Plasma estriol levels after intramuscular injection of estriol and two of its esters

Twelve female volunteers from Berlin and 9 from Stockholm, all using a contraceptive pill (30 micrograms ethinyl estradiol and 150 micrograms levonorgestrel), received an intramuscular injection of estriol (E3; 1 mg in oil) on day 5 of withdrawal bleeding. Blood samples were collected at increasing time intervals during 4 weeks. Three months later, on day 5 of their withdrawal bleeding, 6 women were given intramuscularly (in oil) estriol 3,17-dipropionate (E3-prop) and 15 women estriol 3,17-dihexanoate (E3-hex). The doses were equivalent to 5 mg of estriol, i.e. 6.94 and 8.90 mg, respectively. Blood samples were collected during a period of 9 weeks. Estriol was analyzed by radioimmunoassay in all plasma samples. The average half-life of E3 ranged from 1.5 to 5.3 h after the administration of E3. It was 12.7 h and between 187 and 221 h after the administration of E3-prop and E3-hex, respectively. The average areas under the curve (in nmol.l-1.h) of E3 were between 82.5 and 161 after the administration of E3-prop or E3-hex, and between 27.1 and 37.9 when E3 had been given. As E3 was administered in a 5-fold lower dose than the esters, the areas under curve appeared to be comparable. Thus, the total exposure to E3 seemed to be almost independent of the type of E3 derivatization, while the time and intensity of exposure were very different.     

Alterations in osteoclast morphology following long-term 17beta-estradiol administration in the mouse

Background  

Although the role of the osteoclast in bone resorption is becoming better understood, much remains to be learned about osteoclastogenesis and the exact mechanism of action of anti-resorbing agents such as 17β-estradiol. This study investigated bone and morphologic osteoclast alterations following long-term estrogen administration to the B6D2F1 mouse. B6D2F1 mice aged 4-5 weeks were exposed to high levels of estrogen via implanted silastic tubing for at least 12 weeks; controls received empty tubing. Femurs of control and treated mice were assessed with radiology, quantitative histomorphometry and transmission electron microscopy.     

Acute effects of 17beta-estradiol on ventricular and vascular hemodynamics in postmenopausal women

Because premenopausal women have lower cardiovascular morbidity than postmenopausal women, it has been proposed that estrogen may have a protective role. Estrogen is involved in smooth muscle relaxation both through its specific receptor as well as through calcium channel blockade. This study examined the acute effect of estradiol on invasive cardiovascular hemodynamics in 18 postmenopausal women (age 62.6 +/- 7.6 years, means +/- SD). The effect of estradiol on left ventricular chamber performance was studied in 9 women using simultaneous left ventricular pressure-volume recordings. In a further group of 9 women, the acute effect of estradiol on arterial function was assessed using input impedance (derived from simultaneous aortic pressure and flow recordings), pressure waveform analysis, and pulse wave velocity. After 2 mg micronized 17beta-estradiol was administered, serum estradiol levels increased from 50.9 +/- 21.9 to 3,190 +/- 2,216 pmol/l, P < 0.0001. There was no effect of estradiol on either left ventricular inotropic or lusitropic function. There was no acute effect of estradiol on arterial impedance, reflection coefficient, augmentation index, or pulse wave velocity. There was a trend to decreased heart rate and cardiac output in both groups of 9 women. Because heart rate and cardiac output were common to both hemodynamic data sets, results for these parameters were pooled. Across all 18 women, there was a small but significant decrease in heart rate (69.2 +/- 10.4 vs. 67.2 +/- 9.9 beats/min, P = 0.02), as well as a significant decrease in cardiac output (4.82 +/- 1.77 vs. 4.17 +/- 1.56 l/min, P = 0.002). Despite achieving supraphysiological serum levels, this study found no significant effect of acute 17beta-estradiol on ventricular or large artery function.     

Effects of 17 beta-estradiol on levels and distribution of metallothionein and zinc in squirrelfish

Females of the squirrelfish family (Holocentridae) accumulate higher levels of zinc in the liver than any other known animal. This zinc accumulation is made possible by high expression of the zinc-binding protein, metallothionein (MT). In the present study, the squirrelfish (Holocentrus ascensionis) MT cDNA was cloned and sequenced. The deduced amino acid sequence was very similar to other teleost MT. The role of estrogens on zinc metabolism was investigated by injecting male and immature female squirrelfish with 17 beta-estradiol (E(2)). E(2) treatment triggered transient increases in plasma zinc and vitellogenin (VTG) levels, and both of these variables showed very similar time courses. These results suggest that E(2) is responsible for the large hepatoovarian translocation of zinc observed in female squirrelfish and that VTG might be a vehicle for zinc. E(2) did not directly alter the levels of zinc or MT mRNA in the liver. However, the hepatic MT protein concentration increased differentially in the nuclear fraction. Thus E(2) is probably responsible for the association of MT with the nuclear fraction previously observed in untreated mature female squirrelfish.     

Plasma hormone levels and central c-Fos expression in ferrets after systemic administration of cholecystokinin

Posterior pituitary hormone secretion and central neural expression of the immediate-early gene product c-Fos was examined in adult ferrets after intravenous administration of CCK octapeptide. Pharmacological doses of CCK (1, 5, 10, or 50 microg/kg) did not induce emesis, but elicited behavioral signs of nausea and dose-related increases in plasma vasopressin (AVP) levels without significant increases in plasma oxytocin (OT) levels. CCK activated neuronal c-Fos expression in several brain stem viscerosensory regions, including a dose-related activation of neurons in the dorsal vagal complex (DVC). Activated brain stem neurons included catecholaminergic and glucagon-like peptide-1-positive cells in the DVC and ventrolateral medulla. In the forebrain, activated neurons were prevalent in the paraventricular and supraoptic nuclei of the hypothalamus and also were observed in the central nucleus of the amygdala and bed nucleus of the stria terminalis. Activated hypothalamic neurons included cells that were immunoreactive for AVP, OT, and corticotropin-releasing factor. Comparable patterns of brain stem and forebrain c-Fos activation were observed in ferrets after intraperitoneal injection of lithium chloride (LiCl; 86 mg/kg), a classic emetic agent. However, LiCl activated more neurons in the area postrema and fewer neurons in the nucleus of the solitary tract compared with CCK. Together with results from previous studies in rodents, our findings support the view that nauseogenic treatments activate similar central neural circuits in emetic and nonemetic species, despite differences in treatment-induced emesis and pituitary hormone secretion.     

Elimination kinetic of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate ester metabolites in calves' urine

Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C(18) SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CCalpha) for steroids were below 0.1 microg L(-1), except for 19-norandrosterone (CCalpha=0.7 microg L(-1)) and estrone (CCalpha=0.3 microg L(-1)). Kinetics of elimination of the administered 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate were established by monitoring 17beta-estradiol, 17alpha-estradiol, estrone and 17beta-nandrolone, 17alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17alpha-estradiol and 17alpha-nandrolone (>20 and 2 mg L(-1), respectively after 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate administration) whereas 17beta-estradiol, estrone, 17beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the microg L(-1) level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17beta form.     

Steroid levels after intramuscular injection of radioactive estradiol-17beta, estrone, progesterone and testosterone in the bovine

Eight 2 year old Hereford cows from days 8 to 12 of the estrous cycle were injected intramuscularly with 5 ml of corn oil containing 5 mg of estradiol-17beta (two cows), estrone (two cows), progesterone (two cows) or testosterone (two cows). Each cow treated with estradiol received 494 microc of estradiol-17beta-6, 7 H3 and each cow treated with estrone received 492 microc of estrone-6, 7 H3. Each cow treated with progesterone or testosterone received 400 muc of H3 compound labeled in the 7 position. Total urine was collected by urethral catheterization of the cows treated with estrogens. Blood samples for plasma and serum were collected via jugular cannulae. Blood and urine samples from estrogen-treated cows were collected hourly for the first 24 hr, at 2 hr intervals for the next 26 hr, at 4 hr intervals for the next 12 hr and at 12 hr intervals until background was reached. Blood samples were collected hourly from 1 to 8 hr after injection from progesterone or testosterone-treated cows. Plasma and serum levels of radioactive estradiol-17beta, estrone, progesterone and testosterone were similar. Blood levels of radioactivity peaked at 2 hr post-injection in cows receiving estradiol-17beta and at 3 hr in cows receiving estrone. Blood levels of labeled estradiol-17beta and estrone were nondetectable by 54 hr and 83 hr, respectively. Peak urinary excretion of radioactivity was reached at 7 hr for estradiol-17beta and at 14 hr for estrone and nondetectable levels were reached by 95 hr for estradiol-17beta and 14 hr for estrone. At these times, 15.5% of the total dose of radioactive estradiol-17beta and 17.5% of the injected estrone had been excreted in the urine. Peak blood and urinary excretion levels were reached earlier for radioactive estradiol-17beta than for estrone, and excretion of estradiol-17beta was completed more rapidly. No difference was found in plasma and serum levels for any steroid studies; thus, endogenous steroid titers in blood plasma and serum are not different in the cow.     

Histo-autoradiographic study of a testosterone target organ, the epididymis of the lizard (Lacerta vivipara), after administration of 3H-17 beta-estradiol]

[6,7 3H] oestradiol-17 beta was injected into castrated male viviparous lizards in order to compare retention of this steroid to that of testosterone during the period of sexual activity. Retention of the isotope in epididymis was greater than in blood, lung and stomach. Radioautographs of epididymis indicated that oestradiol or a metabolite was concentrated in cell nuclei and over discharged secretory granules as was observed with testosterone. Reason of such binding is not yet known.     

Docosahexaenoic acid and 17 beta-estradiol co-treatment is more effective than 17 beta-estradiol alone in maintaining bone post-ovariectomy

Bone-protective effects of combined treatment with long chain polyunsaturated fatty acids (LCPUFAs) and estrogenic compounds following ovariectomy have previously been reported. Recent evidence suggests the n-3 LCPUFA docosahexaenoic acid (DHA, 22:6n-3) is particularly bone-protective. The aim of this study was to determine whether combined treatment with DHA and estrogenic compounds has a beneficial effect on bone mass in ovariectomized (OVX) rats. Rats were randomized into 9 groups and either ovariectomized (8 groups) or sham-operated (1 group). Using a 2 x 4 factorial design approach, OVX animals received either no estrogenic compound, genistein (20 mg/kg body weight/day), daidzein, (20 mg/kg body weight/day) or 17 beta-estradiol (1 microg/day) with or without DHA (0.5 g/kg body weight/day) for 18 weeks. Bone mineral content (BMC), area (BA), and density (BMD), plasma osteocalcin and IL-6 concentrations, and red blood cell (RBC) fatty acid composition were measured. Femur BMC was significantly greater in animals treated with DHA or 17 beta-estradiol than in ovariectomized controls. Plasma carboxylated osteocalcin was significantly higher in DHA-treated animals and total osteocalcin significantly lower in 17 beta-estradiol-treated animals compared with ovariectomized controls. There were significant interactions between treatment with estrogenic compounds and DHA for femur BMC, plasma IL-6 concentration, and RBC fatty acid composition. Combined treatment with 17beta-estradiol+DHA was more effective than either treatment alone at preserving femur BMC and lowering circulating concentrations of pro-inflammatory IL-6. The percentage of n-3 LCPUFAs in RBCs was significantly greater in animals receiving 17 beta-estradiol+DHA compared with either treatment alone. There was no beneficial effect of combined DHA and phytoestrogen treatment on bone. Results from this study raise the possibility that co-treatment with 17 beta-estradiol and DHA may allow a lower dose of 17 beta-estradiol to be used to provide the same bone-protective effects as when 17 beta-estradiol is administered alone.     

Effects of 17beta-estradiol and flutamide on splenic macrophages and splenocytes after trauma-hemorrhage

Since splenic immune functions are depressed in metestrus females following trauma-hemorrhage, we hypothesized that administration of the androgen receptor antagonist flutamide at the onset of resuscitation will maintain the immune function of the spleen following trauma-hemorrhage. Female C57BL6/J mice (metestrus state, 8-12 weeks old), underwent laparotomy and hemorrhagic shock (35.0+/-5.0 mm Hg for 90 min) and received 17beta-estradiol (50 microg/25 g), flutamide (625 microg/25 g) or 17beta-estradiol+flutamide. Four hours after resuscitation, the in vitro productive capacity of different cytokines (TNF-alpha, IL-6, IL-10, and IFN-gamma) by splenic MPhi and splenocytes were determined by flow cytometry. A significantly decreased cytokine production by both splenocytes and splenic MPhi was observed following trauma-hemorrhage compared to shams. Administration of 17beta-estradiol, flutamide and 17beta-estradiol+flutamide following trauma-hemorrhage resulted in a significant increase in the in vitro IL-6 release by splenic MPhi. The TNF-alpha productive capacity, however, was only restored by 17beta-estradiol and 17beta-estradiol+flutamide administration following trauma-hemorrhage. No significant effect of either treatment was observed with regard to the suppressed splenic MPhi IL-10 release. Anti-CD3 stimulation, administration of 17beta-estradiol and 17beta-estradiol+flutamide, but not the administration of flutamide alone resulted in a significant increased release of TNF-alpha, IL-6 and IFN-gamma compared to vehicle-treated animals. No significant effect of either treatment was found on IL-10 productive capacity. These results collectively suggest that flutamide administration following trauma-hemorrhage in females has beneficial effects on splenic immune function. However, flutamide administration in combination with estrogen does not provide any significant, additional effects over 17beta-estradiol administration alone.     

Changes in the mutagenic and estrogenic activities of 17beta-estradiol after treatment with nitrite

We determined the changes in the mutagenic and estrogenic activities of 17beta-estradiol after a nitrite treatment. Nitrite-treated 17beta-estradiol showed mutagenic activities toward Salmonella typhimurium strains TA 100 and TA 98. We confirmed that nitrite-treated 17beta-estradiol generated radicals from the results of an analysis of electron spin resonance. By applying an instrumental analysis, we identified 2-nitro-17beta-estradiol to have been formed in the reaction mixture. 2-Nitro-17beta-estradiol did not exhibit mutagenic activities toward Salmonella typhimurium strains, suggesting that other mutagens might have been formed in the reaction mixture. The clastogenic properties of nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol were analyzed by a micronucleus test with male ICR mice. Nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol induced a significantly higher frequency of micronucleated reticulocytes in mice. The estrogenic activity of 2-nitro-17beta-estradiol was found to be lower than that of 17beta-estradiol. These data suggest that a daily oral intake of 17beta-estradiol and nitrite might induce the formation of mutagenic compounds in our body.     

Metabolism of radioactive 17 beta-estradiol 3-methyl ether by humans.

A mixture of 2-3H and 4-14C-17beta-estradiol 3-methyl ether was administered orally to a man and to a woman. 34 and 35 percent of the 3H was liberated into the body water of the man and of the woman, respectively, reflecting reactions involving position 2. The metabolism of estradiol methyl ether was qualitatively similar to that observed previously for radioactive estradiol administered intravenously to the same subjects, as judged by the measurement of various urinary metabolites by reverse isotope dilution. Evidence was obtained for hydroxylation at position 2 without demethylation by the isolation of urinary 2-hydroxyestrone 3-methyl ether which retained 33% of the original 3H. This 3H was presumably at position 1, resulted from an NIH shift which does not occur during hydroxylation of estrone or estradiol. This was confirmed by subsequent administration of a mixture of 4-14C and 3H-(methoxyl)-estradiol 3-methyl ether to the man. There was no evidence (by reverse isotope dilution) for 1-hydroxyestrone, 1-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether or 4-hydroxyestradiol 3-methyl ether as urinary metabolites of estradiol 3-methyl ether.