Effects of maternal androgens and their metabolite etiocholanolone on prenatal development in birds.

Offspring phenotypes can be affected by maternal testosterone and androstenedione (A4), which are considered a tool of mothers to adjust offspring to a fluctuating environment. Yet testosterone and A4 are very rapidly metabolized by developing avian embryos, suggesting that either the maternal testosterone and A4 have potent organizational effects on the embryos extremely early before being metabolized or it is the metabolites that evoke phenotypic variation in the offspring. One of the metabolites, etiocholanolone, increases substantially during early embryonic development and is a likely candidate for mediating maternal effects as it can promote erythropoiesis. To investigate and compare the effects of testosterone and A4 with the possible effects of etiocholanolone during prenatal embryonic development, we increased their levels in black-headed gull eggs (Larus ridibundus), and used sham-injected eggs as controls. This species usually has 3-egg clutches in which maternal androgen levels increase with the egg-laying sequence. We analysed embryonic heart rate, peri-hatching biometric traits, the ratio of white to red blood cells (W/R ratio) and bursa development. We found that testosterone and A4 treatment increased embryonic heart rate irrespective of egg-laying sequence and decreased bill length and W/R ratio, whereas etiocholanolone did not mimic these effects. Instead, etiocholanolone treatment decreased tarsus length and brain mass. Our finding that etiocholanolone does not mimic the effects induced by testosterone and A4 suggests that the embryonic metabolism of maternal testosterone and A4 can potentially diversify the function of these maternal androgens.

Characterizing the timing of yolk testosterone metabolism and the effects of etiocholanolone on development in avian eggs.

Maternal transfer of steroids to eggs can elicit permanent effects on offspring phenotype. Although testosterone was thought to be a key mediator of maternal effects in birds, we now know that vertebrate embryos actively regulate their exposure to maternal testosterone through steroid metabolism, suggesting testosterone metabolites, not testosterone, may elicit the observed phenotypic effects. To address the role steroid metabolism plays in mediating yolk testosterone effects, we used European starling (Sturnus vulgaris) eggs to characterize the timing of testosterone metabolism and determine whether etiocholanolone, a prominent metabolite of testosterone in avian embryos, is capable of affecting early embryonic development. Tritiated testosterone was injected into freshly laid eggs to characterize steroid movement and metabolism during early development. Varying levels of etiocholanolone were also injected into eggs, with incubation for either 3 or 5 days, to test whether etiocholanolone influences the early growth of embryonic tissues. The conversion of testosterone to etiocholanolone was initiated within 12 h of injection, but the increase in etiocholanolone was transient, indicating that etiocholanolone is also subject to metabolism, and that exposure to maternal etiocholanolone is limited to a short period during early development. Exogenous etiocholanolone manipulation had no significant effect on the growth rate of the embryos or extra-embryonic membranes early in development. Thus, the conversion of testosterone to etiocholanolone may be an inactivation pathway that buffers the embryo from maternal steroids, with any effects of yolk testosterone resulting from testosterone that escapes metabolism; alternatively, etiocholanolone may influence processes other than growth or take additional time to manifest.

Individual stress responses to Sarcoptes scabiei infestation in Iberian ibex, Capra pyrenaica.

In this study we have monitored the stress of Iberian ibex at individual level within the course of an experimental infection with Sarcoptes scabiei mites. For this purpose we have measured faecal 11-ketoetiocholanolone (11-k) using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). We used linear mixed models to explore the effects of host sex and age, clinic (mange status) and time (number of days post-infection) on the concentration of faecal 11-k. The most parsimonious model included clinic, time and host age, which explained 76.6% of the variance of the response variable. Moreover, the concentration of faecal 11-k varied greatly between individuals. Our results evidence the stressor nature of the disease and highlight the negative effects on hosts due to cortisol release and activity.

Inhibitory action of thymol on fecal microbial activity in Tamandua tetradactyla and its effect on glucocorticoid metabolite measurement.

Faecal glucocorticoid measurement is a potentially important tool for improving wildlife conservation, but its use is still limited by methodological issues including the need to avoid modifications of steroids by faecal microorganisms during storage. The freezing of faeces is recommended as a means of avoiding such alterations, but this is costly under non-controlled environmental conditions. The present study was designed to determine whether the application of thymol reduced the proliferation of microorganisms in the faeces of Tamandua tetradactyla and whether it influenced faecal glucocorticoid metabolite (FGM) measurements. Tamandua tetradactyla faeces were individually collected after defaecation, divided into fractions (5.5 g each) and kept in sealed glass Petri dishes at 22 ±â€¯2 °C. A thymol solution (550 µL; 5 mg g-1 feces; 80% ethanol) or an 80% ethanol solution (550 µL, control) was added before storage of faeces. Negative controls for FGM consisted of samples without thymol or ethanol solutions. All samples were evaluated at 0, 24, 48 and 72 h post-defaecation. Thymol was first incubated with a glucocorticoid standard in a faeces-free tube or in a faecal sample in order to determine whether it interfered with FGM measurements. Data showed that thymol did not affect FGM measurements. Post-defaecation time caused a significant reduction in FGM measurements in the negative control, an increment at 48 h in the control, and no change in FGM measurements in thymol treatment. FGM measurements were significantly different between groups (negative control > control - treatment). Thymol caused a significant reduction of up to three orders of magnitude in total coliforms, total aerobic and anaerobic heterotrophic mesophilic bacteria, mold and yeast per gram of faeces at 24, 48 and 72 h. The reduction in microbial activity presumably contributed to the stability of FGM over time. Spore-forming bacteria (SFB) in faeces were not reduced by thymol. We propose thymol as an alternative to freezing since it stabilizes FGMs for at least 3 days after collection in the faeces of Tamandua tetradactyla.

Evaluating capture stress in wild gray mouse lemurs via repeated fecal sampling: method validation and the influence of prior experience and handling protocols on stress responses.

Reliable measurements of physiological stress are increasingly needed for eco-physiological research and for species conservation or management. Stress can be estimated by quantifying plasma glucocorticoid levels, but when this is not feasible, glucocorticoid metabolites are often measured from feces (FGCM). However, evidence is accumulating on the sensitivity of FGCM measurements to various nuisance factors. Careful species- and context-specific validations are therefore necessary to confirm the biological relevance and specificity of the method. The goals of this study were to: (1) establish and validate sampling methods and an enzymeimmunoassay to measure FGCM in the gray mouse lemur (Microcebus murinus); (2) explore causes of variability in the FGCM measurements, and; (3) assess the consequences of capturing and handling for free-living individuals by quantifying their stress responses via repeated fecal sampling within capture sessions. We further assessed the influence of different handling protocols and the animals' previous capture experience on the magnitude of the physiological response. Our validations identified the group-specific measurement of 11ß-hydroxyetiocholanolone as the most suitable assay for monitoring adrenocortical activity. The sample water content and the animal's age were found to significantly influence baseline FGCM-levels. Most captured animals exhibited a post-capture FGCM-elevation but its magnitude was not related to the handling protocol or capture experience. We found no evidence for long-term consequences of routine capturing on the animals' stress physiology. Hence the described methods can be employed to measure physiological stress in mouse lemurs in an effective and relatively non-invasive way.

SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy.

AIMS: Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes. METHODS AND RESULTS: Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed (i) marked DD not associated to cardiac fibrosis, (ii) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, (iii) reduced LV SERCA2 protein level and activity and (iv) slower SR Ca2+ uptake rate, (v) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, (vi) increased diastolic Ca2+, and (vii) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. CONCLUSIONS: SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment.

Responses of round goby (Neogobius melanostomus) olfactory epithelium to steroids released by reproductive males.

The wild perciform teleost Neogobius melanostomus (the round goby) originated from the Ponto-Caspian region and is now a highly successful invasive species in the Laurentian Great Lakes. Males may attract females into their nests for spawning by releasing reproductive pheromones, and it has been previously shown that reproductive males synthesize and release the 5ß-reduced and 3α-hydroxyl steroids 3α-hydroxy-5ß-androstane-11,17-dione (11-oxo-etiocholanolone; 11-O-ETIO) and 3α-hydroxy-5ß-androstane-11,17-dione 3-sulfate (11-oxo-etiocholanolone-3-sulfate; 11-O-ETIO-3-s) and 3α,17ß-dihydroxy-5ß-androstan-11-one 17-sulfate. In this study, we investigated properties of these released steroids by recording field potential responses from the olfactory epithelium (electro-olfactogram, EOG). The steroid 3α,17ß-dihydroxy-5ß-androstan-11-one 17-sulfate did not elicit olfactory responses while both 11-O-ETIO and 11-O-ETIO-3-s stimulated olfactory field potentials in the round goby, but not in the goldfish. Cross-adaptation analysis demonstrated that round gobies discriminated between11-O-ETIO and 11-O-ETIO-3-s (as well as etiocholanolone, ETIO) at the sensory level. Second messenger cascades depending on both cAMP and IP(3) were inferred for steroids from pharmacological inhibition studies, while the canonical teleost odors taurocholic acid (a bile acid) and L: -alanine (an amino acid) used only cAMP and IP(3), respectively. The round goby presents itself as an excellent species for the study of olfactory function of fish in the wild, given its possible use of these released steroids as pheromones.

The effect of elevated steroids released by reproductive male round gobies, Neogobius melanostomus, on olfactory responses in females.

The round goby, Neogobius melanostomus, is a highly successful invasive species in the Laurentian Great Lakes. Previous behavioral studies implied that females are attracted by pheromones to the nests of reproductive males, and that males release putative steroidal pheromones--unconjugated as well as conjugated forms of 3α-hydroxy-5ß-androstane-11,17-dione (11-O-ETIO)-following stimulation of the hypothalamic--gonadal axis with salmon gonadotropin releasing hormone analog (sGnRHa). In this study, we tested the olfactory system of females in response to extracts containing these released steroids. We compared electrical field potential responses from the olfactory epithelium (electro-olfactogram, EOG) of non-reproductive females to methanol extracts of water that previously held males, collected before and after injection of the males with sGnRHa or saline. The females showed increased EOG responses to the post-injection extracts when males were treated with sGnRHa but not saline. This finding provides further evidence for interactions between male and female N. melanostomus via steroidal reproductive pheromones.

Studies in porphyria. I. A defect in the reductive transformation of natural steroid hormones in the hereditary liver disease, acute intermittent porphyria.

A variety of 5beta steroid metabolites derived from hormones natural to man are potent inducers experimentally of delta-aminolevulinate synthetase, the rate-limiting enzyme in porphyrin-heme formation. This mitochondrial enzyme is found at high levels of activity in the livers of patients with the genetic disease, acute intermittent porphyria (AIP). In this study the metabolism of (14)C-labeled testosterone was examined in AIP patients to determine whether there was a disproportionate conversion of the hormone to its 5beta, compared to its 5alpha metabolite. The results indicate that AIP subjects do generate a substantially greater than normal fraction of 5beta metabolite from this steroid; the excessive degree of ring A reduction of testosterone taking place via the 5beta pathway in the porphyric patients averages 350% greater than in the nonporphyric subjects. In one asymptomatic AIP patient the disproportionate generation of 5beta metabolite from the hormone reached a level 10 times the normal mean. Studies with a second (14)C-labeled hormone, dehydroisoandrosterone, whose metabolism in man resembles that of testosterone, confirmed the derangement in reductive transformation of steroids found in the individuals carrying the genetic lesion of AIP. These findings define a new endocrine abnormality in AIP patients and raise the possibility that endogenously derived 5beta steroids may contribute by an induction mechanism to the increased levels of hepatic delta-aminolevulinate synthetase activity found in AIP patients.

The effects of sex, age and commensal way of life on levels of fecal glucocorticoid metabolites in spiny mice (Acomys cahirinus).

We studied levels of fecal glucocorticoid metabolites (GCM) in a social rodent - Egyptian spiny mouse. As breeding adults are socially dominant over subadults, and adolescent males are driven away by the dominant males, we addressed the question whether animals within extended families are stressed differently depending upon their social category. In addition, we evaluated whether there are differences between non-commensal (outdoor) and commensal (adapted to human settlements) populations. Concentrations of fecal GCM were assessed from samples collected in a special cage that allowed continuous individual sampling of undisturbed mice housed as a semi-natural social unit. First we performed an ACTH challenge test to validate two enzyme immunoassays (EIA): a 5alpha-pregnane-3beta,11beta,21-triol-20-one EIA and an 11-oxoetiocholanolone EIA to measure a group of fecal GCM in this species. Next we monitored concentrations of fecal GCM in 68 individuals belonging to 10 family groups and two populations. Commensal spiny mice showed higher fecal GCM levels than non-commensal ones. No effect of age (i.e., social dominance) and only a small effect of sex (in the commensal population only, with males exhibiting lower values) on fecal GCM levels were found. On the other hand, considerable variations in measured fecal GCM between family groups were revealed, indicating that the social settings of the particular group play an important role.

Glucocorticoid-binding proteins in human acute lymphoblastic leukemic blast cells.

The first known step in steroid hormone action is the association of the steroid with specific cytoplasmic steroid-binding proteins (SBP). Using a competitive binding assay, we detected, quantified, and partially characterized such a SBP in cytosol from glucocorticoid-sensitive human lymphoblastic leukemic blasts. The affinity of steroids for the SBP was directly related to their known killing potency. For example, steroids without glucocorticoid effect such as androstenedione, etiocholanolone, and tetrahydrocortisol were unable to displace radiolabeled dexamethasone from the SBP in the binding reaction. The dose-response curve for in vitro inhibition of [(3)H]thymidine uptake in leukemic blasts correlated closely with the binding affinity of glucocorticoids to the SBP, providing additional support for an essential physiologic role for SBP in steroid action. SBP activity was either greatly diminished or absent in glucocorticoid-resistant cells. Six patients who intially had SBP in their blasts and were responsive to combinations of drugs including glucocorticoids no longer had SBP activity detectable at a time when they no longer responded to combinations of drugs including glucocorticoids. In vitro [(3)H]thymidine uptake was not inhibited by steroids in leukemic blast cells lacking SBP activity. Other patients who had received some antileukemic therapy including glucocorticoids and who still had SBP in their leukemic blasts, were still responsive to drug combinations that included glucocorticoids. This appears to be the first study demonstrating glucocorticoid receptors in a human tissue.

Effect of diet on plasma and urinary hormones in South African black men with prostatic cancer.

Epidemiological evidence suggests that the incidence and death rate from prostatic cancer, an endocrine-associated disease, are related to environmental factors including diet. In this study, a comparison of serum and urinary levels of steroid hormones was carried out in healthy elderly rural vegetarian South African black men, a low-risk population, and a comparable group of men with prostatic cancer. In these prostatic cancer patients, plasma androgen levels decreased, while estrogen levels increased. Concomitantly, the androsterone:etiocholanolone ratio increased, and a greater proportion of estrogens was excreted as estriol. When transferred to a Western diet, plasma androgens showed a further decrease and a greater increase in estrone in prostatic cancer patients. In prostatic cancer patients, the total urinary androgen and estrogen levels were unaltered. However, in elderly healthy men, the Western diet decreased the excretion of estrogens and androgens. Thus, a Western diet supplemented the decrease in plasma androgens initially present in these patients. Evidence suggests that the decrease in plasma androgens increases the estrogen: androgen ratio, which may lead to hyperplasia of the prostatic ductal epithelia, a change enhanced by a Western diet. Changes in urinary steroid hormone levels in South African black patients comparable to those reported in white prostatic cancer patients indicate that hormonal changes must be related to several environmental factors, apart from diet. A simultaneous study of the steroid hormone composition of blood and prostatic fluid in this low-risk population is suggested.

Functional characterization and anti-cancer action of the clinical phase II cardiac Na+/K+ ATPase inhibitor istaroxime: in vitro and in vivo properties and cross talk with the membrane androgen receptor.

Sodium potassium pump (Na+/K+ ATPase) is a validated pharmacological target for the treatment of various cardiac conditions. Recent published data with Na+/K+ ATPase inhibitors suggest a potent anti-cancer action of these agents in multiple indications. In the present study, we focus on istaroxime, a Na+/K+ ATPase inhibitor that has shown favorable safety and efficacy properties in cardiac phase II clinical trials. Our experiments in 22 cancer cell lines and in prostate tumors in vivo proved the strong anti-cancer action of this compound. Istaroxime induced apoptosis, affected the key proliferative and apoptotic mediators c-Myc and caspase-3 and modified actin cystoskeleton dynamics and RhoA activity in prostate cancer cells. Interestingly, istaroxime was capable of binding to mAR, a membrane receptor mediating rapid, non-genomic actions of steroids in prostate and other cells. These results support a multi-level action of Na+/K+ ATPase inhibitors in cancer cells and collectively validate istaroxime as a strong re-purposing candidate for further cancer drug development.

Structure and activity of the murine type 5 17beta-hydroxysteroid dehydrogenase gene(1).

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) play a crucial role in the control of active sex steroid intracellular levels. Seven types of 17beta-HSD have been described. In this study, we report the cloning and characterization of the mouse type 5 17beta-HSD belonging to the aldo-keto reductase superfamily, in contrast with types 1, 2, 3, 4, 6, and 7 17beta-HSD which belong to the short-chain alcohol dehydrogenase family. The gene spans 16 kb and contains 9 exons separated by 8 introns. Primer extension analysis identified a major transcription start site beginning 50 nucleotides upstream from the ATG initiation codon. Northern blot analysis showed a high mRNA expression level in the liver and a weaker signal in the kidney. To determine more precisely the substrate specificity of the enzyme, we established a stable cell line expressing mouse type 5 17beta-HSD in transformed human embryonic kidney (293) cells. The transfected cell line preferentially catalyzes the transformation of 4-androstenedione (4-dione) and androstanedione (A-dione) into testosterone (T) and dihydrotestosterone (DHT), respectively. This data is somewhat in contradiction with a previous study that described the enzyme as estradiol 17beta-dehydrogenase. Our results indicate that the rate of transformation of estradiol (E(2)) to estrone (E(1)) represents only 1% of the rate of transformation of 4-dione to T. Mouse type 5 17beta-HSD shares 76% amino acid sequence identity with human type 5 17beta-HSD; 71%, 76%, 76% with rat 3alpha-HSD and human types 1 and 3 3alpha-HSDs, respectively; and 71%, 69% and 77% with mouse, rat and human 20alpha-HSD, respectively.

Androgen metabolism by isolated hairs from women with idiopathic hirsutism is usually normal.

We have tested the hypothesis that idiopathic hirsutism (IH) may be due to abnormality of androgen-responsive hair follicles. Because androgen metabolism within target cells is an important determinant of androgen action, we have analyzed the rates of formation and disposition of the major mediators of androgen action, testosterone (T) and dihydrotestosterone (DHT). In normal women, the pattern of androgen metabolism by growing hairs favors T predominance over DHT and inactivation of both these 17 beta-hydroxysteroids to 17-ketosteroids. This pattern results greatly from predominance of 17 beta-hydroxysteroid dehydrogenation. For example, in normal women's scalp hair, DHT disposition to 5 alpha-androstanedione proceeded at the rate of 8.6 +/- 2.0 (SEM) %/micrograms DNA/min, whereas DHT was formed from T at a rate of 0.14 +/- 0.02, and T was formed from androstenedione at a rate of 0.60 +/- 0.12, all significantly different from one another. Both the formation of 17-ketosteroids and the apparent 5 alpha-reductase activity were exaggerated in the pubic hair of men; whether these differences are site-, sex-, or androgen-related, remains to be determined. Pubic hairs tended to metabolize androgens at a greater rate than did scalp hair. This was related to the significantly greater DNA content of plucked pubic hairs, a difference unrelated to sex or androgen levels. Women with IH had heterogeneous pubic hair abnormalities. Only 1 of the 4 IH patients studied had abnormal pubic hair follicle androgen metabolism, with the greatest abnormality being an exaggerated rate of 17 beta-hydroxysteroid inactivation to 17-ketosteroids. Two of the other 3 IH cases had increased DNA content of plucked pubic hairs, a different kind of exaggeration of normal, which suggests an abnormality of hair follicle growth unrelated to androgen sensitivity. We favor the concept that IH is related to various distinct types of sexual hair abnormalities which reflect fundamental defects in the regulation of hair growth.

Aromatase activity in quail brain: correlation with aggressiveness.

Testosterone (T) triggers aggressive behavior in males of many vertebrate species; however, the neural and hormonal basis of individual differences in the frequency or intensity of aggressive behavior is still debated. Using the Japanese quail (Coturnix coturnix japonica), a species in which individuals exhibit a wide range of aggressiveness in nature and the laboratory, together with a newly devised test procedure for quantifying aggressiveness, we recently demonstrated that aggression is estrogen dependent. Here we extend these studies by testing the hypothesis that aromatization in brain is a rate-limiting step in the expression of individual differences in aggressiveness. Using procedures previously validated for this species, aromatase and 5 alpha- and 5 beta-reductase activities were estimated in selected brain regions of reproductively active male quail by measuring conversion of [3H]androstenedione to [3H]estrone, [3H]5 alpha-androstanedione, and [3H]5 beta-androstanedione, respectively. In Exp 1, behaviorally inexperienced test birds were killed 90 sec after a single behavioral test. Aggressiveness of individuals in this group, as determined by pecking and locomotor activity in response to visualization of a conspecific, ranged 3- to 4-fold from high to low. Aromatase activity in the posterior hypothalamus (PHYP) was significantly higher in males rated high for aggressiveness than in animals rated low (1.04 vs. 0.59 pmol/h.mg protein; P less than 0.02). Similar differences were observed in the anterior hypothalamus/preoptic area (AHPOA) but were not significant. In Exp 2, sexually mature males were behaviorally tested eight times over 22 days and killed 24 h after the final test. Aggressiveness varied 5-fold from high to low, although the rating in a given bird remained constant with time and repeat testing. Aromatase activity in the AHPOA was significantly greater in birds rated high for aggressiveness than in low aggressiveness birds (3.77 vs. 2.80 pmol/h.mg protein; P less than 0.02). In addition, when AHPOA aromatase in all birds was plotted against behavioral intensity, there was a 2-fold variation and a significant positive correlation (r = 0.556; P less than 0.02). Similar differences were observed in PHYP, but these were of borderline significance. By contrast, aromatase levels outside the AHPOA and PHYP were unrelated to behavior. Moreover, in both Exp 1 and 2, 5 alpha- and 5 beta-reductase activities in AHPOA, PHYP, and other brain regions; plasma T, 5 alpha-dihydrotestosterone, and total estrogens; and relative testicular weights were not consistently related to aggression.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolism of 19-hydroxyandrostenedione in human subjects. Urinary excretion of conjugates.

A mixture of [19-3H]hydroxyandrostenedione and [14C]androstenedione was administered intravenously to 3 women and urine was collected. Only negligible radioactivity could be extracted from the untreated urine. Most of the 14C but only 11% of the 3H was rendered solube in organic solvents by beta-glucuronidase. [3H-19]hydroxyandrostenedione was recovered from this fraction. The conjugates remaining in the urine were extracted into CHCl3 as their pyridinium salts. After solvolysis of the extract with HCLO4 in tetrahydrofuran, neutral metabolites were obtained. Substances extractable from water with organic solvents were obtained by solvolysis of the conjugates with perchloric acid in tetrahydrofuran. [3H-19]hydroxyandrostenedione was identified by isotopic dilution as the major product of solvolysis. Thus, 19-hydroxyandrostenedione undergoes conjugation with glucuronic acid and probably sulfuric acid, most likely at C-19. The major urinary metabolite is the sulfate-like conjugate. Reduction in ring A is less important than for other steroids.

Androstenedione metabolism in human alveolar macrophages.

The metabolism of [3H]androstenedione by human alveolar macrophages was investigated. Alveolar macrophages were obtained by bronchopulmonary lavage by use of a heparinized saline solution devoid of Ca++ and Mg++. After purification, the macrophages were incubated at 37 C in RPMI-1640 medium that contained glucose and [1,2,6,7-3H]androstenedione under various experimental conditions. Control incubations were conducted without macrophages. After incubation, 14C-labeled steroids that corresponded to the metabolites were added as internal recovery standards. The metabolites were characterized by chromatography and crystallization to constant 3H to 14C ratios. Human alveolar macrophages convert [3H]androstenedione to 5 alpha-androstane-3,17-dione, testosterone, 5 alpha-dihydrotestosterone, androsterone, and isoandrosterone. Unidentified polar metabolites also were formed. Therefore, the following enzymes are present in these cells: 5 alpha-reductase, 17 beta-hydroxysteroid dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, and unknown hydroxylase(s). The rates of formation of the principal metabolites, 5 alpha-androstanedione and testosterone, remained linear up to 4 h of incubation and with macrophage number up to 1.5 X 10(7) cells/ml. These findings suggest that alveolar macrophages may be involved in the peripheral metabolism of androstenedione to potent androgens in man. It is possible that androgens, formed from blood-borne androstenedione within alveolar macrophages, may modulate phagocytic and other activities in these cells.

The influence of age and sex on the metabolism of testosterone.

The influence of age and sex on the peripheral metabolism of testosterone was studied by giving intravenous tracers of 14C-testosterone to 21 prepubertal children (13 boys and 8 girls), 39 young adults 18-43 years old (23 men and 16 women), and 10 elderly adults 68-86 years old (6 men and 4 women). Studies were also carried out in 2 sexually immature young adults, one 18-year-old 45 XO phenotypic female with gonadal agenesis and one 18-year-old 45 XO, 46 XX mosaic female with gonadal dysgenesis; the latter was restudied after prolonged estrogen-progestagen therapy. Age and sex influences were observed only with respect to the androsterone/etiocholanolone (A/E) ratio; a sex difference in diol metabolite formation was not observed. Prepubertal children showed no sex difference in A/E ratio, which averaged 1.7 +/- 0.28 in boys and 1.9 +/- 0.42 in girls. Young adult men showed a slightly lower A/E ratio, averaging 1.5 +/- 0.10, while females showed a much greater decrease in A/E ratio, to 0.9 +/- 0.09, so that there was a highly significant (P less than .001) sex difference in this age group. The decreased averages were due to disappearance of the higher end of the ranges seen in prepubertal children; the lower limit of the ranges remained the same. Elderly adult men showed a further fall in the A/E ratio, to 1.0 +/- 0.11, and elderly women also showed a further fall, to 0.4 +/- 0.04; a highly significant (P less than .005) sex difference remained. Once again, the fall in average A/E ratio from young adults to elderly adults was due to disappearance of the higher end of the ranges in the former, the lower limits of the ranges were the same in both groups. Of the 2 sexually immature young women, one showed an A/E ratio of 1.3, just below the upper limit for young adult women, and the other showed a ratio of 1.8, well above that limit and thus typical of prepubertal girls. Estrogen-progestagen therapy of the second girl decreased the A/E ratio to 1.4, the upper limit for young adult women. It was concluded that there is a fundamental aging effect in both sexes which causes a gradual progressive decrease of the mean A/E ratio as a result of progressive disappearance of the higher individual A/E values while the lower end of the range of values remains constant; superimposed on this gradual decrease is an acute pubertal decrease in females, probably mediated by the development of the estrogen-progestagen milieu characteristic of sexually mature women.