Insights from the Structure of Estrogen Receptor into the Evolution of Estrogens: Implications for Endocrine Disruption

In the last decade, there has been important progress in understanding the origins and evolution of receptors for adrenal steroids (aldosterone, cortisol) and sex steroids (estradiol, progesterone, testosterone) due to the sequencing of genomes from animals that are at key sites in vertebrate evolution. Although the estrogen receptor [ER] appears to be the ancestral vertebrate steroid receptor and estradiol [E2] is the physiological ligand for vertebrate ERs, the identity of the ancestral ligand(s) for the ER remains unknown. Here, using an analysis of crystal structures of human ER[alpha] with E2 and other chemicals and 3D models of human ER[alpha] with 27-hydroxycholesterol and 5-androsten-3[beta],17[beta]-diol, we propose that one or more [DELTA]5 steroids were the ancestral ligands for the ER, with E2 evolving later as the canonical estrogen. The evidence that chemicals with a [beta]-hydroxy at C3 in a saturated A ring can act as estrogens and the conformational flexibility of the vertebrate ER can explain the diversity of synthetic chemicals that disrupt estrogen responses by binding to vertebrate ERs

Steroid Receptors and Vertebrate Evolution

Considering that life on earth evolved about 3.7 billion years ago, vertebrates are young, appearing in the fossil record during the Cambrian explosion about 542 to 515 million years ago. Results from sequence analyses of genomes from bacteria, yeast, plants, invertebrates and vertebrates indicate that receptors for adrenal steroids (aldosterone, cortisol), and sex steroids (estrogen, progesterone, testosterone) also are young, with receptors for estrogens and 3-ketosteroids first appearing in basal chordates (cephalochordates: amphioxus), which are close ancestors of vertebrates. An ancestral progesterone receptor and an ancestral corticoid receptor, the common ancestor of the glucocorticoid and mineralocorticoid receptors, evolved in jawless vertebrates (cyclostomes: lampreys, hagfish). This was followed by evolution of an androgen receptor and distinct glucocorticoid and mineralocorticoid receptors in cartilaginous fishes (gnathostomes: sharks). Adrenal and sex steroid receptors are not found in echinoderms: and hemichordates, which are ancestors in the lineage of cephalochordates and vertebrates. The presence of steroid receptors in vertebrates, in which these steroid receptors act as master switches to regulate differentiation, development, reproduction, immune responses, electrolyte homeostasis and stress responses, argues for an important role for steroid receptors in the evolutionary success of vertebrates, considering that the human genome contains about 22,000 genes, which is not much larger than genomes of invertebrates, such as Caenorhabditis elegans (~18,000 genes) and Drosophila (~14,000 genes).Comment: 18 pages, 5 figure

Investigation of urinary steroid metabolites in calf urine after oral and intramuscular administration of DHEA

DHEA (3 beta-hydroxy-androst-5-en-17-one) is a natural steroid prohormone. Despite a lack of information on the effect, DHEA and other prohormones are frequently used as a food supplement by body-builders. DHEA is suspected for growth promoting abuse in cattle as well. Considering the latter, urine samples from a previous exposure study in which calves were exposed to 1 g DHEA per day for 7 days, were used. The calves were divided in three groups: one orally treated, one intramuscularly injected, and a control group. The effect of this treatment on the urinary profile of several precursors and metabolites of DHEA was investigated. Urine samples were collected several days before and during the 7 days of administration and were submitted to a clean-up procedure consisting of a separation of the different conjugates (free, glucuronidated, and sulfated forms) of each compound on a SAX column (Varian). An LC-MS/MS method was developed for the detection and quantification of several metabolites of the pathway of DHEA including 17 alpha- and 17 beta-testosterone, 4-androstenedione, 5-androstenediol, pregnenolone, and hydroxypregnenolone. Elevated levels of DHEA, 5-androstenediol, and 17 alpha-testosterone were observed in the free and sulfated fraction of the urine of the treated calves, thus indicating that the administered DHEA is metabolized mainly by the a dagger(5)-pathway with 5-androstenediol as the intermediate. Sulfoconjugates of DHEA and its metabolites were found to constitute the largest proportion of the urinary metabolites. The free form was also present, but in a lesser extent than the sulfated form, while glucuronides were negligible

Investigation of urinary steroid metabolites in calf urine after oral and intramuscular administration of DHEA

DHEA (3ß-hydroxy-androst-5-en-17-one) is a natural steroid prohormone. Despite a lack of information on the effect, DHEA and other prohormones are frequently used as a food supplement by body-builders. DHEA is suspected for growth promoting abuse in cattle as well. Considering the latter, urine samples from a previous exposure study in which calves were exposed to 1 g DHEA per day for 7 days, were used. The calves were divided in three groups: one orally treated, one intramuscularly injected, and a control group. The effect of this treatment on the urinary profile of several precursors and metabolites of DHEA was investigated. Urine samples were collected several days before and during the 7 days of administration and were submitted to a clean-up procedure consisting of a separation of the different conjugates (free, glucuronidated, and sulfated forms) of each compound on a SAX column (Varian). An LC-MS/MS method was developed for the detection and quantification of several metabolites of the pathway of DHEA including 17a- and 17ß-testosterone, 4-androstenedione, 5-androstenediol, pregnenolone, and hydroxypregnenolone. Elevated levels of DHEA, 5-androstenediol, and 17a-testosterone were observed in the free and sulfated fraction of the urine of the treated calves, thus indicating that the administered DHEA is metabolized mainly by the ¿5-pathway with 5-androstenediol as the intermediate. Sulfoconjugates of DHEA and its metabolites were found to constitute the largest proportion of the urinary metabolites. The free form was also present, but in a lesser extent than the sulfated form, while glucuronides were negligible

Androgenic supplementation in men: effects of age, herbal extracts, and mode of delivery

Ingesting androstenedione alone or with herbal extracts, or androstenediol with herbal extracts purportedly increases serum [testosterone] in men. The addition of herbal extracts is purported to prevent the conversion of testosterone precursors into estrogens or dihydrotestosterone. The effects of ingesting androstenedione, androstenedione + herbal extracts, and androstenediol + herbal extracts on serum [testosterone], [estradiol], [dihydrotestosterone], [prostate specific antigen] (PSA), and blood lipids was investigated in 30 to 59 year old men. Subjects were randomly assigned to consume ASD (300 mg/day androstenedione; n = 28), DION (300-mg androstenedione, 150-mg dehydroepiandrosterone, 540-mg saw palmetto, 300-mg indole-3-carbinol, 625-mg chrysin, and 750-mg Tribulus terrestris per day; n = 28), AND-HB (300-mg androstenediol, 480-mg saw palmetto, 450-mg indole-3-carbinol, 300-mg chrysin, 1,500 mg gamma-linolenic acid, and 1,350-mg Tribulus terrestris per day; n = 28), or placebo (n = 27) for 28 days. Serum [free testosterone], [total testosterone], [androstenedione], [dihydrotestosterone], [estradiol], [PSA], and [lipids] were measured before and throughout the 4-week supplementation period. Serum [total testosterone] and [PSA] were unchanged by supplementation. Serum [androstenedione] (174%, 342%, and 300%), [free testosterone] (37%, 38%, and 45%), [dihydrotestosterone] (57%, 71%, and 83%), and [estradiol] (86%, 103%, 68%) were elevated (P \u3c 0.05) for AND-HB, DION, and ASD, respectively. Serum [HDL-C] was reduced (P \u3c 0.05) similarly by 0.13 mmol·l-1 in ASD, DION and AND-HB. There is no difference in the serum [testosterone], [estradiol], [dihydrotestosterone], [PSA], and [lipid] response to ingesting androstenedione alone, androstenedione + herbal extracts, or androstenediol + herbal extracts.;Orally ingested androgens undergo a large degree of digestive and hepatic breakdown. Therefore, we evaluated the hormonal response to sublingual cyclodextrin androstenediol. Eight males (22.9 +/- 1.2 y) consumed either 20 mg androstenediol in a sublingual cyclodextrin tablet (SL-DIOL) or placebo. Blood samples were collected before supplementation and every 30-min for 3 h after supplementation. Serum [androstenedione] was increased 125% at 120 min, serum [free testosterone] was increased 118% at 60 min, serum [total testosterone] was increased 110% at 60 min, and serum [estradiol] was 71% elevated at 180 min in SL-DIOL. These data indicate that, in contrast to ingested androstenediol, sublingual cyclodextrin androstenediol intake increases serum [androstenedione], [free testosterone], [total testosterone], and [estradiol]

A pilot study comparing the metabolic profiles of elite-level athletes from different sporting disciplines

Background: The outstanding performance of an elite athlete might be associated with changes in their blood metabolic profile. The aims of this study were to compare the blood metabolic profiles between moderate- and high-power and endurance elite athletes and to identify the potential metabolic pathways underlying these differences. Methods: Metabolic profiling of serum samples from 191 elite athletes from different sports disciplines (121 high- and 70 moderate-endurance athletes, including 44 high- and 144 moderate-power athletes), who participated in national or international sports events and tested negative for doping abuse at anti-doping laboratories, was performed using non-targeted metabolomics-based mass spectroscopy combined with ultrahigh-performance liquid chromatography. Multivariate analysis was conducted using orthogonal partial least squares discriminant analysis. Differences in metabolic levels between high- and moderate-power and endurance sports were assessed by univariate linear models. Results: Out of 743 analyzed metabolites, gamma-glutamyl amino acids were significantly reduced in both high-power and high-endurance athletes compared to moderate counterparts, indicating active glutathione cycle. High-endurance athletes exhibited significant increases in the levels of several sex hormone steroids involved in testosterone and progesterone synthesis, but decreases in diacylglycerols and ecosanoids. High-power athletes had increased levels of phospholipids and xanthine metabolites compared to moderate-power counterparts. Conclusions: This pilot data provides evidence that high-power and high-endurance athletes exhibit a distinct metabolic profile that reflects steroid biosynthesis, fatty acid metabolism, oxidative stress, and energy-related metabolites. Replication studies are warranted to confirm differences in the metabolic profiles associated with athletes’ elite performance in independent data sets, aiming ultimately for deeper understanding of the underlying biochemical processes that could be utilized as biomarkers with potential therapeutic implications

Steroid metabolism in utero and in the neo-natal period

1. Two steroids were identified for the first time as major steroid components of urine collected from infants and plasma obtained from the umbilical blood vessels. These compounds have the formula 3p,l6|3- dihydroxyandrost-5-en-17-one and 3(3,17a-dihydroxyandrost-5-ene, but their importance in the metabolism of steroids by the foeto-placental unit is not known. 2. A method was developed for measuring steroids in plasma obtained from the umbilical blood vessels and urine collected from infants. This depends upon the separation of steroids by thin-layer chromatography, and their assay on the thin-layer plates by developing colours with various spray reagents and subsequent densitometric scanning. The accuracy and specificity of the method is discussed. 3. This method was used to establish normal ranges for the excretion of several 3|3-hydroxy-A steroids found in urine specimens obtained from infants, and to determine the effects upon the excretion of steroids of administering corticotrophin and human chorionic gonadotrophin to newborn infants. 4. The enzyme defects present in three infants with abnormal adrenal glands were investigated by analysis of steroids in urine specimens obtained from these patients. 5. Several 3(3-hydroxy-A"* steroids were measured in plasma samples prepared from venous and arterial blood obtained from the umbilical cord. The concentrations present in arterial plasma were higher than in the umbilical vein, indicating a net uptake of these steroids by the placenta, where it is thought that they are converted into 3-oxo-A steroids and oestrogens. The relationship between the levels of 3(3-hydroxy-Asteroids in plasma obtained from the umbilical blood vessels and oestrogen excretion by the mother was also investigated

The acute effects of transdermal testosterone precursor administration on serum steroid hormone levels in females

Most governing sports authorities have banned the use of anabolic steroids as ergogenic aids. Recently, a transdermal testosterone precursor dietary supplement (AndrosteDERMRTM) has become available; athletes can apply the cream to their skin with the belief that it will increase serum testosterone levels, muscle mass, and strength. The purpose of this study was to measure the effects of one milliliter of AndrosteDERMRTM) applied to the inner surface of the upper arm on the serum levels of androstenedione, and free and total testosterone in female subjects. Serum levels were measured before application and every 45 minutes thereafter for six hours. Serum androstenedione, and free and total testosterone levels were analyzed using radioimmunoassay. Although the trend seemed to indicate that serum levels did not rise after application, several subjects had physiologically impossible values, which appear to be due to methodological errors. That information, along with the initial small number of subjects made the use of statistical treatment unwise and inferences about the population impossible