5-ene Steroids Research Articles

Hydroxylative activity of Aspergillus niger towards androst-4-ene and androst-5-ene steroids

Aspergillus niger, one of fungal species most frequently used for experimental and industrial-scale biotransformations of various organic compounds, is generally known to transform steroids at 16β position. In this work, application of the strain A. niger KCH910 to bioconversion of dehydroepiandrosterone (DHEA), androstenediol and testosterone is described, with emphasis on the metabolic steps leading to the products. Evidence from this study indicated that incubated 5-ene steroids underwent bioconversion within two metabolic pathways: oxidation by the action of 3β-HSD (3β-hydroxysteroid dehydrogenase) to 4-ene steroids, and minor allylic hydroxylation to epimeric 7-alcohols. Further transformation of the 3-oxo-4-ene metabolites resulted in non-selective 16-hydroxylation. It is the first report on an A. niger strain able to introduce not only 16β- but also 16α-hydroxyl function into steroids.

Novel metabolites of dehydroepiandrosterone and progesterone obtained in Didymosphearia igniaria KCH 6670 culture

Abstract Dehydroepiandrosterone (DHEA) (10) and its five derivatives: testosterone (1), androstenedione (2), 17α-methyltestosterone (6), progesterone (13) and pregnenolone (14) were subjected to microbial transformation by the filamentous fungus Didymosphaeria igniaria KCH 6670. The predominant metabolism of the incubated 5-ene steroids (10 and 14) occurred through 3β-hydroxy-steroid dehydrogenase/5,4-en isomerase pathways resulting in the generation of a 4-en-3-oxo system on ring-A. The transformations of C19 steroids (1, 2, and 10) included a hydroxylation at 7α position, ketone–alcohol interconversion at C-17 and reduction of the double bond at C-4 and 3-keto group to the 3β-alcohol with 5α-stereochemistry at A/B ring. D. igniaria also carried out 6(7)-dehydrogenation and 6,7β-epoxidation during transformation of DHEA. Under these conditions transformation of DHEA (10) gave four products: 7α-hydroxyandrost-4-en-3,17-dione (4), 17β-hydroxyandrost-4,6-dien-3-one (11), 17β-hydroxyandrost-6β-epoxy-4-en-3-one (12) and 3β,17β-dihydroxy-5α-androstane (5). The compounds 11 and 12 are identified as DHEA metabolites for the first time. The transformation of C21 steroids (13 and 14) led to the mixture of mono- (mainly 11α- and 15β-) and dihydroxy- (7α,15β-; 14α,15β-; 11α,15β-; 11α,14α-) products. 7α,15β-Dihydroxypregnan-4-en-3,20-dione (18) and 14α,15β-dihydroxypregnan-4-en-3,20-dione (19) were found to be new compounds. The main product of transformation of 17α-methyltestosterone (6) was 12β-hydroxy-17α-methyltestosterone (7). The results of these transformations demonstrate the dependence of hydroxylation position on the structure of steroid nucleus.

The influence of low dose finasteride, a type II 5α-reductase inhibitor, on circulating neuroactive steroids.

Finasteride is a 5α-reductase inhibitor that has received clinical approval for the treatment of human benign prostatic hyperplasia and androgenetic alopecia. The treatment is practically without side effects, although some occasional cases of depression syndrome have been reported. 5α-Reductase is an enzyme responsible for the reduction of testosterone, progesterone or deoxycorticosterone to their 5α-reduced derivatives possessing anticonvulsant, antidepressant, and anxiolytic activity. Therefore, the formation of GABAergic neuroactive steroids is likely to be impacted by finasteride. The objective of the study was to show how the treatment of premature androgenetic alopecia with low doses (1mg/day) of finasteride influences the broad spectrum of steroids with potential neuroactivity. A group of 12 men with premature androgenetic alopecia participated in the present study. The steroid hormone profile was determined for all individuals. Finasteride was administered for 4 months at a daily dose of 1mg. After the treatment, the same hormonal profile was determined again. 5α-Reduced steroids, e.g., 5α-dihydrotestosterone, androsterone, epiandrosterone, 5α-androstene-3α,17β-diol, allopregnanolone, isopregnaolone, and some 5-ene steroids, such as dehydroepiandrosterone and pregnenolone, decreased gradually during treatment. The decrease of 5α-reduced steroids, especially of allopregnanediol, dihydrotestosterone, and pregnenolone, is probably one of the factors responsible for the increased occurrence of depression in men treated with finasteride, even at low doses.

Transformation of 5-ene steroids by the fungus Aspergillus tamarii KITA: Mixed molecular fate in lactonization and hydroxylation pathways with identification of a putative 3β-hydroxy-steroid dehydrogenase/Δ 5–Δ 4 isomerase pathway

The fungus Aspergillus tamarii metabolizes progesterone to testololactone in high yield through a sequential four step enzymatic pathway which, has demonstrated flexibility in handling a range of steroidal probes. These substrates have revealed that subtle changes in the molecular structure of the steroid lead to significant changes in route of metabolism. It was therefore of interest to determine the metabolism of a range of 5-ene containing steroidal substrates. Remarkably the primary route of 5-ene steroid metabolism involved a 3β-hydroxy-steroid dehydrogenase/Δ 5–Δ 4 isomerase (3β-HSD/isomerase) enzyme(s), generating 3-one-4-ene functionality and identified for the first time in a fungus with the ability to handle both dehydroepiansdrosterone (DHEA) as well as C-17 side-chain containing compounds such as pregnenolone and 3β-hydroxy-16α,17α-epoxypregn-5-en-20-one. Uniquely in all the steroids tested, 3β-HSD/isomerase activity only occurred following lactonization of the steroidal ring-D. Presence of C-7 allylic hydroxylation, in either epimeric form, inhibited 3β-HSD/isomerase activity and of the substrates tested, was only observed with DHEA and its 13α-methyl analogue. In contrast to previous studies of fungi with 3β-HSD/isomerase activity DHEA could also enter a minor hydroxylation pathway. Pregnenolone and 3β-hydroxy-16α,17α-epoxypregn-5-en-20-one were metabolized solely through the putative 3β-HSD/isomerase pathway, indicating that a 17β-methyl ketone functionality inhibits allylic oxidation at C-7. The presence of the 3β-HSD/isomerase in A. tamarii and the transformation results obtained in this study highlight an important potential role that fungi may have in the generation of environmental androgens.

Aromatase Reaction of 3-Deoxyandrogens: Steric Mode of the C-19 Oxygenation and Cleavage of the C10−C19 Bond by Human Placental Aromatase

Aromatase is a cytochrome P-450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione (AD) to estrone and formic acid through three sequential oxygenations of the 19-methyl group. To gain insight into the catalytic function of aromatase as well as the mechanism of the hitherto uncertain third oxygenation step, we focused on the aromatase-catalyzed 19-oxygenation of 3-deoxyandrogens: 3-deoxy-AD (1), which is a very powerful competitive inhibitor but poor substrate of aromatase, and its 5-ene isomer 4, which is a good competitive inhibitor and effective substrate of the enzyme. In incubations of their 19S-(3)H-labeled 19-hydroxy derivatives 2 and 5 and the corresponding 19R-(3)H isomers with human placental microsomes in the presence of NADPH under air, the radioactivity was liberated in both water and formic acid. The productions of (3)H(2)O and (3)HCOOH were blocked by the substrate AD or the inhibitor 4-hydroxy-AD, indicating that these productions are due to a catalytic function of aromatase. A comparison of the (3)H(2)O production from S-(3)H substrates 2 and 5 with that from the corresponding R-(3)H isomers revealed that the 19-pro-R hydrogen atom was stereospecifically (pro-R:pro-S = 100:0) removed in the conversion of 5-ene substrate 5 into the 19-oxo product 6, whereas 75:25 stereoselectivity for the loss of the pro-R and pro-S hydrogen atoms was observed in the oxygenation of the other substrate, 2. The present results reveal that human placental aromatase catalyzes three sequential oxygenations at C-19 of 3-deoxyandrogens 1 and 4 to cause the cleavage of the C(10)-C(19) bond through their 19-hydroxy (2 and 5) and 19-oxo (3 and 6) intermediates, respectively, where there is a difference in the stereochemistry between the two androgens in the second 19-hydroxylation. It is implied that the aromatase-catalyzed 19-oxygenation of 5-ene steroid 4 but not the 4-ene isomer 1 would proceed in the same steric mechanism as that involved in the AD aromatization.

Structure–activity relationships of 3-deoxy androgens as aromatase inhibitors. synthesis and biochemical studies of 4-substituted 4-ene and 5-ene steroids

As part of our investigation into the structure–activity relationship of a novel class of aromatase inhibitors, two series of 3-deoxy androgens, androst-5-en-17-ones with a non-polar alkoxy ( 5 and 6), alkyl ( 20− 22), or phenylalkyl ( 23 and 24) group at C-4β and 4-acyloxyandrost-4-en-17-ones ( 29– 32, and 34) were synthesized and evaluated. The 4β-alkyl and 4β-phenylalkyl compounds were obtained through reaction of 4α,5α-epoxy steroid ( 8) with RMgBr (R: alkyl and phenylalkyl) followed by dehydration of the 4β-substituted 5α-hydroxy products ( 15− 19) with SOCl 2 as key reactions. Acylation of 4α,5α-diol ( 25) with (RCO) 2O in pyridine and subsequent dehydration with SOCl 2 gave the 4-acyloxy steroids. All of the steroids studied, except for 4-acetoxy-19-ol ( 34) that was a non-competitive inhibitor of human placental aromatase, blocked aromatase activity in a competitive manner. 4-Benzoyloxy- and 4-acetoxy steroids ( 31) and ( 32) were the most powerful inhibitors of aromatase ( K i=70 and 60 nM, respectively). Elongation of an acetoxy group in a series of 4-acyloxy steroids or a methyl group in a series of 4β-alkyl steroids decreased affinity for aromatase principally in relation to carbon number of the acyl or alkyl function. The present findings are potentially useful for understanding the spatial and electronic nature of the binding site of aromatase as well as for developing effective aromatase inhibitors.

Accumulation of 4- and 5-ene steroid sulfates in human breast cyst fluids

Gross cystic disease of the breast is one of the most common diseases of adult females. Breast cyst fluid contains various steroid hormones. In order to obtain more information about the concentrations of 4- and 5-ene steroids in human breast cyst fluids, levels of pregnenolone sulfate (PREGS), pregnenolone (PREG), dehydroepiandrosterone sulfate (DHEAS) and dehydroepiandrosterone (DHEA) were determined by high-performance liquid chromatography (HPLC). A total of 35 human breast cyst fluid samples, obtained from 35 patients (28–54 years old) were analyzed. Cyst fluid electrolytes were simultaneously determined. Levels of PREGS (mean±S.D.) were 26.9±20.0 μmol/l ( N=35) and of PREG were <0.1 μmol/l. Levels of DHEAS and DHEA were 89.1±111.7 μmol/l ( N=35) and 0.3±0.2 μmol/l ( N=35), respectively. Cyst fluids were divided into two groups (types I and II) according to their electrolyte ratio (K +/Na +). The cysts of the type I group (K +/Na + >1.5) contained significantly higher levels of PREGS (39.9±21.1 μmol/l) and DHEAS (133.2±87.9 μmol/l) than those of the type II group (K +/Na + <1.5), the mean levels of which were 19.8±16.2 μmol/dl for PREGS, and 36.3±29.0 μmol/dl for DHEAS ( P<0.05). PREGS and DHEAS levels in the cysts were significantly correlated ( r=0.49; P<0.01). Human breast cyst fluids contain high concentration of DHEAS and PREGS, especially in the cyst fluids containing high K +/Na + ratios.

Transformation in the PC-aided biochemical data analysis.

Data transformations enable expression of original data in a new scale, more suitable for data analysis. In computer-aided interactive analysis of biochemical and clinical data an exploratory data analysis often finds that the sample distribution is systematically skewed or does not accept a sample homogeneity. Under such circumstances the original data should be transformed. The power transformation and the Box-Cox transformation improve sample symmetry and also stabilize variance. Both the Hines-Hines selection graph and the plot of logarithm of a maximum likelihood function allow selection of an optimum transformation parameter. The proposed procedure of data transformation in univariate data analysis is illustrated on a determination of 17-hydroxypregnenolone in umbilical blood of a population of newborns. Lower levels of free 5-ene steroids in umbilical blood and elevated levels of 5-ene steroid sulfates indicate a congenital sex-specific placental sulfatase insufficiency. After examination of statistical assumptions by diagnostic plots of an exploratory data analysis the best estimate of a mean value of 17-hydroxypregnenolone is derived.

19-oxygenations of 3-deoxy androgens, potent competitive inhibitors of estrogen biosynthesis, with human placental aromatase

Aromatase is a cytochrome P450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione (AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the ability of 3-deoxy derivative of AD, compound 1, and its 5-ene isomer 4, which are potent competitive inhibitors of aromatase, to serve as a substrate, we studied their 19-oxygenation by human placental aromatase and the metabolites isolated were analyzed by gas chromatography–mass spectrometry. Inhibitors 1 and 4 were found to be oxygenated with aromatase to produce the corresponding 19-hydroxy derivatives 2 and 5 and 19-oxo derivatives 3 and 6 as well as the 17β-reduced 19-hydroxy compounds 7 and 8. Kinetic studies indicated that the 5-ene steroid 4 was surprisingly a good substrate for the aromatase-catalyzing 19-oxygenation with the V max value of 45 pmol/min per mg prot which was approx. four times higher than that of the other. The relative K m value for steroids 1 and 4 obtained in this study is opposite from the relative K i value obtained previously in the inhibition study. The results reveal that there is a difference between a binding suitable for serving as an inhibitor of aromatase and a binding suitable for serving as a substrate of the enzyme in the 3-deoxy steroid series and the C-3 carbonyl group of AD is essential for a proper binding as a substrate to the active site of aromatase.

Biotransformation XLVII: transformations of 5-ene steroids in Fusarium culmorum culture

The course of the transformation of six 5-ene steroids with varying substituents at C-17 or/and C-3: dehydroepiandrosterone (DHEA), 5-androsten-3β,17β-diol, 17α-methyl-5-androsten-3β,17β-diol, 5-androsten-17-one, 5-androsten-3β-ol and pregnenolone by Fusarium culmorum was investigated. Three substrates with oxygen functions at C-3 and C-17 i.e. DHEA, 5-androsten-3β,17β-diol and 17α-methyl-5-androsten-3β,17β-diol were hydroxylated entirely at 7α-axial, allylic position. The mixture of 7α-hydroxy- and 7α,15α-dihydroxyderivatives was formed during the transformation of pregnenolone and 5-androsten-17-one, from the latter 2α,7α-dihydroxyderivative was also obtained. 7α,15α- Dihydroxyderivative was the only product isolated from the 5-androsten-3β-ol post-transformation mixture. The time-course of the DHEA transformation by F. culmorum shows that the substrate induces 7α-hydroxylase activity. DHEA was transformed by androstenedione induced F. culmorum cultures to a larger extent than by a noninduced microorganism; the selectivity of the transformation remained unchanged.

4-Oxygenated androst-5-en-17-ones and their 7-oxo derivatives as aromatase inhibitors.

A series of androst-5-ene-4,7-diones and 4-oxygenated androst-5-enes were synthesized and tested for their ability to inhibit aromatase in human placental microsomes. All of the steroids examined inhibited the enzyme in a competitive manner. The inhibitory activity of 4beta-hydroxy-5-ene steroid 7 (Ki = 25 nM) was much more powerful than that of the parent 5-ene steroid 11 (Ki = 78 nM), whereas 4beta-acetate 8 and 4-oxo analog 5 (Ki = 90 and 120 nM, respectively) were less potent than compound 11. This indicates that a hydrogen bonding between a hydroxy group of the 4beta-ol 7 and a residue of the active site of aromatase plays an important role in its binding. The 5-en-4-one steroid 5 did not cause a time-dependent inactivation of aromatase. In contrast, 5-ene-4,7-dione 13 as well as its 19-hydroxy and 19-oxo analogs 19 and 20 caused the time-dependent inactivation only in the presence of NADPH in air with the k(inact) values ranging from 0.057 to 0.192 min(-1), although their affinities for the enzyme were not high (Ki = 430-6300 nM). The inactivation was prevented by androstenedione, and no significant effect of L-cysteine on the inactivation was observed in each case. These results suggest that oxygenation at C-19 would be at least in part involved in the inactivation caused by the inhibitor 13.

A key enzyme in the biosynthesis of neurosteroids, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD), is expressed in rat brain

In rat brain, the presence of pregnenolone and progesterone, not attributable to peripheral glandular sources, has been demonstrated and thus the two compounds can be classified as neurosteroids. In vitro experiments have shown the conversion of pregnenolone, a 3 beta-hydroxy-delta 5-ene steroid, into progesterone, a delta 4-oxo steroid, thus demonstrating a 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) enzymatic activity. The conversion of 3 beta-hydroxy-delta 5-derivatives into the corresponding delta 4-oxo steroids by 3 beta-HSD is an essential step in the biosynthesis of all steroid hormones in endocrine glands. To date, four isoforms of 3 beta-HSD have been characterized in the rat. We report here the selective expression of a 3 beta-HSD isoform in rat brain. An in situ hybridization study, using an oligonucleotide common to the 4 known isoforms, demonstrated 3 beta-HSD mRNA in neurons of the olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, habenula, hippocampus and cerebellum. The cerebellum showed the highest level of 3 beta-HSD mRNA corresponding to a transcript of 1.8 kb. Nucleotide sequencing of PCR-amplified cDNA fragments from cerebellar mRNA indicated the expression of an isoform of 3 beta-HSD cDNA very closely related to the isoform I expressed in the adrenals and gonads. Further evidence for the expression of 3 beta-HSD gene in the brain was demonstrated utilizing anti-peptide 3 beta-HSD antibodies which revealed an immunoreactive protein of approximately 45 kDa in the cerebellum. Our results demonstrate for the first time the expression of the enzyme 3 beta-HSD in the brain, at both the mRNA and protein levels. Since several neuroactive neurosteroids are substrates or products of the 3 beta-HSD enzymatic activity, our findings offer new possibilities to study the regulatory mechanisms governing their biosynthesis in the brain.

Serum and Urinary Steroids in Girls with Precocious Pubarche and/or Hirsutism due to Mild 3-Beta-Hydroxysteroid Dehydrogenase Deficiency

To obtain data on the correlation of serum and urinary steroids in nonclassical 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency, 9 girls with precocious pubarche and 33 adolescent girls with mild to severe hirsutism were studied. Urinary steroid profiles were analyzed by capillary gas chromatography. Serum 17-OH-pregnenolone (17-OHPreg) and 17-OH-progesterone (17-OHP) were determined by RIA after column-chromatographic separation. One out of 9 girls with precocious pubarche and 4/33 girls with hirsutism had elevated ratios of 17-OHPreg to 17-OHP after ACTH stimulation in serum and elevated urinary excretion of 5-ene steroids under basal conditions. These patients were defined to have decreased adrenal 3 beta-HSD activity. Basal and ACTH-stimulated serum 17-OHPreg levels in patients with mild 3 beta-HSD deficiency overlapped those of healthy controls and peripubertally virilized female patients without enzyme deficiency. Post-ACTH 17-OHPreg/17-OHP ratios in serum discriminated patients with and without 3 beta-HSD deficiency using a cutoff value of 13 instead of mean + 2 SD for age-related control values (6.7 and 11.6 for girls with Tanner stage II-III and IV-V, respectively). Sums of urinary 5-ene steroids in patients with 3 beta-HSD deficiency overlapped those in patients without enzyme deficiency. Results showed that an abnormal post-ACTH serum 17-OHPreg/17-OHP ratio may not be associated with elevated urinary 5-ene steroid excretion, and vica versa. In conclusion, patients with simultaneous elevation of post-ACTH serum 17-OHPreg/17-OHP ratio and basal urinary 5-ene steroid excretion are supposed to have mild 3 beta-HSD deficiency.

Plasma 3α-androstanediol glucuronide in precocious adrenarche

The aim was to study 3 alpha-androstanediol glucuronide (3AG) plasma levels and its relationship with 5-ene and 4-ene steroids in children with the benign form of precocious pubarche (precocious adrenarche). Sixty-five children with precocious adrenarche (PA), aged 3.6-8.2 yr (55 girls and 10 males) and 15 normal age-matched children were studied. We evaluated plasma androstenedione (A), dehydroepiandrosterone (DHA), its sulfate (DHA-S), testosterone (T), dihydrotestosterone (DHT), its glucuronide (DHTG), 3 alpha-androstanediol (3Ad) and its glucuronide (3AG) in all subjects. All androgens are expressed as mean +/- SD. We found significantly higher plasma levels not only in glandular androgens but also in peripheral androgens (A, 2.4 +/- 1.5 nM vs 0.79 +/- 0.46 nM, p < 0.001; DHA, 9.8 +/- 4.9 vs 2.7 +/- 0.76 ng/dl, p < 0.001; DHA-S, 3.4 +/- 2 microM vs 2.4 +/- 0.65 microM, p < 0.05; T, 0.74 +/- 0.5 nM vs 0.4 +/- 0.1 nM, p < 0.001; DHT, 0.36 +/- 0.13 nM vs 0.12 +/- 0.05 nM, p < 0.001; 3Ad, 0.13 +/- 0.1 nM vs 0.054 +/- 0.03 nM, p < 0.001; DHTG, 0.5 +/- 0.3 nM vs 0.26 +/- 0.09 nM p < 0.01). As far as the plasma 3AG levels are concerned we found significantly higher values in PA with respect to controls (1.17 +/- 0.7 nM vs 0.61 +/- 0.04 nM, p < 0.01), suggesting that 3AG may be considered a marker of skin androgen utilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of correlation between sex hormone binding globulin, adrenal and peripheral androgens in precocious adrenarche

Sex hormone binding globulin (SHBG) is a specific steroid-binding plasma glycoprotein regulated by several different factors. Sex steroids are currently considered to be the main physiological regulators of this protein. Testosterone (T) in adults seems to be the main hormone active in lowering SHBG. The role of dihydrotestosterone (DHT) in such regulation, particularly in the prepubertal age, is not well understood, and no data exist about the role of 3 alpha-androstanediol (3A alpha) and its glucuronide. In adulthood, in addition to T, 5-ene steroids seems to play a role in the regulation of SHBG plasma concentration. To assess the effect of adrenal and peripheral androgens in modulating SHBG levels in the prepubertal age, we studied subjects with precocious pubarche secondary to precocious adrenarche (PA). PA represents, in fact, a good model of study as it is characterized by an increased production and action of adrenal androgen in females under 8 yr of age and in males under 9. Sixty-five subjects (55 females and 10 males; chronologic age: 3.6 - 8.2 yr (6.9 +/- 1.3, SD); bone age: 3.6 - 11 yr (7.6 +/- 1.9); BMI 17.9 +/- 3 kg/m2) were studied. Fifteen age-matched normal children (BMI 15.2 +/- 0.8 kg/m2) were studied as controls. Androstenedione (A), dehydroepiandrosterone (DHA) and its sulphate (DHA-S), T, DHT, 3Ad and its glucuronide (3AG) and SHBG were evaluated in all subjects. In PA cases serum SHBG levels (50 +/- 27 nM) were significantly lower (p less than 0.05) with respect to normal prepubertal patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of steroid and steroid sulfate production and aromatase activity in cultured human granulosa-luteal cells

The regulation of the production of steroids and steroid sulfates and the activity of aromatase in human luteinized granulosa cells were investigated. The cells were cultured for 48 h in the presence or absence of hCG and FSH. Basal production of pregnenolone (Pre, 0.3 ± 0.03 ng/μg protein) and progesterone (P 19.3 ± 17. ng/μg protein) were high compared with that of other steroids beyond P in the steroidogenic pathway. The concentration of 17α-hydroxyprogesterone (17-OHP) was lower 0.17 ± 0.06 ng/μg and that of other steroids in the 4-ene and 5-ene pathways and steroid sulfates <0.05 ng/μg. Both hCG and FSH (100 ng/ml) stimulated the production of Pre and P 3- to 5-fold, but only minimal stimulation of other steroids and steroid sulfates was observed. Aromatase activity of granulosa-luteal cells was measured from the rate of formation of 3H 2O from 1β-[ 3H]androstenedione (1β[ 3H]A) after exposing the cells to hCG, FSH or estradiol (E 2) for 48 h. Basal aromatase activity was relatively low, but hCG and FSH stimulated aromatase 8- and 4-fold, respectively. The incubation of granulosa-luteal cells with E 2 did not affect basal aromatase activity, but E 2 augmented FSH-stimulated aromatase 1.4-fold ( P < 0.025). The results suggest that there is low 17α-hydroxylase and steroid sulfokinase activity in human granulosa-luteal cells. Aromatase activity in these cells is regulated by both hCG and FSH, and intra-ovarian estrogens may regulate granulosa cell aromatase activity.

Effects of dihydrotestosterone treatment on adrenal gland function and morphology in adult female guinea-pigs

The effect of chronic treatment of female guinea-pigs with dihydrotestosterone (DHT) on growth and function of the adrenal gland and, in particular, on the reticular zone is described. Two groups of 6 young adult, female guinea-pigs were treated with DHT (l mg/kg dissolved in peanut oil and injected s.c.) for 30 and 60 days. Two other groups of animals, treated only with oil, were used as controls. At the end of treatment, animals were killed and adrenal glands were quickly removed. Plasma levels of pregnenolone, dehydroepiandrosterone (DHA) and its sulfate (DHA-S), 17α-hydroxyprogesterone, androstenedione, testosterone, estradiol, 11-deoxycortisol, androstenedione, DHT and 3α-androstanediol were determined by R.I.A. following celite microcolumn chromatography. Animals treated for 30 days showed only elevated DHT and 3α-androstanediol plasma levels, whereas animals treated for 60 days also showed increased values of pregnenolone (251 ± 62 vs 193 ± 51 ng/dl; P < 0.05), DHA-S (12,046 ± 4110 vs 2780 ± 888 ng/dl; P < 0.001) and slightly increased values of DHA (110 ± 31 vs 86.5 ± 55.4). In the 30-day-treated animals no histological changes were observed, but in the 60-day-treated group the total size as well as cell volumes of the zona reticularis were significantly increased. Normal estrous cycles were observed in the 30-day-treated animals whereas the 60-day-treated animals showed a progressive acyclicity during the second month of treatment. These results indicate that in guinea-pigs, prolonged treatment with DHT induces a growth of the zona reticularis of the adrenal gland associated with increased levels of 5-ene steroids, particularly DHA-S. The mechanisms inducing these modifications are probably mediated by a DHT effect at the hypothalamic-pituitary level. A direct effect of DHT on the zona reticularis, however, cannot be excluded.

Changes in steroid pattern following acute and chronic adrenocorticotropin administration in premature adrenarche

Biochemically adrenarche is characterized by increased production of 5-ene steroids, in particular Dehydroepiandrosterone (DHA) and its sulphate (DHA-S). It is still not clear if ACTH is responsible for this adrenal steroid production. The aim of the present study was to evaluate the effect of acute and chronic ACTH administration, without dexamethasone pretreatment, on hormonal patterns in 20 patients (5 males aged between 6 1 2 and 7 10 12 years and 15 females aged between 5 9 12 and 7 6 12 years) with idiopathic premature adrenarche. Pregnenolone (5P), DHA, DHA-S, 17-hydroxyprogesterone (17-OHP), androstenedione (A), 11-deoxycortisol (S) and Cortisol (F) have been determined by Radioimmunoassay. The results of the hormonal evaluation (means ± standard error) showed high plasma levels of DHA [329.2 ±41.7 ng/100 ml (dl)] and DHA-S (169.1 ± 54 μg/dl) and slightly increased levels of 5P (74.4 ± 7.1 ng/dl), of A (45.4 ± 4.6 ng/dl) and 17-OHP (69.3 ± 11.3 ng/dl) in comparison to those of controls, thus indicating a decrease in 3β-hydroxysteroid dehydrogenase activity and an increase in 17–20-lyase and 17-hydroxylase activities, characteristic for adrenarche. Acute and chronic ACTH stimulation did not amplify the characteristic basal hormonal pattern, but they induced a shift of adrenal steroid metabolism to 4-ene pathway, suggesting that the basal hormonal pattern in premature adrenarche may be independent or, at least, not exclusively dependent on ACTH control.

Phospholipases modulate the rat testicular androgen biosynthetic pathway in vitro.

The role of membrane phospholipids in testicular androgen biosynthesis was investigated by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Androgen biosynthesis in untreated rat testicular microsomes was examined by monitoring the temporal appearance of pregnenolone metabolites and was found to proceed through the 4-ene route. When phospholipase A2 was included, the 5-ene steroids 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) were formed in greater quantities, and the production of 4-ene steroids was reduced indicating that the conversion of 5-ene steroids to the 4-ene configuration was inhibited by phospholipase A2 treatment. Phospholipase C, in addition to inhibiting this step, also inhibited the conversion of C21 steroids to C19 steroids. When the enzymatic steps were measured individually, phospholipase A2 inhibited 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-Isomerase) with an ED50 of 73 mU/ml but had no effect on the activities of 17-hydroxylase, C-17, 20 lyase, or 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). However, though phospholipase C treatment inhibited 3 beta-HSD-Isomerase, it caused less inhibition (the ED50 value was 149 mU/ml). Furthermore, 17-hydroxylase and C-17, 20 lyase activities were also inhibited by phospholipase C treatment (ED50 values were 410 and 343 mU/ml, respectively), but no effect on 17 beta-HSD was observed. The differences in the apparent phospholipid requirements of the steroidogenic enzymes provides the possibility that the metabolic fate of pregnenolone may be regulated by changes in the phospholipid composition of the microenvironment.

Further evidence that there is more than one adrenal 21-hydroxylase system

The 21-hydroxylase activity of microsomes isolated from bovine adrenal cortex have been assayed using [21- 3H]17-hydroxypregnenolone and [1,2- 3H]17-hydroxyprogesterone as substrates. When the assays are performed in the presence of an NADH regenerating system, to inhibit steroid 3β-hydroxy isomerase-dehydrogenase activity, the microsomes oxidize the 3β-hydroxy-5-ene steroid at a rate of 0.37nmol/min·nmol cytochrome P-450 and the 3-keto-4-ene steroid at a rate of 6.4 nmol/min · nmol. When the microsomes are solubilized with Triton CF-54 they lose the ability to oxidize the 3-hydroxy-5-ene steroid, while the specific activity of the microsomes for the 3-keto-4-ene steroid is enhanced 3-fold. In contrast, when the microsomes are solubilized with sodium cholate, their specific activity towards the 4-ene steroid is decreased by 50% while the specific activity for a low concentration of the 5-ene steroid, 1 μM, is unchanged. In addition, when the oxidations of the labeled steroids (at 1 μM) by the microsomes, are examined in the presence of unlabeled 17-hydroxyprogesterone (at 20 μM) the oxidation of the 3-keto-4-ene steroid is inhibited by 92% while the oxidation of the 3β-hydroxy-5-ene steroid is only inhibited by 20%. These results all suggest that there are at least two 21-hydroxylases in bovine adrenal tissue, one of which can utilize the 3-keto-4-ene steroids only, the other of which, in addition, can utilize the 3β-hydroxy-5-ene steroids as substrates.