Estrogen metabolism in neural tissues of rabbits: 17 beta - hydroxysteroid oxidoreductase activity.

Incubation of adult rabbit neural tissue homogenates with (3H)-estrone and (3H)-estradiol revealed that the conversion of estrone to estradiol is higher in both male and female animals than estradiol to estrone. Both 17 oxidation and reduction are higher in male animals than in females. However, it is observed that the quotient of estrone leads to estradiol/estradiol leads to estrone for pituitary tissue and hypothalamus are higher in females than in males. There is no such dimorphism in cerebral cortex. The overall metabolism in pituitary is higher than in hypothalamus and cortex in both sexes. These results suggest that 17 beta hydroxysteroid oxidoreductase activity may play an important role in the regulation of estrogen function in neuroendocrine tissues.     

Effects of 4-hydroxy-4-androstene-3,17-dione and 10-propargylestr-4-ene-3,17-dione on the metabolism of androstenedione in human breast carcinoma and breast adipose tissues

The effects of 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 10-propargylestr-4-ene-3,17-dione (PED) on the aromatization of androstenedione (A) and the conversion of A to testosterone (T) were studied in incubations with breast carcinoma and breast adipose tissues. Parallel studies were carried out to determine the effects of 4-OH-A and PED on A metabolism in tissue from 5 patients with breast carcinoma. At 11 μM, both compounds fully inhibited aromatization, whereas the conversion of A to T was decreased in only 2 incubations.Studies with varying concentrations of 4-OH-A and PED demonstrated that both compounds inhibited estrone (E₁) formation by 80% at a concentration of 0.085 μM, with maximum effect at 0.34 μM. 90% inhibition of estradiol (E₂) formation was observed at inhibitor concentrations of 0.17 μM or greater. T formation was slightly affected at 0.67 μM, but was progressively inhibited with increasing 4-OH-A or PED concentrations, reaching 70% at 11 μM.Similar experiments with 4-OH-A in breast adipose tissue homogenates showed that a concentration of 0.1 μM was sufficient to inhibit aromatization while T inhibition required 11 μM.4-OH-A and PED are selective inhibitors of aromatization in human breast tissues and may provide a mechanism for controlling estrogen responsive processes.     

Androgen metabolism in male and female breast tissue

Incubation studies have been carried out using normal breast tissue and breast tissue from patients with gynecomastia, mammary dysplasia and breast carcinoma to determine the pattern of androstenedione metabolism. All tissues formed estrone (E₁) and testosterone (T) in all incubations. Estradiol (E₂) was isolated in incubations of tissue from 1 to 6 patients with mammary dysplasia, 5 of 6 patients with gynecomastia and in all incubations with normal and carcinoma tissue. Estrone formation was lowest in mammary dysplasia and gynecomastia, and higher in apparently normal breast tissue. The greatest E₁ formation was found in incubations with breast carcinoma tissue, although there was considerable variation within this tissue group. Estradiol formation was low in all tissues, with the highest conversion rates in carcinoma tissue. Testosterone formation in carcinoma tissue was greater than in mammary dysplasia or gynecomastia, but similar to apparently normal tissue. These results indicate that breast tissue from different pathological states varies in its capacity to aromatize androstenedione (A) to estrogenic products and to convert it to other androgens. They have also shown that the pattern of metabolism is distinctive for the nature of the pathological abnormality.     

Estrogen metabolism in nonhuman primates I. In vitro biosynthesis of estrogen glucosiduronates in rhesus monkey liver

Estrone glucosiduronate, 17β-estradiol-3-glucoslduronate, 17β-estradiol-17-glucosiduronate and estriol-16α-glucoslduronate have been biosynthesized in substantial yield by incubation of radioactive estrone, 17β-estradiol, estriol and uridlne diphosphoglucosiduronic acid with rhesus monkey liver homogenates. The metabolites were characterized by chromatography on Celite and DEAE-Sephadex, enzyme hydrolysis, derivative formation and crystallization to constant specific activity. The percent conversion to 17β-estradiol-17-glucosiduronate from 17β-estradlol ranged between 56–71%; from estrone, the conversion was 49–54%. Other metabolites formed from estradiol were estrone glucosiduronate(12–21%) and 17β-estradiol-3-glucosiduronate(5–12%). The same metabolites derived from estrone accounted for 18–28% and 10–14% respectively. After estriol incubation, more than 90% of the steroid was converted to estriol-16α-glucosiduronate with no detectable estriol-3-glucosiduronate. This report represents the first time that 17β-estradiol-17-glucosiduronate has been reported as a metabolite in the rhesus monkey and this is the only known species which forms 17β-estradiol-17-glucosiduronate as the predominant metabolite of either estrone or estradiol $\text{in vitro}$.Rhesus monkey liver is similar to the human and baboon in that it metabolizes estriol exclusively to estriol-16-glucosiduronate.     

Blood production rates of estrogens in women with differing ratios of urinary estrogen conjugates.

On the basis of the ratios of the estrogen conjugates in their urine (estriol/estrone + estradiol: E3/[E1+E2]), 19 women were divided into two groups: 9 women had ratios less than 0.6 and 10 women had ratios greater than 1.3. All women had measurements made of endogenous estrogens in their plasma by radioimmunoassay. They were then given constant infusions of 3H-estrone, 3H-estradiol and 14C-estriol during days 5-7 and days 20-22 of their cycles, and metabolic clearance rates (MCR) and blood production rates (PB) of estrone, estradiol and estriol were determined. Despite the wide disparity in their ratios of urinary estrogens, no differences could be found between the groups for the MCR's and PB's for all estrogens at either time of the cycle. The mean ratios of PB's (PB3/[PB2+PB1]) of estrone, estradiol and estriol ranged from 0.07 to 0.10 for each group during the cycle. The amounts of estriol entering the blood are small compared to the amounts of estrone and estradiol and do not correlate with the ratios of their urinary conjugates.     

In vitro effects of estrogens on rat prostatic 5 alpha-reduction of testosterone

The inhibitory effects of a variety of estrogens on rat prostate testosterone Δ4–5α-reductase activity were measured by a specific $\text{in vitro}$ assay. The conversion of ³H-testosterone (initial concentration 2.8 × 10^?9 M) to labelled 5α-dihydrotestosterone and 5α-androstane-3α, 17β-diol was used as a measure of Δ4?5a-reductase activity. At a concentration of 1.8 × 10^?6 M, estradiol was the most potent inhibitor (83.4%) of the estrogens tested. Various ester derivatives, e.g. 3-acetate, 3-phosphate, were effective inhibitors. The 17-glucuronide and 3-sulfate conjugates were less effective inhibitors. The estriol isomers exerted similar degrees of inhibition (40–60%). The 3-methoxy derivatives of estradiol and estriol were poor inhibitors. The introduction of certain groups into the steroid structure, e.g. 15α-hydroxy and 6-ketone, greatly decreased the inhibitory effect of estradiol. The nature of the oxygen function at carbon 17 did not greatly influence the inhibitory effects.     

Synthesis of estrogen methyl ethers by extractive alkylation.

A rapid method for the quantitative preparation of a number of estrogen methyl ethers is described. Estrogen in aqueous base is extracted as an ion pair with the tetrahexylammonium oin into methylene chloride where irreversible alkylation (extractive alkylation) by methyl iodide occurs. Gas chromatography (GC) - mass spectrometry (MS) provided the basis for identification of the methylated products. Estrone (1) and estradiol were easily 3-0-methylated whereas estriol gave a dimethylated product. Further experiments suggested that dimethylation of 2-hydroxyestrone in reasonable yield was possible.     

Failure of breast cancer cells in long-term culture to aromatize androstenedione

The ability of breast cancer cells in long-term tissue culture to aromatize androstenedione to estrone and estradiol was examined. (3H) androstenedione (1.1 x 10(-7)M) was incubated with 9 cell lines of human breast cancer and one line derived from a dimethylbenz(a)anthracene induced rat mammary tumor. No conversion to estrone or estradiol was detected. The findings are discussed in light of previous studies showing aromatization of androgens in breast cancer tissues.     

Placental and luteal steroidogenesis in the pregnant rhesus monkey.

Progesterone, 20alpha-dihydroprogesterone, estrone and estradiol-17beta concentrations were estimated by radioimmunoassay in blood plasma from uterine, uteroovarian and femoral veins of rhesus monkeys (Macaca mulatta) on days 22, 49, 128 and 160 of gestation. Steroids were consistently more concentrated in uterine and uteroovarian that in femoral venous plasma and in many cases levels in the uteroovarian vein were also higher than those in the uterine vein indicating luteal secretion of both progestins and estrogens thoughout gestation. In some animals, however, the corpus luteum appeared quiescent. As reflected in the decline in the uterine venous progesterone/estradiol-17beta concentration ratio, a shift in steroid contribution from the uterus and its contents occurred between days 22 and 49 of gestation with progesterone declining more rapidly than estradiol-17beta. Progesterone/20alpha-dihydroprogesterone was higher in both uterine and uteroovarian than in femoral venous plasma suggesting peripheral metabolism of progesterone to 20alpha-dihydroprogesterone.     

Changes in plasma levels of estrone sulfate and estrone in the pregnant ewe around parturition

A method for the extraction, separation and measurement of estrone sulfate and estrone in a single plasma sample is described. The method has been applied to the determination of plasma levels of estrone sulfate and estrone in pregnant ewes over the period 60 hr before to 20 hr after parturition. The study revealed that the plasma levels of estrone sulfate and estrone began to increase about 40 hr before parturition, reached a peak at parturition and then declined rapidly to levels below the sensitivity of the method by 15 hr postpartum. The peak level of estrone sulfate recorded at parturition was 103 pmol (38 ng) per ml of plasma which was approximately 30 times greater than the corresponding peak level of estrone.     

In vivo studies on the metabolism of estrone and estradiol-17beta by the brain

³H-Estrone and H-estradiol-17β were infused in separate experiments into the jugular veins of each of 4 ewes. During the infusions blood samples were obtained from the ipsilateral jugular vein and common carotid artery. The blood samples were analyzed for radioactivity as free estrone and estradiol-17β and the conversion of infused precursor to product steroid by brain tissue, the transtissue conversion (ρ^PRE-PRO_AV) and the extraction by brain tissue of infused precursor, the transtissue extraction (1-ρ^PRE-PRE_AV) and the metabolic clearance rates were calculated.The mean ± SE for ρ_AV^1,2 (precursor, estrone = 1; product, estradiol = 2) was 0.09 ±0.03 and the mean ± SE for ρ_AV^2,1 (precursor, estradiol = 2; product, estrone = 1) was 0.08 ±0.02. The mean trans-tissue extraction of estrone was 0.13 ± 0.02 and of estradiol-17β was 0.14- ± 0.02. The transtissue extractions of estrone and estradiol-17β were greater than ρ_AV^1,2 ρ_AV^2,1 respectively in 2 of the 4 ewes.Brain metabolism of estrogens can account for only 2–4% of the total metabolism of these free estrogens from the blood pool.     

Canine testicular 17beta-hydroxysteroid oxidoreductase activity. I. Substrate specificity.

The substrate specificity of 17beta-hydroxysteroid oxidoreductase (17beta-HOR) activity was investigated in microsomal preparations of canine testes. Enzyme activity was measured by quantitating the conversion of radioactive substrates to products. The apparent Michaelis constants were determined to be 1.3 x 10(-6)M for androstenendione, 3--10 x 10(-6)M for dehydroepiandrosterone and 25 x 10(-6)M for estrone. These data are similar to those reported for human testicular 17beta-HOR activity and suggest that the canine activity may serve as an animal model for the study of testicular 17beta-HOR.     

Synthesis of new steroid haptens for radioimmunoassay. part I. 15β-Carboxyethylmercaptotestosterone-bovine serum albumin conjugate. measurement of testosterone in male plasma without chromatography

A radioimmunoassay (RIA) procedure has been developed for measurement of testosterone in male plasma after ether:chloroform (4:1) extraction of the plasma sample without resorting to chromatography. The highly specific anti-testosterone serum was generated from both rabbits and sheep immunized with 15β-carboxyethylmercapto-testosterone-BSA conjugate. The synthesis of 15β-carboxyethylmercaptotestosterone and the preparation of its BSA conjugate are described. The high affinity (K_a = 2.38 × 10⁹ liters/mole) antiserum binds 50% of 50 picograms of tritiated testosterone at working dilutions of 1:100,000 to 1:200,000. Both 5α and 5β-dihydrotestosterone compounds exhibited less than 2% cross-reaction. The only other steroids that showed minor cross-reaction were 11β-hydroxytestosterone (3.8%), progesterone (2.1%), corticosterone (1.6%), and deoxycorticosterone (7.7%).     

Variations in the concentrations of androstenedione in peripheral plasma of women the day and during the menstrual cycle

The target tissues (e.g., hypothalamus, pituitary, uterus and vagina) of mature female ovariectomized rats show selective uptake of radioactivity in one hour after the injection of 6,7, ³H-estradiol-17β in a dose of 0.1 μg per 100 g body weight. Injection of 100 μg norethindrone or norgestrel per 100 g body weight 15 min before or 15 min after the administration of tritiated estradiol reduced the radioactivity in most target tissues, and also in the non-target tissues to a lesser extent. The uptake of radioactivity in the pituitary and uterus is reduced more by norethindrone than by norgestrel treatment when these Steroids were injected 15 min after estradiol-17β injection. It appears that there exists a competitive inhibition of estradiol-17β by these contraceptive Steroids in the rat. It is speculated that such competition with estradiol-17β may be an inherent property of the 17-substituted 19-nortestosterone group of Steroids.     

The in vitro synthesis of 7-hydroxy dehydroepiandrosterone by human mammary tissues

Normal and tumorous human mammary tissues were incubated in vitro with [7α-³H] dehydroepiandrosterone and [7α-³H] dehydroepiandrosterone sulphate. Tritium-labelled 7α-hydroxy dehydroepiandrosterone was identified as a principal metabolite from four of the five studies with normal tissue and all seven studies with tumorous tissue.     

Conjugation of androgens and estrogens by human breast tumors in vitro

The metabolism of ³H-androstenedione (Δ⁴ -A) and ³H-estriol (E³) was studied in 12 human breast tumors. Part of each tumor was analyzed for estrogen receptor content. Aliquots of tumor homogenates were incubated for 2 hr separately with ³H-δ⁴-A and ³H-E₃ in the presence of appropriate cofactors. No distinct differences emerged in the profiles of the unconjugated metabolites of ³H-δ⁴-A, the major compounds in the approximate order of descendence being androsterone, androstanedione, testosterone, 5α-androstane-3α,17β-diol, epiandrosterone, and dihydrotestosterone. One tumor homogenate from an infiltrating lobular carcinoma converted ³H-Δ⁴-A to glucosiduronate metabolites (11%), of which androsterone, 6.4%; testosterone, 1.6%; and androstanediol, 0.6% predominated. The homogenate of this tumor and two other tumors converted ³H-E₃ to ³H-E₃-3S. Conversions of E₃ to E₃-3S In the other tumor homogenates were less than 0.6%. No correlation between receptor content and the capability of the tumor to conjugate Δ⁴-A or E₃ evolved. However, correlations between steroid hormone metabolism and tumor histopathology may exist.     

A radioimmunoassay of plasma estradiol-17beta with the use of polyethylene glycol to separate free and antibody-bound hormone

A radioimmunoassay for plasma estradiol-17β was developed using polyethylene glycol to separate free from antibody-bound hormone. Specificity for estradiol-17β was achieved by a modified celite microcolunm procedure in which estradiol was.separated from interfering estrogens, including estrone. Using trace ³H-estradiol to monitor procedural losses, the method was shown to be sensitive and accurate. Intra- and inter-assay coefficient of variation of the method was 8.7 and 10.6%, respectively. Polyethylene glycol used for antibody precipitation appears to be a generally applicable method for steroid hormone radioimmunoassays. The simplicity, precision and rapid analysis, coupled with its lack of time dependence and ease in automation, makes this a convenient and practical method.     

The in vitro biosynthesis and metabolism of progesterone and estrogens by chimpanzee placental tissue

The biosynthesis and metabolism of progesterone and estrogens have been studied in chimpanzee placental tissue in vitro. The conversion of androstenedione-4-14C to estrone and estradiol-17β and of pregnenolone-7α-3H to progesterone has been demonstrated. In addition, the following metabolites were isolated following incubation of either pregnenolone-7α-3H or progesterone-4-¹⁴C: 20α-dihydroprogesterone, 20β-dihydroprogesterone, 6β-hydroxyprogesterone, 5α-pregnane-3,20 dione. The compound 5α-pregnan-3β o1-20-one was identified only after incubation with pregnenolone-7α-3H, while 5β-pregnane-3, 20 dione was identified only after incubation with progesterone-4-¹⁴C. No estrogens could be demonstrated following the incubation of placental preparations with either of the C₂₁ substrates.     

Effect of estradiol-17β on the conversion of pregnenolone to progesterone in human term placenta incubated in vitro

The effect of different doses of estradiol-17β (E₂) on the netabolic pregnenolone to progesterone pathway in fragments of human term placenta incubated $\text{in vitro}$ was studied. Doses considered as being physiological of 0.09 and 0.9 μM had a stimulatory effect on the conversion (p < 0.008 to 0.0l6). However, a supraphysiological dose of 45 μM showed an inhibitory activity related to the maximal stimulation (p < 0.03). A dose of 0.9 μM E₂ favoured the accumulation of (³H)-progesterone in the tissue (p < 0.05). These results suggest that E₂ may regulate the synthesis of progesterone in human term placenta.