Showing papers on "Pregnenolone published in 2007"

Steroid profiling in brain and plasma of male and pseudopregnant female rats after traumatic brain injury: analysis by gas chromatography/mass spectrometry.

Abstract: Steroids in brain arise from the peripheral endocrine glands and local synthesis. In traumatic brain injury (TBI), the endogenous circulating hormones at the time of injury are important for neuroprotection. In particular, pseudopregnant females recover better than males from TBI. We investigated the effect of pseudopregnancy and TBI on steroid levels in plasma and in three brain regions (within, adjacent, and distal to the lesion site), 6 and 24 h after prefrontal cortex injury. The following steroids were analyzed by gas chromatography/mass spectrometry: pregnenolone, progesterone, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone, 3beta,5alpha-tetrahydroprogesterone, dehydroepiandrosterone, Delta(4)-androstenedione, testosterone, 5alpha-dihydrotestosterone, 3alpha,5alpha-tetrahydrotestosterone, 3beta,5alpha-tetrahydrotestosterone, and 17beta-estradiol. Corticosterone was assayed in plasma to account for stress in the rats. We found different steroid profiles in brain and plasma of male and pseudopregnant female rats and specific profile changes after TBI. In sham-operated pseudopregnant females, much higher levels of progesterone, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone, and 3beta,5alpha-tetrahydroprogesterone were measured in both brain and plasma, compared with sham-operated males. Plasma levels of corticosterone were high in all groups, indicating that the surgeries induced acute stress. Six hours after TBI, the levels of pregnenolone, progesterone, and 5alpha-dihydroprogesterone increased, and those of testosterone decreased in male brain, whereas levels of 5alpha-dihydroprogesterone and 3beta,5alpha-tetrahydroprogesterone increased in brain of pseudopregnant female rats. Plasma levels of 5alpha-dihydroprogesterone did not change after TBI, suggesting a local activation of the 5alpha-reduction pathway of progesterone in both male and pseudopregnant female brain. The significant increase in neurosteroid levels in the male brain after TBI is consistent with their role in neuroprotection. In pseudopregnant females, high levels of circulating progestagens may provide protection against TBI.

Rv1106c from Mycobacterium tuberculosis is a 3β-hydroxysteroid dehydrogenase

Abstract: New approaches are required to combat Mycobacterium tuberculosis (Mtb), especially the multi-drug resistant and extremely drug resistant organisms (MDR-TB and XDR-TB). There are many reports that mycobacteria oxidize 3beta-hydroxysterols to 3-ketosteroids, but the enzymes responsible for this activity have not been identified in mycobacterial species. In this work, the Rv1106c gene that is annotated as a 3beta-hydroxysteroid dehydrogenase in Mtb has been cloned and heterologously expressed. The purified enzyme was kinetically characterized and found to have a pH optimum between 8.5 and 9.5. The enzyme, which is a member of the short chain dehydrogenase superfamily, uses NAD+ as a cofactor and oxidizes cholesterol, pregnenolone, and dehydroepiandrosterone to their respective 3-keto-4-ene products. The enzyme forms a ternary complex with NAD+ binding before the sterol. The enzyme shows no substrate preference for dehydroepiandrosterone versus pregnenolone with second-order rate constants (kcat/Km) of 3.2 +/- 0.4 and 3.9 +/- 0.9 microM-1 min-1, respectively, at pH 8.5, 150 mM NaCl, 30 mM MgCl2, and saturating NAD+. Trilostane is a competitive inhibitor of dehydroepiandrosterone with a Ki of 197 +/- 8 microM. The expression of the 3beta-hydroxysteroid dehydrogenase in Mtb is intracellular. Disruption of the 3beta-hydroxysteroid dehydrogenase gene in Mtb abrogates mycobacterial cholesterol oxidation activity. These data are consistent with the Rv1106c gene being the one responsible for 3beta-hydroxysterol oxidation in Mtb.

An acetylation/deacetylation cycle controls the export of sterols and steroids from S. cerevisiae

Abstract: Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. Here we report the identification of a novel reversible sterol modification in yeast, the sterol acetylation/deacetylation cycle. Sterol acetylation requires the acetyltransferase ATF2, whereas deacetylation requires SAY1, a membrane-anchored deacetylase with a putative active site in the ER lumen. Lack of SAY1 results in the secretion of acetylated sterols into the culture medium, indicating that the substrate specificity of SAY1 determines whether acetylated sterols are secreted from the cells or whether they are deacetylated and retained. Consistent with this proposition, we find that acetylation and export of the steroid hormone precursor pregnenolone depends on its acetylation by ATF2, but is independent of SAY1-mediated deacetylation. Cells lacking Say1 or Atf2 are sensitive against the plant-derived allylbenzene eugenol and both Say1 and Atf2 affect pregnenolone toxicity, indicating that lipid acetylation acts as a detoxification pathway. The fact that homologues of SAY1 are present in the mammalian genome and functionally substitute for SAY1 in yeast indicates that part of this pathway has been evolutionarily conserved.

Inhibitors of steroidal cytochrome p450 enzymes as targets for drug development.

Abstract: Cytochrome P450's are enzymes which catalyze a large number of biological reactions, for example hydroxylation, N-, O-, S- dealkylation, epoxidation or desamination. Their substrates include fatty acids, steroids or prostaglandins. In addition, a high number of various xenobiotics are metabolized by these enzymes. The enzyme 17alpha-hydroxylase-C17,20-lyase (P450(17), CYP 17, androgen synthase), a cytochrome P450 monooxygenase, is the key enzyme for androgen biosynthesis. It catalyzes the last step of the androgen biosynthesis in the testes and adrenal glands and produces androstenedione and dehydroepiandrosterone from progesterone and pregnenolone. The microsomal enzyme aromatase (CYP19) transforms these androgens to estrone and estradiol. Estrogens stimulate tumor growth in hormone dependent breast cancer. In addition, about 80 percent of prostate cancers are androgen dependent. Selective inhibitors of these enzymes are thus important alternatives to treatment options like antiandrogens or antiestrogens. The present article deals with recent patents (focus on publications from 2000 - 2006) concerning P450 inhibitor design where steroidal substrates are involved. In this context a special focus is provided for CYP17 and CYP19. Mechanisms of action will also be discussed. Inhibitors of CYP11B2 (aldosterone synthase) will also be dealt with.

A Conserved Tyrosine in the β2Subunit M4 Segment Is a Determinant of γ-Aminobutyric Acid Type A Receptor Sensitivity to Propofol

Abstract: Background: The -aminobutyric acid type A receptor (GABAA-R) subunits are critical targets for the actions for several intravenous general anesthetics, but the precise nature of the anesthetic binding sites are unknown. In addition, little is known about the role the fourth transmembrane (M4) segment of the receptor plays in receptor function. The aim of this study was to better define the propofol binding site on the GABAA-R by conducting a tryptophan scan in the M4 segment of the 2 subunit. Methods: Seven tryptophan mutations were introduced into the C-terminal end of the M4 segment of the GABAA-R 2 subunit. GABAA-R subunit complementary DNAs were transfected into human embryonic kidney 293 cells grown on glass coverslips. After transfection (36‐72 h), coverslips were transferred to a perfusion chamber to assay receptor function. Cells were whole cell patch clamped and exposed to GABA, propofol, etomidate, and pregnenolone. Chemicals were delivered to the cells using two 10channel infusion pumps and a rapid solution exchanger. Results: All tryptophan mutations were well tolerated, and with one exception, all resulted in minimal changes in receptor activation by GABA. One mutation, 2(Y444W), selectively suppressed the ability of propofol to enhance receptor function while retaining normal sensitivity to etomidate and pregnenolone. Conclusions: This is the first report of a mutation that selectively reduces propofol sensitivity without altering the action of etomidate. The reduction in propofol sensitivity is consistent with the loss of a hydrogen bond within the propofol binding site. These results also suggest a possible orientation of the propofol molecule within its binding site.

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

Abstract: It is now clearly established that the brain has the capability of synthesizing various biologically active steroids including 17-hydroxypregnenolone (17OH-Δ5P), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA) and androstenedione (Δ4). However, the presence, distribution and activity of cytochrome P450 17α-hydroxylase/C17, 20-lyase (P450C17), a key enzyme required for the conversion of pregnenolone (Δ5P) and progesterone (P) into these steroids, are poorly documented. Here, we show that P450C17-like immunoreactivity is widely distributed in the frog brain and pituitary. Prominent populations of P450C17-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, P450C17-like immunoreactivity was almost exclusively located in neurons. In several hypothalamic nuclei, P450C17-positive cell bodies also contained 3β-hydroxysteroid dehydrogenase-like immunoreactivity. Incubation of telencephalon, diencephalon, mesencephalon, metencephalon or pituitary explants with [3H]Δ5P resulted in the formation of several tritiated steroids including 17OH-Δ5P, 17OH-P, DHEA and Δ4. De novo synthesis of C21 17-hydroxysteroids and C19 ketosteroids was reduced in a concentration-dependent manner by ketoconazole, a P450C17 inhibitor. This is the first detailed immunohistochemical mapping of P450C17 in the brain and pituitary of any vertebrate. Altogether, the present data provide evidence that CNS neurons and pituitary cells can synthesize androgens.

Transcriptional regulation of human CYP11A1 in gonads and adrenals

Abstract: The CYP11A1 gene encodes the cholesterol side-chain cleavage enzyme, also termed cytochrome P450scc, which catalyzes the conversion of cholesterol to pregnenolone in the first step of steroid biosynthesis in mitochondria. The adrenal- and gonad-selective, hormonally and developmentally regulated expression of CYP11A1 is principally driven by its 2.3 kb promoter. Multiple trans-acting factors like SF-1, Sp1, AP-2, TReP-132, LBP-1b, LBP-9, AP-1, NF-1, and Ets control CYP11A1 transcription either through DNA-protein interaction with their specific cis-acting elements or through protein-protein interaction between each other, wherein SF-1 plays a central role in adrenals and testes. In addition to binding with its proximal and upstream motifs, SF-1 also physically interacts with TFIIB, CBP/p300, TReP-132, and c-Jun/AP-1 to specifically transmit the regulatory signals of cAMP. Other factors like Sp1 family members, AP-2, and LBP-1b/LBP-9 may be other factors that play a role in CYP11A1 transcription, particularly in placental cells. The TATA sequence could also contribute to tissue-specificity and hormonal regulation of CYP11A1 transcription. This article reviews recent studies focusing on adrenals and gonads.

Luteinizing hormone receptor mediates neuronal pregnenolone production via up-regulation of steroidogenic acute regulatory protein expression.

Abstract: The functional consequences of luteinizing hormone/human chorionic gonadotropin signaling via neuronal luteinizing hormone/human chorionic gonadotropin receptors expressed throughout the brain remain unclear. A primary function of luteinizing hormone (LH) in the gonads is the stimulation of sex steroid production. As LH can cross the blood–brain barrier, present in cerebrospinal fluid and is expressed by neuronal cells, we tested whether LH might also modulate steroid synthesis in the brain. Treatment of differentiated rat primary hippocampal neurons and human M17 neuroblastoma cells with LH (100 mIU/mL) resulted in a twofold increase in pregnenolone secretion in both cell types, suggesting an increase in P450scc-mediated cleavage of cholesterol to pregnenolone and its secretion from neurons. To explore how LH might regulate the synthesis of pregnenolone, the precursor for steroid synthesis, we treated rat primary hippocampal neurons with LH (0, 10 and 100 mIU/mL) and measured changes in the expression of LH receptor and steroidogenic acute regulatory protein (StAR). LH induced a rapid (within 30 min) increase in the expression of StAR, but induced a dose-dependent decrease in LH receptor expression. Consistent with these results, the suppression of serum LH in young rats treated with leuprolide acetate for 4 months down-regulated StAR expression, but increased LH receptor expression in the brain. Taken together, these results indicate that LH induces neuronal pregnenolone production by modulating the expression of the LH receptor, increasing mitochondrial cholesterol transport and increasing P450scc-mediated cleavage of cholesterol for pregnenolone synthesis and secretion.

Neurosteroids: synthesis and functions in the central and peripheral nervous systems.

Abstract: Some steroids are synthesized within the central and peripheral nervous systems, mostly by glial cells. These are known as neurosteroids. In the brain, neurosteroids have been shown to act directly on membrane receptors for neurotransmitters. For example, progesterone inhibits the neuronal nicotinic acetylcholine receptor, whereas its 3 alpha,5 alpha-reduced metabolite 3 alpha,5 alpha-tetrahydroprogesterone (allopregnanolone) activates the type A gamma-aminobutyric acid receptor complex. Besides these effects, neurosteroids also regulate important glial functions, such as the synthesis of myelin proteins. Thus, in cultures of glial cells prepared from neonatal rat brain, progesterone increases the number of oligodendrocytes expressing the myelin basic protein (MBP) and the 2',3'-cyclic nucleotide-3'-phophodiesterase (CNPase). An important role for neurosteroids in myelin repair has been demonstrated in the rodent sciatic nerve, where progesterone and its direct precursor pregnenolone are synthesized by Schwann cells. After cryolesion of the male mouse sciatic nerve, blocking the local synthesis or action of progesterone impairs remyelination of the regenerating axons, whereas administration of progesterone to the lesion site promotes the formation of new myelin sheaths.

Steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage (P450scc) as molecular and cellular targets for 17alpha-ethynylestradiol in salmon previtellogenic oocytes.

Abstract: Gonadal steroids are known to modulate both the synthesis and the release of gonadotropins by the pituitary and influence several brain functions that are apparently responsible for gender-specific differences in the regulation of the hypothalamus-pituitary-gonadal (HPG) axis. It is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and subsequent conversion to pregnenolone by P450-mediated cholesterol side chain cleavage (P450 scc). In the present study, we have evaluated the effects of 17alpha-ethynylestradiol (EE2) on salmon previtellogenic oocytes using an in vitro culture system and molecular, histological, and physiological methods. The in vitro culture technique was based on an agarose floating method recently validated for xenoestrogens in our laboratory. Tissue was cultured in a humidified incubator at 10 degrees C for 3, 7, and 14 days with different concentrations of EE2 [0 (control), 0.01, 0.1, and 1 microM] dissolved in ethanol (0.1%). The StAR, P450 scc, P450 arom isoforms, and insulin-like growth factor 2 (IGF-2) mRNA expressions were performed using validated real-time polymerase chain reaction (PCR) with specific primers, and immunohistochemistry of the StAR and P450 scc proteins was performed using antisera prepared against synthetic peptide for both proteins and estradiol-17beta (E2); testosterone (T) and 11-ketotestosterone (11-KT) tissue levels were performed using enzyme immunoassay (EIA). Our data show that EE2 produced time- and concentration-specific effects on the StAR protein, P450 scc, P450 arom isoforms, and IGF-2 gene expressions in salmon gonadal tissues. Cellular expression of the StAR and P450 scc proteins was mainly demonstrated in follicular cells of the oocyte membrane, showing time- and EE2 concentration-dependent differences in staining intensities. Tissue levels of E2, T, and 11-KT in salmon were differentially modulated by EE2 in a time- and concentration-specific manner. Although an apparent negative relationship between E2 and T that reflected aromatization of T to E2 was observed at day 3 postexposure, T and 11-KT showed an apparent concentration-dependent effect after EE2 exposure at day 14. The consistencies between our data at day 14 postexposure suggest that the EE2 modulates steroidogenesis by targeting the initial and rate-limiting step that involves the StAR protein. In general, these findings show that the synthetic pharmaceutical endocrine disruptor and ubiquitous environmental pollutant also produce variations in key gonadal steroidogenic and growth-regulating pathways. These effects and the hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleosts.

Neurosteroids: endogenous modulators of neuronal excitability and plasticity.

Abstract: Neurosteroids are steroids synthesized in glial cells and in some neurons independently of the adrenal glands and gonads.1 Whereas circulating steroids have readily access to the CNS, neurosteroids may act as paracrine signals affecting neuronal function by modulating neurotransmitter-gated ion channels and G-protein-coupled receptors.2,3 Paracrine signals are chemical signals that are released from neurons or glial cells and diffuse via the extracellular fluid to affect neighboring target cells. Neurosteroids have been implicated in mechanisms of epileptogenesis,4 hepatic encephalopathy,5 neurodegeneration,6–8 neuroprotection,9,10 and psychiatric disorders.11–13 Drugs that affect synthesis or metabolism of neurosteroids provide a new therapeutic opportunity for treatment of these disorders.4,8 Neurosteroids are synthesized in astrocytes, oligodendrocytes, Schwann cells, and a few neurons such as Purkinje cells, hippocampal neurons, and retinal amacrine and ganglion cells1,3,4 (figure). The rate-limiting step in neurosteroid biosynthesis is the transport of cholesterol into the mitochondria, which involves the peripheral-type benzodiazepine receptor.11 At the inner mitochondrial membrane, cholesterol is converted to pregnenolone by action of the P450 cholesterol side-chain cleavage enzyme. Pregnenolone then passes to the cytosol, where it serves as the precursor of all neurosteroids. In the cytosol, pregnenolone is converted, via successive enzymatic steps, to potent neurosteroids such as tetrahydroprogesterone (or allopregnanolone) and dehydroepiandrosterone (DHEA) (figure). DHEA is the precursor of testosterone, which, via action of aromatase, is converted to estradiol. All the enzymes required for neurosteroid biosynthesis and metabolism are expressed in astrocytes, oligodendrocytes, neurons, and Schwann cells.14 Steroidogenesis in the nervous system is regulated by interactions between neurons and glial cells. Figure. Main steps in the biosynthesis of neurosteroids and effects of neurosteroids on γ-aminobutyric acid (GABA) A and N-methyl-d-aspartate (NMDA) receptor–mediated neurotransmission. Neurosteroids are primarily synthesized in astrocytes, and the rate-limiting step is the incorporation of cholesterol in the mitochondria …

The neurosteroids dehydroepiandrosterone sulfate and pregnenolone sulfate inhibit the UNC-49 GABA receptor through a common set of residues.

Abstract: Neurosteroids are endogenous neuromodulators that bind and allosterically regulate GABA(A) receptors Residues were recently identified in the first transmembrane domain (M1) of GABA(A) receptor subunits that are important for neurosteroid modulation We are studying the inhibition of GABA(A) receptors by sulfated neurosteroids One of these neurosteroid, pregnenolone sulfate (PS), depends on six identified M1 residues to inhibit the UNC-49 GABA receptor, a homomeric GABA receptor from Caenorhabditis elegans that is homologous to the mammalian GABA(A) receptor Here, we investigate the inhibition of the UNC-49 GABA receptor by another sulfated neurosteroid, dehydroepiandrosterone sulfate (DHEAS) DHEAS is identical to PS except that it contains a carbonyl oxygen instead of an acetyl group at C17 on the steroid D ring UNC-49 mutations that affect PS inhibition had broadly parallel effects on DHEAS, suggesting the two neurosteroids act through similar mechanisms However, certain M1 mutations affected DHEAS differently than PS Considering that first, the D ring contains the only structural difference between PS and DHEAS, and second, the strongest chemical and steric effects of a mutation are likely to be felt in the local environment of the altered residues, this result implies that the steroid D ring may contact M1 near the mutated residues This possibility is interesting because current models of neurosteroid interactions with GABA(A) receptors, based on pregnane steroids, suggest that the steroid A ring binds M1, whereas the D ring binds M4 Our findings suggest that there may be considerable diversity in the way different classes of neurosteroids interact with GABA(A) receptors

Cloning, tissue expression, and inducibility of CYP 3A79 from sea bass (Dicentrarchus labrax).

Abstract: Multiple members of the CYP3A subfamily have been identified and intensively studied in mammals as they represent prominent CYP enzymes involved in drug metabolism. Also in fish, some CYP3A genes have been identified by cDNA cloning and immunological techniques, but relatively little is known about their function, distribution, and inducibility. In this study, a novel CYP3A, designated as CYP3A79 was isolated from adult male sea bass, an economically valuable species in fisheries. The sea bass CYP3A79 that was cloned contained an open-reading frame of 1512 bp that encoded a 504 amino acid protein and shared a high-sequence identity with medaka, killifish, and trout CYP3As. Interestingly, CYP3A79 also shares five of six substrate recognition sites (SRS) with the SRS of other fish CYP3As, suggesting an evolutionary conservation of the function of these enzymes. In this fish, we also investigated the expression of CYP3A79 and its susceptibility to induction by various compounds including clotrimazole and dehydroepiandrosterone, two strong ligands of zebrafish PXR. The expression of CYP3A79 mRNA was detected by RT-PCR only in the intestine and liver. The immunoblot analysis by antitrout CYP3A27 confirmed the presence of a CYP3A-like protein in the microsomes of these tissues, but, in addition, a immunoreactive protein with this antibody was also observed in the heart microsomes, suggesting the presence of other CYP3A isoforms in this fish. Accordingly, the southern blot analysis of genomic DNA indicated that multiple CYP 3As may be present in sea bass. All attempts to induce 6beta-testosterone hydroxylase, as a marker of CYP3A79, by dexametasone, 17beta-estradiol, pregnenolone 16alpha-carbonitrile, corticosterone, clotrimazole, and dehydroepiandrosterone failed. On the contrary, the administration of 17beta-estradiol, pregnenolone 16alpha-carbonitrile, and corticosterone strongly inhibited this activity and, in parallel, reduced the expression of CYP3A79 transcript. Thus, the sea bass CYP3A79 appears to be resistant to induction, suggesting that this enzyme and likely other CYP3As are regulated differently compared to those of mammals.

Monobutyl phthalate inhibits steroidogenesis by downregulating steroidogenic acute regulatory protein expression in mouse Leydig tumor cells (MLTC-1).

Abstract: Di-n-butyl phthalate (DBP) and its active metabolite, monobutyl phthalate (MBP), display no binding affinity for the androgen receptor, yet exert antiandrogenic effects by altering steroid biosynthesis However, the mechanisms underlying this observed effect are not known The purpose of this study was to determine the site of MBP action on steroidogenesis in vitro using mouse Leydig tumor cells (MLTC-1) Various concentrations of MBP (0, 50, 100, 200, 400, or 800 micromol/L) were added to the medium for 24 h followed by stimulation with some compounds such as human chorionic gonadotrophin (hCG), cholera toxin (CT), cAMP analog 8-Br-cAMP, 22(R)-hydroxycholesterol (22R-HC), and pregnenolone Data showed that MBP inhibited the increases in progesterone production induced by hCG and CT In contrast, the levels of intracellular cAMP remained unaltered In addition, 8-Br-cAMP-stimulated progesterone production was also suppressed by MBP These results suggested that the site in the steroid biosynthesis pathway affected by MBP occurs downstream of PKA activation in MLTC-1 cells Moreover, incubation with 22R-HC and pregnenolone as progesterone precursors for P-450 side-chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase (3betaHSD) respectively resulted in no marked change in progesterone production, indicating that MBP did not influence P450scc and 3betaHSD but did exert an effect on cholesterol transportation into mitochondria, the rate-limiting step These results were supported by the downregulated StAR expression seen with MBP administration, as StAR is a key factor in this process Data indicate that MBP interfered with steroid hormone production by affecting StAR expression in MLTC-1 cells

Comparison of the 17α-Hydroxylase/C17,20-Lyase Activities of Porcine, Guinea Pig and Bovine P450c17 Using Purified Recombinant Fusion Proteins Containing P450c17 Linked to NADPH-P450 Reductase

Abstract: The cDNAs for cytochrome P450c17 (P450c17) of three species, pig, guinea pig, and cow, representing three families of mammals (suidae, procaviidae, and bovidae, respectively) were each engineered into an expression plasmid (pCWori+). The P450c17 domain of the coding sequence was connected to a truncated form of rat NADPH-P450 reductase by a linker sequence encoding two amino acids (SerThr). These fusion proteins were expressed in E. coli and purified for use in enzymatic assays to determine similarities and differences in 17α-hydroxylase and lyase activities. The fusion proteins were found to catalyze both the 17α-hydroxylation of progesterone (P4) and pregnenolone (P5) to 17α-hydroxylated P4 and P5 (17α-OH P4 and 17α-OH P5) followed by the C17,20-lyase reaction for the conversion of these C21-17α-hydroxylated steroids to C19-steroids (the C17,20-lyase reaction). These in vitro studies show that (a) porcine P450c17 possesses cytochrome b5 (b5)-stimulated C17,20-lyase activity that converts 17αOH-P4 to and...

Steroidogenic Acute Regulatory Protein Expression and Pregnenolone Synthesis in Rat Astrocyte Cultures

Abstract: Neurosteroids are steroids synthesised by brain cells. The molecular mechanism of neurosteroidogenesis from cholesterol has not yet been revealed. We studied the potential role of the steroidogenic acute regulatory (StAR) protein in neurosterodogenesis by using rat brain astrocytes. The novelty of the study is that regulation of StAR is described in primary cultures from embryonic mesencephalon and cerebellum regions of the brain. Dibutyryl cyclic AMP (dbcAMP) treatment increased StAR protein expression in astrocyte cultures. This was observed in immunoblots of mitochondrial fractions and by immunocytochemistry. Dual-labelling showed that the cyclic AMP-induced increase in StAR immunofluorescence was localised to mitochondria. In addition, mitochondrial cytochrome P450-side chain cleavage enzyme was demonstrated with a specific antibody, indicating the potential for pregnenolone production in these cells. Radioimmunoassay on ether-extracted conditioned media of control and dbcAMP treated cells demonstrated pregnenolone production by mesencephalic and cerebellar astrocyte cultures. Furthermore, 24-h pregnenolone levels, in the presence of inhibitors of further pregnenolone metabolism, were significantly increased by dbcAMP exposure. A murine StAR promoter-luciferase fusion plasmid was activated by dbcAMP in transiently transfected mesencephalic and cerebellar astrocytes. These novel results indicate that cyclic AMP signalling can regulate StAR expression and pregnenolone production in brain astrocytes, and provide additional insight into the role of StAR in neurosteroidogenesis.

Effects of 17α-methyltestosterone exposure on steroidogenesis and cyclin-B mRNA expression in previtellogenic oocytes of Atlantic cod (Gadus morhua)

Abstract: Steroid hormone (estrogens and androgens) synthesis and regulation involve a large number of enzymes and potential biochemical pathways. In the context of these biochemical pathways, it is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and the subsequent conversion to pregnenolone by cytochrome P450-mediated side-chain cleavage (P450scc) enzyme. Oocyte development is a complex process that is triggered by the maturation-promoting factor (MPF) involving cyclin-B as a regulatory factor. In the present study, we evaluated the endocrine effects of 17alpha-methyltestosterone (MT) on steroidogenic pathways of Atlantic cod (Gadus morhua), using an in vitro previtellogenic oocyte culture technique that is based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 1, 5, 10 and 20 days with different concentrations of the synthetic androgen MT (0 (control), 1, 10, 100 and 1000 microM) dissolved in ethanol (0.3%). Gene expressions for StAR, P450scc, aromatase-alpha (P450aromA) and cyclin-B were detected using validated real-time PCR with specific primer pairs. Cellular localization of the StAR protein and P450scc were performed using the immunohistochemical technique with antisera prepared against synthetic peptide for both proteins. Steroid hormones (estradiol-17beta: E2 and testosterone: T) levels were estimated using enzyme immunoassay. Our data showed significant concentration-specific increase (at day 1 and 5) and decrease (at day 10 and 20) of the StAR mRNA expression after exposure to MT. P450scc expression showed a MT concentration-specific decrease during the exposure periods and cyclin-B mRNA expression was decreased in MT concentration-dependent manner at days 10 and 20 (reaching almost total inhibition after exposure to 1000 microM MT). MT exposure produced variable effects on the P450aromA mRNA expression that can be described as concentration-specific increase (day 1) and decrease (days 5 and 10). Cellular localization of the StAR protein and P450scc demonstrated their expression mainly in ovarian follicular cells. MT produced an apparent concentration-and time-dependent increase of E2 and T levels. Thus, the present study reveals some novel effects of pharmaceutical endocrine disruptor on the development of previtellogenic oocytes in cod. The impaired steroidogenesis and hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleosts.

Aging and the Decline of Androgen Production

Abstract: Human male aging is associated with progressive decreases in serum concentrations of testosterone, which are not in response to decreased circulating basal luteinizing hormone (LH) concentrations, suggesting that reduced testosterone results from a primary deficit at the gonadal rather than the hypothalamic-pituitary level. This also is true of Brown Norway rats, a strain that has become widely used for studies of Leydig cell aging. Age-related reduced testosterone was found to result from reduced Leydig cell steroidogenesiss, but not by their loss. This chapter deals with the cellular changes, which are associated with reduced testosterone, and their causes. Age-related reductions have been reported in Leydig cell LH receptor numbers, intracellular cyclic adenosine monophosphate (cAMP) formation, steroidogenic acute regulatory protein, peripheral benzodiazapine receptor, the conversion of cholesterol to pregnenolone within the mitochondria and the subsequent conversion of pregnenolone to progesterone, 17a-hydroxyprogesterone, androstenedione, and ultimately testosterone in the smooth endoplasmic reticulum. Culturing isolated Leydig cells with LH maintained high levels of testosterone production by young cells but did not restore old cells to “young” levels. In contrast, culturing old cells with dibutyryl cAMP restored testosterone production to high levels, suggesting a deficit in the signal transduction mechanism between the LH receptor and cAMP production. Long-term suppression of steroidogenesis, accomplished by administering exogenous testosterone to middle-aged rats, prevented the steroidogenic aging of the cells, suggesting that ultimately steroidogenesis itself might result in age-related reductions in steroidogenesis. As measured with lucigenin, reactive oxygen production by old Leydig cells was found to be significantly more than by young Leydig cells. Microarray and Northern blot analysis revealed that the expression of genes for Cu-Zn Superoxide dismutase 1, Cu-Zn Superoxide dismutase 2, catalase, and glutathione peroxidase, the products of which protect Leydig cells from oxidative stress, are reduced as the Leydig cells age, as does their activities and protein levels. Depletion of glutathione with buthionine sulfoximine resulted in reduced testosterone production by young adult Leydig cells. Incubation of the cells with vitamin E delayed reduced testosterone production. Taken together, the available data suggests that changes in reactive oxygen and thus, an altered redox environment in aging Leydig cells might cause the changes in Leydig cells that result in age-related reduced testosterone production, and that the reactive oxygen might derive, at least in part, from steroidogenesis itself.

Effects of endocrine disruptors on dehydroepiandrosterone sulfotransferase and enzymes involved in PAPS synthesis: genomic and nongenomic pathways.

Abstract: Endocrine disruptors (EDs) have been studied quite extensively in environmental biology (Toppari et al. 1996); however, their exact effects on humans are uncertain. Rising rates of hormone-dependent cancers, such as breast, ovary, and testicular cancer, appear to be linked to rising levels of EDs contaminating the environment (Weir et al. 2000), although a causal link between observed abnormalities and chemical exposure has not been established (Baker 2001). These compounds may modulate both the endocrine, neuronal, and immune systems resulting in alteration of homeostasis, reproduction, development, and behavior (Amaral Mendes 2002). EDs are not classical poisons or carcinogens (Colborn et al. 1996); the detailed mechanisms by which EDs exert their effects are gradually being elucidated (Tapiero et al. 2002). They can directly initiate or inhibit actions mediated by members of the superfamily of zinc finger nuclear receptors, such as estrogen, androgen, and triiodothyronine receptors, although their chemical structures may not resemble those of steroids or related hormones. Alternatively, they may exert indirect genomic effects by modulating the metabolism of hormones. In this context, sulfation plays a key role because the cellular availability of steroid and thyroid hormones is modulated by their sulfation and desulfation by sulfotransferases and sulfatases, respectively. Sulfation of hydroxyl residues in these hormones renders them inactive and speeds exit from the cell, whereas desulfation regenerates their endocrine potential. A decrease in the sulfation/desulfation ratio could therefore lead to inappropriately high levels of hormones within cells (Falany et al. 2002; Kirk et al. 2001; Qian et al. 1998). Dehydroepiandrosterone (DHEA) and its sulfate-ester (DHEAS) are neurosteroids secreted mainly by the adrenal cortex and gonads, although synthesis of DHEAS can also take place in the central nervous system (CNS). In humans, plasma concentrations of DHEAS peak during the teenage years and thereafter decline by about 10% per decade (Ravaglia et al. 1996). It has been shown that any disruption of sulfation of DHEA or pregnenolone can block memory processes in rodents (Vallee et al. 2001), probably because these neurosteroids modulate acetylcholine release and γ-aminobutyric acid (GABA) and N-methyl-D-aspartate (NMDA) receptor action in the cortex and hippocampus (Dubrovsky 2005; Mayo et al. 2003). Results from studies in humans have been rather less conclusive because in vivo experiments are not possible. The situation is confounded because the brain can synthesize DHEA independently of the adrenal gland; therefore, measurement of DHEA and DHEAS in the plasma may not give a true indication of levels in the CNS (Racchi et al. 2003). Reduced levels of both plasma DHEAS and pregnenolone sulfate have been linked with decreased cognitive function (Armanini et al. 2003; Mayo et al. 2003). However, there is evidence that DHEA levels may exert subtle effects, particularly in Alzheimer’s disease (Vallee et al. 2001). DHEA sulfation is catalyzed by SULT 2A1, a member of the SULT 2 subfamily of the sulfotransferases, which mainly acts on endogenous hydroxysteroids, including pregnenolone, but can also sulfate various xenoestrogens and drugs. SULT 2A1 is unusual in that, although it has a Km for DHEA of about 2 μM (Chang et al. 2001), the physiologic concentration of DHEA ranges from 1–20 nM (Heuser et al. 1998). The essential co-factor in sulfation reactions is PAPS, which is synthesized in vivo by oxidation of sulfur-containing amino acids (Do and Tappaz 1996; Griffith 1987). Four enzymes play a key role in the synthesis of PAPS from cysteine. These are cysteine dioxygenase type I (CDO1), sulfite oxidase (SUOX), and the bifunctional enzymes 3′-phosphoadenosine 5′-phosphosulfate synthase 1 and 2 (PAPSS1 and PAPSS2). In humans, the supply of PAPS appears to be rate limiting for sulfation of both endogenous and exogenous compounds. Chemicals produced from the plastics and detergent industries, such as alkylphenols and bisphenol A, have been discovered to be estrogenic (Jobling et al. 1995). Plasticizers are ubiquitous in the environment at low levels and may be present in mixtures that result in additive, antagonistic, and/or synergistic effects (Guenther et al. 2002). In this article we show that a range of plasticizers commonly encountered in the environment act as competitive inhibitors of SULT 2A1 when sulfating physiologic concentrations of DHEA. In addition, they reduce steady-state mRNA levels of enzymes involved in PAPS synthesis.