Role of microsomal steroid hydroxylases in Δ7-steroid biosynthesis

CYP17 (steroid 17α-hydroxylase/17,20-lyase) is a key enzyme in steroid hormone biosynthesis. It catalyzes two independent reactions at the same active center and has a unique ability to differentiate Δ⁴-steroids and Δ⁵-steroids in the 17,20-lyase reaction. The present work presents a complex experimental analysis of the role of CYP17 in the metabolism of 7-dehydrosteroids. The data indicate the existence of a possible alternative pathway of steroid hormone biosynthesis using 7-dehydrosteroids. The major reaction products of CYP17 catalyzed hydroxylation of 7-dehydropregnenolone have been identified. Catalytic activity of CYP17 from different species with 7-dehydropregnenolone has been estimated. It is shown that CYP21 cannot use Δ⁵–Δ⁷ steroids as a substrate.     

Are gonadal steroid hormones involved in disorders of brain aging?

Human aging is associated with a decrease of circulating gonadal steroid hormones. Since these hormones act as trophic factors for neurones and glia, it is possible that the decrease in sex steroid levels may contribute to the increased risk of neurodegenerative disorders with advanced age. Sex steroids are neuroprotective in several animal models of central and peripheral neurodegenerative diseases, and clinical data suggest that these hormones may reduce the risk of neural pathology in aged humans. Potential therapeutic approaches for aged-associated neural disorders may emerge from studies conducted to understand the mechanisms of action of sex steroids in the nervous system of aged animals. Alterations in the endogenous capacity of the aged brain to synthesize and metabolize sex steroids, as well as possible aged-associated modifications in the signalling of sex steroid receptors in the nervous system, are important areas for future investigation.     

Specificity in steroid histochemistry,with special reference to the use of steroid solvents. distribution of 11 β-hydroxysteroiddehydrogenase in kidney and thymus from the mouse

Summary Several factors influencing the steroiddehydrogenase histochemistry were investigated: diffusion of enzyme; inactivation of enzyme; effects of the steroid solvents commonly used; the validity in localization of the enzyme activity; nothing dehydrogenase reaction. 1. The importance in controlling the diffusion of each enzyme system to be studied is emphasized. Provided that the presence of SH-groups in the active centre of the dehydrogenase can be proved, a control experiment using a double-section incubation method should be carried out. 2. A comparison between the use of unfixed and briefly prefixed sections is recommended in order to avoid a possible distortion of the tissue during the incubation. The influence of prefixation on diffusion of enzymes or reaction products as well as on inactivation of enzymes must be studied. 3. The steroid solvents—especially dimethyl formamide caused a morphological distortion, and an inactivation and/or extraction of reaction products (the red monoformazan) in fresh frozen sections, these solvents should therefore be handled with caution. A special mixture of dimethyl formamide and propylene glycol is recommended. 4. The steroid should be completely soluble in the incubation medium in order to secure zero order kinetics. 5. Avoidance of sulphydryl nothing dehydrogenase reaction, since the reaction predominantly manifests itself as a red formazan obscuring sites with low dehydrogenase activity. 6. The localization of the NAD(P)H oxidase systems must be controlled, in order to ensure that they should not be a limiting factor in the detection of the dehydrogenase activity. Secondly, this investigation may act as a control on diffusion of dehydrogenase and/or reduced coenzyme. 7. That the investigation of the incubation time needed for initial visual reaction allows a certain quantitative estimation of the concentration of enzyme localized at different sites in the same section. The investigation should also include the red formazan, since it has recently been proved to be an intermediary step in the enzymic reduction of Nitro BT, and as such may reflect sites with low enzyme concentration.Further, some of the functional aspects of the activity of 11-hydroxysteroiddehydrogenaseNAD(P)H oxidase systems in the thymus were discussed, and lastly the localization of these systems in the kidney was revised.This work was supported by a grant from Statens almindelige Videnskabsfond, Copenhagen.     

Insight into the orientational versatility of steroid substrates—a docking and molecular dynamics study of a steroid receptor and steroid monooxygenase

Numerous steroids are essential plant, animal, and human hormones. The medical and industrial applications of these hormones require the identification of new synthetic routes, including biotransformations. The metabolic fate of a steroid can be complicated; it may be transformed into a variety of substituted derivatives. This may be because a steroid molecule can adopt several possible orientations in the binding pocket of a receptor or an enzyme. The present study, based on docking and molecular dynamics, shows that it is indeed possible for a steroid molecule to bind to a receptor binding site in two or more orientations (normal, head-to-tail reversed, upside down). Three steroids were considered: progesterone, dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. Two proteins were employed as hosts: the human mineralocorticoid receptor and a bacterial Baeyer–Villiger monooxygenase. When the steroids were in nonstandard orientations, the estimated binding strength was found to be only moderately diminished and the network of hydrogen bonds between the steroid and the host was preserved.     

3β-Hydroxy steroid dehydrogenase activity in the mitochondria of rat adrenal homogenates

The activity of 3beta-hydroxy steroid dehydrogenase (EC 1.1.1.51) in the mitochondrial fraction of rat adrenal homogenates was approx. 31% of the total activity recovered after differential centrifugation and washing of the particulate fractions. Some 45% of the total activity was found in the microsomal fraction. The activity was assayed by a radioisotopic method devised in this laboratory for the purpose of studying small quantities of tissue and cell fractions. Satisfactory separation of the two fractions was demonstrated by electron microscopy of the pellets and by comparative recoveries of RNA, steroid 21-hydroxylase and cytochrome c oxidase in the various compartments. Analyses of the kinetics of the enzyme activity in the two fractions revealed no significant differences in apparent K(m) for pregnenolone, dehydroepiandrosterone or NAD(+), but demonstrated a distinct difference in the K(m) for NADP(+). pH optima and susceptibility to cyanoketone inhibition were similar in both fractions.     

Cholesterol transport in steroid biosynthesis: Role of protein–protein interactions and implications in disease states

The transfer of cholesterol from the outer to the inner mitochondrial membrane is the rate-limiting step in hormone-induced steroid formation. To ensure that this step is achieved efficiently, free cholesterol must accumulate in excess at the outer mitochondrial membrane and then be transferred to the inner membrane. This is accomplished through a series of steps that involve various intracellular organelles, including lysosomes and lipid droplets, and proteins such as the translocator protein (18 kDa, TSPO) and steroidogenic acute regulatory (StAR) proteins. TSPO, previously known as the peripheral-type benzodiazepine receptor, is a high-affinity drug- and cholesterol-binding mitochondrial protein. StAR is a hormone-induced mitochondria-targeted protein that has been shown to initiate cholesterol transfer into mitochondria. Through the assistance of proteins such as the cAMP-dependent protein kinase regulatory subunit Iα (PKA-RIα) and the PKA-RIα- and TSPO-associated acyl-coenzyme A binding domain containing 3 (ACBD3) protein, PAP7, cholesterol is transferred to and docked at the outer mitochondrial membrane. The TSPO-dependent import of StAR into mitochondria, and the association of TSPO with the outer/inner mitochondrial membrane contact sites, drives the intramitochondrial cholesterol transfer and subsequent steroid formation. The focus of this review is on (i) the intracellular pathways and protein–protein interactions involved in cholesterol transport and steroid biosynthesis and (ii) the roles and interactions of these proteins in endocrine pathologies and neurological diseases where steroid synthesis plays a critical role.     

Oligomeric amyloid-β peptide affects the expression of genes involved in steroid and lipid metabolism in primary neurons

Amyloid-β peptide (Aβ) is the principal component of plaques in the brains of patients with Alzheimer's disease (AD), and the most toxic form of Aβ may be as soluble oligomers. We report here the results of a microarray study of gene expression profiles in primary mouse cortical neurons in response to oligomeric Aβ(1-42). A major and unexpected finding was the down-regulation of genes involved in the biosynthesis of cholesterol and other steroids and lipids (such as Fdft1, Fdps, Idi1, Ldr, Mvd, Mvk, Nsdhl, Sc4mol), the expression of which was verified by quantitative real-time RT-PCR (qPCR). The ATP-binding cassette gene Abca1, which has a major role in cholesterol transport in brain and other tissues and has been genetically linked to AD, was notably up-regulated. The possible involvement of cholesterol and other lipids in Aβ synthesis and action in Alzheimer's disease has been studied and debated extensively but remains unresolved. These new data suggest that Aβ may influence steroid and lipid metabolism in neurons via multiple gene-expression changes.     

Is steroid therapy needed in the treatment of destructive thyrotoxicosis induced by alpha-interferon in chronic hepatitis C?

OBJECTIVE: Treatment with interferon (IFN) of patients affected by chronic hepatitis C (CH-C) may produce alterations in thyroid function, such as hypothyroidism, Graves'-like hyperthyroidism and destructive thyrotoxicosis (DT). IFN-induced DT is characterized by suppressed serum TSH levels, normal or elevated FT4 and FT3 concentrations, with the presence or absence of thyroid peroxidase antibodies and antithyroglobulin antibodies, the absence of thyroid receptor antibodies and radioactive iodine uptake suppressed or <5%. DESIGN: IFN-induced DT is a mild clinical disease, because thyroid-destructive processes last for a short time and involve a small portion of the gland. At present, the therapeutic approach in DT suggests IFN withdrawal and 1-2 months of methylprednisolone treatment. METHODS: In consideration of possible untoward side effects of steroid treatment in patients with CH-C, we studied two groups of patients with CH-C who developed DT after treatments with various preparations of recombinant IFN (with or without ribavirin). Patients sequentially entered the study during a 4-year period, at the time of DT diagnosis, when IFN therapy was discontinued. The first 12 subjects (group A) were treated with 8-16 mg/day methylprednisolone for 30-40 days after IFN withdrawal; in the following 15 patients (group B), IFN withdrawal was not followed by any additional treatment. All patients underwent clinical and laboratory controls of thyroid function at 1, 2, 3 and 6 months after DT diagnosis. RESULTS: The results showed restoration of euthyroidism in both group A and group B patients at 6 months after DT diagnosis, regardless of steroid treatment. CONCLUSIONS: The simple withdrawal of IFN therapy in patients with CH-C, who had developed DT, appears to be effective in the treatment of the thyroid disease. This therapeutic approach should be preferred in order to avoid possible undesired side effects of steroid therapy in patients with CH-C.     

Indole and benzimidazole derivatives as steroid 5α-reductase inhibitors in the rat prostate

A novel series of indole and benzimidazole derivatives were synthesized and evaluated for their inhibitory activity of rat prostatic 5α-reductase. Among these compounds, 4-{2-[1-(4,4′-dipropylbenzhydryl)indole-5-carboxamido]phenoxy}butyric acid (15) and its benzimidazole analogue 25 showed potent inhibitory activities for rat prostatic 5α-reductase (IC₅₀ values of 9.6 ± 1.0 and 13 ± 1.5nM, respectively), with the potency very close to that of finasteride. Compound 30, in which the moiety between the benzene ring and amide bond was replaced by quinolin-4-one ring, showed almost equipotent activity (IC₅₀ = 19 ± 6.2nM) with the correspondent amide derivative 13. This result was consistent with the previous observation that the coplanarity of this moiety might contribute to the potent inhibitory activity.     

The influence of androgenic steroid hormones on female aggression in ‘atypical’ mammals

Dimorphism on dominance and agonistic behaviour in mammals tends to be strongly biased toward males. In this review, we focus on a select few species of mammals in which females are as or more aggressive than males, and/or are dominant to males, and explore the role of androgenic hormones in mediating this important difference. While the data are not as clear-cut as those published on traditional laboratory mammals, our review highlights important endocrine substrates for both organizational and activational influences of steroids on female aggressive behaviour. We highlight areas in which further observations and experiments are crucial, especially the potential facilitative effects of androgens on female aggression. Finally, new and innovative techniques, including molecular genetics and receptor pharmacology, portend important insights into the ways in which androgenic hormones regulate aggressive behaviour in ‘atypical’ female mammals.     

Regulation of 17β-hydroxysteroid dehydrogenases in cancer: regulating steroid receptor at pre-receptor stage

Increased intracelullar hormone concentration levels have been shown to be the cause of several endocrine-related cancers including breast, prostate, endometrial, ovarian, cervix, testicular, thyroid, and osteosarcoma. Deregulated expression of steroidogenic enzymes in these tumors seems to be the source of a positive balance in active steroids that bind to the corresponding nuclear receptor, thus ultimately stimulating cell proliferation. Among these enzymes, 17β-hydroxysteroid dehydrogenases catalyze the interconversion between 17-ketosteroids and 17-hydroxysteroids on the last steps of sex hormones biosynthesis and metabolism. To date, 14 isoforms have been identified in vertebrates although only 13 are present in humans. Development and clinical evaluation of specific inhibitors to block their activity is currently under progress especially against the best characterized members 1 to 5. Selectivity and potency of these drugs constitute the main challenge in this new approach to cancer and steroid-dependent diseases treatment at the "pre-receptor level". Here we review the current state of knowledge regarding the deregulation of the expression of some of these enzymes in endocrine-related tumors.     

Equilenin: a specific fluorescent probe for steroid—protein interactions in sex steroid-binding protein

Equilenin, a naturally fluorescent steroid, has high binding affinity for human sex steroid-binding protein (SBP). At 4°C the equilibrium association constant is ～6 × 10⁷ M^?1. The fluorescence excitation and emission spectra of the steroid—protein complex indicate that both hydrophobic interactions and hydrogen bonding of the 3'-hydroxyl group of the estrogen are important in its binding to the protein. Equilenin has a substantially different 3-dimensional spatial configuration compared with the normally bound androgens, and yet exhibits very tight binding to SBP. This suggests that SBP undergoes a conformational change to accomodate equilenin.     

Sex and seasonal differences in aggression and steroid secretion in Lemur catta: are socially dominant females hormonally 'masculinized'?

Female social dominance characterizes many strepsirrhine primates endemic to Madagascar, but currently there is no comprehensive explanation for how or why female lemurs routinely dominate males. Reconstructing the evolutionary pressures that may have shaped female dominance depends on better understanding the mechanism of inheritance, variation in trait expression, and correlating variables. Indeed, relative to males, many female lemurs also display delayed puberty, size monomorphism, and 'masculinized' external genitalia. As in the spotted hyena (Crocuta crocuta), a species characterized by extreme masculinization of the female, this array of traits focuses attention on the role of androgens in female development. Consequently, I examined endocrine profiles and social interaction in the ringtailed lemur (Lemur catta) to search for a potential source of circulating androgen in adult females and an endocrine correlate of female dominance or its proxy, aggression. I measured serum androstenedione (A(4)), testosterone (T), and estradiol (E(2)) in reproductively intact, adult lemurs (10 females; 12 males) over four annual cycles. Whereas T concentrations in males far exceeded those in females, A(4) concentrations were only slightly greater in males than in females. In both sexes, A(4) and T were positively correlated, implicating the Delta(4)-biosynthetic pathway. Moreover, seasonal changes in reproductive function in both sexes coincided with seasonal changes in behavior, with A(4) and T in males versus A(4) and E(2) in females increasing during periods marked by heightened aggression. Therefore, A(4) and/or E(2) may be potentially important steroidal sources in female lemurs that could modulate aggression and underlie a suite of masculinized features.     

Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes

A system has been developed which allows the stimulation in vitro of prostatic RNA polymerase by prostatic 5alpha-dihydrotestosterone-protein receptor complexes prepared from the tissues of castrated rats. The reconstitution in vitro of such a system necessitates the purification of several subcellular components. Two 5alpha-dihydrotestosterone-receptor complexes are located in the prostatic soluble supernatant fraction, separable by selective ammonium sulphate fractionation, and one complex can be isolated from the nuclear fraction. In the presence of all these complexes, stimulation of RNA polymerase in intact nuclei and nucleoli was observed. The complexes also increased the activity of the enzyme solubilized from whole nuclei. Greater stimulation of this system was noted in the presence of prostatic chromatin as template, as compared with that observed with calf thymus DNA or liver chromatin as template. The effects of the complexes on subnuclear forms of RNA polymerase, of nucleolar and extranucleolar origin, are also described. RNA polymerase solubilized from nucleoli is more susceptible to stimulation by the 5alpha-dihydrotestosterone-receptor complexes than is the ;nucleoplasmic' enzyme. Stimulation occurs less readily in the presence of Mn(2+) and at high ionic strength than in the presence of Mg(2+) and at low ionic strength. Preliminary experiments show that prostatic nucleolar RNA polymerase transcribes prostatic chromatin poorly as compared with the nucleoplasmic enzyme. The observations reported indicate an involvement of non-histone proteins associated with DNA in the process by which stimulation of enzyme activity by the 5alpha-dihydrotestosterone-receptor complexes is achieved. The implications of these findings in the mechanism of steroid hormone action is considered.     

Deletions of the steroid sulphatase gene in “classical” X-linked ichthyosis and in X-linked ichthyosis associated with Kallmann syndrome

Summary We have studied 16 men, from 10 unrelated Italian families, affected by steroid suphatase (STS) deficiency, which is the basic defect of X-linked ichthyosis (XLI). The patients' clinical diagnoses were of either isolated ichthyosis or ichthyosis associated with Kallmann syndrome (KS) (hypogonadotropic hypogonadism and anosmia). DNA from patients and their relatives was analysed by Southern blotting followed by hydridization with an STS cDNA probe. None of the patients affected by either XLI or XLI/KS showed any hybridization signal, thus revealing a deletion in the STS gene. We suggest that a gene deletion may be the most common molecular defect involved in XLI and that the syndrome XLI/KS may be due to a deletion of both the STS and the KS loci.     

Serotonin modulates anxiety-like behaviors during withdrawal from adolescent anabolic–androgenic steroid exposure in Syrian hamsters

From the U.S. to Europe and Australia anabolic steroid abuse remains high in the adolescent population. This is concerning given that anabolic steroid use is associated with a higher incidence of pathological anxiety that often appears during withdrawal from use. This study uses pubertal Syrian hamsters (Mesocricetus auratus) to investigate the hypothesis that adolescent anabolic/androgenic steroid (AAS) exposure predisposes hamsters to heightened levels of anxiety during AAS withdrawal that is modulated by serotonin (5HT) neural signaling. In the first two sets of experiments, adolescent AAS-treated hamsters were tested for anxiety 21 days after the cessation of AAS administration (i.e., during AAS withdrawal) using the elevated plus maze (EPM), dark/light (DL), and seed finding (SF) tests and then examined for differences in 5HT afferent innervation to select areas of the brain important for anxiety. In the EPM and DL tests, adolescent AAS exposure leads to significant increases in anxiety-like response during AAS withdrawal. AAS-treated hamsters showed long-term reductions in 5HT innervation within several areas of the hamster brain implicated in anxiety, most notably the anterior hypothalamus and the central and medial amygdala. However, no differences in 5HT were found in other anxiety areas, e.g., frontal cortex and lateral septum. In the last experiment, adolescent AAS-treated hamsters were scored for anxiety on the 21st day of AAS withdrawal following the systemic administration of saline or one of three doses of fluoxetine, a selective serotonin reuptake inhibitor. Saline-treated hamsters showed high levels of AAS withdrawal-induced anxiety, while treatment with fluoxetine reduced AAS withdrawal-induced anxiety. These findings indicate that early AAS exposure has potent anxiogenic effects during AAS withdrawal that are modulated, in part, by 5HT signaling.     

Characterization and application of fusidane antibiotic biosynethsis enzyme 3-ketosteroid-?1-dehydrogenase in steroid transformation

Microbial ?(1)-dehydrogenation is one of the most important transformations in the synthesis of steroid hormones. In this study, a 3-ketosteroid-?(1)-dehydrogenase (kstD(F)) involved in fusidane antibiotic biosynthesis from Aspergillus fumigatus CICC 40167 was characterized for use in steroid transformation. KstD(F) encodes a polypeptide consisting of 637 amino acid residues. It shows 51% amino acid identity with a kstD from Thermomicrobium roseum DSM 5159. Expression of kstD(F) in Escherichia coli and Pichia pastoris showed that all kstD(F) activity is located in the cytoplasm. This indicates that it is a soluble intracytoplasmic enzyme, unlike most kstDs from bacteria, which are membrane-bound. The expression of kstD(F) was performed in P. pastoris, both intracellularly and extracelluarly. The intracellularly expressed protein displayed good activity in steroid transformation, while the extracellularly expressed protein showed nothing. Interestingly, the engineered P. pastoris KM71 (KM71(I)) and GS115 (GS115(I)) showed different transformation activities for 4-androstene-3,17-dione (AD) when kstD(F) was expressed intracellularly. Under the same conditions, KM71(I) was found capable of transforming 1.0 g/l AD to 1,4-androstadiene-3,17-dione (ADD), while GS115(I) could transform 1.5 g/l AD to both ADD and boldenone (BD). The production of BD is attributed to a 17β-hydroxysteroid dehydrogenase in P. pastoris GS115(I), which catalyzes the reversible reaction between C17-one and C17-alcohol of steroids. The conversion of AD by GS115(I) and KM71(I) may provide alternative means of preparing ADD or BD. In brief, we show here that kstD(F) is a promising enzyme in steroid ?(1)-dehydrogenation that is propitious to construct genetically engineered steroid-transforming recombinants by heterologous overexpression.     

Prolonged treatment with the anabolic–androgenic steroid stanozolol increases antioxidant defences in rat skeletal muscle

Testosterone and its synthetic derivatives anabolic–androgenic steroids have been shown to increase skeletal muscle work capacity and fatigue resistance, but the molecular basis for these effects remains uncertain. Since muscle performance has been related to redox status of exercising muscles, this investigation was aimed at testing whether a treatment with suprapharmacological doses of the anabolic–androgenic steroid stanozolol, (2 mg/kg body weight, 5 days/week, for 8 weeks), either alone or in conjunction with treadmill training (12 weeks), enhanced antioxidant defences in rat muscles. Stanozolol treatment did not modify thiobarbituric acid reactive substances and glutathione content in soleus and extensor digitorum longus (EDL) homogenates. In soleus from sedentary rats, superoxide dismutase and glutathione reductase activities were increased by 25% (P < 0.05) and by 40% (P < 0.01) after stanozolol administration, whereas catalase and glutathione peroxidase activities were not modified. This response was similar to that induced by training alone. In EDL from sedentary rats, stanozolol increased only superoxide dismutase activity (20%, P < 0.05). In no case, the effects of steroid administration and training were additive. HSP72 levels were up-regulated in soleus (1.5-fold, P < 0.01) and EDL (threefold, P < 0.001) following training but remained unchanged after stanozolol treatment. Endurance capacity, assessed in a treadmill endurance test, was similar for treated and control rats. We conclude that stanozolol treatment increases antioxidant capacity in selected skeletal muscles from sedentary rats. However, the steroid was not effective in improving endurance capacity or enhancing the training effects on muscle antioxidant defence systems.