scispace - formally typeset
Search or ask a question

Showing papers on "Steroid biosynthesis published in 1994"


Journal ArticleDOI
TL;DR: Ketoconazole remains the only available steroid-inhibitory drug for a therapeutic trial in patients with Cushing's syndrome who cannot be treated definitively by surgery.

133 citations


Journal ArticleDOI
TL;DR: It is demonstrated that anti-LHRH immunization was effective in reducing the level of androstenone, a boar taint-related compound, although having a limited effect on the performance of the animals.
Abstract: The effect of a newly developed anti-LH-RH vaccine on the performance, sexual development, and incidence of boar taint-related compounds was investigated in young intact male pigs. At 29 kg BW, 40 crossbred intact males and 20 castrates were allocated to three groups. Castrates and half of the intact males were untreated. The remaining intact males were immunized against LH-RH at 29 kg and again at 89 kg BW. All pigs were slaughtered at 105 kg BW. Compared with control intact males, feed efficiency in castrates was decreased by 10%, muscle content was reduced by 5%, and carcass fat content was increased by 26%. Growth performance and carcass traits did not differ significantly between immunized and control intact males. Genital tract weight, measured at slaughter, was decreased (P < or = .002) by immunization. Plasma testosterone concentrations were not significantly affected at 89 kg BW, whereas they were sevenfold lower (P < .001) in immunized than in control intact males at 105 kg BW. Fat androsterone levels, measured at slaughter, were substantially reduced (P < .001) from .66 +/- .07 microgram/g in control to .21 +/- .01 microgram/g in immunized intact males. Rates of testicular steroid biosynthesis, measured in vitro, were decreased by immunocastration. Fat skatole levels were very low and did not differ significantly between the three groups. The present results demonstrate that anti-LHRH immunization was effective in reducing the level of androstenone, a boar taint-related compound, although having a limited effect on the performance of the animals.

131 citations


Journal ArticleDOI
TL;DR: The chemical consequences of involving either an FeV==O or an FeIII--OOH species for five different C--C bond cleavage reactions, including the formation of 17 alpha-hydroxyandrogen from pregnenolone, presents an interesting contrast.
Abstract: It is now well-known that conventional cytochrome P-450s catalyze hydroxylation reactions using an iron mono-oxygen species, the structure of which, as inferred from chemical model studies, may be drrepresented by the following canonical forms: FeV==O (.+)FeIV==O FeIV--O(.). Certain multifunctional P-450s, notably those involved in steroid biosynthesis, catalyze, in addition to hydroxylation reactions, an acyl-carbon cleavage process in which the participation of an iron peroxide intermediate, FeIII--OOH, has been suggested. However the possibility still exists that the C--C bond cleavage may also occur using the FeV==O species. We have scrutinized the chemical consequences of involving either an FeV==O or an FeIII--OOH species for five different C--C bond cleavage reactions. With respect to the status as well as the origin of hydrogen and oxygen atoms, in four of the examples the mechanism involving the FeV==O species makes the same prediction as that using the iron peroxide intermediate, that is, the incorporation of an atom of oxygen from O2 into acyl part of the cleaved fragment. The fifth example, however, involving the formation, with pig testes microsomes, of 17 alpha-hydroxyandrogen (androst-5-ene-3 beta,17 alpha-diol) from pregnenolone, presents an interesting contrast--in this case different outcomes are predicted by the two mechanisms. These possibilities have been experimentally evaluated using substrates stereo- and regiospecifically labeled with heavy isotopes and incubated with pig testes microsomes under either 16O2 or 18O2.(ABSTRACT TRUNCATED AT 250 WORDS)

108 citations


Journal ArticleDOI
TL;DR: It is concluded that IrCRH is present in normal human ovaries and follicular fluid, suggesting that this neuropeptide may play a regulatory role in one or more of the various functions of this gonad, such as ovulation and/or luteolysis, through its proinflammatory properties and/ or its auto/paracrine regulation of steroid biosynthesis.
Abstract: Recently, we demonstrated the presence of immunoreactive (Ir) CRH and its receptors in the rat ovary. To determine whether CRH is also present in human ovaries, we examined ovaries from normal women and patients with the polycystic ovarian syndrome (PCOS). Immunoreactive CRH in normal human ovaries had a similar distribution to that of rat ovarian IrCRH, as determined by immunohistochemistry. Thus, immunoreactivity was intense in the cytoplasm of thecal cells surrounding the ovarian follicles, in luteinized cells of the stroma, and in a subpopulation of cells within the corpora lutea. No IrCRH was present in oocytes of primordial follicles. Polycystic ovaries also had IrCRH in thecal cells; however, CRH immunostaining was less prominent or completely absent from the stroma or the sparsely present corpora lutea and was clearly detected in oocytes of primordial follicles. Using a specific RIA, the IrCRH content in extracts of normal ovaries was higher than that in polycystic ovaries (mean +/- SD, 0.075 +/- 0.02 vs. 0.038 +/- 0.009 pmol/g wet tissue, respectively; P < 0.05). Human follicular fluid samples collected from women undergoing ovarian hyperstimulation for assisted reproduction had low, but detectable, levels of IrCRH (mean +/- SD, 4.975 +/- 1.179 pmol/L), whereas IrCRH was undetectable in concurrently drawn plasma samples. IrCRH detected in normal and polycystic ovaries and in follicular fluid had similar chromatographic mobility to that of rat/human CRH-(1-41) by reverse phase HPLC. We conclude that IrCRH is present in normal human ovaries and follicular fluid, suggesting that this neuropeptide may play a regulatory role in one or more of the various functions of this gonad, such as ovulation and/or luteolysis, through its proinflammatory properties and/or its auto/paracrine regulation of steroid biosynthesis, in analogy to its action on testosterone secretion by the Leydig cell. Its decreased concentration and localization in primary oocytes of polycystic ovaries may be related to the increased androgen biosynthesis by the theca and stroma and/or to the oocyte dysfunction observed in women with the polycystic ovarian syndrome, respectively.

84 citations


Journal ArticleDOI
TL;DR: It is suggested that growth factors may differentially regulate cAMP-dependent processes in human thecal and granulosa cells of the developing follicle.
Abstract: In this report we examined the effects of growth factors and phorbol esters on steroid hydroxylase activity in cultured human thecal and granulosa-lutein cells Treatment of thecal cells with epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor-beta (TGF beta), and tetradecanoyl phorbol acetate (TPA) resulted in the inhibition of forskolin- and dibutyryl cAMP-stimulated 17 alpha-hydroxylase activity and 17 alpha-hydroxyprogesterone and dehydroepiandrosterone production In contrast, cAMP-stimulated 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity was enhanced by FGF and TGF beta, and treatment with EGF enhanced cAMP-stimulated progesterone production cAMP stimulated 3 beta HSD activity was unaffected by TPA (10 nmol/L) treatment, yet TPA inhibited cAMP-stimulated progesterone production Basal 3 beta HSD activity and progesterone production were inhibited by TPA In contrast to the inhibitory actions of EGF, FGF, and TGF beta on 17 alpha-hydroxylase expression, insulin and insulin-like growth factor-I enhanced forskolin-stimulated 17 alpha-hydroxylase activity In granulosa-lutein cells, forskolin-stimulated aromatase activity was suppressed by EGF, FGF, and TPA TGF beta had no effect on forskolin-stimulated aromatase activity EGF, FGF, and TGF beta did not affect forskolin-stimulated progesterone production, whereas treatment with TPA inhibited cAMP-stimulated progesterone secretion These data suggest that growth factors may differentially regulate cAMP-dependent processes in human thecal and granulosa cells of the developing follicle

70 citations


Journal ArticleDOI
Eric Dupont1, J. Simard1, Van Luu-The1, Fernand Labrie1, Georges Pelletier1 
TL;DR: The present data strongly suggest that a restricted population of neurons might be involved in steroid biosynthesis in the adult rat brain and the role of the rat 3 beta-HSD family in the nuclei of two cranial nerves remains to be investigated.

66 citations


Journal ArticleDOI
TL;DR: In MA-10 cells, the most rapid effect described thus far of hCG and cAMP, is the transient induction of a higher affinity benzodiazepine-binding site, which occurs concomitantly with an increase in the rate of steroid formation.
Abstract: We previously demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) is coupled to hormone-activated steroidogenesis by regulating the intramitochondrial cholesterol transport, the rate-determining step of steroid biosynthesis. In the present study we examined whether PBR is the site of hormone action using the hCG-responsive MA-10 mouse Leydig tumor cell line as a model system. Within 15 sec of the addition of hCG to Leydig cells a 3-fold cAMP-dependent increase in PBR binding was observed. This rapid increase returned to basal levels within 60 sec. No effect was observed after 1 min in the continued presence of hCG. Scatchard analysis revealed that in addition to the known high affinity (5.0 nM) benzodiazepine-binding site, a second, hormone-induced, higher affinity (0.2 nM) benzodiazepine-binding site appeared. We then examined whether in such a short time frame steroid synthesis occurs. Fifteen-second incubation of MA-10 cells with the inhibitor of cholesterol metabolism aminoglutethimide together with hCG also resulted in an increased rate of pregnenolone formation by their isolated mitochondria that were washed and incubated in aminoglutethimide-free buffer. The dose response of benzodiazepine binding to hCG closely parallels the increase in steroid formation by the mitochondria of stimulated cells. Addition of the selective inhibitor of cAMP-dependent protein kinase, H-89, completely blocked hormone-induced PBR binding and steroid formation, whereas addition of the inactive analog H-85 was without any effect. The addition of flunitrazepam, a benzodiazepine previously shown to inhibit the trophic hormone action on steroidogenesis, completely abolished the hCG-induced rapid stimulation of steroid synthesis. These results demonstrate that in MA-10 cells, the most rapid effect described thus far of hCG and cAMP, is the transient induction of a higher affinity benzodiazepine-binding site, which occurs concomitantly with an increase in the rate of steroid formation. This, in turn, suggests that these hormones alter PBR to activate cholesterol delivery to the inner mitochondrial membrane and subsequent steroid formation.

59 citations



Journal ArticleDOI
TL;DR: A 5-year-old XY pseudohermaphrodite was found to have a defect of steroid biosynthesis consistent with a partial deficiency of the enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and three related heterozygous adult females showed evidence of a small over-production of delta 5 steroids and steroid metabolites and a variable reduction in ovarian function.
Abstract: A 5-year-old XY pseudohermaphrodite was found to have a defect of steroid biosynthesis consistent with a partial deficiency of the enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). Circulating concentrations of delta 5 steroids and delta 5 urinary steroid metabolites were elevated and remained elevated after orchidectomy. There was no evidence of salt loss, plasma renin being within normal limits, and no detectable glucocorticoid abnormality. The coding sequences of the genes for 3 beta-HSD types I and II were amplified by PCR and screened for mutations by denaturing gradient gel electrophoresis (DGGE) and manual and automatic DNA sequencing. A mutation in the gene for 3 beta-HSD type II was observed at codon 173 (CTA-->CGA), leading in the affected patient to a homozygous substitution in which the leucine at residue 173 was altered to an arginine (L173R). The propositus's 2-year-old XX sister was also homozygous for L173R and showed the biochemical characteristics of partial 3 beta-HSD deficiency without clinical symptoms or signs. The mutation segregated as an autosomal recessive. Three related heterozygous adult females showed evidence of a small over-production of delta 5 steroids and steroid metabolites and a variable reduction in ovarian function. Concentrations of delta 5 steroids and steroid metabolites in the heterozygous father of the propositus were within the normal range. These data are discussed in relation to the endocrine causes of pseudohermaphroditism and hirsutism. Evidence for tight linkage between the genes for 3 beta-HSD types I and II was obtained using a microsatellite polymorphism in the third intron of the gene for 3 beta-HSD type II and synonymous and non-synonymous mutations and polymorphisms in the gene for 3 beta-HSD type I. The latter polymorphisms were located 88 bp apart at the 3' end of the type I coding sequence and could be physically resolved as haplotypes using DGGE. The application of DGGE to the analysis of mutations in members of a multigene family is discussed.

54 citations


Journal ArticleDOI
TL;DR: The present findings, which describe the novel missense mutation Y254D in the human type II 3beta HSD gene, provide useful information on the structure-activity relationships of the 3 beta HSD superfamily.
Abstract: Three beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) deficiency is a form of congenital adrenal hyperplasia characterized by severe impairment of steroid biosynthesis in the adrenals and gonads. To better understand the molecular basis of the phenotypic heterogeneity found in 3 beta HSD deficiency, we analyzed the structure of type I and II 3 beta HSD genes in a female patient with nonsalt-losing 3 beta HSD deficiency diagnosed at puberty. We directly sequenced DNA fragments generated by polymerase chain reaction amplification of the four exons, the exon-intron boundaries, and the 5'-flanking regions of each gene. No mutation was detected in the type I 3 beta HSD gene, which is the predominant species expressed in the placenta and peripheral tissues. We detected a novel missense mutation, Y254D, in one allele of the patient's type II 3 beta HSD gene, which is the almost exclusive type expressed in the adrenals and gonads. The influence of the Y254D mutation on enzymatic activity ...

50 citations


Book ChapterDOI
TL;DR: Cholesterol desmolase and 3 beta-hydroxysteroid dehydrogenase deficiencies affect synthesis of all classes of steroids in both the adrenals and gonads, suggesting that the inability to synthesize placental estrogens may adversely affect fetal survival.
Abstract: All major classes of biologically active steroid hormones (progestins, mineralocorticoids, glucocorticoids, and sex steroids) are synthesized from cholesterol through 11 different bioconversions. With the exception of 5 alpha-reductase, all the enzymes mediating these reactions fall into two classes, cytochromes P450 and short-chain dehydrogenases. Cytochromes P450 are heme-containing membrane-bound proteins with molecular weights of approximately 50,000 that utilize molecular oxygen and electrons from NADPH-dependent accessory proteins to hydroxylate substrates. Short-chain dehydrogenases have molecular weights of 30,000-40,000, have tyrosine and lysine residues at the active site, and remove a hydride from the substrate, transferring the electrons of the hydride to NAD+ or NADP+. In most cases, this reaction is reversible so that the dehydrogenase can also function as a reductase under appropriate conditions. Inherited disorders in enzymes required for steroid biosynthesis have varying effects. Defects that prevent cortisol from being synthesized are referred to collectively as congenital adrenal hyperplasia. Because the enzymes required for cortisol biosynthesis in the adrenal cortex are in many cases required for the synthesis of mineralocorticoids and/or sex steroids, these classes of steroids may also not be synthesized normally. Thus, cholesterol desmolase and 3 beta-hydroxysteroid dehydrogenase deficiencies affect synthesis of all classes of steroids in both the adrenals and gonads. Steroid 21-hydroxylase deficiency, the most common cause (> 90% of cases) of congenital adrenal hyperplasia, can affect both mineralocorticoid and glucocorticoid synthesis, but androgen secretion is usually abnormally high due to shunting of accumulated precursors into this pathway. Excessive secretion of androgens and mineralocorticoids occurs in 11 beta-hydroxylase deficiency (the second most frequent form of congenital adrenal hyperplasia). Mineralocorticoid excess is also seen in 17 alpha-hydroxylase deficiency, but in this disorder sex steroid synthesis is defective. All defects that affect estrogen synthesis (deficiencies of cholesterol desmolase, 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, aromatase, and 17 beta-hydroxysteroid dehydrogenase) are very rare, suggesting that the inability to synthesize placental estrogens may adversely affect fetal survival. A number of enzymes are expressed at sites of steroid action and regulate the amount of active steroid available to steroid receptors. Steroid 5 alpha-reductase converts testosterone to the more active dihydrotestosterone. Deficiency of this activity leads to incomplete development of male genitalia; 17 beta-hydroxysteroid dehydrogenase deficiency has similar phenotypic effects.(ABSTRACT TRUNCATED AT 400 WORDS)

Journal ArticleDOI
TL;DR: It is found that only the vitamin D receptor (VDR) mRNA concentration was influenced by the hormones, and both 1,25(OH)2D3 and retinoic acid separately stabilized the VDR mRNA levels increasing the apparent half-life by 11 h and 6 h, respectively.

Journal ArticleDOI
TL;DR: The expression of testicular hormone‐sensitive lipase mRNA and protein coincided with the onset of spermatogenesis and was dependent on scrotal localization of the testis, suggesting a temperature‐dependent, pretranslational regulation of expression.

Journal ArticleDOI
TL;DR: RS-21607 is shown to be an effective and selective inhibitor of lanosterol 14 alpha-demethylase, both in vivo and in vitro, and the ligand attached to the imidazole in RS-216 07 is a good non-sterol substitute for DHL.
Abstract: The discovery of selective lanosterol 14 alpha-demethylase inhibitors may lead to novel hypolipidemic drugs. RS-21607, (2S,4S)-cis-2[1H-imidazol-1-yl)methyl]-2-[2-(4-chlorophenyl)ethyl]-4- [[(4-aminophenyl)thio]methyl]-1,3-dioxolane, was characterized as a tight-binding, competitive inhibitor of lanosterol 14 alpha-demethylase purified from rat liver. The apparent Ki was determined to be 840 pM and found to be similar in hepatic microsomes from human, rat, and hamster. RS-21607, which contains two chiral centers, was a more effective lanosterol 14 alpha-demethylase inhibitor than its three stereoisomers. In vitro, RS-21607 had a greater affinity for lanosterol 14 alpha-demethylase than the other cytochromes P450 evaluated: CYP7, CYP27, CYP11A1, CYP19, CYP17, CYP11B1, CYP21, CYP3A4, CYP4A, CYP2D6, CYP1A2, CYP2C9, and 27-hydroxycholesterol 7 alpha-hydroxylase. The other stereoisomers were not as selective as RS-21607. Doses of 3-30 mg/kg RS-21607 given orally to hamsters caused a dose-dependent decrease in cholesterol biosynthesis with a corresponding accumulation of 24,25-dihydrolanosterol. RS-21607 inhibited the enzyme and cholesterol biosynthesis in hamster liver by 50% at 18 h following a 30 mg/kg oral dose. This was interpreted to indicate that RS-21607 is able to distribute to the site of action in hamsters and inhibit the target enzyme. In the same dose range, the plasma concentrations of testosterone, corticosterone, and progesterone, the endpoints for the cytochromes P450 involved in steroid biosynthesis, were relatively unaffected. These data show RS-21607 to be an effective and selective inhibitor of lanosterol 14 alpha-demethylase, both in vivo and in vitro. RS-21607 interacted with the purified enzyme to produce a type II binding spectrum, consistent with an interaction between the imidazole moiety and the heme. The electrostatic contribution of the imidazole binding was investigated using the desimidazole analog of RS-21607. The apparent Ki for the desimidazole compound (65 microM) was similar to the apparent Km for the substrate DHL (79 microM). Together, these data confirm that the ligand attached to the imidazole in RS-21607 is a good non-sterol substitute for DHL, i.e., binding to the enzyme with similar affinity, and that the coordination of the imidazole to the heme provides a major electrostatic contribution for the inhibition of lanosterol 14 alpha-demethylase by RS-21607. RS-21607 was also observed to increase the accumulation of 3 beta-hydroxy-24,25-dihydrolanost-8-en-32-al, the second intermediate in the multistep oxidation, but not the first intermediate. 24,25-dihydrolanost-8-ene-3 beta,32-diol.(ABSTRACT TRUNCATED AT 400 WORDS)

Journal ArticleDOI
TL;DR: Clinical data from several groups show that the administration of ketoconazole normalizes the urinary excretion of cortisol in the mean in about 70% of patients with Cushing's disease, and the antimycotic drug was effective in many patients with a benign primary adrenal form of Cushed's syndrome.
Abstract: About one-third of all patients with Cushing's syndrome cannot be cured by surgery (at the pituitary or adrenal level) or radiation therapy and are therefore candidates for medical treatment. As a conservative therapeutic approach to lower hypercortisolism, the use of steroid biosynthesis blocking substances has the greatest importance. Trilostane, an inhibitor of the adrenal 3β-hydroxysteroid dehydrogenase Δ5,4-isomerase system, has been studied in only a few patients with Cushing's syndrome and was not potent enough to normalize hypercortisolism, especially in patients with pituitary-dependent Cushing's disease. Aminoglutethimide, predominantly blocking side-chain cleavage, normalized elevated serum or urinary cortisol levels in only a minority of patients with Cushing's disease and showed adverse reactions in the majority. Metyrapone, a strong inhibitor of adrenal 11β-hydroxylase activity, has only an insufficient blocking effect on elevated cortisol levels in some patients with various forms of Cushing's syndrome and shows side effects in a significant number of patients. Ketoconazole in vitro blocks predominantly adrenal 17,20-desmolase activity and to a lesser extent 17α- and 11β-hydroxylase activity. Therefore the substance in vivo more markedly suppresses serum androgen levels (dehydroepiandrosterone sulfate, androstenedione, testosterone) than cortisol. However, clinical data from several groups show that the administration of ketoconazole normalizes the urinary excretion of cortisol in the mean in about 70% of patients with Cushing's disease. Furthermore, the antimycotic drug was effective in many patients with a benign primary adrenal form of Cushing's syndrome, in about 50% of patients with ectopic ACTH syndrome, but rarely in patients with adrenocortical carcinoma. The main side effect of ketoconazole is liver toxicity, in about 10% of all cases. Etomidate has strong inhibiting properties on adrenal 11β-hydroxylase activity and in vivo is the most potent substance to normalize hypercortisolism. However, its widespread use is prevented by the necessity of intravenous administration. Mitotane inhibits various pathways of adrenal steroid biosynthesis, but its main effect is a cytolytic effect especially on adrenocortical cells. It is therefore a special cytostatic drug for patients with adrenocortical carcinoma. High doses of the substance lower or normalize elevated cortisol parameters in the majority of these patients, but objective tumor regression has been documented in only in few cases.


Journal ArticleDOI
TL;DR: The results show that, under conditions preserving membrane integrity, the "impermeance" of suramin meant that concentrations similar to the plasma-levels reached in treated patients induced only slight inhibition of the final intramitochondrial steps in aldosterone synthesis, but suramin strongly inhibited mitochondrial respiration.

Journal ArticleDOI
TL;DR: An immunocytochemical investigation of angiotensin II in developing ovarian follicles of trout has been carried out and there was strong immunoreactivity in the ooplasm of endogenous vitellogenic follicles.