Sites of in vivo extraction and interconversion of estrone and estradiol in the dog

The interconversion and extraction of estrone and estradiol-17β across and within different tissues or areas have been studied in the dog by the constant infusion technique. The results were calculated using the ³H/¹⁴C ratios and radioactive concentrations of estrone and estradiol obtained from afferent and efferent blood and tissues at equilibrium. From these results it is concluded that: (1) there is no significant difference between metabolic clearance rates of estrone and estradiol, (2) blood transfer constants indicate a higher conversion of estradiol to estrone than of estrone to estradiol, (3) the transtissue interconversion favors the formation of estrone while the intratissue interconversion favors the formation of estradiol, (4) no interconversion of the two estrogens is observed in adipose tissue, (5) the extraction of estradiol entering a tissue was lower than the extraction of estradiol formed in these tissues, (6) calculation of the tissue metabolic clearance rates show that 63% and 61% of the total metabolism of estrone and estradiol, respectively, occurs in the splanchnic bed, and (7) the contribution of each tissue to the total interconversion of estrone and estradiol show that more than 90% of this interconversion occurs extrahepatically.     

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.     

Metabolism and conjugation of [4-14C]progesterone by bovine liver and adipose tissues, in vitro

The ability of bovine liver and fat to metabolize progesterone and also to form glucuronide conjugates with these progestins $\text{in vitro}$ was investigated. Tissue supernatants were incubated with [4-¹⁴C] progesterone, UDP-glucuronic acid, and a NADPH generating system for 5 hr, at 37°C. Steroids were identified by thin-layer chromatography, high performance liquid chromatography, and recrystallization to a constant specific activity. The total original radioactivity which could not be removed by exhaustive ether extraction (presumptive conjugates) was 44.7 ± 14.2% in liver, 5.0 ± 3.6% in subcutaneous fat, and 3.7 ± 2.2% in kidney fat samples. Progestins identified in liver samples include 5β-pregnane-3α, 20α-diol (free and conjugate), 5β-pregnane-3α, 20β-diol (free and conjugate), 3α-hydroxy-5sB-pregnan-20-one (free and conjugate), 3β-hydroxy-5β-pregnan-20-one (free), 5β-pregnane-3, 20-dione (free), and progesterone (conjugate). Progestins identified in both the free and conjugate fractions of subcutaneous fat and kidney fat samples include progesterone, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-4-pregnen-3-one, and 20α-hydroxy-4-pregnen-3-one. Differences due to sex of bovine used were noted. These results confirm the ability of bovine liver to readily metabolize progesterone and form glucuronide conjugates of these compounds and suggest that adipose tissues take an active role in these actions in cattle.     

Aromatization of androgens by human breast cancer.

The metabolism of dehydroepiandrosterone and testosterone by human mammary tumor was investigated. Estrogen synthesis from dehydroepiandrosterone was observed in 9 of 10 estrogen-receptor-negative tumors and only in 2 of 8 receptor-positive tumors (p less than 0.025). Conversion of testosterone to estrogens was observed in 7 of 8 receptor-negative and 2 of 7 receptor-positive tumors. Tumors which are capable of transforming dehydroepiandrosterone to estrogens were also able to aromatize testosterone suggesting that the presence of the aromatase enzyme is inherent to certain tumor cells. No estrogen formation was detected by the mitochondrial-microsomal fraction of normal breast cells while fractions from both fat cell and tumor cell showed estrogen synthesis. Estrogen formation by tumor cell fraction ranged from 5 to 190 times that observed for fat cells. The physiological significance of these results in the neoplastic tissue and its relationship to hormone dependence are discussed.     

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.     

Steroid delta 4-5 beta-reductase in human mammary tumors.

Steroid delta 4-5 alpha- and delta 4-5 beta-reductase activity was determined in 16 human mammary tumors and 8 DMBA-induced rat mammary tumors using a spectrophotometric assay. Steroid delta 4-5 alpha-reductase was present in all tumors investigated while delta 4-5 beta-reductase was detected in only 6 estrogen receptor negative human breast tumors and absent in all estrogen receptor positive human breast tumors as well as in all rat mammary tumors. Further support for the presence of delta 4-5 beta-reductase was established by using a dual-labelling technique consisting of incubating tumor slices with [14C] testosterone and adding [3H] etiocholanolone, [3H] testosterone and [3H]-5 alpha-dihydrotestosterone at the end of the reaction. Following extraction and chromic acid oxidation, 4-androstenedione, 5 beta-androstanedione and 5 alpha-androstanedione were isolated and purified, and the constancy of the 14C/3H ratio was used as proof of 5 alpha-reductase and 5 beta-reductase. These results were shown to be consistent with the data obtained using the spectrophotometric assay.     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

Influence of age on the formation of 5alpha-androstanediol and 7alpha-hydroxy-testosterone by incubated rat testes.

Testes from rats of different ages were indubated with or without tritiated testosterone. The exogenously-added or endogenously-produced testosterone is mainly metabolized to 7alpha-hydroxylated testosterone in adult animals, and to 5alpha-reduced metabolites (especially 5alpha-androstanediol) in immature animals.     

The conversion of testosterone to 7α-hydroxy-testosterone by incubated rat testes

Incubations of testes of adult rats with testosterone yield rather important amounts of a very polar metabolite which is identified as 7α-hydroxytestosterone. The identification of the metabolite is based on chromatography, spectrophotometry, fluorimetry, counter current distribution and NMR spectrometry.     

Chemiluminescence enzyme immunoassay of dehydroepiandrosterone and its sulfate using peroxidase as label

We report a highly sensitive enzyme immunoassay for dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) using horseradish peroxidase as the label enzyme. Separation of free and bound DHEA-peroxidase conjugate was by insolubilized antibody, prepared by coupling purified IgG of goat anti-rabbit IgG serum with Sepharose 4B or a polystyrene tube. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. The sensitivity was 25 pg/assay tube for DHEA and 100 pg/assay tube for DHEA-S. Upon comparison, results obtained by radioimmunoassay and this method showed good agreement; r = 0.86 for free DHEA, r = 0.92 for acid-hydrolyzed DHEA-S and r = 0.91 for solvolyzed DHEA-S. The present method is applicable in the routine determination of DHEA and DHEA-S in biological fluid.     

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.     

In vitro metabolism of 4-androstene-3,17-dione and testosterone by rat submaxillary gland.

Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of male and female rats. The metabolism was predominately reductive. In 15 and 180 min incubations submaxillary tissue converted 4-androstene-3,17-dione chiefly to androsterone. Less testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one, 5 alpha-androstane-3,17-dione, 5 alpha-androstane-3 alpha, 17 beta-diol, and 4-androstene-3 alpha, 17 beta-diol were also identified. Testosterone was converted to the same products plus 4-androstene-3,17-dione. 5 alpha-Androstane-3 alpha, 17 beta-diol was the major testosterone metabolite. Qualitatively the metabolism by male and female submaxillary gland was similar.     

Developmental pattern of Δ5 3β-hydroxysteroid dehydrogenase activity in isolated rat Leydig cells

A direct method for determination of Δ⁵ 3β-hydroxysteroid dehydrogenase (3β-HSD) activity was employed in isolated Leydig cells (LC) derived from rats on fetal day 19 (F₁₉) and postnatal (N) days 1,12,24, 34 and 45 and adults. The activity of 3β-HSD in the adult LC was 1.15 ± 0.02 (μmole/μg DNA/hr, mean ± SEM, n = 73). Activities in the other groups, expressed as a percentage of the respective adult control, were: F₁₉-38%; N₁-39%; N₁₂-8%; N₂₄-89%; N₃₄-166%; and N₄₅-118%. A good correlation was found between histochemical staining for 3β-HSD and the quantitive method employed. Using (³H)-DHA as a substrate, LC isolated from F₁₉, n₁ and N₁₂ produced testosterone in appreciable amounts (41%, 55% and 20% of the toal products respectively) whereas at advanced stages of development (N₂₄ to adulthood) the major product was androstenedione (93 ± 1%). These findings may be explained by the observed decrease in 17β-hydroxysteroid dehydrogenase (17β-HSD) activity, due to an insufficient supply of NADPH, in the older vs. earlier stages of development. This study indicates the presence of steroidogenic enzymatic activity in LC throughout development in the rat. It also provides a relatively simple in vitro model for studies of testicular regulation during development.     

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.     

N-hydroxymethylpentamethylmelamine,a major in vitro metabolite of hexamethylmelamine

N-Hydroxymethylpentamethylmelamine (HMPMM) was identified by HPLC and by GLC-MS after derivatization, as a metabolite of the anticancer drug hexamethylmelamine (HMM) in incubation mixtures with fortified mouse liver 9000 × g and microsomal preparations. HMPMM formation was dependent on the presence of NADPH and oxygen. N-demethylated metabolites were also found. HMPMM displays appreciable chemical stability and 29% was recovered after 60 min incubation in buffer. HMPMM constituted more than 50% of total HMM metabolites in 30 min incubations. The known chemical reactivity of carbinolamines means that HMPMM could be involved in the pharmacological or toxic effects of HMM.     

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.     

Sizing of two bacteriophage T4 specific messenger ribonucleic acids formed in vitro and in vivo

Messenger RNA for two T4 specific enzymes, deoxynucleotide kinase and α glucosyltransferase, have been sized by sedimentation on sucrose density gradients. The sedimentation constants of transferase and kinase mRNAs formed $\text{in vitro}$ were 21.5S and 14.5S respectively, regardless of the duration of incubation up to 20 min. Although the kinase mRNA isolated from cells infected with T4 phage for 10 min was the same size as found $\text{in vitro}$ (14.5S), the transferase mRNA was found in a segment approximating the size of the kinase mRNA (14.5S). The experiments indicate that α glucosyltransferase mRNA is formed first as a polycistronic message and is then processed to the smaller unit.     

Simultaneous radioimmunoassay of testosterone,dihydrotestosterone, 5α-androstane-3α, 17β-diol and 5α-androstane-3β, 17β-diol in the plasma of adult male rats

A single thin layer chromatography and three antibodies were used for the specific radioimmunoassay of four androgens in pooled rat plasma (Sprague-Dawley adult males). The following values were found (pg/ml ± SD). Testosterone : 3, 138 ± 173; dihydrotestosterone : 374 ± 20; 5α-androstane-3α 17β-diol : 284 ± 24; 5α-androstane-3β, 17β-diol : 223 ± 11.