Estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase from rabbit liver--II. Mechanisms of enzyme-steroid interaction

Binding of [3H]estradiol, [3H]testosterone and [3H]progesterone to purified NADP-dependent estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase (EHSD) from rabbit liver cytosol has been examined. The three steroids bind to the enzyme with moderate [corrected] affinity (Ka congruent to 10(7) [corrected] M-1 at 4 degrees C) and equal binding capacity. High-rates were shown for both association and dissociation processes. The steroids competitively inhibited the binding of each other to EHSD. At the same time, their relative binding affinities (RBA) were dependent on the nature of [3H]ligand. The results of RBA determinations for 72 steroids and their analogues by inhibition of [3H]progesterone binding to EHSD suggest that androgens and gestagens bind preferentially to the same site on EHSD molecule, while estrogens (at least by their D-ring) bind to another site. The assumption that EHSD molecule has more than one binding site for steroids is corroborated by (i) substrate inhibition revealed for a number of steroids; (ii) the estrogen ability to potentiate 20 alpha-reduction of progesterone; (iii) stimulatory effect of 5 alpha (beta)-androstane-3 alpha (beta), 17 beta-diols on [3H]testosterone and progesterone binding; and (iv) reciprocal effect of NADP on [3H]estradiol and [3H]testosterone binding to EHSD. Significant differences in sensitivity to pH and changes in NaCl concentration upon metabolism and binding of various steroids have been found. At concentrations of 16 mM dithiothreitol potentiated catalytic conversion of some steroids and had no effect on metabolism of others. Both the affinity for steroids and binding capacity of EHSD are found to be cofactor-dependent. It is speculated that EHSD has a complex active center including at least two mutually influencing steroid-binding sites tightly related with cofactor-binding site. The polyfunctionality of EHSD may be due to both the excess of functional protein groups that form individual constellations upon binding of any steroid and also to conformational lability of EHSD molecule implying alternative orientations of steroids at the binding site.     

Reversed-phase high-performance liquid chromatographic screening method for serum steroids using retention index and diode-array detection

A multisteroid screening method has been developed based on the use of 1-[4-(2,3-dihydroxypropoxy)phenyl]-1-alkanones as retention index standards and UV absorbance spectra recorded on-line with a diode-array detector using reversed-phase high-performance liquid chromatographic gradient elution with acetonitrile and water. The effect of chromatographic conditions on retention indices of steroids were studied. The method was tentatively applied to profiling of steroids in serum samples.     

Binding of glucocorticoid antagonists to androgen and glucocorticoid hormone receptors in rat skeletal muscle

The binding of ten steroids possessing antiglucocorticoid activity has been studied in rat skeletal muscle cytosol. The affinity of these steroids for both the androgen and the glucocorticoid receptors was determined by competition with radioactive R1881 (methyltrienolone, metribolone) and dexamethasone, respectively. The antiglucocorticoid activity of these compounds was assessed in rat hepatoma (HTC) cells by measuring their inhibitory effect on the glucocorticoid-induced tyrosine aminotransferase activity. This led to identification of five novel in vitro glucocorticoid antagonists. All the steroids tested bound to both the glucocorticoid and the androgen receptors in muscle. Four steroids had an affinity for the glucocorticoid receptor higher than for the androgen receptor. The assumption is made that the steroids tested also behave as antagonists when binding to the glucocorticoid receptor in muscle and behave as agonists when binding to the androgen receptor. On this basis, the data allow one to compute a potential anticatabolic (PAG) and a potential anabolic (PAA) index for each compound. These indices might be of predictive value to determine whether these steroids exert their anabolic action in muscle through the glucocorticoid receptor or through the androgen receptor. The data also make it unlikely that satellite cells are a preferential target for anabolic steroids in muscle.     

Tachykinins and ovarian function in mammals

Tachykinins are bioactive peptides whose presence has been demonstrated in endocrine glands, where they likely exert a paracrine modulatory activity on hormonal secretions. In the ovary, tachykinins have been shown to be present in nerve fibers, blood vessels, and in granulosa, luteal and interstitial cells. Tachykinin gene expression was shown in granulosa and luteal cells. Tachykinins have also been found in the follicular fluid. Substance P (SP) has been demonstrated to significantly affect the release of hormonal steroids by ovarian cells in vitro. While some authors found that SP stimulated the release of steroids, others found an inhibitory effect by the same tachykinin. Gonadotropins decrease tachykinin concentrations in the ovary. The neonatal treatment of rats with capsaicin, a drug that depletes SP in primary afferent neurons, resulted in a modest reduction in the reproductive success in rats. The experimental results listed in this review suggest that tachykinins are synthesized in the ovary, in the granulosa and luteal cells. Tachykinins are likely intraovarian modulators of the secretion of hormonal steroids. Their stores in the ovary are likely regulated by pituitary gonadotropins.     

Anabolic steroids: a review of their effects on the muscles, of their possible mechanisms of action and of their use in athletics.

Anabolic steroids are synthetic molecules developed in the hope of obtaining a complete separation of the androgenic and myotrophic (anabolic) actions of testosterone. Such a goal has never been fully achieved. However, some synthetic steroids present a partial dissociation between these two activities. Since a single hormonal receptor apparently mediates the androgenic as well as the anabolic actions of testosterone, differences in patterns of androgen metabolization in the muscles and the sex accessory organs have been proposed as a possible cause of this phenomenon. Undoubtedly, androgens are able to exert a trophic effect on skeletal and cardiac muscle fibres in subjects with low circulating levels of testosterone such as prepubertal or hypogonadal males and females; however, the widespread use of anabolic steroids in male athletes to increase their physical performances poses the question of whether these compounds are active in the presence of normal circulating levels of testosterone. Most experimental animal studies indicate that anabolic steroids are ineffective in this situation. Since the results of the experiments performed in humans are largely contradictory, it is still not clear whether anabolic steroids are able to improve athletic performances. These and other relevant issues are reviewed.     

THE OUTWARD TRANSPORT OF CORTISOL BY MAMMALIAN CELLS IN VITRO

It has been determined that cortisol and a few other steroids are transported outward from certain mammalian cells growing in vitro. The extrusion process is temperature dependent, glucose dependent, saturable, and operates for only a few selected steroids. Many, but not all, steroids are able to block the extrusion process but are not themselves transported. The outward transport process for steroids has been found in mouse fibroblasts, mouse lymphoma cells, and functional mouse adrenal gland tumor cells growing in vitro. The transport process is not present in two varieties of cells cultured from human sources—HeLa or diploid fibroblasts, WI-38.     

Method development for corticosteroids and anabolic steroids by micellar liquid chromatography

A systematic optimization of the HPLC separation of a complex mixture containing urinary steroids (anabolics and corticoids), boldenone and bolasterone (synthetic anabolics) by micellar liquid chromatography has been carried out. The isocratic micellar mobile phases (from binary to quaternary) consisted of sodium dodecyl sulphate and organic modifiers such as acetonitrile, tetrahydrofuran, propanol, butanol or pentanol. The effect of the organic modifiers, surfactant concentration, temperature, ionic strength and flow-rate on the separation has been studied. A micellar mobile phase made of 5% propanol and 40 mM surfactant allowed the separation of 13 steroids in about 23 min. A bivariant optimization method for the micellar mobile phase surfactant-propanol corroborated the above results. The separations obtained show good perspectives for future developments.     

Role of cyclic AMP in steroid action in rat intestine

We have investigated the effect of mineralocorticoids on intestinal fluid and electrogenic glucose-linked Na+ transport across isolated sections of the rat small intestine. A rapid in vitro response is observed that contrast with the delay normally associated with steroid hormone responses. Cyclic AMP is known to affect intestinal glucose, water and Na+ transport and the effects of the steroids may be understood in terms of an inhibitory effect on intestinal cyclic AMP production. An inhibitory effect of the steroids on membrane-bound adenylate cyclase has been demonstrated and dose-response effects suggest the presence of specific membrane-bound glucocorticoid receptors.     

Neuroactive steroids alter the circadian system of the Syrian hamster in a phase-dependent manner

Several steroid compounds affect neuronal function, primarily by modulating the GABAA receptor complex. A circadian variation in the brain concentration of neurosteroids has been reported in rats and humans. We have previously reported that natural occurring or synthetic neuroactive steroids such as androsterone and alphaxalone also have a rhythmic effect on behavior (anesthetic and anticonvulsant activity) and GABAergic activity. In the present work, we have tested the ability of neuroactive steroids to phase shift circadian rhythms in hamsters. The GABA(A) negative modulator dehydroepiandrosterone sulphate (DHEAS) elicited phase advances when administered at CT 6, while the positive modulator androsterone lacked any effect at this time. A complete phase response curve for DHEAS revealed a nonphotic-like effect. DHEAS also blocked the circadian effects of light, while androsterone induced photic-like responses. There is also evidence that neurosteroids may be present and even synthesized in the SCN. Collectively, the results so far indicate that some neuroactive steroids might modulate the activity of the circadian clock.     

Peripheral oestrogen metabolism in postmenopausal women with or without breast cancer: the role of dietary lipids and growth factors

Studies we have carried out have revealed significant differences in oestrogen production and metabolism between normal women and postmenopausal women with breast cancer. The free, biologically available fraction of oestradiol is elevated in plasma from women with breast cancer and we have found that metabolic clearance rates and production rates of oestradiol are also increased. In vitro studies have suggested that lipids can influence the distribution of sex steroids in plasma and we have therefore examined the effect of dietary lipids on the distribution of sex steroids in plasma in vivo. Consumption of a meal with a high saturated fat content or the oral or i.v. administration of "Intralipid", a stabilised emulsion of soya bean oil that is high in unsaturated free fatty acids, had little effect on the available fractions of oestradiol in plasma. However, results from a preliminary study suggest that long-term changes in dietary fat intake can alter the distribution of steroids in plasma. It is concluded that dietary lipids may influence the availability of sex steroids to tissues. Such a mechanism could account for the significant correlation that has been found between dietary fat consumption and the incidence of breast cancer on a world-wide basis.     

Comparison of different high-performance liquid chromatographic systems for the purification of adrenal and gonadal steroids prior to immunoassay

The high-performance liquid chromatography of nineteen hormonal steroids with special respect to its suitability for routine purification of these steroids from crude, organic extracts of biological fluids prior to final quantitation by immunoassay has been studied. In all systems the gradient elution technique was applied. Separation of steroids has been investigated using different stationary phases chemically coated with non-polar, hydroxyl, NO₂ and CN groups. Reproducibility of retention times was studied on a stationary phase coated with hydroxyl groups (DIOL column) using different organic eluents. Coefficients of variation range from 0.76 to 8.16%. Reproducibility was shown to be unequivocally better in the gradient part than in the isocratic part of the chromatographic run. In contrast to the other steroids, 18-hydroxylated steroids were more or less unstable in certain systems studied. As to resolution and reproducibility, the DIOL column run with an n-hexane—dioxane gradient has been shown to be superior to the other systems studied.     

Optimization of the separation of a complex mixture of natural and synthetic anabolic steroids by micellar liquid chromatography

A systematic optimization of the HPLC separation of a complex mixture containing natural and synthetic anabolic steroids by micellar liquid chromatography using a Hypersil (150 mm x 3.0 mm i.d., 5 microm) C18 column and UV detection at 245 nm (exception is made for oxymetolone and danazol which were monitorized at 280 nm) has been carried out. The isocratic micellar mobile phases (from binary to quaternary) consisted of sodium dodecyl sulphate and organic modifiers such as acetonitrile, tetrahydrofuran, propanol, butanol or pentanol. The effect of the organic modifiers, surfactant concentration, temperature, ionic strength and flow-rate on the separation has been studied. A micellar mobile phase 5% propanol and 40 mM surfactant allowed the separation of 12 steroids out of 14 tested in about 20 min. A bivariant optimization method for the micellar mobile phase propanol-surfactant corroborated the above results.     

The pH and temperature dependence of the interaction of steroid hormones with the transport system of glucose in human erythrocytes.

The thermodynamic parameters deltaH degrees, deltaF degrees, deltaS degrees and the pH dependence of the interaction between steroids and the glucose carrier in human erythrocytes have been studied. The results indicate that, according to the structure of the steroids, hydrophilic as well as hydrophobic bonds can be involved in the association. For the binding of the C-21-steroids to the carrier the polarity rule has been observed to be valid. On the basis of the thermodynamic parameters it has been found that in the association process of the steroids with the carrier, simultaneous randomization of ordered water molecules is important.     

Metabolism and excretion of anabolic steroids in doping control--new steroids and new insights

The use of anabolic steroids in sports is prohibited by the World Anti-Doping Agency. Until the 1990s, anabolic steroids were solely manufactured by pharmaceutical companies, albeit sometimes on demand from national sports agencies as part of their doping program. Recently the list of prohibited anabolic steroids in sports has grown due to the addition of numerous steroids that have been introduced on the market by non-pharmaceutical companies. Moreover, several designer steroids, specifically developed to circumvent doping control, have also been detected. Because anabolic steroids are most often intensively subjected to phase I metabolism and seldom excreted unchanged, excretion studies need to be performed in order to detect their misuse.

This review attempts to summarise the results of excretion studies of recent additions to the list of prohibited steroids in sports. Additionally an update and insight on new aspects for “older” steroids with respect to doping control is given.     

Hormone-induced Maturation of Xenopus laevis oocytes: Effects of Different Steroids and Study of the Properties of a Progesterone Receptor

The ability of various steroids to induce maturation of Xenopus laevis oocytes after incubation or injection has been studied.
Only two of these hormones, hydrocortisone and testosterone, are active by injection as well as by incubation. Progesterone can induce maturation only when administered from the outside medium, which suggests the presence of a receptor in or near the membrane.
This progesterone-receptor has been localized in the melanosomes, and its properties and affinity for other steroids have been studied. The morphological and biochemical effects of the injection of the progesterone-receptor complex have been studied.     

Do mollusks use vertebrate sex steroids as reproductive hormones? II. Critical review of the evidence that steroids have biological effects

In assessing the evidence as to whether vertebrate sex steroids (e.g. testosterone, estradiol, progesterone) have hormonal actions in mollusks, ca. 85% of research papers report at least one biological effect; and 18 out of 21 review papers (published between 1970 and 2012) express a positive view. However, just under half of the research studies can be rejected on the grounds that they did not actually test steroids, but compounds or mixtures that were only presumed to behave as steroids (or modulators of steroids) on the basis of their effects in vertebrates (e.g. Bisphenol-A, nonylphenol and sewage treatment effluents). Of the remaining 55 papers, some can be criticized for having no statistical analysis; some for using only a single dose of steroid; others for having irregular dose–response curves; 40 out of the 55 for not replicating the treatments; and 50 out of 55 for having no within-study repetition. Furthermore, most studies had very low effect sizes in comparison to fish-based bioassays for steroids (i.e. they had a very weak ‘signal-to-noise’ ratio). When these facts are combined with the fact that none of the studies were conducted with rigorous randomization or ‘blinding’ procedures (implying the possibility of ‘operator bias’) one must conclude that there is no indisputable bioassay evidence that vertebrate sex steroids have endocrinological or reproductive roles in mollusks. The only observation that has been independently validated is the ability of estradiol to trigger rapid (1–5 min) lysosomal membrane breakdown in hemocytes of Mytilus spp. This is a typical ‘inflammatory’ response, however, and is not proof that estradiol is a hormone – especially when taken in conjunction with the evidence (discussed in a previous review) that mollusks have neither the enzymes necessary to synthesize vertebrate steroids nor nuclear receptors with which to respond to them.     

Rapid and efficient method for extraction and separation of glucocorticosteroids and sex steroids from urines

The influence of different pH values on the recoveries of glucocorticosteroids and sex steroids from Kieselguhrfilled minicolumns has been investigated. While the recoveries of all steroids tested were similar if samples had acidic or neutral pH values, sex steroids could effectively be separated from glucocorticosteroids by increasing the pH value of 13.7: recoveries were 1.7% for glucocorticosteroids and 56–76% for sex steroids. For the determination of sex steroids in biological samples it is recommended to adjust samples to a strong alkaline pH before extraction; this holds especially true for samples with very high glucocorticosteroid levels.     

Role of Luteinizing Hormone in Regulation of the Rat Estrous Cycle

The role of luteinizing hormone (LH) in regulation of secretionof ovarian steroids during the preovnlatory phase of the ratestrous cycle has been discussed. Evidence indicating an involvementof these steroids in control of the uterine growth, vaginalcornification, mating behavior and LH secretion which occurduring this phase has been reviewed. A positive feedback controlsystem has been proposed in which both ovarian and adrenal steroidsparticipate to initiate the preovulatory "LH surge" which culminatesin ovulation and luteinization     

Partial characterization of the cytosol 3 alpha-hydroxysteroid: NAD(P)+oxidoreductase of rat ventral prostate.

The cytosol 3alpha-hydroxysteroid dehydrogenase of rat ventral prostate has been partially purified. The rates of both the oxidation and reduction by crude and partially purified enzymes have been measured with a variety of radioactive substrates, and the effects of several inhibitor steroids have been assessed. Four conclusions have been drawn from the study. First, no detectable 3beta-androstanediol was formed from dihydrotestosterone, and the oxidation of 3beta-androstanediol was undetectable. Second, the cytosol enzyme exhibits a distinct and unique substrate specificity in that steroids with keto or hydroxyl substitution on the 11th carbon of the steroid cannot serve as substrates or as inhibitors of the enzyme. Third, either 5alpha or 5 beta reduction of delta4,3-keto steroids must take place before the steroids can serve as substrates of the enzyme. Fourth, many delta4,3-keto steroids that cannot act as substrates for the enzyme inhibit the enzyme competitively and may well serve as physiological regulators of the reaction in intact cell.