derivatives, glycyrrhetinic acid, corticosterone and the “end products” cortisone and 11-dehydrocorticosterone were potent inhibitors of the NAD-dependent conversion of cortisol to cortisone. Finally high levels of 11β-HSD2 mRNA and activity were observed in term placentae, which correlated positively with foetal weight. The tissue-specific distribution of the 11β-HSD isoforms is in keeping with their differential roles, 11β-HSD1 regulating glucocorticoid hormone action and 11β-HSD2 mineralocorticoid hormone action. The correlation of 11β-HSD2 activity in the placenta with foetal weight suggests, in addition, a crucial role for this enzyme in foetal development, possibly in mediating ontogeny of the foetal hypothalamo-pituitary-adrenal axis.     

The subcellular localization of 17β-hydroxysteroid dehydrogenase type 4 and its interaction with actin

The porcine 17β-hydroxysteroid dehydrogenase type 4 is the key enzyme for the inactivation of estradiol. Its localization in peroxisomes was proven by immunogold electron microscopy. Interactions of the 17β-hydroxysteroid dehydrogenase with cytoskeletal proteins might be mandatory for a topical assignment of enzymatic activity to defined subcellular compartments.     

Gossypolone suppresses progesterone synthesis in bovine luteal cells

Gossypolone, a proposed major metabolite of gossypol, was synthesized and investigated for its effect on progesterone synthesis in cultured bovine luteal cells. Gossypolone inhibited human chorionic gonadotropin(hCG)-stimulated progesterone secretion, reduced substrate-enhanced conversions of 25-hydroxycholesterol to pregnenolone and of pregnenolone to progesterone in a dose-dependent fashion. These findings indicate that gossypolone inhibits not only 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, as gossypol does, but also side-chain cleavage enzyme complex (cytochrome P450scc activity. However, the two compounds appear to have a similar potency in inhibiting progesterone secretion. Both gossypolone and gossypol (8.5 μM) induced morphological changes in cellular organelles.     

Expression of the 11beta-hydroxysteroid dehydrogenase type II enzyme in breast tumors and modulation of activity and cell growth in PMC42 cells

Manipulating the metabolism of glucocorticoids may serve as a useful adjunct in the treatment of breast cancer. The 11β-hydroxysteroid dehydrogenase type 2 enzyme (11βHSD2) potently inactivates glucocorticoids thereby protecting the non-selective mineralocorticoid receptor (MR) in fluid transporting tissues. In the present study, Western blot analysis showed the presence of 11βHSD2 in 66% of the breast tumor samples. The 11βHSD2 and MR are also present in the breast tumor cell line PMC42. Glycyrrhetinic acid abolished glucocorticoid metabolism and inhibited cell growth by 40%, the latter at concentrations consistent with glucocorticoid receptor (GR) and MR binding studies. Metabolism was increased by glucocorticoids, the anti-glucocorticoid RU 38486 and anti-mineralocorticoid spironolactone, while aldosterone had no effect. Neither cortisol nor aldosterone affected cell proliferation, but both RU 38486 and spironolactone caused a significant decrease in cell number. The effects of RU 38486 were only observed at micromolar concentrations and are inconsistent with an action via GR or progesterone receptor (PR). This study shows that 11βHSD2 activity and cell proliferation of PMC42 cells can be modulated via steroid receptors.     

Antidiabetic activity of N-(6-substituted-1,3-benzothiazol-2-yl)benzenesulfonamides

N-(6-Substituted-1,3-benzothiazol-2-yl)benzenesulfonamide derivatives 1–8 were synthesized and evaluated for their in vivo antidiabetic activity in a non-insulin-dependent diabetes mellitus rat model. Several compounds synthesized showed significant lowering of plasma glucose level in this model. As a possible mode of action, the compounds were in vitro evaluated as 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors. The most active compounds (3 and 4) were docked into the crystal structure of 11β-HSD1. Docking results indicate potential hydrogen bond interactions with catalytic amino acid residues.     

Bis-aryl triazoles as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1

3-Aryl-5-phenyl-(1,2,4)-triazoles were identified as selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). They are active in both in vitro and an in vivo mouse pharmacodynamic (PD) model. The synthesis and structure activity relationships are presented.     

Differential inhibition of dehydrogenase and 5-ene→4-ene isomerase activities of purified 3β-hydroxysteroid dehydrogenase. Evidence for two distinct sites

The success in synthesis of [³H]5-androstene-3,17-dione, the intermediate product in the transformation of DHEA to 4-androstenedione by 3β-hydroxysteroid dehydrogenase/ 5-ene→4-ene isomerase (3β-HSD) offers the opportunity to determine whether or not the two activities reside in one active site or in two closely related active sites. The finding that N,N-dimethyl-4-methyl-3-oxo-4-aza-5-androstane-17β-carboxamide (4-MA) inhibits competitively and specifically the dehydrogenase activity whereas a non-competitive inhibition type with a K_i value 1000 fold higher was observed for the isomerase activity, indicated that dehydrogenase and isomerase activities belong to separate sites. Using 5-dihydro-testosterone and 5-androstane-3β,17β-diol, exclusive substrates for dehydrogenase activity, it was shown that dehydrogenase is reversible and strongly inhibited by 4-MA and that thus the irreversible step in the transformation of DHEA to 4-androstenedione is due to the isomerase activity.     

Localization of 17beta-hydroxysteroid dehydrogenase in Mycobacterium sp. VKM Ac-1815D mutant strain

The localization of mycobacterial 17β-hydroxysteroid dehydrogenase (17β-OH SDH) was studied using cell fractionation and cytochemical investigation. Mycobacterium sp. Et1 mutant strain derived from Mycobacterium sp. VKM Ac-1815D and characterized by increased 17β-OH SDH activity was used as a model organism.

Subcellular distribution study showed both soluble and membrane-bound forms of mycobacterial 17β-hydroxysteroid dehydrogenase. The cytochemical method based on a copper ferrocyanide procedure followed by electron microscopic visualization was applied in order to investigate the intracellular localization of bacterial 17β-OH SDH in more detail. The enzyme was found to be located in the peripheral cytoplasmic zone adjoining the cytoplasmic membrane (CM). 17β-OH SDH was loosely membrane bound and easily released into the environment under the cell integrity failure.     

Metabolism of steroid hormones by Taenia solium and Taenia crassiceps cysticerci

Previous in vitro experiments showed that both, Taenia crassiceps and Taenia solium cysticerci have the ability to metabolize exogenous androstenedione to testosterone. Here we evaluate on the capacity of both cysticerci to synthesize several sex steroid hormones, using different hormonal precursors. Experiments using thin layer chromatography (TLC) showed that both cysticerci were able to produce ³H-hydroxyprogesterone, ³H-androstenedione and ³H-testosterone when ³H-progesterone was used as the precursor. They also synthesized ³H-androstenediol and ³H-testosterone when ³H-dehydroepiandrosterone was the precursor. In addition, both cysticerci interconverted ³H-estradiol and ³H-estrone. These results, strongly suggest the presence and activity of the Δ4 and Δ5 steroid pathway enzymes, 3β-hydroxysteroid dehydrogenase/Δ^5-4 isomerase-like enzyme (3β-HSD), that converts androstenediol into testosterone; and the 17β-hydroxysteroid dehydrogenase that interconverts estradiol and estrone, in both types of cysticerci.     

The molecular biology of androgenic 17β-hydroxysteroid dehydrogenases

The enzyme 17β-hydroxysteroid dehydrogenase (17β-HSD) catalyzes the 17β-oxidation/reduction of C₁₈- and C₁₉-steroids in a variety of tissues. Three human genes encoding isozymes of 17β-HSD, designated 17β-HSD types 1, 2 and 3 have been cloned. 17β-HSD type 1 (also referred to as estradiol 17β-dehydrogenase) catalyzes the conversion of estrone to estradiol, primarily in the ovary and placenta. The 17β-HSD type 2 is expressed to high levels in the liver, secretory endometrium and placenta. The type 2 isozyme catalyzes the oxidation of androgens and estrogens equally efficiently. Also, the enzyme possesses 20-HSD activity demonstrated by its ability to convert 20-dihydro-progesterone to progesterone. Testicular 17β-HSD type 3 catalyzes the conversion of androstenedione to testosterone, dehydroepiandrosterone to 5-androstenediol and estrone to estradiol. The 17β-HSD3 gene is mutated in male pseudohermaphrodites with the genetic disease 17β-HSD deficiency.     

Lack of effect of nicotine or ethanol on the activity of 11β-hydroxysteroid dehydrogenase type 2

Low birth weight in combination with a large placenta predicts human hypertension. The pathophysiological link remains unclear, but glucocorticoid excess impairs fetal growth and leads to offspring hypertension. A key controller of fetal glucocorticoid exposure and local tissue availability is 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). The activity of placental 11β-HSD2 correlates with fetal growth in animals and humans. Ethanol abuse and smoking are known to retard fetal growth which may relate to altered glucocorticoid action or dynamics. This study has examined whether nicotine or ethanol modulate glucocorticoid action in the placenta or fetus by inhibiting 11β-HSD2, using clonal cell cultures, freshly isolated dually perfused intact human placentas and placentas from in vivo treated rats. No significant effect on the activity of 11β-HSD2 by pathophysiologically relevant nicotine or ethanol concentrations was observed. The mechanism of action of nicotine and ethanol relevant to reduced fetal growth requires further study.     

Expression and characterization of isoforms of 3β-hydroxysteroid dehydrogenase/Δ-isomerase in the hamster

The enzyme 3β-hydroxysteroid dehydrogenase/Δ^5→4-isomerase (3β-HSD) is essential for the production of all classes of steroid hormones. Multiple isozymes of this enzyme have been demonstrated in the kidney and liver of both the rat and the mouse, although the function of the enzyme in these tissues is unknown. We have characterized three isozymes of 3β-HSD expressed in various tissues of the hamster. Both western and northern blot analyses demonstrated very high levels of 3β-HSD in the adrenal, kidney and male liver. Conversely, there were extremely low levels of enzyme expression in the female liver. cDNA libraries prepared from RNA isolated from hamster adrenal, kidney and liver were screened with a full-length cDNA encoding human type 1 3β-HSD. Separate cDNAs encoding three isoforms of 3β-HSD were isolated from these libraries. To examine the properties of the isoforms, the cDNAs were ligated into expression vectors for over-expression in 293 human fetal kidney cells. The type 1 isoform, isolated from an adrenal cDNA library, was identified as a high-affinity 3β-hydroxysteroid dehydrogenase. A separate isoform, designated type 2, was isolated from the kidney, and this was also a high-affinity dehydrogenase/isomerase. Two cDNAs were isolated from the liver, one identical in sequence to type 2 of the kidney, and a distinct cDNA encoding an isoform designated type 3. The type 3 3β-HSD possessed no steroid dehydrogenase activity but was found to function as a 3-ketosteroid reductase. Thus male hamster liver expresses a high-affinity 3β-HSD (type 2) and a 3-ketosteroid reductase (type 3), whereas the kidney of both sexes express the type 2 3β-HSD isoform. These differ from the type 1 3β-HSD expressed in the adrenal cortex.     

Progestin regulation of 11beta-hydroxysteroid dehydrogenase expression in T-47D human breast cancer cells

This study examined the enzymatic characteristics and steroid regulation of the glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) in the human breast cancer cell line T-47D. In cell homogenates, exogenous NAD significantly increased the conversion of corticosterone to 11-dehydrocorticosterone, while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11β-HSD2 in T-47D cells, while 11β-HSD1 mRNA levels were undetectable. In T-47D cells treated for 24 h with medroxyprogesterone acetate (MPA), 11β-HSD catalytic activity was elevated 11-fold, while estrone (E₁), estradiol (E₂) and the synthetic glucocorticoid dexamethasone (DEX) were ineffective. The antiprogestin mifepristone (RU486) acted as a pure antagonist of the progestin-enhanced 11β-HSD activity, but did not exert any agonistic effects of its own. In addition, RT-PCR analysis demonstrated that MPA was a potent inducer of 11β-HSD2 gene expression, increasing the steady-state levels of 11β-HSD2 mRNA. Taken together, these results demonstrate that 11β-HSD2 is the 11β-HSD isoform expressed by T-47D cells under steady-state conditions and suggest the existence of a previously undocumented mechanism of action of progestins in breast cancer cells.     

Reproductive studies of Brahman cattle III. Comparison of weight, progesterone content, histological characteristics, and 3β-hydroxysteroid dehydrogenase activity in corpora lutea of Brahman, Hereford, and Brahman × Hereford heifers

Sixty corpora lutea (CL), 30 from day 8 and 30 from day 13 of the estrous cycle were collected from 10 Brahman, 10 Hereford and 10 Brahman × Hereford F-1 (B×H) heifers and compared for weight, progesterone concentration and progesterone content. 3β-hydroxysteroid dehydrogenase (3β-HSD) activity and histological and morphological differences were evaluated in CL from 10 animals from each breed at each day.

The Brahman CL were significantly smaller than either Hereford or B×H Cl, 2.616, 3.836 and 4.211 g, respectively. No statistically significant differences were detected for luteal progesterone concentration or content, however, Brahman and B×H CL tended to have less progesterone per CL than did Hereford CL. The histology and morphology of the luteal tissue appeared similar for the three breeds, since there were no detectable differences in the organization, apparent population of cells per area, or the cell types present in the CL. Brahman CL had significantly higher 3β-HSD activity than Hereford or B×H. Day 13 corpora from all breeds had higher 3β-HSD enzyme than CL from day 8 of the cycle. It is evident from this study that major differences exist in CL from Brahman and B×H as compared to Hereford.     

NMR studies of the stereospecificity of 20 -hydroxysteroid dehydrogenase

We (3,4) previously observed the reduction of 21-dehydrocorticosteroids in the presence of 20β-hydroxysteroid dehydrogenase proceeded at a faster rate than the reduction of the corresponding corticosteroids. The presence of adjacent carbonyl groups suggested the possibility that the increased rate of reduction of the 20-one,21-a1 steroid analogs resulted from a lack of specificity of the enzyme 20β-hydroxysteroid dehydrogenase for either the aldehyde or ketone group. Nuclear magnetic resonance spectroscopy indicated that the angular methyl groups of the steroid were sensitive probes for the constituents on the basic steroid skeleton. The C₁₈ methyl resonance of 17,21-dihydroxy-4-pregnene-3,20-dione and 17-hydroxy-3,20-dioxo-4-pregnene-21-a1 were 0.722 ppm and 0.728 ppm respectively. The magnitude and sign of the change in chemical shift of the C₁₈ methyl resonance for the enzymatic products of 17,21-dihydroxy-4-pregnene-3,20-dione and 17-hydroxy-3,20-dioxo-4-pregnene-21-a1 (+0.135 ppm and +0.144 ppm respectively) were consistent with a stereochemical assignment of 20β-hydroxyl.