Effects of different steroid-biosynthesis inhibitors on the testicular steroidogenesis of the toad Bufo arenarum

Testis fragments from Bufo arenarum were incubated with [7(n)-(3)H]pregnenolone (P5), [1,2-(3)H]dehydroepiandrosterone (DHEA) and [1,2,6.7-(3)H]testosterone (T), and different steroid-biosynthesis inhibitors. The inhibitors used were: cyanoketone (CNK), spironolactone (SPNL) and finasteride (FIN). CNK significantly increased the recovery of 3beta-hydroxy-5-ene steroids while SPNL reduced the metabolism of P5 and the production of C19-steroids. The metabolism of C19-substrates was only modified by CNK, which reduced the transformation of DHEA without modifying the metabolism of T. To determine the degree of inhibition exerted by the inhibitors used, the activities of the enzymes were estimated as the percentage of their contribution to the total steroid metabolism. CNK strongly inhibited the activity of hydroxysteroid dehydrogenase/isomerase if its contribution was estimated using both P5 and DHEA. If the analysis was made considering both activities associated to cytochrome P450 17chi-hydroxylase, C17-20 lyase (P450c17), it became evident that SPNL inhibited both of them. The percent contribution of 17beta-hydroxysteroid dehydrogenase (17betaHSD) activity diminished in the presence of CNK only if it was estimated considering P5 and DHEA metabolism. SPNL produced a significant inhibition of 17betaHSD when its contribution was estimated considering P5 metabolism. However, SPNL was insufficient if DHEA or T were considered. The effect of SPNL on the contribution of 17betaHSD could be due to the reduction of C19-substrates. The activity of 5chi-reductase was inhibited by CNK only if results from P5 and DHEA were considered.     

Cellular distribution and bioactivity of the key steroidogenic enzyme,cytochrome P450side chain cleavage,in sensory neural pathways

Neurosteroids are steroids produced within the nervous system. Based on behavioural responses evoked in animals by synthetic steroid injections, several studies suggested neurosteroid involvement in important neurophysiological processes. These observations should be correlated only to neuroactive effects of the injected steroids. Neurosteroids mostly control the CNS activity through allosteric modulation of neurotransmitter receptors within concentration ranges used by neurotransmitters themselves. Therefore, neurosteroid production within pathways controlling a neurophysiological process is necessary to consider neurosteroid involvement in that process. Because of the increasing speculation about pain modulation by neurosteroids based on pharmacological observations, we decided to clarify the situation by investigating neurosteroidogenesis occurrence in sensory pathways, particularly in nociceptive structures. We studied the presence and activity of cytochrome P450side chain cleavage (P450scc) in rat pain pathways. P450scc-immunoreactive cells were localized in dorsal root ganglia (DRG), spinal cord (SC) dorsal horn, nociceptive supraspinal nuclei (SSN) and somatosensory cortex. Incubation of DRG, SSN or SC tissue homogenates with [3H]cholesterol yielded the formation of radioactive metabolites including [3H]pregnenolone of which the synthesis was reduced in presence of aminogluthetimide, a P450scc inhibitor. These first neuroanatomical and neurochemical results demonstrate the occurrence of neurosteroidogenesis in nociceptive pathways and strongly suggest that neurosteroids may control pain mechanisms.     

Dehydroepiandrosterone formation is independent of cytochrome P450 17α-hydroxylase/17, 20 lyase activity in the mouse brain

Cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) is a microsomal enzyme reported to have two distinct catalytic activities, 17α-hydroxylase and 17, 20 lyase, that are essential for the biosynthesis of peripheral androgens such as dehydroepiandrosterone (DHEA). Paradoxically, DHEA is present and plays a role in learning and memory in the adult rodent brain, while CYP17 activity and protein are undetectable. To determine if CYP17 is required for DHEA formation and function in the adult rodent brain, we generated CYP17 chimeric mice that had reduced circulating testosterone levels. There were no detectable differences in cognitive spatial learning between CYP17 chimeric and wild-type mice. In addition, while CYP17 mRNA levels were reduced in CYP17 chimeric compared to wild-type mouse brain, the levels of brain DHEA levels were comparable. To determine if adult brain DHEA is formed by an alternative Fe²⁺-dependent pathway, brain microsomes were isolated from wild-type and CYP17 chimeric mice and treated with FeSO₄. Fe²⁺ caused comparable levels of DHEA production by both wild-type and CYP17 chimeric mouse brain microsomes; DHEA production was not reduced by a CYP17 inhibitor. Taken together these in vivo studies suggest that in the adult mouse brain DHEA is formed via a Fe²⁺-sensitive CYP17-independent pathway.     

Immunohistochemical localization and biological activity of the steroidogenic enzyme cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450C17) in the frog brain and pituitary

It is now clearly established that the brain has the capability of synthesizing various biologically active steroids including 17-hydroxypregnenolone (17OH-Delta(5)P), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA) and androstenedione (Delta(4)). However, the presence, distribution and activity of cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450(C17)), a key enzyme required for the conversion of pregnenolone (Delta(5)P) and progesterone (P) into these steroids, are poorly documented. Here, we show that P450(C17)-like immunoreactivity is widely distributed in the frog brain and pituitary. Prominent populations of P450(C17)-containing cells were observed in a number nuclei of the telencephalon, diencephalon, mesencephalon and metencephalon, as well as in the pars distalis and pars intermedia of the pituitary. In the brain, P450(C17)-like immunoreactivity was almost exclusively located in neurons. In several hypothalamic nuclei, P450(C17)-positive cell bodies also contained 3beta-hydroxysteroid dehydrogenase-like immunoreactivity. Incubation of telencephalon, diencephalon, mesencephalon, metencephalon or pituitary explants with [(3)H]Delta(5)P resulted in the formation of several tritiated steroids including 17OH-Delta(5)P, 17OH-P, DHEA and Delta(4). De novo synthesis of C(21) 17-hydroxysteroids and C(19) ketosteroids was reduced in a concentration-dependent manner by ketoconazole, a P450(C17) inhibitor. This is the first detailed immunohistochemical mapping of P450(C17) in the brain and pituitary of any vertebrate. Altogether, the present data provide evidence that CNS neurons and pituitary cells can synthesize androgens.     

Expression of cytochrome P450c17 and other steroid-converting enzymes in the rat kidney throughout the life-span

We have investigated the metabolism of [14C]-labelled progesterone (P4) and dehydroepiandrosterone (DHEA) by kidney tissues of newborn and 7-, 15-, 30-, 60- and 365-day-old rats of both sexes. The following enzymes were revealed at all ages by radiochemical identification of the corresponding products: 5alpha-reductase, cytochromes P450c17 and P450c21, 3beta-hydroxysteroid dehydrogenase (HSD)/delta5-delta4 isomerase, and 17beta- and 20alpha-HSDs, catalyzing reductive reactions. The major P4 metabolites were 5alpha-reduced C21 steroids, whose formation was almost completely suppressed by the 5alpha-reductase 4-azasteroid inhibitor, PNU 156765. Androstenedione and testosterone were also formed via 17alpha-hydroxyprogesterone, together with 11-deoxycorticosterone and 20alpha-dihydroprogesterone. DHEA was mainly converted to androst-5-ene-3beta,17beta-diol, with smaller amounts of the above androgens. Cytochrome P450c17 mRNA and protein were demonstrated by Northern blotting and Western blotting analyses, respectively. P450c17 mRNA, assessed by Northern blotting, protein and catalytic activity all peaked in the kidney samples at 15 days of life and declined thereafter. Cytochrome P450arom was below the level of detection of semi-quantitative RT-PCR. Since the rat kidney has been previously shown to contain cytochrome P450scc as well as androgen and estrogen receptors, it is suggested that it is capable of autonomous hormonal steroidogenesis and that renal steroids, or nephrosteroids, may act locally, in a paracrine or autocrine fashion.     

Steroid metabolism in the corpus luteum of the ferret

Implantation in the ferret is believed to be induced by a luteal substance which acts in concert with progesterone (P4) and which is secreted sometime between Days 6 and 8 of pregnancy. This experiment was designed to identify the steroid products synthesized by ferret corpora lutea (CL) on these 2 days of pregnancy. CL were dissected from ferrets on Day 6 or 8 of pregnancy and incubated with [3H] pregnenolone (P3), [3H] P4, or [3H] dehydroepiandrosterone (DHEA). Controls with no tissue or with 50 microliters packed blood cells were incubated at the same time. After incubation of Day 6 CL with [3H] P3 for 180 min, 39% of the added label was found incorporated into P4, 3% into 17 alpha-hydroxyprogesterone (17 alpha-OHP4) and 1% into androstenedione (A). Incubation of Day 8 CL with the same precursor resulted in 35%, 1% and 0.65% of the label being incorporated into the previously mentioned products, respectively. Incubations of Days 6 and 8 ferret CL with [3H] P4 or [3H] DHEA confirmed these results, demonstrating activity of C21-steroid, 17 alpha-hydroxylase and delta 5-isomerase, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). These results suggest that ferret CL primarily accumulate steroids of the delta4 pathway on both Days 6 and 8 of pregnancy, with P4, 17 alpha-OHP4, A and testosterone (T) being the most abundant products after in vitro incubation. Thus, ferret CL appear to metabolize steroids in a manner similar to that observed in rats, sows and mares.     

Conversion of pregnenolone to DHEA by human 17alpha-hydroxylase/17, 20-lyase (P450c17). Evidence that DHEA is produced from the released intermediate, 17alpha-hydroxypregnenolone.

Most previous studies using reconstituted systems and fast kinetics suggest that the conversion of pregnenolone to dehydroepiandrosterone (DHEA; the precursor of androgen and estrogen biosynthesis) by P450c17 does not require the release of the intermediate 17alpha-OHPreg (a precursor of cortisol biosynthesis). With such a mechanism, it is difficult to conceive how high amounts of DHEA may be produced in some cells or tissues, such as the testis and cells from the adrenal reticularis, while in other tissues such as the fasciculata zone, high levels of 17alpha-OHPreg are synthesized. In this report, we address this matter using intact transfected cells, which better reflect the actual cellular conditions. Furthermore, by using transfected cells, we can conveniently analyze human enzymes, as we are not restricted by the availability of human tissues as in the case of methods using purified or partially purified enzymes. Using intact HEK-293 cells transfected with human P450c17 in culture, we showed, in a time course study of the transformation of pregnenolone, that there is an accumulation of 17alpha-OHPreg, and that, subsequently, the accumulated 17alpha-OHPreg decreases with a concomitant increase in DHEA production. The DHEA/17alpha-OHPreg ratio changes from 0.1 :1 after 1 h incubation to 50 : 1 after 20 h. This result strongly suggests that the transformation of Preg to DHEA proceeds through two steps in which DHEA is produced from the released intermediate 17alpha-OHPreg. We also show that high levels of substrate vs. enzyme concentration will lead to high hydroxylase activity whereas the reverse will increase the lyase activity. The result is in good agreement with recent observations suggesting that surrounding enzymes and steroids could modulate the lyase activity. Cotransfection of vectors expressing cytochrome b5 and NADPH cytochrome P450 reductase indicates that both are required for an optimum production of DHEA.     

Deletion of the mouse P450c17 gene causes early embryonic lethality

Dehydroepiandrosterone (DHEA), a 19-carbon precursor of sex steroids, is abundantly produced in the human but not the mouse adrenal. However, mice produce DHEA and DHEA-sulfate (DHEAS) in the fetal brain. DHEA stimulates axonal growth from specific populations of mouse neocortical neurons in vitro, while DHEAS stimulates dendritic growth from those cells. The synthesis of DHEA and sex steroids, but not mouse glucocorticoids and mineralocorticoids, requires P450c17, which catalyzes both 17 alpha-hydroxylase and 17,20-lyase activities. We hypothesized that P450c17-knockout mice would have disordered sex steroid synthesis and disordered brain DHEA production and thus provide phenotypic clues about the functions of DHEA in mouse brain development. We deleted the mouse P450c17 gene in 127/SvJ mice and obtained several lines of mice from two lines of targeted embryonic stem cells. Heterozygotes were phenotypically and reproductively normal, but in all mouse lines, P450c17(-/-) zygotes died by embryonic day 7, prior to gastrulation. The cause of this early lethality is unknown, as there is no known function of fetal steroids at embryonic day 7. Immunocytochemistry identified P450c17 in embryonic endoderm in E7 wild-type and heterozygous embryos, but its function in these cells is unknown. Enzyme assays of wild-type embryos showed a rapid rise in 17-hydroxylase activity between E6 and E7 and the presence of C(17,20)-lyase activity at E7. Treatment of pregnant females with subcutaneous pellets releasing DHEA or 17-OH pregnenolone at a constant rate failed to rescue P450c17(-/-) fetuses. Treatment of normal pregnant females with pellets releasing pregnenolone or progesterone did not cause fetal demise. These data suggest that steroid products of P450c17 have heretofore-unknown essential functions in early embryonic mouse development.     

Electron factors in the properties of cytochrome P-450 and mechanism of activation of molecular oxygen

A quantum-chemical calculation was carried out for the electronic structures of coordination compounds of general formula: FeP(L1)(L2) (P--porphin; L1 = SHCH3, [SCH3]-, [SC6F4H]-; L2 = CO, NO, O2), modeling the active site of cytochrome P450. It was shown that Coulomb repulsion between the electrons of the sulfur lone pair leads to the transfer of the electronic density from the ligands L1 = [SCH3]- or [SC6F4H]- to the porphyrin of/and to the L2 ligand. This explains the origin of the band at 450 nm in the absorption spectra of the complexes of cytochrome P450 with CO, the absence of such a band in those with O2, and the strong activation of dioxygen by cytochrome P450.     

Functional receptors for vasoactive intestinal polypeptide in cultured astroglia from neonatal rat brain

The effects of vasoactive intestinal polypeptide (VIP) were assessed on astroglia cultured from rat CNS. In these cultures VIP (500 nM) promoted the hydrolysis of [3H]glycogen newly synthesized from [3H]glucose. This effect on [3H]glycogen levels was also observed with the structurally related peptide PHI-27 and with other substances which had been demonstrated to promote glycogenolysis in rodent CNS in vitro such as: norepinephrine (NE), serotonin, histamine, adenosine, K+ and dibutyryl cyclic-AMP (dbcAMP). Furthermore, VIP (500 nM) and PHI 27 (500 nM), when applied to astroglial cultures in serum-free medium, displayed marked effects on the morphological appearance of the cell population: they converted the flat cells present in the cultures into cells with typical astrocytic morphology. As previously reported, this effect on the cellular morphology of the cultures was also observed, under identical experimental conditions, after NE and dbcAMP application. These studies demonstrate that cultured rat neonatal astroglia possess receptors for VIP, and suggest that a cyclic AMP accumulation may mediate both the metabolic and morphologic components of this response.     

Studies on nuclear binding of dehydroepiandrosterone in hepatocytes.

Experiments were conducted to determine if dehydroepiandrosterone (DHEA) specifically binds to liver nuclei or interferes in the binding of other steroid hormones. Hepatocytes were isolated from obese, female Zucker rats that were treated with and without 0.6% DHEA in their diets for 2 weeks. The hepatocytes were incubated with either [3H]DHEA, [3H]estrone, or [3H]corticosterone. The nuclei isolated from the incubated cells from either control or DHEA-treated rats had no binding with [3H]DHEA, but had the expected binding with [3H]estrone and [3H]corticosterone. Furthermore, increasing concentrations of cold, unlabeled DHEA in the incubation media with [3H]estrone or [3H]corticosterone failed to have any effect on the binding of either of these steroid hormones to the nuclei. These results suggest that DHEA treatment does not exert its effects on cellular metabolism through a receptor-mediated mechanism like other steroid hormones or by interfering in the expression of steroid hormones such as estrone and corticosterone.     

Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse

In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous [³²P]ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using [³H]methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the [³H] methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.     

In vitro metabolism of androgens by mammalian cauda epididymal spermatozoa.

The in vitro metabolism of [3H] testosterone (17beta-hydroxy-4-androsten-3-one), [3H] androstenedione (4-androstene-3,17-dione) and [3H] dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) by cauda epididymal spermatozoa from the rat, rabbit, hamster, guinea-pig and ram, varied between species. There were differences in the androgens utilized, the extent of their conversion and the identities of the metabolites formed. Of the steroid substrates tested rat spermatozoa metabolized testosterone preferentially while spermatozoa from guinea-pig transformed [3H] dehydroepiandrosterone (DHEA) almost exclusively. Rabbit spermatozoa converted all three [3H] androgens while hamster sperm utilized [3H] testosterone and [3H] DHEA. Spermatozoa collected from rams killed at the abattoir metabolized both [3H] androstenedione and [3H] DHEA but this capacity was dramatically reduced in spermatozoa collected from rams subjected to short-term anaesthesea. The results are discussed in relation to the possible direct roles of androgens in sperm physiology.     

Production of sex steroid hormones from DHEA in articular chondrocyte of rats

Dehydroepiandrosterone (DHEA), a precursor of sex steroid hormones, is synthesized by cholesterol side-chain cleavage cytochrome P-450 and 17alpha-hydroxylase cytochrome P-450 mainly from cholesterol and converted to testosterone and estrogen by 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17beta-HSD, and aromatase cytochrome P-450. Although sex steroid hormones have important effects in the protection of articular cartilage, it is unclear whether articular cartilage has a local steroidogenic enzymatic machinery capable of metabolizing DHEA. This study was aimed to clarify whether steroidogenesis-related enzymes are expressed in articular chondrocytes, whether expression levels are changed by DHEA, and whether articular chondrocytes are capable of synthesizing sex steroid hormones from DHEA. Articular chondrocytes isolated from adult rats were cultured with DHEA for 3 days. All of the mRNA expressions of steroidogenesis-related enzymes were detected in cultured articular chondrocytes of rats, but the mRNA expression levels of testosterone and estradiol in cultured media increased after the addition of DHEA. These findings provided the first evidence that articular chondrocytes expressed steroidogenesis-related enzyme genes and that they are capable of locally synthesizing sex steroid hormones locally from DHEA.     

Mechanism of dissociation of cortisol and adrenal androgen secretion after removal of adrenocortical adenoma in patients with Cushing's syndrome

We investigated the mechanism of dissociation of cortisol and dehydroepiandrosterone sulfate (DHEA-S) secretion by the adrenal glands after the removal of an adrenal gland containing an adrenocortical adenoma in a patient with Cushing's syndrome. After removal of the adrenocortical adenoma, the serum cortisol rapidly decreased from 24.6 +/- 6.4 micrograms/dl (mean +/- SD, n = 6) to 0.7 +/- 0.5 micrograms/dl. Serum DHEA-S levels were 15 +/- 14 micrograms/dl and 6 +/- 9 micrograms/dl before and after surgery, respectively, and significantly lower than the control values. Serum cortisol levels reverted to normal levels 1.5 to 3 years after the surgery. On the other hand, DHEA-S levels reverted to normal 5 to 7 years after the serum cortisol levels had normalized. Monolayer cultures of normal human adrenal cells obtained at adrenalectomy in patients with advanced breast cancer and atrophic adrenal cells adjacent to the adrenocortical adenoma in patients with Cushing's syndrome were used to study the mechanism of the dissociation of cortisol and DHEA-S secretion. ACTH caused significant increases in the productions of pregnenolone (P5), progesterone (P4), 17-hydroxypregnenolone (17-OH-P5), 17-hydroxyprogesterone (17-OH-P4), DHEA, DHEA-S, androstenedione (delta 4-A), and cortisol. The amounts of 17-OH-P5 and 17-OH-P4 produced by ACTH in atrophic adrenal cells were significantly greater than those in normal adrenal cells. The amounts of DHEA, DHEA-S and delta 4-A produced by ACTH in atrophic adrenal cells were significantly smaller than those of normal adrenal cells. The conversion rate of 17-OH-[3H]P5 to 17-OH-[3H]P4 and 11-deoxy-[3H] cortisol was higher in atrophic adrenal cells than in normal adrenal cells, but the conversion rate to [3H]DHEA, [3H]DHEA-S and [3H]delta 4-A was significantly lower in atrophic adrenal cells than in normal adrenal cells. These results suggest that the dissociation of cortisol from DHEA-S after the removal of adrenocortical adenoma is a probably due to diminished C17,20-lyase activity in the remaining atrophic adrenal gland.     

Characterization, development, and localization of the deoxycytidine phosphorylating systems in mammalian brain

The accumulation of deoxycytidine by rabbit and mouse brain was studied in vitro. Brain slices from brain stem, cerebellum, and forebrain of rabbits of various ages (1 day to 2.5 years) and forebrain from adult mice were incubated for various times in artificial CSF containing 6 nM [3H]deoxycytidine at 37 degrees C under 95% O2/5% CO2. Rabbit and mouse brain slices of all ages accumulated [3H]deoxycytidine by a saturable system (IC50 = 4 microM) and converted it to [3H]deoxycytidine phosphates and [3H]DNA. When slices from all brain regions of 1-day-old rabbits were incubated in 6 nM [3H]deoxycytidine for 30 min, tissue-to-medium ratios of 3H were between 1.2 and 2.5 and declined with age, except in cortex; the percentages of total 3H in perchloric acid homogenates of brain slices as [3H]DNA were 10-24% and declined to low levels in middle age. However, at all ages and in all regions tested, 30-85% of the [3H]deoxycytidine within the slices was phosphorylated. After homogenization and subcellular fractionation of the brain slices incubated in [3H]deoxycytidine for 30 min, the highest percentage of [3H]deoxycytidine phosphates plus [3H]DNA was present in the nuclear and mitochondrial fractions of all brain regions. Deoxycytidine phosphates were synthesized from deoxycytidine in all brain regions tested into middle age.     

Mass measurements of inositol(1,4,5)trisphosphate in rat cerebral cortex slices using a radioreceptor assay: effects of neurotransmitters and depolarization

The specific binding of [3H] and [32P]Ins(1,4,5)P3 to a particulate preparation of bovine adrenal cortex has been used as a radioreceptor assay to determine the concentration of Ins(1,4,5)P3 in agonist- and depolarization-stimulated rat cerebral cortex slices. The resting concentration of Ins(1,4,5)P3 in slices that had been preincubated in a physiological medium was 18.8 +/- 2.6 pmol/mg prot. Carbachol evoked a rapid and dose-related increase in the concentration of Ins(1,4,5)P3. Maximal stimulation (80%) was already seen at the earliest point (10 sec) examined and was maintained for at least 5 min. The EC50 for carbachol was 75 +/- 17 microM and the response was totally suppressed by the muscarinic antagonist atropine. A direct comparison in the same slices was made between mass determinations and [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 accumulation determined by h.p.l.c. Although an identical time course was observed for cold and radiolabelled Ins(1,4,5)P3, the greater stimulation of [3H]Ins(1,4,5)P3 may indicate changes in specific radioactivity. Of a variety of other receptor agonists studied, only the glutamate receptor agonist quisqualate, and noradrenaline significantly increased the mass of Ins(1,4,5)P3 in cerebral cortical slices. However, depolarizing concentrations of K+ were as effective as carbachol at elevating this second messenger.