Journal of steroid biochemistry最新文献

Steroidogenesis was studied in guinea-pig glomerulosa-fasciculata cells maintained in primary culture for up to 7 days. The basal secretion which remained stable for the first 2 days in culture rapidly rose to reach a plateau on day 4 at levels 6–7-fold higher than those observed during the first 2 days of culture while the maximal response to ACTH in terms of cortisol and androstenedione secretion was fairly stable throughout the 7-day period. Exposure of glomerulosa-fasciculata cells to ACTH caused a stimulation of pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione, 11β-hydroxyandrostenedione and aldosterone while, after 48 h of incubation, a marked accumulation of end-products, namely cortisol and 11β-hydroxyandrostenedione, was observed. The half-maximal steroidogenic response to ACTH occurred at concentrations varying between 1.7 × 10⁻¹ and 1.1 × 10⁻¹⁰ mol/l for the 12 steroids examined. Addition of 8-bromoadenosine 3', 5'-cyclic monophosphate stimulated steroid secretion in a dose-dependent manner. Maximal response to 8-bromoadenosine 3', 5'-cyclic monophosphate was obtained at 1 mmol/l, and no further rise of steroid secretion was observed after addition of ACTH. Incubation of glomerulosa-fasciculata cells with labeled corticosterone, cortisol and androstenedione indicates that only androstenedione can be converted into 11β-hydroxyandrostenedione, thus suggesting that this end-product is a good parameter of the C-19 steroid production by guinea-pig glomerulosa-fasciculata cells in primary culture. The present data confirm that guinea-pig glomerulosa-fasciculata cells in primary culture provide an interesting model for the study of the regulation of C-19 steroid formation by the adrenals.

Human placental 17β-hydroxysteroid dehydrogenase (17-HSD) was purified to apparent homogeneity using ammonium sulfate precipitation and chromatography on Red-Agarose and DEAE-Sepharose columns. Electrophoresis on polyacrylamide gels under denaturing conditions and using silver staining showed a single protein with an apparent molecular weight of 37,800. Antibodies to the purified protein were raised in rabbits and were found by immunoblotting to be specific to 17-HSD.

A sensitive radioimmunoassay was established using ¹²⁵I-labeled 17-HSD as a tracer, an appropriate dilution of the antibody, and a kaolin-coupled double antibody for separating the antibody-bound and free fractions. The detection limit of the assay was approximately 150 pg/tube (1.5 μg/l). The cytosol fraction (105,000 g) of term placental tissue contained approximately 0.7 mg of 17-HSD per gram of protein, and the concentrations of 17-HSD measured by immunoassay and enzymatic activity proved to be strictly parallel in different partly purified placental preparations.

The supernatants from centrifugations of human endometrial homogenates at 800 g and 105,000 g (after detergent treatment) displayed cross-reactivity with the antibody. The mean concentration of the cross-reacting substance in the radioimmunoassay was 14.1 μg/g protein (range 2–62.3) in specimens taken on different days in the cycle. These concentrations showed a significant correlation with the 17-HSD activities measured in the endometrial specimens (r = 0.722, P < 0.001, n = 21). Mean concentrations of substance were 8.3 μg/g protein in endometrial specimens taken during the follicular phase (days 4–12, n = 8) and 22.9 μg/g protein during the luteal phase (days 16–22, n = 6) were obtained using the radioimmuoassay. There was excellent parallelism between the competition curves for [¹²⁵I]iodo-17-HSD with purified 17-HSD standards and placental and endometrial homogenate dilutions. These data strongly suggest that the substance measured in the endometrial specimens was 17-HSD.

A comparative study of the tissue distribution of five tritium-labeled androgens was done in rats to determine the efficiency and selectivity of their uptake by target tissue. Testosterone (T), 5α-dihydrotestosterone (DHT), 19-nortestosterone (nor-T), mibolerone (Mib) and methyltrienolone (R1881) all showed selective uptake by the ventral prostate in one-day castrated rats (250 g) that was 61–90% displaceable by co-injection of an excess of unlabeled steroid. The greatest uptake was with R1881 (0.69% injected dose per gram prostate tissue (%ID/g) at 1 h), and Mib (0.56% ID/g); the other three showed lower uptake (approx. 0.4% ID/g). The target tissue activity remained high for all compounds up to 4 h after injection, and at 2–4 h the prostate to blood ratio for Mib and R1881 exceeded 10 and 20, respectively. The uptake efficiency and selectivity of these five androgens appear to be related to their affinity for the androgen receptor and their resistance to metabolism. Mib and R1881 have substantial affinity for other steroid receptors, which might account for some of their prostate uptake. However, co-administration of triamcinolone acetonide, which has high affinity for progesterone and corticosteroid receptors but not for the androgen receptor, failed to block their uptake significantly, whereas co-administration of DHT, the most selective ligand for the androgen receptor, blocked their uptake as completely as the unlabeled tracer itself. The prostate uptake of Mib and R1881 in intact animals was significantly lower than in castrated animals, but treatment of the intact animals with diethylstilbestrol restored their uptake nearly to the level seen in castrated animals. These uptake patterns are consistent with earlier studies of in vivo androgen uptake and with known changes in androgen receptor content and occupancy as a result of castration or diethylstilbestrol treatment. They further suggest that high affinity androgens labeled with suitable radionuclides—particularly derivatives of mibolerone (Mib) or methyltrienolone (R1881)—may be effective receptor-based imaging agents for androgen target tissues and tumors, even when patients are already receiving hormonal therapy.

The mechanisms by which ethanol (EtOH) inhibits the human chorionic gonado-tropin (hCG)-stimulated testosterone synthesis was studied in isolated rat Leydig cells in vitro. EtOH inhibited steroidogenesis, but this inhibition was reversed by l-glutamate (Glu) and an uncoupler of the oxidative phosphorylation, 2,4-dinitrophenol (DNP). The mechanism of EtOH-induced inhibition was studied by measuring steroidogenic precursors and comparing them with the cytosolic and mitochondrial NADH redox states during uncoupling or in the presence of Glu. DNP had a dual effect. Low concentrations abolished the EtOH-induced inhibition of progesterone to testosterone formation suggesting that the inhibitory step was at or before progesterone formation. A large concentration led to an overall decrease in steroidogenesis indicating toxic effects on steroidogenesis. The mitochondrial NADH/NAD⁺ ratio, measured as the 3-hydroxybutyrate/acetoacetate ratio, decreased simultaneously when steroidogenesis was stimulated, either during uncoupling or in the presence of Glu, whereas cytosolic NADH/NAD⁺ ratio, measured as lactate/pyruvate ratio showed no response. These results demonstrate that the rise in the mitochondrial NADH/NAD⁺ ratio rather than in the cytosolic one is connected with the inhibition of testosterone synthesis by EtOH in isolated Leydig cells. The EtOH-induced high mitochondrial NADH/NAD⁺ ratio may deplete mitochondrial oxalacetate concentrations. This can decrease the activity of several transport shuttles and interrupt the flow of mitochondrial citrate into the smooth endoplasmic reticulum, which then reflects to decreased rate of steroidogenesis in the presence of ethanol.

The functional relationship between the microsomal cytochrome P450 and 17β-hydroxysteroid oxidoreductase (HSOR) enzymes involved in steroid metabolism was investigated in rat liver. In male and female rat hepatic microsomes the NADPH-dependent conversion of androstenedione (AD) to testosterone (T) was approx. 4-fold greater at 6 weeks of age than in 1 week old animals. In hepatic microsomes from 15 week old rats the activity of the HSOR pathway was greater in males than in females (1.51 compared to 0.80 nmol T formed/min/mg protein). However, oestradiol administration to intact adult male rats did not decrease HSOR activity. Thus, androgen is not essential for maintenance of HSOR enzymes. Instead, it is likely that irreversible androgen imprinting of the HSOR enzyme occurs during the prepubertal period.

The in vitro characteristics of HSOR activity were also assessed. The K_m for NADH-dependent reduction of AD to T was 9.2 μM and the V_max was 3.0 nmol/min/mg protein but the NAD-mediated formation of AD from T did not follow Michaelis-Menton kinetics. pH markedly influenced HSOR-mediated AD/T interconversion with 17-ketosteroid reduction facilitated at low pH, and 17β-hydroxysteroid dehydrogenation about 2-fold more efficient at pH 8.0 than at pH 5.5. Product steroid activation of HSOR activity was noted. 17β-Hydroxy-steroids, including T and oestradiol, activated the rate of conversion of AD to T and 17-ketosteroids such as oestrone and AD activated the NAD-dependent dehydrogenation of T. Activation was not observed at low steroid substrate concentrations so that it was not possible to analyse this phenomenon by a conventional kinetic approach.

For many years, hypersecretion of estrogens has been suspected of being one of the major risk factors of breast cancer for premenopausal women.

Seventeen premenopausal women, who had undergone lumpectomy because of breast cancer (Tla No Mo) 3 yr before entering the study, were compared to 9 normal women of similar age, parity and body weight. A chemiluminescent method was used for the determination of estrone-3-glucuronide (E1-3G) and pregnanediol-3-glucuronide (Pd-3G) in early morning urine samples collected for an entire menstrual cycle of each of the 26 subjects.

During the follicular phase, no significant differences in E1-3G and/or Pd-3G excretion were found between the two groups. During the luteal phase the E1-3G/Pd-3G ratio in the early, middle and late luteal phase had significantly increased in the women with breast cancer, in spite of normal Pd-3G excretion. Therefore, the measurement of glucuronoconjugate metabolites of ovarian hormones in overnight urine might be conveniently applied to the study of ovarian function in subjects with breast cancer. Furthermore, the results of this study may indicate that an estrogen/progesterone imbalance is an additional risk factor for the premenopausal breast cancer patient.

Rat uterus fixed overnight in buffered formalin retains the ability to specifically bind estradiol. However, the estrogen binding property of fixed tissue appears preferentially localized in the nuclear fraction regardless of hormonal status. Furthermore, the quantity of the nuclear estrogen receptor in fresh or fixed uterus is virtually identical in the presence or absence of estrogenic hormone. Yet, while both tissue preparations exhibit equivalent increases in the total nuclear receptor occupancy after hormone exposure, only the fresh uterus contains a major cytosolic estrogen binder which decreases in availability upon the estrogen-induced elevation of the nuclear bound steroid. However, the cytosolic estrogen receptor exhibits a significant loss in its ligand binding property after formalin exposure. Thus, the preferential localization of estrogen binding in the nuclear fraction of fixed whole tissue may just reflect that only the tightly bound nuclear estrogen receptor's functional and/or structural integrity survives long-term formalin fixation. Our observation of estrogen binding in preserved tissue may also be a clinically useful tool in therapy analysis.

The effects of a physiological dose of IGF I (40 ng/ml ≈ 5 × 10⁻⁹ M) on steroidogenesis were studied in bovine adrenal fasciculata cells cultured in serum-free McCoy's medium. They were compared with those of a single dose of ACTH (0.25 ng/ml ≈ 10⁻¹⁰ M) at approximately the concentration inducing half-maximal stimulation. With IGF I, steroidogenesis commenced after 48 h culture and progressively increased throughout the 96-h test period. Expressed as stimulated level/control level ratios, glucocorticoid (cortisol + corticosterone) responses to IGF I after 4 days' culture (2.41 ± 0.20 (SEM) n = 9) were similar to those obtained with ACTH (2.59 ± 0.18, n = 9). A combination of the two peptides had a synergistic effect (5.95 ± 0.79, n = 5).

The cortisol/corticosterone ratio increased in the presence of IGF I from 1 ± 0.19 to 1.76 ± 0.45 (n = 7, P < 0.02), although less so than in the presence of ACTH (5.50 ± 0.98). Moreover, cortisol production was accompanied by adrostenedione production (2.36ng/10⁶ cells, n = 3) similar to that induced by ACTH (2.10 ng/10⁶ cells, n = 3). These findings together suggest stimulation of 17α-hydroxylase activity. Cell multiplication was unaffected by IGF I. [³H]Thymidine incorporation into DNA reached only 193% ± 17 (SEM) (n = 4) of control levels, whereas with ACTH it dropped to 60% ± 5.

Our findings show that IGF I alone has no mitogenic effect on adrenocortical cells in vitro, but that it is capable of inducing differentiated steroidogenesis.

Cobalt-protoporphyrin (CoPP) administration to adult male rats results in a profound reduction in hepatic cytochrome P-450 concentration and activity, and decreased plasma concentrations of testosterone and luteinizing hormone (LH). The metabolism of progesterone by rat testicular microsomes isolated 48 h after treatment in vivo with CoPP was compared to that in microsomes from control rats. The conversion of progesterone to 17α-hydroxyprogesterone and 4-androstenedione, which is NADPH-dependent, was reduced by approximately 40% in testicular microsomes following treatment with CoPP (50 μmol/kg body weight) and this inhibition was dose-dependent. The concentration of cytochrome P-450 in testicular microsomes and the activity of 7-ethoxycoumarin de-ethylase (a cytochrome P-450 dependent function) were also reduced following treatment with CoPP in contrast to two other functional assays of cytochrome P-450, aryl hydrocarbon hydroxylase and ethylmorphine demethylase, which were unaffected by treatment with CoPP. Thus, the profound effect of CoPP on androgen homeostasis has been extended to include decreased testicular synthesis of 4-androstenedione in addition to increased hepatic metabolism of testosterone, attenuated pituitary LH release in response to luteinizing hormone-releasing hormone, and failure of testicular response to LH.

Binding of [³H]estradiol, [³H]testosterone and [³H]progesterone to purified NADP-dependent estrophilic 3α,3β,17β,20α-hydroxysteroid dehydrogenase (EHSD) from rabbit liver cytosol has been examined. The three steroids bind to the enzyme with mode-rate affinity (K_a −; 10⁻¹⁷ M⁻¹ at 4°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 20α-reduction of progesterone; (iii) stimulatory effect of 5α(β)-androstane-3α (β), 17β-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.