Steroid dynamics in the rabbit

Male rabbits were infused at a constant rate with 3H-androstenedione/14C-estrone (n = 5) or 3H-testosterone/14C-estradiol-17 beta (n = 3) for 3 1/2 hr and blood samples were obtained over the last hour and analyzed for radioactivity as androstenedione (A), testosterone (T), estrone (E1), estradiol-17 beta (E2 beta) and estradiol-17 alpha (E2 alpha). The mean value for the metabolic clearance rate of androstenedione (MCRA) was 85 +/- 10 l/day/kg, which was significantly greater than the mean MCRE1 59 +/- 10 l/day/kg. MCRT, 42 +/- 8 l/day/kg, and MCRE2 beta, 45 +/- 9 l/day/kg were not different. The conversion ratio of androstenedione to testosterone (CRA,T) was greater than CRT,A but for the estrogens, CRE2 beta, E1 was greater than CRE1,E2 beta. CRE2 beta, E2 alpha was greater than CRE1,E2 alpha. The overall aromatization of androstenedione to estrone, the fraction of 3H-androstenedione infused into the blood and measured as 3H-estrone in blood [( rho]A,E1BB) was 0.0005 +/- 0.0001 and for [rho]T,E2 beta BB was 0.0012 +/- 0.0006. In the rabbit both sex hormone binding globulin (SHBG) and albumin binding may effect the MCRs, and peripheral aromatization of androgens occurs to a far lesser degree than in humans and primates.     

Estrogen metabolism as measured in blood and urine in female rhesus monkeys

In order to measure the interconversions of estrone (E1) and estradiol (E2) and their conversion to the 16 alpha-hydroxylated estrogens, 16 alpha-hydroxy estrone (16 alpha-OHE1) and estriol (E3), we infused 11 female rhesus monkeys with [3H]E2 and [14C]E1 and measured radioactivity in the blood as E1, E2 and 16 alpha-OHE1 (n = 9) and in the urine as the glucuronides of E1, E2, 16 alpha-OHE1, and E3 (n = 11). The mean conversion of E1 to E2 as measured in blood (percent of infused E1 measured in blood as E2, [rho]1.2BB) was 29.2 +/- 1.6% and as measured in the urine of the same animals, [rho]1.2BM, was 77.4 +/- 5.9%. The mean conversion of E2 to E1, [rho]2.1BB was 21.5 +/- 1.0% and as measured in urine, [rho]2.1BM was 67.7 +/- 4.6%. Thus for both estrone and estradiol only 30-35% of the interconversions occurred in pools which were in equilibrium with the blood pool of these estrogens. The remaining 65-70% occurred in a pool, probably liver, in which glucuronidation occurred immediately after conversion. The conversion ratios (the ratio of the concentration in the blood of radioactivity as 16 alpha-OHE1 to its precursor, CRPrec,16 alpha-OHE1) was 0.036 +/- 0.008 for CRE1,16 alpha-OHE1 and 0.0039 +/- 0.0010 for CRE2,16 alpha-OHE1. The percentages of administered E1 excreted in the urine as the glucuronides of E1, E2, 16 alpha-OHE1 and E3 were 20.1 +/- 1.5, 1.6 +/- 0.2, 0.96 +/- 0.20 and 0.76 +/- 0.07 respectively. The percentages of administered E2 excreted in the urine as E1, E2, 16 alpha-OHE1 and E3 were 14.4 +/- 1.0, 2.2 +/- 0.3, 0.57 +/- 0.05 and 0.68 +/- 0.11 respectively. Thus there are minor differences in the patterns of excreted metabolites of E1 and E2. Furthermore, 16 alpha-OHE1 and E3 are not major metabolites of E1 or E2 in the female rhesus monkey.     

Androgen and estrogen dynamics in the female baboon (Papio anubis)

Androgen and estrogen dynamics were studied in 5 female baboons (Papio anubis) using constant infusions of [3H]androstenedione/[14C]estrone and [3H]testosterone/[14C]estradiol. Blood samples were obtained prior to the infusions and both blood and plasma was used for measurements of androstenedione (A), testosterone (T), dihydrotestosterone (DHT), estrone (E1), estradiol (E2). Plasma was used for measurements of sex-hormone binding globulin (SHBG), and the percents of T and E2 free, bound to SHBG, and to albumin. Blood samples obtained during the infusions were analyzed for radioactivity as purified androgens and estrogens. Metabolic clearance rates (MCR), and transfer factors ([rho]BB; fraction of steroid infused which is converted to and measured in blood as product) and blood production rates were calculated from whole blood data. All urine was collected for 96 h and an aliquot analyzed for radioactivity as the glucuronides of estrone and estradiol and the % peripheral aromatization calculated. The MCR's, calculated in whole blood, of A, E1, E2 and T were 53 +/- 6 1/day/kg, 39.3 +/- 3 1/day/kg, 29.9 +/- 5.2 1/day/kg and 10.1 +/- 2.3 1/day/kg, respectively. Each MCR was different (P less than 0.05) from the others. The PB of E1 was 15 +/- 2 micrograms/day and was not different from that of E2 (12 +/- 3 micrograms/day). The PB of A, 231 +/- 55 micrograms/day, was greater than that of T, 13 +/- 5 micrograms/day. The interconversions of both the androgens (18.9 +/- 3.4% vs 3.9 +/- 1.0%) and the estrogens (48.8 +/- 10.7% vs 4.0 +/- 0.8%) favored the oxidative pathway, i.e. conversion of 17-OH to 17-oxo steroids. The conversion ratio of A to DHT was greater than that of T to DHT (16.4 +/- 2.1% vs 5.3 +/- 0.7%), and A is a more important source of DHT than is T. The percent of T bound to SHBG (80.7 +/- 0.9%) was greater than percent of E2 (36.9 +/- 9.8%) and inversely the percents of T bound to albumin and free (17.5 +/- 0.8% and 1.65 +/- 0.16%) were less than the respective percents for estradiol (60.5 +/- 9.5% and 2.40 +/- 0.27%). The mean SHBG concentration was 54 +/- 6 nM. The peripheral aromatization of androstenedione, 1.36 +/- 0.05%, was greater than of testosterone, 0.18 +/- 0.02%. This difference is, in part, due to the lack of SHBG-binding of androstenedione. The general pattern of androgen and estrogen dynamics is similar to that in women. This similarity is due, in part, to the presence of SHBG in both baboons and women.     

Regulation of sex hormone-binding globulin secretion in human hepatoma G2 cells.

Our purpose was to examine the roles of natural (estradiol (E2) and estrone (E1)) and synthetic estrogens (ethinyl estradiol (EE), moxestrol (MOX), and tamoxifene (TAM)) in regulating production of sex hormone-binding globulin (SHBG) by human hepatoma G2 (Hep G2) cells, the rationale being that synthetic estrogens are less rapidly metabolized than natural estrogens and, thus, may alter SHBG levels more readily. In Hep G2 cells, E2, E1, and EE at 10(-7) M did not result in significantly greater SHBG secretion compared to control cells. The synthetic estrogens, MOX and TAM, caused significant, P < 0.001, increases of 30% and 51% in SHBG secretion at 10(-7) M compared to controls. However, when TAM and E2 were added together, each at 10(-7) M, no increase in SHBG secretion was noted. We conclude that natural estrogens at physiologic concentrations do not increase SHBG secretion by Hep G2 cells, but the increase of SHBG secretion caused by MOX and TAM suggests that the lack of effect of E2 and E1 may, in part, be due to their rapid metabolism. In addition, TAM stimulates SHBG secretion by interaction with the genome that is different, in certain respects, from that of E2.     

Contraceptive steroid treatment affects steroid binding proteins and the percentage of free 17 beta-estradiol and testosterone in the cynomolgus monkey (Macaca fascicularis)

The levels of steroid binding globulins were characterized in cynomolgus monkeys that were treated with contraceptive steroid preparations delivered either by intravaginal rings (CVR) or orally (OC) in the diet. Levonorgestrel (dNG) was the bioactive progestin and the estrogen was either 17 beta-estradiol (E2) in the CVR treatment or ethinyl estradiol (EE) in the OC treatment. Both contraceptive treatments lowered sex hormone binding globulin (SHBG) levels below those observed in males (P less than 0.05) and normal females (P less than 0.01). Corticosteroid binding globulin (CBG) was elevated (P less than 0.01) in the OC treatment, demonstrating the potency of EE. The distribution of E2 and testosterone (T) between binding to SHBG or albumin and the unbound fraction was calculated after the determination of the percentage of free steroid by centrifugal ultrafiltration. Both contraceptive treatments increased the percentage of free T and E2 (P less than 0.01) in the subset of monkeys that were evaluated, but the percentage bound to SHBG and albumin were different only for the CVR group (P less than 0.05). Decreased total T concentrations in the treatment groups offset any increase in free T concentrations associated with an increase in the percentage of free T. The differences in the distribution of binding to SHBG associated with these contraceptive steroid treatments was influenced more by the reduction in the binding capacity of SHBG than by the displacement of E2 and T from SHBG by dNG.     

Sex steroid hormone binding globulin levels and free 17 beta-estradiol and testosterone in cynomolgus monkeys during different reproductive states

The effect of differences in sex hormone binding globulin (SHBG) levels associated with reproductive status on free 17 beta-estradiol (E2) and testosterone (T) was characterized in cynomolgus monkeys. Cycling female cynomolgus monkeys had higher SHBG levels than males, pregnant and lactating females (P less than 0.05). Ovariectomized females were not different than control females, suggesting no hypoestrogenic effect. The percentage of free T was elevated in pregnant animals (P less than 0.05) compared to normal males and females, but the percentage of free E2 was similar between these groups. Although a gender difference in the percentages of free E2 and T was not detected, there was a gender difference in the free T and E2 concentrations due to endogenous secretion. Increased free E2 concentrations during pregnancy were the result of endogenous secretion rather than the decreased binding capacity of SHBG; the increased percentage of free T during pregnancy significantly increased free T concentrations. These data suggest that the gender difference in SHBG levels in cynomolgus monkeys is due to androgenic influences and that estrogens have minimal influence. Furthermore, the decrease in SHBG levels during pregnancy and lactation may not be entirely dependent on these androgenic influences.     

A novel spreadsheet method for calculating the free serum concentrations of testosterone, dihydrotestosterone, estradiol, estrone and cortisol: With illustrative examples from male and female populations

In humans, testosterone (T), dihydrotestosterone (DHT), estradiol (E2), estrone (E1) and cortisol (C) bind to the serum proteins sex hormone-binding globulin (SHBG), albumin (Alb) and corticosteroid-binding globulin (CBG). Equilibrium dialysis is considered to be the “gold standard” for measuring the free concentrations of these steroids but is technically difficult and not widely available. Based on a mathematical model of the 5-ligand/3-protein binding equilibria, we developed a novel spreadsheet method for calculating the free and bioavailable (free + Alb-bound) concentrations of each steroid in terms of the total steroid and protein concentrations. The model uses 15 association constants K_SHBG-X, K_Alb-X, and K_CBG-X (X = T, DHT, E2, E1 and C) that have been estimated from a systematic review of published binding studies. The computation of the free and bioavailable concentrations uses an iterative numerical method that can be readily programmed on a spreadsheet. The method is illustrated with six examples corresponding to young men (YM), old men (OM), obese men (Ob M), young women (YM), pregnant women in the 3rd trimester (Preg T3) and oophorectomized women on oral conjugated equine estrogens (CEE). The resulting free hormone concentrations for YM and YW fall within the normal references ranges obtained by equilibrium dialysis for all five hormones. The model also accounts for the competitive binding effects of high estrogen levels on the free T levels in Preg T3. This novel spreadsheet method provides a “user-friendly” approach for estimating the free concentrations of circulating sex hormones and cortisol in men and women.     

The effect of ketoconazole related imidazole drugs and antiandrogens on [3H] R 1881 binding to the prostatic androgen receptor and [3H]5 alpha-dihydrotestosterone and [3H]cortisol binding to plasma proteins

Ketoconazole an orally active imidazole drug and bifonazole, clotrimazole, econazole, isoconazole, miconazole and tioconazole are known inhibitors of cytochrome P-450 dependent steroidogenic enzymes. The aim of the present study was to determine whether these imidazole drugs also have an effect on [3H]R1881 binding to the human prostatic androgen receptor, [3H]5 alpha-dihydrotestosterone (5 alpha-DHT) binding to plasma sex hormone binding globulin (SHBG) and [3H]cortisol binding to plasma corticosteroid binding globulin (CBG). In comparison the effect of both steroidal (cyproterone acetate; CPA) and non-steroidal (anandron, flutamide, hydroxyflutamide, ICI 176344) antiandrogens on these steroid binding proteins was also determined. The results of the present study show that the imidazole drugs were without effect on [3H]R1881 binding to the androgen receptor and on [3H]cortisol binding to CBG up to 100 mumol/l. However, they were weak competitors of [3H]5 alpha-DHT binding to SHBG inhibiting 20-53% of binding at 100 mumol/l. In comparison the antiandrogens were strong competitors of [3H]R1881 binding to the androgen receptor, the order of decreasing potency, determined from ID50 (mumol/l) values were CPA (0.073) greater than ICI 176344 (0.4) greater than anandron (0.63) greater than hydroxyflutamide (1) greater than flutamide (greater than 100). The non-steroidal antiandrogens were without effect on [3H]cortisol binding to CBG whereas CPA caused 36% inhibition of binding at 100 mumol/l. Of the antiandrogens studied CPA was the strongest competitor of [3H]5 alpha-DHT binding to SHBG with an ID50 of 23 mumol/l, in contrast the non-steroidal antiandrogens were weak competitors causing less than 40% inhibition at 100 mumol/l. It is concluded that the primary mode of action of the imidazole drugs is through the inhibition of cytochrome P-450 dependent steroidogenic enzymes with little or no effect on steroid binding proteins. In comparison, the antiandrogens were strong competitors of [3H] binding to the androgen receptor but relatively weaker competitors of [3H] steroids binding to plasma binding proteins.     

Equivalent affinity of aldosterone and corticosterone for type I receptors in kidney and hippocampus: direct binding studies

In studies from several laboratories evidence has been adduced that renal Type I (mineralocorticoid) receptors and hippocampal "corticosterone-preferring" high affinity glucocorticoid receptors have similar high affinity for both aldosterone and corticosterone. In all these studies the evidence for renal mineralocorticoid receptors is indirect, inasmuch as the high concentrations of transcortin (CBG) in renal cytosol make studies with [3H]corticosterone as a probe difficult to interpret, given its high affinity for CBG. We here report direct binding studies, with [3H]aldosterone and [3H]corticosterone as probes, on hippocampal and renal cytosols from adrenalectomized rats, in which tracer was excluded from Type II dexamethasone binding glucocorticoid receptors with excess RU26988, and from CBG by excess cortisol 17 beta acid. In addition, we have compared the binding of [3H]aldosterone and [3H]corticosterone in renal cytosols from 10-day old rats, in which CBG levels in plasma and kidney are extremely low. Under conditions where neither tracer binds to type II sites or CBG, they label an equal number of sites (kidney 30-50 fmol/mg protein, hippocampus approximately 200 fmol/mg protein) with equal, high affinity (Kd 4 degrees C 0.3-0.5 nM). Thus direct tracer binding studies support the identity of renal Type I mineralocorticoid receptors and hippocampal Type I (high affinity, corticosterone preferring) glucocorticoid receptors.     

Positive cooperativity of [3H]dexamethasone binding to chick corticosteroid-binding globulin.

1. Dilution of chick plasma with Tris-EDTA buffer containing dithiothreitol (TED buffer) induced a positive cooperativity in the interaction between corticosteroids and CBG. 2. Positive cooperativity binding is probably induced by the polymerization of chick CBG in the presence of DTT. 3. As the increase of the value of B/F in Scatchard plot analysis was dependent on the concentration of [3H]dexamethasone, it is possible that the polymerization of CBG is steroid-dependent. 4. Positive cooperativity was also observed in TED buffer containing 0.2 M KCl and 0.5 M KCl. Addition of KCl seemed to enhance the polymerization of chick CBG.     

The effects of sex hormone binding globulin (SHBG) on testosterone transport into the cerebrospinal fluid.

The movement of testosterone (T) from blood across the blood-brain barrier (BBB) is thought to reflect the combined effects of T's lipid solubility and the presence of circulating binding proteins for T such as albumin or sex hormone binding globulin (SHBG). Since the adult rat lacks a circulating specific high affinity sex steroid binding protein, examination of the disappearance from serum and uptake into cerebrospinal fluid (CSF) of [3H]T before and after SHBG or albumin infusion should provide insight into the function of these two proteins with respect T transport. Three groups of adult male Sprague-Dawley rats were cannulated at the femoral vein and cisterna magna. In a control group (n = 8), [3H]T was given as an intravenous bolus beginning at time zero; multiple serum and CSF collections were assayed for counts per min (cpm) during the subsequent 45 min. Data from these animals were then compared to those seen in animals that received either purified human SHBG (hSHBG) (n = 7) or human albumin (hALB) (n = 6) 10 min prior to the [3H]T infusion. High performance liquid chromatography was used to monitor the metabolic fate of the steroid infusate at the end of each study period. Infusion of hSHBG increased serum concentrations from undetectable to 93.8 nM/l (mean +/- SEM, n = 6). Administration of hALB significantly increased (25.0 +/- 1.2 g/l at baseline, 33.4 +/- 1.6 g/l post-infusion, mean +/- SEM, P less than 0.03, n = 5) the circulating albumin concentration. Comparison of data from each group of animals demonstrated that (1) following an i.v. injection of radiolabeled T, the initial decline in serum [3H]T was significantly reduced (P less than 0.03) in the presence of hSHBG, (2) hALB did not affect the movement of [3H]T out of serum, (3) the time to peak appearance of [3H]T in the CSF was significantly delayed (P less than 0.02) by the presence of circulating hSHBG, and (4) the net quantity of [3H]T found in the cSF under steady-state conditions was not affected by serum SHBG or albumin levels. This study demonstrates that high-affinity steroid binding proteins do modulate the transport of sex steroids across the BBB. Specifically, SHBG delays the clearance of T from serum and slows the rate of T uptake into the CSF during non-equilibrium conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Circadian variations of serum sex hormone binding globulin binding capacity in normal adult men and women

Diurnal variations of serum sex hormone binding globulin (SHBG), testosterone (T) and estradiol (E2) in five normal adult men and five normal adult women were investigated. SHBG binding capacity was measured by both polyacrylamide gel electrophoresis and dextran-coated charcoal technique (DCC); T and E2 were assayed by RIA and free T and free E2 were determined by means of equilibrium dialysis. In male subjects the variations of SHBG binding capacity was associated with the changes of total T, free T and T/SHBG index, which had the highest concentrations in the morning and the lowest levels in the evening during the 24 h test period, but percentage free T remained unchanged. Serum protein concentrations did not change significantly during 24 h. No significant diurnal changes of SHBG binding capacity, total E2, free E2, percentage free E2 and percentage free T were found in female subjects in the mid-luteal phase of the menstrual cycle, although significant fluctuations of total T, free T and T/SHBG index were observed throughout the day. The results suggested that SHBG may play a buffer role in the presence of fluctuations of testosterone production during 24 h period, allowing stabilization of a bioactive fraction of the hormone both in normal adult male and female. However, the concentrations of T in normal adult women may be too low to drive any change of SHBG levels while there were no significant variations of E2 throughout a day in the mid-luteal phase of the menstrual cycle.     

Hormonal effects on the secretion and glycoform profile of corticosteroid-binding globulin

Corticosteroid-binding globulin (CBG) is a plasma glycoprotein that is primarily synthesized in the liver and binds cortisol and progesterone with high affinity. In this study, a CBG secreting hepatocellular carcinoma derived cell line (HepG2) was used to investigate the hormonal regulation of hepatic CBG synthesis. HepG2 cells were grown for 72 h in 30, 300 and 3000 nM concentrations of estradiol (E₂), testosterone (T), insulin, thyroxin (T₄) and dexamethasone (DMZ) and the secreted CBG quantified by a novel enzyme-linked immunosorbent assay (ELISA). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was carried out to determine the effects of these hormones on the relative distribution of CBG glycoforms.

Insulin, T₄ and high concentrations of E₂ decreased the secretion of CBG by HepG2 cells (p < 0.05). Ethanol, the solvent used for E₂, T and DMZ, also significantly attenuated CBG secretion. 2D-PAGE resolved 13–14 glycoforms of CBG produced by HepG2 cells. Insulin caused a reduction in the synthesis of more acidic, while T₄ and DMZ decreased the production of more basic CBG glycoforms. Stimulation with E₂ resulted in the synthesis of additional isoforms of increased acidity, which may represent a type of CBG only seen during pregnancy in vivo. Possible physiological implications of these findings are discussed.     

Characteristics of estrogen-2/4-hydroxylase of porcine ovarian follicles: influence of steroidal and non-steroidal agents on the activity of the enzyme in vitro

The conversion of [3H]estradiol to 2-hydroxyestradiol (2-OH-E2) by homogenates of porcine ovarian follicles was assayed in vitro in the presence and absence of 10 and 100 microM concentrations of the following potential substrates or inhibitors of estrogen-2/4-hydroxylase (E-2/4-H): (1) estrogens; estrone (E1), estriol (E3) and 17 alpha-estradiol (17 alpha-E2), (2) catecholestrogens; 2-hydroxyestradiol (2-OH-E2), 4-hydroxyestradiol (4-OH-E2) and 2-hydroxyestrone (2-OH-E1); (3) 2-methoxyestradiol (2-MeO-E2); (4) halogenated estrogens; 2-bromoestradiol, (2-Bromo-E2) 4-bromoestradiol and 2,4-dibromoestradiol; (5) androgens; testosterone (T), dihydrotestosterone (DHT) and androstenedione; (6) progesterone; (7) epinephrine; (8) inhibitors of steroid aromatase; aminoglutethimide and 4-hydroxyandrostenedione and (9) SKF 525A, an inhibitor of cytochrome P-450. Progesterone and 2-Bromo-E2 were the two most effective inhibitors (2-OH-E2 formation = 4 and 5% of control at 100 microM and 29.6 and 17.4% at 10 microM of progesterone and 2-Bromo-E2, respectively). 2-MeO-E2 at 100 microM was nearly as effective as progesterone in inhibiting E-2/4-H activity but only caused about 50% inhibition at 10 microM. The three catecholestrogens reduced 2-OH-E2 formation to about the same degree (21-23% of control at 100 microM). The 2,4-dibromo-E2 was equipotent with the catecholestrogens while 4-bromo-E2 was about half as effective. The phenolic estrogens, potential substrates for the enzyme, reduced 2-OH-E2 formation to different degrees, with E3 being the most effective. Among the androgens, DHT was almost as effective an inhibitor as the catecholestrogens, T was about half as effective while androstenedione had no effect. Epinephrine and the two inhibitors of aromatase did not inhibit E-2/4-H activity. SKF 525A inhibited E-2/4-H activity but with a potency only about 1/10th that reported for liver.     

Protein binding of the contraceptive steroids gestodene, 3-keto-desogestrel and ethinylestradiol in human serum

The protein binding of ethinylestradiol (EE2), gestodene (GEST) and 3-keto-desogestrel (KDG) has been determined by ultrafiltration in the serum of women who had either taken a gestodene (n = 37) or desogestrel (n = 28) containing oral contraceptive for a time period of at least 3 months. GEST and KDG were analyzed in individual serum pools whereas EE2 was repeatedly measured in two serum pools, each one representing one treatment group. The respective free fractions of the three steroids were 0.6 +/- 0.1% (GEST), 2.5 +/- 0.2% (KDG), 1.7 +/- 0.6% (EE2, in the gestodene-group) and 1.5 +/- 0.2% (EE2, in the desogestrel-group). EE2 was exclusively bound to albumin, whereas GEST and KDG were also bound to sex-hormone-binding globulin (SHBG). The distribution of the two progestins over the serum binding proteins was determined after heat-treatment of serum samples. For GEST, the contribution of albumin and SHBG was 24.1 +/- 9.1 and 75.3 +/- 9.1%, respectively and for KDG it was 65.9 +/- 11.9 and 31.6 +/- 12.0%, respectively. SHBG and corticosteroid-binding globulin (CBG) concentrations were measured in the serum samples obtained from both treatment groups. In the gestodene-group 180 +/- 61 nmol/l (SHBG) and 89 +/- 13 mg/l (CBG) were measured, the corresponding values in the desogestrel-group were 226 +/- 64 nmol/l (SHBG) and 93 +/- 14 mg/l (CBG). SHBG concentrations were correlated with the total concentration of GEST and its free fraction and a positive (r = 0.395) and negative (r = -0.491) correlation respectively was found. Only a weak negative correlation (r = -0.291) was found for SHBG and the free fraction of KDG in the serum. These data demonstrate that the three contraceptive steroids EE2, GEST and KDG were all bound extensively to serum proteins, however, with pronounced differences concerning their distribution over the various binding proteins.     

Estrogen and androgen regulation of sex hormone binding globulin secretion by a human liver cell line

Both estrogens and androgens have been shown to stimulate sex hormone binding globulin (SHBG) secretion in vitro in the hepatocellular carcinoma cell line, Hep G2, in contrast to the expected inhibition by androgens from in vivo studies. However, such in vitro stimulation was only demonstrated at high steroid doses, generally in serum-containing medium, with added Phenol Red. In the present study, Hep G2 cells were grown in serum-free medium, without Phenol Red, under the influence of testosterone (T) (0, 0.5-500 nM) and ethinyl estradiol (EE2) (0, 50 pM-500 nM). Levels of secreted SHBG and albumin were correlated with androgen receptors in cytosolic (ARc) and nuclear (ARn) fractions and with DNA levels. In the presence of increasing T levels, SHBG levels fell to 39% of control values at 5 nM T (P = 0.047), rising to 97% of control at 500 nM. Conversely, incubation with EE2 produced a rise in SHBG secretion of more than 100% at 0.5 nM (P less than 0.02) which was sustained to 50 nM (P less than 0.005). DNA levels did not change with the addition of testosterone or EE2, with the exception of a 15% reduction at 5 nM EE2 (P less than 0.05). Albumin levels in the medium were not significantly altered by either steroid. However, in response to T, androgen receptor (AR) levels were reduced in cytosolic (42% of control) and nuclear (22%) fractions at 5 nM, and these changes in ARc and ARn correlated with SHBG levels over the range of T concentrations (P = 0.04 and P = 0.017, respectively). Nuclear estrogen receptor (ER) increased over 10-fold at 5 and 50 pM EE2 (P less than 0.001) and maintained 50 nM (P less than 0.001). Cytosolic ER was reduced at 0.5 and 5 nM but recovered at 50 nM, correlating with SHBG levels (P less than 0.001). These findings are consistent with the hypothesis that estrogens and androgens regulate SHBG synthesis in man by direct, specific, probably receptor-mediated effects on hepatocytes. Hep G2 cells grown in serum-free medium are a suitable experimental system for further study of this phenomenon.     

Reducing estrogen synthesis does not affect gonadotropin secretion in the developing boar

Boars have high concentrations of plasma and testicular estrogens, but how this hormone is involved in feedback regulation of the gonadotropins and local regulation of testicular hormone production is unclear. The present study examined the effects of reducing endogenous estrogens by aromatase inhibition on concentrations of plasma LH and FSH and on testicular and plasma concentrations of testosterone (T) and immunoreactive inhibin (INH). Thirty-six littermate pairs of boars were used. One boar from each pair was assigned to the control group (vehicle); the other boar to the treatment group (aromatase enzyme inhibitor, Letrozole, 0.1 mg/kg body weight [BW]). Weekly oral treatment started at 1 wk of age and continued until castration at 2, 3, 4, 5, 6, 7, or 8 mo. Plasma concentrations of gonadotropins, INH, T, estradiol (E2), and estrogen conjugates (ECs) were determined. Testicular tissue was collected at castration for determination of INH and T and for confirmation of reduced aromatase activity. The acute effects of aromatase inhibition on gonadotropins were monitored in two adult boars treated once with Letrozole (0.1 mg/kg BW). Treatment with the aromatase inhibitor reduced testicular aromatase activity by 90% and decreased E2 and ECs without changing acute, long-term, or postcastration LH and FSH. Plasma T, testicular T, and circulating INH concentrations did not change. Testicular INH was elevated in treated boars compared with controls. In conclusion, estrogen does not appear to play a regulatory role on gonadotropin secretion in the developing boar. This is in direct contrast to findings in males of several other species.     

Central hypogonadism in burned men

Serum samples were obtained from 30 burned men at different times up to the fourth month after injury. Mean concentrations of estradiol (E2) were elevated above those for healthy control subjects. Mean serum total testosterone (T), sex hormone-binding globulin (SHBG), bioassayable luteinizing hormone (LH), thyroxine (T4), triiodothyronine (T3) and their free indices (FT4I and FT3I) were depressed below those of controls during the first postburn week. Mean values for T and LH were progressively higher in samples taken from later time periods but remained depressed. Mean SHBG and thyroid hormones rose and were not significantly different from control values during later periods of the study. Calculated non-SHBG-bound T (NSBT) was below normal in each time period. The close correlation of SHBG values with those of T3 and FT3I in the patients suggests that SHBG responds to the altered thyroid hormone milieu of burn injury. It is postulated that elevated serum E2 perhaps from adrenal precursors promotes an alteration of hypothalamic function resulting in a markedly reduced secretion of bioactive LH and diminished Leydig cell function.     

Interaction of ring B unsaturated estrogens with estrogen receptors of human endometrium and rat uterus

The present investigation was undertaken to compare the binding affinities (Ka) of the ring B unsaturated equine estrogens (equilin [Eq], equilenin [Eqn], 17 beta-dihydroequilin [17 beta-Eq], 17 beta-dihydroequilenin [17 beta-Eqn], 17 alpha-dihydroequilin [17 alpha-Eq], and17 alpha-dihydroequilenin [17 alpha-Eqn]) and the classic estrogens (estrone [E1], 17 beta-estradiol [17 beta-E2], and 17 alpha-estradiol [17 alpha-E2]) for estrogen receptors in human endometrium and rat uterus. In both species, the ring B unsaturated estrogens bind with cytosol and nuclear receptors with high affinity (Ka x 10(9) M-1). The relative binding affinities of these estrogens were measured by determining the amount of unlabeled estrogen required to reduce by 50% the specific binding of [3H]17 beta-Eq to endometrial cytosol receptors. The order of activity found was 17 beta-Eq greater than 17 beta-E2 greater than 17 beta-Eqn greater than E1 greater than Eq greater than 17 alpha-Eq greater than 17 alpha-E2 greater than 17 alpha-Eqn greater than Eqn. Essentially the same order of activity was observed when the apparent affinity constants of these estrogens for human and rat cytosol and nuclear receptors were determined by a competitive (inhibition) binding assay. Sucrose density gradient analysis indicated that these estrogens form protein complexes with cytosol and nuclear preparation that sediment at approximately 8S and 4S, respectively. The affinity constants for 17 beta-Eq were approximately two- to six-fold higher than E2 in both species. In a rat uterotropic assay, all nine estrogens were uterotropic. These data indicate that all ring B unsaturated estrogens present in conjugated equine estrogen preparations are biologically active and they express their biologic effects in the human endometrium by mechanisms similar to those described for the classic estrogens.     

Effect of free fatty acids on the bioavailability of plasma testosterone and dihydrotestosterone

Free fatty acids (FFA) are known to interfere with the binding of thyroid hormone and estrogens to circulating proteins, but their effect on androgen binding is unknown. The effect of linoleic, oleic and palmitic acids at physiological concentrations on the binding of testosterone (T) and dihydrotestosterone (DHT) to circulating proteins was evaluated in vitro, using equilibrium dialysis and ammonium sulfate precipitation techniques. The results indicate that FFA can inhibit T binding to albumin and SHBG. They also can inhibit DHT binding to albumin, whereas DHT binding to SHBG is not altered, suggesting that FFA at physiological concentrations may be important regulators of bioavailability of T to tissues.