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This paper summarizes the most recent data obtained in the authors' laboratory on the metabolism of testosterone and progesterone in neurons, in the glia, and in neuroblastoma cells. The activities of the 5 alpha-reductase (the enzyme that converts testosterone into dihydrotestosterone, DHT), and of the 3 alpha-hydroxysteroid dehydrogenase (the enzyme that converts DHT into 5 alpha-androstane-3 alpha, 17 beta-diol, 3 alpha-diol) have been first evaluated in primary cultures of neurons, oligodendrocytes and type-1 and -2 astrocytes, obtained from the fetal or neonatal rat brain. All the cultures were used on the fifth day. The formation of DHT of 3 alpha-diol was evaluated incubating the different cultures with labeled testosterone or DHT as substrates. The results obtained indicate that the formation of DHT takes place preferentially in neurons; however, type-2 astrocytes and oligodendrocytes also possess considerable 5 alpha-reductase activity, while type-1 astrocytes show a much lower enzymatic concentration. A completely different localization was observed for 3 alpha-hydroxysteroid dehydrogenase; the formation of 3 alpha-diol appears to be prevalently, if not exclusively, present in type-1 astrocytes; 3 alpha-diol is formed in very low yields by neurons, type-2 astrocytes and oligodendrocytes. The compartmentalization of two strictly correlated enzymes (5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase) in separate central nervous system (CNS) cell populations suggests the simultaneous participation of neurons and glial cells in the 5 alpha-reductive metabolism of testosterone. Subsequently it has been shown that, similarly to what happens when testosterone is used as the substrate, the 5 alpha-reductase which metabolizes progesterone into 5 alpha-pregnane-3,20-dione (DHP) shows a significantly higher activity in neurons than in glial cells; however, type-1 and -2 astrocytes as well as oligodendrocytes also possess some ability to 5 alpha-reduce progesterone. On the other hand, 3 alpha-hydroxysteroid dehydrogenase, the enzyme which converts DHP into 5 alpha-pregnane-3 alpha-ol-20-one, appears to be present mainly in type-1 astrocytes; much lower levels of this enzyme are present in neurons and in type-2 astrocytes. At variance with the previous results obtained using androgens as precursors, oligodendrocytes show considerable 3 alpha-hydroxysteroid dehydrogenase activity, even if this is statistically lower than that present in type-1 astrocytes. The existence of isoforms of the enzyme involved in androgen and progesterone metabolism is discussed.(ABSTRACT TRUNCATED AT 400 WORDS)
J Steroid Biochem Mol Biol 1993 Dec
PMID:Androgen and progesterone metabolism in the central and peripheral nervous system. 827 36

Doses equivalent to 18, 72 and 216 mg testosterone (T)/week were administered for 6 months to eugonadal men. Urinary excretions of androgen glucuronides (G) were quantitatively analyzed by gas chromatography-mass spectrometry with stable isotope dilution during periods of control (without hormone treatment), T administration and recovery. The lowest dosage T administration did not affect the androgen profile, while higher dosages generally increased urinary excretions of T metabolites (TG, T sulfate, glucuronides of androsterone, etiocholanolone, 5 alpha- and 5 beta-androstane-3 alpha,17 beta-diol) and decreased excretions of conjugates of epitestosterone (ET) and its precursor androgen 5-androstene-3 beta,17 alpha-diol. A dose-dependent decrease of urinary LH in response to T was also observed. The ratio (T/ET)G, which is the sole official criterium for assessment of T self-administration by athletes, increased above the threshold value of 6 in most of the subjects, but not all, after the two highest dosage T regimens, and returned to normal during the recovery period. False positive or negative testing emphasizes the need for improvement of testing procedures. In this regard, valuable complementary information may be gained from ratios such as TG/ET(Total), TG/LH, (T/5-androstene-3 beta,17 alpha-diol)G, (5 alpha/5 beta)androstane-3 alpha,17 alpha-diol and (5 alpha/5 beta)androstane-3 alpha,17 beta-diol.
J Steroid Biochem Mol Biol 1993 Feb
PMID:Long-term administration of testosterone enanthate to normal men: alterations of the urinary profile of androgen metabolites potentially useful for detection of testosterone misuse in sport. 843 22

To study mechanisms of aromatase inhibition in brain cells, a highly effective non-steroidal aromatase inhibitor (Fadrozole; 4-[5,6,7,8-tetra-hydroimidazo- (1,5-a)-pyridin-5-yl] benzonitrile HCl; CGS 16949A) was compared with endogenous C-19 steroids, known to be formed in the preoptic area, which inhibit oestrogen formation. Using a sensitive in vitro tritiated water assay for aromatase activity in avian (dove) preoptic tissue, the order of potency, with testosterone as substrate was: Fadrozole (Ki < 1 x 10(-9) M) > 4-androstenedione > 5 alpha-androstanedione > 5 alpha-dihydrotestosterone (Ki = 6 x 10(-8) M) > 5 beta-androstanedione > 5 beta-dihydrotestosterone (Ki = 3.5 x 10(-7) M) > 5 alpha-androstane-3 alpha,17 beta-diol (Ki = 5 x 10(-6) M) > 5 beta-androstane-3 beta,17 beta-diol. Five other steroids, 5 beta-androstane-3 alpha,17 beta-diol, 5 alpha-androstane-3 beta,17 beta-diol, progesterone, oestradiol and oestrone, showed no inhibition at 10(-4) M. The kinetics indicate that endogenous C-19 steroids show similar competitive inhibition of the aromatase as Fadrozole. Mouse (BALB/c) preoptic aromatase was also inhibited by Fadrozole. We conclude that endogenous C-19 metabolites of testosterone are effective inhibitors of the brain aromatase, and suggest that they bind competitively at the same active site as Fadrozole.
J Steroid Biochem Mol Biol 1993 Mar
PMID:Action of endogenous steroid inhibitors of brain aromatase relative to fadrozole. 847 76

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD)/delta 5-->4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3 beta-HSD/delta 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 3 beta-HSD activity. For this purpose, we compared the efficiencies of a 3 beta-hydroxy-5-ene steroid (DHEA) and a 3 beta-hydroxy-5 alpha-reduced steroid (5 alpha-androstane-3 beta,17 beta-diol, 5 alpha-A-diol) as substrates for the enzyme. The apparent Michaelis constant (Km) for 5 alpha-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 Km of placental 3 beta-HSD for 5 alpha-A-diol was in the range of 18 to 40 mumol/l (n = 3) vs 0.45 to 4 mumol/l for DHEA (n = 3); for the liver enzyme, 17 mumol/l for 5 alpha-A-diol and 0.60 mumol/l for DHEA, and for the skin enzyme 14 and 0.18 mumol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5 alpha-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to Kms and rates of product formation was obtained by use of purified placental 3 beta-HSD with DHEA, pregnenolone, and 3 beta-hydroxy-5 alpha-androstan-17-one (epiandrosterone) as substrates: the Km of 3 beta-HSD for DHEA was 2.8 mumol/l, for pregnenolone 1.9 mumol/l, and for epiandrosterone 25 mumol/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 3 beta-HSD, DHEA at a constant level of 5 mumol/l behaved as a competitive inhibitor when the radiolabeled substrate, [3H]5 alpha-A-diol, was present in concentrations of 20 to 60 mumol/l, but at 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 5 alpha-A-diol (40 mumol/l).(ABSTRACT TRUNCATED AT 400 WORDS)
J Steroid Biochem Mol Biol 1993 Jun
PMID:3 beta-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5 alpha-androstane-3 beta,17 beta-diol and dehydroepiandrosterone as substrates. 851 7

Recent findings obtained by our group showed that incubation of LNCaP cells with labeled steroids leads to the formation of 3- and 17-hydroxysteroid glucuronides. In this study, the specificity and the kinetic properties of 3-hydroxy-C19steroid uridine diphospho-glucuronosyltransferase (3-OH-UGT) and 17-hydroxy-C19steroid UGT (17-OH-UGT) activities in LNCaP cells were investigated. Results indicate that the UGT has a high affinity for testosterone, dihydrotestosterone (DHT), androsterone (ADT) and androstane-3 alpha, 17 beta-diol (3 alpha-DIOL), with Km values ranging from 0.25 to 0.68 microM. The Km values are approx. 10-fold higher for androst-5-ene-3 beta,17 beta-diol (5-ene-DIOL) and androstane-3 beta,17 beta-diol (3 beta-DIOL). The relative specificities (Vmax/Km) also showed higher turnover rates for testosterone, DHT, ADT and 3 alpha-DIOL with values ranging from 2.93 to 5.71, than for 3 beta-DIOL and 5-ene-DIOL with ratios of 0.41 and 1.10, respectively. Dixon plot and Cornish-Bowden analysis demonstrate that testosterone, DHT, ADT, and 3 alpha-DIOL inhibit the glucuronidation of DHT and ADT in a competitive fashion. In contrast, when the studies are performed with 3 beta-diol and 5-ene-DIOL the inhibition of ADT glucuronidation is uncompetitive while the glucuronidation of DHT is inhibited competitively, suggesting the presence of two UGT enzymes, one for glucuronidation of the 17 beta-OH group and a second for the 3 alpha-OH group. Further evidence for the presence of two UGTs in LNCaP cells was obtained by incubation with a variety of 3 beta-OH-C19 steroids which caused a marked inhibition of DHT-G formation but had no effect on the glucuronidation of ADT. In summary, our data demonstrate the presence of at least two UGTs in the human prostate cancer cell line LNCaP. The relative specificity of the 17-OH-UGT in LNCaP cells is 3 alpha-DIOL > DHT > testosterone, while ADT is glucuronidated by the 3-OH-UGT.
J Steroid Biochem Mol Biol 1995 Dec
PMID:Specificity of glucuronosyltransferase activity in the human cancer cell line LNCaP, evidence for the presence of at least two glucuronosyltransferase enzymes. 854 Dec 32

The isoenzymes of the 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene-isomerase (3 beta-HSD) gene family catalyse the transformation of all 5-ene-3 beta-hydroxysteroids into the corresponding 4-ene-3-keto-steroids and are responsible for the interconversion of 3 beta-hydroxy- and 3-keto-5 alpha-androstane steroids. The two human 3 beta-HSD genes and the three related pseudogenes are located on the chromosome 1p13.1 region, close to the centromeric marker D1Z5. The 3 beta-HSD isoenzymes prefer NAD+ to NADP+ as cofactor with the exception of the rat liver type III and mouse kidney type IV, which both prefer NADPH as cofactor for their specific 3-ketosteroid reductase activity due to the presence of Tyr36 in the rat type III and of Phe36 in mouse type IV enzymes instead of Asp36 found in other 3 beta-HSD isoenzymes. The rat types I and IV, bovine and guinea pig 3 beta-HSD proteins possess an intrinsic 17 beta-HSD activity specific to 5 alpha-androstane 17 beta-ol steroids, thus suggesting that such "secondary" activity is specifically responsible for controlling the bioavailability of the active androgen DHT. To elucidate the molecular basis of classical form of 3 beta-HSD deficiency, the structures of the types I and II 3 beta-HSD genes in 12 male pseudohermaphrodite 3 beta-HSD deficient patients as well as in four female patients were analyzed. The 14 different point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene predominantly expressed in the placenta and peripheral tissues. The mutant type II 3 beta-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, at the exception of L108W and P186L proteins, which have some residual activity (approximately 1%). Mutations found in nonsalt-loser patients have some residual activity ranging from approximately 1 to approximately 10% compared to the wild-type enzyme. Characterization of mutant proteins provides unique information on the structure-function relationships of the 3 beta-HSD superfamily.
J Steroid Biochem Mol Biol 1995 Dec
PMID:Structure-function relationships and molecular genetics of the 3 beta-hydroxysteroid dehydrogenase gene family. 854 74

The steroid specificity of the cell surface progesterone receptor in human sperm was examined with the use of progesterone, testosterone, and androstane analogues. Many compounds were shown to be more effective than progesterone at increasing intracellular free calcium concentration, e.g., 2 alpha-methyl-17beta-methoxy-5 alpha-androstan-3-one. Several testosterone analogues were demonstrated to be antagonists of progesterone, e.g., 9(11)-dehydro-2 alpha,17alpha-dimethyltestosterone. The synthetic potent progestigens, norethynodrel, cyproterone acetate, norethindrone, and megestrol acetate, were found to be only weak stimulators of the sperm cell surface receptor. Furthermore, these compounds were shown to antagonize the effect of progesterone to elevate intracellular free calcium concentration in sperm. It is known that progesterone and some of its analogues bind to the intracellular progesterone nuclear receptor via the alpha-face of the steroid molecule. In stark contrast, it was concluded from the analysis of the steroid analogues examined on human sperm in this study that intimate contact exists between the effective progesterone analogues and the sperm cell surface progesterone receptor across the beta-face of the steroid C/D-ring "upper" edge (C11, C12, and C17). Positioning of the C21 methyl group is also critical for efficacy, and recognition of the steroid A-ring seems not to be involved.
Mol Pharmacol 1996 Apr
PMID:Unusual steroid specificity of the cell surface progesterone receptor on human sperm. 860 3

We established a new squamous cell carcinoma cell line, designated RSS18, from a 7,12-dimethyl-benz[a]anthracene (DMBA)-induced submandibular gland of the female rat, and investigated a testosterone metabolism in the cells. During 6 h incubation of RSS18 cells with testosterone as a substrate, the cells produced a significant amount of 5alpha-dihydrotestosterone (DHT) and three kinds of minor metabolites, and their percentages metabolized against total metabolites were in descending order of DHT (89 %) > 5alpha-androstane-3alpha,17beta-diol (9.0 %) > 5alpha-androstanedione(1.6%) > 4-androstene-3,17-dione (0.69%). Therefore, testosterone in RSS18 cells was predominantly converted to DHT by 5alpha-reductase. Growth of RSS18 cells was stimulated by DHT (10(-11)-10(-9) M) to around 170%. By reverse transcription-polymerase chain reaction, the androgen receptor mRNA was significantly detected in RSS18 cells. As a result of these findings, DHT production from testosterone and expression of androgen receptor mRNA, we concluded that RSS18 proliferation may be stimulated by DHT through 5alpha-reductase from testosterone.
J Steroid Biochem Mol Biol 1996 Mar
PMID:Testosterone metabolism in new squamous cell carcinoma cell line (RSS18) from 7,12-dimethylbenz[a]anthracene-induced submandibular gland of female rat. 863 71

3 beta-Hydroxy-delta 5-steroid dehydrogenase (3 beta-HSD)/steroid delta 5-4-isomerase catalyses the conversion of 3 beta-hydroxy-5-ene steroids (e.g. pregnenolone) to 3-oxo-4-ene-steroids (progesterone) in human placenta. Isotope exchange at equilibrium using NAD+/NADH and the 5 alpha-reduced steroids, 5 alpha-androstane-3 beta, 17 beta-diol and 5 alpha-androstan-17 beta-ol-3-one, determined a cofactor-first order of binding for these 3 beta-HSD substrates [1]. Exchange at equilibrium cannot be performed with 3 beta-hydroxy-5-ene steroids because 3 beta-HSD is not reversible with the 5-ene substrates. To compare their cofactor requirements for binding, 3 beta-hydroxy-5-ene and 3 beta-hydroxy-5 alpha-reduced steroids were tested as protectors against the inactivation of purified human placental 3 beta-HSD by 2 alpha-bromoacetoxyprogesterone (2 alpha-BAP) in the presence or absence of cofactor. In incubations without cofactor, pregnenolone or dehydroepiandrosterone dramatically slowed (protected) the rate of 3 beta-HSD inactivation by 2 alpha-BAP, an affinity alkylator that binds specifically at the 3 beta-HSD substrate site. In contrast, 5 alpha-androstan-3 alpha-ol-17-one, 5 alpha-androstane-3 beta, 17 beta-diol, or 11 alpha-acetoxy-5 alpha-pregnan-3,20-dione protected 3 beta-HSD from inactivation by 2 alpha-BAP only in the presence of NADH (0.3 microM) or NAD+ (10 microM). At these low concentrations, neither NADH nor NAD+ slowed the inactivation of 3 beta-HSD by 2 alpha-BAP in the absence of protector-steroid. Further, the 3-oxo-5 alpha-reduced alkylator, 11 alpha-bromoacetoxy-5 alpha-pregnan-3,20-dione (11 alpha-BA-5 alpha-P), did not inactivate 3 beta-HSD in a specific manner. After pre-incubation with NAD+ (10 microM), 11 alpha-BA-5 alpha-P inactivated 3 beta-HSD rapidly and specifically (t1/2 = 3.7 min). 11 alpha-Bromoacetoxyprogesterone inactivated 3 beta-HSD at the same rate (t1/2 = 5.0 min) in the presence or absence of NAD+. These affinity labelling studies confirm the cofactor-first binding order for 3 beta-hydroxy-5 alpha-reduced steroids, and conclusively show that the more important, physiological 3 beta-hydroxy-5-ene substrates bind to 3 beta-HSD without a cofactor requirement.
J Steroid Biochem Mol Biol 1996 May
PMID:Physiological 3 beta-hydroxy-5-ene steroid substrates bind to 3 beta-hydroxysteroid dehydrogenase without the prior binding of cofactor. 880 3

This paper summarizes the most recent data obtained in the authors' laboratory on the metabolism of testosterone and progesterone in neurons and in the glia. 1. The activities of 5 alpha-reductase (the enzyme that converts testosterone into dihydrotestosterone; DHT) and of 3 alpha-hydroxy steroid dehydrogenase (the enzyme that converts DHT into 5 alpha-androstane-3 alpha, 17 beta-diol; 3 alpha-diol) were first evaluated in primary cultures of neurons, oligodendrocytes, and type-1 and type-2 astrocytes, obtained from the fetal or neonatal rat brain. The formation of DHT and 3 alpha-diol was evaluated incubating the different cultures with labeled testosterone or labeled DHT as substrates. The results obtained indicate that the formation of DHT takes place preferentially in neurons; however, also type-2 astrocytes and oligodendrocytes possess considerable 5 alpha-reductase activity. A completely different localization was observed for 3 alpha-hydroxysteroid dehydrogenase; the formation of 3 alpha-diol appears to be prevalently, if not exclusively, present in type-1 astrocytes; 3 alpha-diol is formed in very low yields by neurons, type-2 astrocytes, and oligodendrocytes. Moreover, the results indicate that, in type 1 astrocytes, both 5 alpha-reductase and 3 alpha-HSD are stimulated by coculture with neurons and by the addition of neuron-conditioned medium, suggesting that secretory products released by neurons might intervene in the control of glial cell function. 2. Subsequently it was shown that, similarly to what happens when testosterone is used as the substrate, 5 alpha-reductase, which metabolizes progesterone into 5 alpha-pregnane-3,20-dione, (DHP), shows a significantly higher activity in neurons than in glial cells; however, also type-1 and type-2 astrocytes as well as oligodendrocytes possess some ability to 5 alpha-reduce progesterone. On the contrary, 3 alpha-hydroxysteroid dehydrogenase, the enzyme which converts DHP into 5 alpha-pregnane-3 alpha-ol-20-one (THP), appears to be present mainly in type-1 astrocytes; much lower levels of this enzyme are present in neurons and in type-2 astrocytes. At variance with the previous results obtained using androgens as precursors, oligodendrocytes show considerable 3 alpha-hydroxysteroid dehydrogenase activity, even if this is statistically lowe than that present in type-1 astrocytes. The existence of isoenzymatic forms of the enzymes involved in androgen and progesterone metabolism is discussed.
Cell Mol Neurobiol 1996 Jun
PMID:Testosterone and progesterone metabolism in the central nervous system: cellular localization and mechanism of control of the enzymes involved. 881 96


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