Testosterone Metabolites Research Articles

Neuroactive steroid levels and psychiatric and andrological features in post-finasteride patients

Recent reports show that, in patients treated with finasteride for male pattern hair loss, persistent side effects including sexual side effects, depression, anxiety and cognitive complaints may occur. We here explored the psychiatric and andrological features of patients affected by post-finasteride syndrome (PFS) and verified whether the cerebrospinal fluid (CSF) and plasma levels of neuroactive steroids (i.e., important regulators of nervous function) are modified. We found that eight out of sixteen PFS male patients considered suffered from a DSM-IV major depressive disorder (MDD). In addition, all PFS patients showed erectile dysfunction (ED); in particular, ten patients showed a severe and six a mild-moderate ED. We also reported abnormal somatosensory evoked potentials of the pudendal nerve in PFS patients with severe ED, the first objective evidence of a neuropathy involving peripheral neurogenic control of erection. Testicular volume by ultrasonography was normal in PFS patients. Data obtained on neuroactive steroid levels also indicate interesting features. Indeed, decreased levels of pregnenolone, progesterone and its metabolite (i.e., dihydroprogesterone), dihydrotestosterone and 17beta-estradiol and increased levels of dehydroepiandrosterone, testosterone and 5alpha-androstane-3alpha,17beta-diol were observed in CSF of PFS patients. Neuroactive steroid levels were also altered in plasma of PFS patients, however these changes did not reflect exactly what occurs in CSF. Finally, finasteride did not only affect, as expected, the levels of 5alpha-reduced metabolites of progesterone and testosterone, but also the further metabolites and precursors suggesting that this drug has broad consequence on neuroactive steroid levels of PFS patients.

6β‐Hydroxytestosterone, a Cytochrome P450 1B1 Metabolite of Testosterone, Contributes to Angiotensin II‐Induced Vascular Dysfunction in Male Mice

Previously, we showed that 6β‐hydroxytestosterone (6β‐OHT), a cytochrome P450 1B1 (CYP1B1)‐derived metabolite of testosterone, contributes to the development of angiotensin II (Ang II)‐induced hypertension and renal dysfunction in male mice. Ang II‐induced hypertension is associated with vascular dysfunction. In the present study we determined the contribution of 6β‐OHT to Ang II action on vascular reactivity, endothelial dysfunction, hypertrophy and fibrosis. In Ang II‐treated Cyp1b1+/+ mice, responses of the thoracic aorta to phenylephrine (PE) and endothelin‐1 (ET‐1), media to lumen ratio were significantly increased compared to Ang II‐treated Cyp1b1−/− or castrated Cyp1b1+/+ mice. Treatment of the later two groups of mice with 6β‐OHT restored the effect of Ang II to increase responses of the thoracic aorta to phenylephrine (PE) and endothelin‐1 (ET‐1) and media to lumen ratio. Ang II infusion caused endothelial dysfunction, as indicated by decreased relaxation to acetylcholine (ACh) of the aorta from Cyp1b1+/+ but not Cyp1b1−/− or castrated Cyp1b1+/+ mice. 6β‐OHT did not alter Ang II‐induced endothelial dysfunction in Cyp1b1+/+ mice but resulted in endothelial dysfunction in Cyp1b1−/− or castrated Cyp1b1+/+ mice. Sodium nitroprusside(SNP)‐induced relaxation of the aorta was not altered in all the groups. Ang II infusion increased vascular fibrosis as indicated by increased α‐smooth muscle actin and collagen deposition, and reactive oxygen species production (ROS) as indicated by increased dihydroethidium (DHE) staining in Cyp1b1+/+ as compared to Cyp1b1−/− or castrated Cyp1b1+/+ mice. 6β‐OHT did not alter Ang II‐induced fibrosis or oxidative stress in Cyp1b1+/+ mice, however in Cyp1b1−/− or castrated mice it restored these effects of Ang II. These data suggest that 6β‐OHT contributes to the development of hypertension, most likely via increased ROS generation and vascular reactivity and hypertrophy, and that CYP1B1 could serve as a novel target for the development of agents that mitigate hypertension and associated pathogenesis.Support or Funding InformationThis work was supported by the National Institutes of Health and National Heart, Lung, and Blood Institute grants R01HL‐19134–40 and R01HL‐079109‐09

Endocrine changes, fetal growth, and uterine artery hemodynamics after chronic estrogen suppression during the last trimester of equine pregnancy.

Equine pregnancy is characterized by very high circulating concentrations of estrogens. The physiological roles of estrogens during equine gestation are largely unknown, although some studies suggest a role in the regulation of uterine artery hemodynamics and a relationship between low circulating estrogen concentrations and late pregnancy loss. The objectives of this experiment were to evaluate the effects of estrogen suppression on uterine artery hemodynamics and on pregnancy outcome. Estrogen synthesis was suppressed using letrozole, a potent aromatase inhibitor. Twelve pregnant mares were randomly assigned to a control (n = 6) or treatment (n = 6; 500 mg letrozole orally every 4 days) group with treatment starting at 240 days of gestation and continuing until parturition. Weekly serum samples were analyzed to determine testosterone, dehydroepiandrosterone sulfate, estradiol, estrone sulfate, progestins, and prostaglandin F2α metabolite concentrations. Ultrasonographic examinations were performed biweekly and measurements included uterine artery hemodynamics (diameter, pulsatility, and resistance indices), fetal growth using the diameter of the fetal eye, and placental evaluation using the combined thickness of the uterus and placenta. At parturition, gestational length, foal weight, and neonatal viability were determined. Letrozole suppressed estrogen synthesis during gestation by approximately 90% compared to control values. This large reduction in circulating estrogens had no effect on uterine artery hemodynamics, normal placental development, maintenance of pregnancy, or neonatal viability. However, neonates from letrozole-treated mares had lower (P < 0.05) birth weights than controls, suggesting that estrogens may play a role in fetal growth that is not mediated through regulation of uterine blood flow.

Interrelationship among steroid hormones, energetics and vocalisation in the Bombay night frog (Nyctibatrachus humayuni)

In vertebrates, the increase in plasma androgens and corticosteroids is essential for the expression of reproductive behaviour. In male anurans, the interaction between hypothalamus-pituitary-gonadal and hypothalamus-pituitary-interrenal axes plays a pivotal role in calling behaviour and energy mobilisation through the secretion of testosterone and corticosterone respectively. To explain the association among body condition, testosterone, corticosterone and calling behaviour the energetic-hormone-vocalisation (EHV) model has been proposed. The model predicts that with continued participation in chorus activity within and across nights, levels of circulating androgens, corticosterone and vocal effort tend to increase and should be positively correlated in calling males. Consequently, decreasing energy reserves should be inversely correlated with corticosterone level in calling males. Depleted energy reserves lead to the peaking of circulating corticosterone, which suppresses androgen production and calling behaviour. In the present study, we used Nyctibatrachus humayuni with unique reproductive behaviour to test the model by quantifying calling behaviour and urinary metabolites of testosterone and corticosterone. We also computed the body condition index (BCI) to assess the association among energetics, levels of testosterone, corticosterone and calling behaviour. The results show that calling males had higher levels of urinary testosterone metabolites (UTM) than non-calling males indicating the importance of testosterone in controlling the calling behaviour. Surprisingly, urinary corticosterone metabolite (UCM) levels were comparable between calling and non-calling males. Further, calling males had higher body condition estimates than non-calling males. The vocal effort was neither associated with UTM, UCM nor BCI. However, a positive association was observed between UTM and UCM levels in calling males indicating the requirement of higher energy for advertisement. Analysis of UTM and UCM levels throughout the breeding season revealed that breeding basal of UTM was significantly higher than that of UCM. Interestingly, UCM levels were maintained at a lower threshold during the breeding season. These observations are in line with some of the predictions of EHV model.

Measuring fecal testosterone metabolites in spotted hyenas: Choosing the wrong assay may lead to erroneous results

ABSTRACTEnzyme-immunoassays (EIA) that detect fecal testosterone metabolites (fTM) are powerful tools to monitor gonadal activity non-invasively. However, a challenge with testosterone EIAs might be their potential for cross-reactivities with structurally similar glucocorticoid metabolites. Therefore, we aimed to verify the capability of four different testosterone EIAs to monitor fTM without reflecting changes in adrenocortical activity in spotted hyenas by analyzing fecal samples following testosterone and ACTH challenge tests. We demonstrated that none of the testosterone EIAs is appropriate to measure fTM as all of them showed substantial cross-reactivities to unknown metabolites. Our study underlines the importance of validating androgen EIAs.

Excretion and measurement of corticosterone and testosterone metabolites in bank voles (Myodes glareolus)

The bank vole is a commonly used model species in behavioral and ecophysiological studies. Thus, presenting a validated method for noninvasive monitoring of corticosterone and testosterone secretion is of high relevance. Here, we evaluated the effect of time of day and an ACTH challenge test on measured fecal corticosterone (FCM) and testosterone (FTM) metabolites in both sexes. Furthermore, we performed radiometabolism experiments for both steroids and sexes to study metabolism and excretion of 3H-corticosterone and 3H-testosterone. FCM and FTM were analysed with a 5α-pregnane-3β,11β,21-triol-20-one enzyme immunoassay (EIA) and a testosterone (measuring 17β-hydroxyandrostanes) EIA, respectively. Males had significantly higher FCM levels than females and their main excretion route was via the feces (∼72%), whereas females excreted nearly equal portions in both feces and urine. For testosterone the main excretion route was via the feces in both sexes (∼80%). The time course of excretion was similar in both sexes, but for the first time a significant difference between injected steroids was found: Corticosterone was excreted faster than testosterone, both in urine (median of peak levels: 4h vs 6h) and feces (6h vs 8h). Several metabolites were present in the feces and the tested EIAs reacted with some of them. Time of day had a significant effect on measured fecal steroid metabolites. As expected, males had significantly higher FTM levels than females. ACTH administration significantly increased FCM values; peaks were observed 4–8h after injection. In conclusion, both tested EIAs proved suited for a noninvasive measurement of glucocorticoids and androgens in bank voles.

Neuroprotective effects of testosterone metabolites and dependency on receptor action on the morphology of somatic motoneurons following the death of neighboring motoneurons.

Partial depletion of spinal motoneuron populations induces dendritic atrophy in neighboring motoneurons, and treatment with testosterone is neuroprotective, attenuating induced dendritic atrophy. In this study we examined whether the protective effects of testosterone could be mediated via its androgenic or estrogenic metabolites. Furthermore, to assess whether these neuroprotective effects were mediated through steroid hormone receptors, we used receptor antagonists to attempt to prevent the neuroprotective effects of hormones after partial motoneuron depletion. Motoneurons innervating the vastus medialis muscles of adult male rats were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Simultaneously, some saporin-injected rats were treated with either dihydrotestosterone or estradiol, alone or in combination with their respective receptor antagonists, or left untreated. Four weeks later, motoneurons innervating the ipsilateral vastus lateralis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Compared with intact normal animals, partial motoneuron depletion resulted in decreased dendritic length in remaining quadriceps motoneurons. Dendritic atrophy was attenuated with both dihydrotestosterone and estradiol treatment to a degree similar to that seen with testosterone, and attenuation of atrophy was prevented by receptor blockade. Together, these findings suggest that neuroprotective effects on motoneurons can be mediated by either androgenic or estrogenic hormones and require action via steroid hormone receptors, further supporting a role for hormones as neurotherapeutic agents in the injured nervous system. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 691-707, 2017.

Activational action of testosterone on androgen receptors protects males preventing temporomandibular joint pain

BackgroundTestosterone protects male rats from Temporomandibular Joint (TMJ) pain. This study investigated whether this protective effect is mediated by an organizational action of testosterone during nervous system development, by central estrogen and androgen receptors and by the 5α-reduced metabolite of testosterone, dihydrotestosterone. MethodsA pharmacological approach was used to assess the ability of the androgen receptor antagonist flutamide, the estrogen receptor antagonist ICI 182 780 and the 5-α reductase inhibitor dutasteride to block the protective effect of testosterone, evaluated through the behavioral response induced by a TMJ injection of 0.5% formalin. Flutamide and ICI 182 780 were injected into the medullary subarachnoid space, and dutasteride and testosterone were systemically administered. ResultsThe TMJ injection of 0.5% formalin induced a significant nociceptive behavioral response in gonadectomized male and naïve female, but not in sham gonadectomized male rats, confirming that endogenous testosterone prevents TMJ nociception in males. Testosterone administration prevented formalin-induced TMJ nociception in males gonadectomized either in the neonatal (at the day of birth) or adult period and in naïve female rats, suggesting that the protective effect of testosterone on TMJ nociception does not depend on its organizational actions during critical periods of development. The administration of flutamide and dutasteride but not of ICI 182 780 blocked the protective effect of testosterone. ConclusionsWe conclude that the protective effect of testosterone on TMJ nociception depends on activational actions of dihydrotestosterone on androgen receptors rather than on organizational androgenic actions during central nervous system development or estrogenic actions.

Abstract 813: The association of androgen metabolizing enzymes and prostate cancer in a multiethnic study

Abstract Introduction: Uridine 5’-diphosphate-glucuronosyltransferase 2B (UGT2B) genes code for enzymes that catalyze the clearance of testosterone, dihydrotestosterone (DHT), and DHT metabolites in the prostate basal and luminal tissue. The expression of the UGT2B15, UGT2B17, and UGT2B28 enzymes has not been evaluated in prostate tissue samples from hormone therapy-naïve patients. Methods: We determined the expression of the three enzymes in 190 prostate tissue samples from surgical specimens of a multiethnic cohort of patients undergoing radical prostatectomy at the Durham Veterans Affairs Medical Center. The association between each biomarker's percent positive and H-score and the risk of biochemical recurrence (BCR) was tested using separate Cox proportional hazards models. In an exploratory analysis, UGT2B17 total positive and H-score were split at the median and we tested the association between UGT2B17 group and risk of BCR. Results: The median follow-up was 118 months (IQR: 85-144). We found no association between UGT2B15 or UGT2B28 and risk of BCR. However, there was a trend between UGT2B17 and BCR (HR = 1.01, 95% CI 1.00-1.02, p = 0.11), though not statistically significant. Upon further investigation, we found patients with UGT2B17 total positive above the median had a significant increased risk of BCR on univariable analysis (HR = 1.57, 95% CI 1.02-2.43, p = 0.041) but this association was attenuated in the adjusted model (HR = 1.50, 95% CI 0.94-2.40, p = 0.088). Conclusions: Our findings suggest that UGT2B17 overexpression was associated with a significant increased risk of BCR. These results are consistent with previous reports that show UGT2B17 significantly expressed in prostate cancer metastases. Citation Format: Delores J. Grant, Lauren Howard, Emily Wiggins, Amanda De Hoedt, Adriana Vidal, Skyla T. Carney, Jill Squires, Clara E. Magyar, Jiaoti Huang, Stephen J. Freedland. The association of androgen metabolizing enzymes and prostate cancer in a multiethnic study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 813.

Differences in gene expression in human monocytes and macrophages from healthy and asthmatic donors in response to IL-4

Abstract The lungs from asthmatics are in a state of Th2-driven inflammation with a shift in macrophage polarization toward an M2 profile induced by IL-4 and IL-13. The number of M2 macrophages in the lung correlates with asthma severity. Women have more lung M2 cells than men and are more likely to have asthma than men. We hypothesized that sex differences affect macrophage polarization in humans. First, we determined that M2-related gene expression was different between healthy and asthmatic donors in response to IL-4 and IL-13. Monocytes from asthmatics expressed more CCL17 in response to IL-4, while CCL22 and MMP12 expression was similar. We found that CD200R was highly expressed in response to IL-4 in monocyte derived-macrophages (MDM) from asthmatics compared with those of healthy donors. Furthermore, this increase was greater in MDM from women compared to men. Moreover, MMP12 and CCL22 expression were not different between the sexes. Interestingly, some genes (TGM2) were only up-regulated in MDM from asthmatics. Second, we analyzed the effect of male and female sex steroid hormones on the IL-4 signaling pathway. In the presence of IL-4, we found that both estrogen (E2) and dihydrotestosterone(active metabolite of testosterone) can induce changes in phosphorylation of Akt but not in STAT6 in MDM. Similar effects on signaling were results were obtained in U937, a human monocytic cell line. The stronger M2 response in female cells could be due to increased expression of estrogen receptors. We found that monocytes from women displayed greater expression of ERa isoforms. These results indicate a potential role for both intrinsic sex differences and E2 as inducers of M2 polarization in macrophages in asthma, leading to worse lung inflammation in women.

BIOCHEMICAL MARKERS OF HYPERANDROGENISM IN CONDITIONS OF INCREASED EXPOSURE OF MANGANESE AND NICKEL

The etiology of polycystic ovaries syndrome (PCOS) may be associated with high levels of exposure manganese and nickel. These trace elements can change the metabolism of androgensObjectives. Determination of biochemical markers of hyperandrogenism in women with PCOS in conditions with elevated levels of manganese and nickel.Methods. 43 women with PCOS were examined. The control group consisted of 27 healthy women of reproductive age. In serum, using chemiluminescent immunoassay method were determined the total (TT) and free (TF) testosterone, dehydroepiandrosterone sulfate (DHEA-S), 17-hydroxyprogesterone (17-HOP), dihydrotestosterone (DHT), Sex hormone-binding globulin (SHBG) and calculated Free Androgen Index (FAI).Results. In 16.27% of women with PCOS hyperandrogenism was not observed. Detected increased levels of TT (36.59%), TF (30%), DHEA-S (36.59%) 17 - HOP (56.41%), DHT (62.5%), FAI (3.85 %). There was only one increase of androgen 17-HOP (15,38%), TT (7.32%), DHT (6.25%) and DHEA-S (2.44%).Conclusions. Elevated levels of manganese and nickel cause accumulation of metabolites of testosterone in the body. Diagnostics of biochemical hyperandrogenism should be based on determination 17-HOP, TT and perhaps DHT.

Valoración de metabolitos de testosterona, progesterona y estrógeno en excretas de venado cola blanca (Odocoileus virginianus) como método para determinar el sexo

We obtained feces of white-tailed deer (Odocoileus virginianus) in captivity from management units for the wildlife conservation, located in Mérida and Ticul, Yucatán, Mexico, in order to measure testosterone, progesterone and estrogen metabolites concentrations using radioinmunoanalysis. We obtained detectable levels of the 3 metabolites, which were analyzed by predictive theory. Indexes progesterone /testosterone -both at 1: 2 dilution and dilution 1: 20/1: 2, respectively-, had the highest efficiency (77.5%) to determine sex in non-breeding season properly. During the breeding season the progesterone /testosterone index -dilution 1: 2/1: 20, respectively, had the highest efficiency (68.75%). These efficiencies were higher than those obtained by indirect methods for determining the sex ratio in white-tailed deer. The method of sex determination by estimating fecal hormone metabolites is likely to be applied to wild populations of white-tailed deer.

Validation of an enzyme-immunoassay for the non-invasive monitoring of faecal testosterone metabolites in male cheetahs (Acinonyx jubatus)

In mammals, the sex hormone testosterone is the major endocrine variable to objectify testicular activity and thus reproductive function in males. Testosterone is involved in the development and function of male reproductive physiology and sex-related behaviour. The development of a reliable androgen enzyme-immunoassay (EIA) to monitor faecal testosterone metabolites (fTM) is a powerful tool to non-invasively assess the gonadal status of males. We validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible cross-reactivities with androgenic metabolites not derived from testosterone metabolism. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations within one day in response to a testosterone injection, (2) a significant increase in fTM concentrations within one day in response to a gonadotropin-releasing hormone (GnRH) injection, which failed following a placebo injection, and (3) significant differences in fTM concentrations between adult male and adult female cheetahs and between adult and juvenile male cheetahs of a free-ranging population. Finally, we demonstrated stability of fTM concentrations measured in faecal samples exposed to ambient temperatures up to 72h. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor testicular activity in male cheetahs.

120 IMPACT OF SEASON ON SEMINAL CHARACTERISTICS AND SPERM CRYOPRESERVATION IN THE TUFTED DEER (ELAPHODUS CEPHALOPHUS)

The Tufted deer (Elaphodus cephalophus), a small deer species native to China, is listed as near threatened on the IUCN Red List and &gt;70 animals are managed in North American zoos as a hedge against extinction. In this study, we 1) characterized the seminal traits, 2) assessed the impact of season on ejaculate traits and testosterone level, and 3) examined sperm sensitivity to cryopreservation. Semen (24 ejaculates) were obtained from five males (1–2 ejaculates/male per season) by electro-ejaculation and evaluated for volume, osmolality, pH as well as sperm concentration, motility (%M), forward progression (FP, scale = 0–5) and acrosomal integrity (%AI). Ejaculates were divided into two aliquots and cryopreserved (4% vol/vol glycerol final concentration; 50–200 × 106 sperm mL–1) over liquid nitrogen vapor using Beltsville extender (BF5F; Howard et al. 1986) or Triladyl® (TRIL; Minitüb GmbH, Germany) extender. Sperm motility and %AI were assessed immediately (subjective) upon thawing and following swim-up processing (SU; 30 min) using computer-assisted semen analysis after 1, 2, 3, and 4 h of incubation (37°C). Fecal samples were collected 3–5 times weekly for 2 years and analyzed for testosterone (T) metabolites using enzyme immunoassay as a function of season (autumn, September–November; winter, December–February; spring, March–May; and summer, June–August). Data were analyzed using Proc GLM or ANOVA with Tukey multiple mean comparison. Significance was set at P &lt; 0.05. Male reproductive and semen traits peaked in autumn (volume, 2.0 mL; concentration, 207.6 × 106 mL–1; pH, 7.6; osmolality, 310.8 mOsm; %M, 76%; FP, 3.5; and %AI, 82.3). Mean testicular length and neck girth in autumn were 4.9 and 43.2 cm, respectively. Mean T concentration (~1.23 µg g–1 of dry feces) was higher (P &lt; 0.05) in summer compared with winter (1.07 µg g–1 of dry feces) or spring (1.06 µg g–1 of dry feces). Sperm motility and %AI were lower (P &lt; 0.05) immediately after thawing (BF5F, 34.6 and 34.7%, respectively; and TRIL, 23.1 and 29.1%, respectively) compared with fresh semen (76.9 and 74.6%, respectively). Motility characteristics immediately after SU (computer-assisted semen analysis) were higher (P &lt; 0.05) in BF5F compared with TRIL: %M (56.4 v. 44.9%), progressive motility (42.5 v. 21.9%), %AI (41.2 v. 31.3%), straight-line velocity (68.0 v. 53.2 µm s–1), straightness (85.1 v. 75.1%), and linearity (54.3 v. 45.0%). At the end of 4 h of incubation, sperm %M and FP declined (P &gt; 0.05) in both BF5F and TRIL (47 and 30%, respectively) but the %AI was higher (P &lt; 0.05) in BF5F (32%) than TRIL (21%). Results indicate that tufted deer ejaculates exhibit seasonal variations in reproductive traits and cryopreservation in BF5F better preserves sperm motility and acrosomal membrane integrity compared with TRIL.

A direct assay for the routine measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone by liquid chromatography tandem mass spectrometry.

The measurement of androgens in many laboratories is often limited to testosterone. To more accurately determine the androgen status in both sexes, the measurement of other androgens such as dihydrotestosterone, the more potent metabolite of testosterone, and androstenendione and dehydroepiandrosterone, the most abundant circulating androgens in women would be informative. We report a combined liquid chromatography tandem mass spectrometry method for the measurement of these androgens. Internal standards in methanol (10 µL) were added to 100 µL serum followed by the addition of zinc sulphate (100 µL). After mixing, 100 µL of acetonitrile was added and was further mixed. The samples were centrifuged and the steroids extracted using an automated online solid phase extraction on a C18 cartridge by a Waters Acquity with online sample manager coupled to a TQS mass spectrometer. Separation of the androgens was achieved by liquid chromatography. The run time was 6.5 min per sample. The lower limit of quantitation was 0.1 nmol/L for testosterone, androstenedione and dihydrotestosterone and 1 nmol/L for dehydroepiandrosterone. The coefficient of variation of the assay in serum for testosterone was <6%, androstenedione <8% and dihydrotestosterone and dehydroepiandrosterone <10%. We have developed a rapid assay for the liquid chromatography tandem mass spectrometry measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone in a routine clinical laboratory. The assay requires a small volume of serum, and all analytes are measured simultaneously. The assay is rapid and simple to execute offering the potential for routine clinical application.

Social and Ecological Correlates of Parasitic Infections in Adult Male Gray-Cheeked Mangabeys (Lophocebus albigena)

Intestinal parasites may constitute an important evolutionary and ecological force. We aimed to identify social, physiological, and environmental factors that correlate with intestinal parasite infections in adult male gray-cheeked mangabeys (Lophocebus albigena). We analyzed 102 fecal samples collected from 18 adult males over 19 mo for the incidence (proportions of samples with parasites) and intensity (total number of parasites per gram of feces) of infection of nematodes relative to social status, fecal glucocorticoid and testosterone metabolites, group size, and rainfall, all of which are factors that earlier studies suggested can be important mediators of parasite load. Parasite incidence was greater in immigrant males compared to low- and high-ranking males whereas parasite intensity was greater in immigrant males and low-ranking males compared to high-ranking males. Fecal samples with more parasites had higher concentrations of fecal glucocorticoid and testosterone metabolites than those with fewer parasites. As immigrant males had a greater incidence of parasites and higher concentrations of both metabolites than resident males, this profile appears to fit immigrant males best. We also found higher nematode intensities in mid-sized groups and during periods with more rainfall. Our results suggest that it will be fruitful for future studies to explore the role of immigrant males as spreaders of intestinal parasites.

Ultra high performance liquid chromatography tandem mass spectrometric detection of glucuronides resistant to enzymatic hydrolysis: Implications to doping control analysis

Controversial results have been reported in the literature regarding the behavior of two testosterone (T) metabolites (3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone) excreted after T administration. Due to their potential as biomarkers of T misuse, a UHPLC–MS/MS method for the direct quantification of these glucuronides was developed and validated. In addition, the main phase II metabolites of T that compose the steroid profile used for doping control purposes (glucuronides of T, epitestosterone, androsterone and etiocholanolone) were included. The method was found to be linear and with suitable LODs and LOQs for all metabolites. The average accuracies were between 86% and 120%, the RSDs for the intra- and inter-day precision were below 15% and 25% respectively. The method showed low matrix effect. Samples obtained before and after the administration of T were analyzed by both the developed UHPLC–MS/MS method and the GC–MS/MS method currently used by anti-doping laboratories. Relevant disagreements between the results obtained for 3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone quantitation were observed. These markers seemed to be more suitable for the screening of T misuse when detected by UHPLC–MS/MS. These discrepancies were further investigated in 50 urine samples from healthy volunteers. The two methods gave highly correlated results for all metabolites that are currently included in the athlete's steroid profile confirming the reliability of the UHPLC–MS/MS method. However, the quantification of 3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone, was only possible by using the UHPLC–MS/MS method since three interfering compounds were observed when performing the GC–MS/MS analysis with the most intense ion transitions. These results confirm the potential of the resistant glucuronides as biomarkers of T misuse. Additionally, they suggest that previously reported reference ranges for these metabolites should be reevaluated.

Social behavior and patterns of testosterone and glucocorticoid levels differ between male chacma and Guinea baboons

In multi-male, multi-female groups of mammals, males usually compete aggressively over access to females. However, species vary in the intensity of male contest competition, which has been linked to differences in testosterone and glucocorticoid profiles. Chacma (Papio ursinus) and Guinea (P. papio) baboons constitute an intriguing model to examine variation in male competition and male endocrine correlates, because of the differences in their social systems. Chacma baboons live in stable female-bonded groups with linear male dominance hierarchies and a high male mating skew, whereas Guinea baboons live in male-bonded, multi-level societies. We recorded male behavior and assayed testosterone (fT) and glucocorticoid metabolite (fGC) levels from fecal samples in one population of each species. Male chacma baboons were more frequently involved in agonistic interactions, and dominance relationships were more consistent than in Guinea baboons, where we could not detect linear hierarchies. Notably, male chacma baboons were also more aggressive towards females, indicating an overall higher aggressiveness in this species. In contrast, male Guinea baboons showed higher levels of affiliative interactions and spatial tolerance. High-ranking and consorting male chacma baboons showed elevated fGC levels and also tended to show elevated fT levels, but there was no effect of consortship in Guinea baboons. Agonism was not related to hormone levels in either species. Thus, predictors of fT and fGC levels in Guinea baboons seem to differ from chacma baboons. Our results support the view that different social systems create differential selection pressures for male aggression, reflected by different hormone profiles.

5Alpha-Reduced Steroids Are Major Metabolites in the Early Equine Embryo Proper and Its Membranes.

Steroid production and metabolism by early conceptuses are very important for the establishment and maintenance of pregnancy in horses. Our earlier work suggested the possible formation of 5alpha-reduced steroids in equine conceptuses. We have now demonstrated the formation of 5alpha-reduced metabolites of androstenedione, testosterone, and progesterone by the embryo and its membranes. A total of 44 conceptuses were collected from 26 mares between 20 and 31 days of pregnancy. Tissues from the embryo proper and from the separated components of the conceptus (bilaminar and trilaminar trophoblast, allantois) were incubated with tritium-labeled substrates. 5Alpha-reduced metabolites (5alpha-dihydro- and 3beta,5alpha-tetrahydro- steroids) as radiolabeled products were identified from a series of chromatographic steps using four solvent systems for high-performance liquid chromatography. Use of a 5alpha-reductase inhibitor confirmed the metabolites were indeed 5alpha-reduced steroids. For the embryo, the only products from androstenedione were 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione, with no evidence of more polar metabolites; there was some 3beta,5alpha-tetrahydrotestosterone but no 5alpha-dihydrotestosterone from testosterone, and formation of androstenedione was followed by the production of 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione. The major 5alpha-reduced product from progesterone was 3beta,5alpha-tetrahydroprogesterone, with lesser amounts of 5alpha-dihydroprogesterone. For the membranes, reductions to tetrahydro, 5alpha-reduced steroids were prominent in most instances, but also present were considerable amounts of products more polar than the substrates. The well-recognized activity of some 5alpha-reduced steroids--for example, 5alpha-dihydrotestosterone in male sexual differentiation--provokes interest in their even earlier appearance, as seen in this study, and suggests a possible role for them in early embryonic development in horses and, more generally, in other species.

Functional characterization of zebrafish cytochrome P450 1 family proteins expressed in yeast

BackgroundZebrafish express five cytochrome P450 1 genes: CYP1A, CYP1B1, CYP1C1, CYP1C2, inducible by aryl hydrocarbon receptor agonists, and CYP1D1, a constitutively expressed CYP1A-like gene. We examined substrate selectivity of CYP1s expressed in yeast. MethodsCYP1s were expressed in W(R) yeast, engineered to over-express P450 reductase, via pYES/DEST52 and via pYeDP60. Microsomal fractions from transformed yeast were examined for activity with fluorogenic substrates, benzo[a]pyrene and testosterone. Modeling and docking approaches were used to further evaluate sites of oxidation on benzo[a]pyrene and testosterone. ResultsCYP1s expressed in yeast dealkylated ethoxy-, methoxy-, pentoxy- and benzoxy-resorufin (EROD, MROD, PROD, BROD). CYP1A and CYP1C2 had the highest rates of EROD activity, while PROD and BROD activities were low for all five CYP1s. The relative rates of resorufin dealkylation by CYP1C1, CYP1C2 and CYP1D1 expressed via pYeDP60 were highly similar to relative rates obtained with pYES/DEST52-expressed enzymes. CYP1C1 and CYP1C2 dealkylated substituted coumarins and ethoxy-fluorescein-ethylester, while CYP1D1 did not. The CYP1Cs and CYP1D1 co-expressed with epoxide hydrolase oxidized BaP with different rates and product profiles, and all three produced BaP-7,8,9,10-tetrol. The CYP1Cs but not CYP1D1 metabolized testosterone to 6β-OH-testosterone. However, CYP1D1 formed an unidentified testosterone metabolite better than the CYP1Cs. Testosterone and BaP docked to CYP homology models with poses consistent with differing product profiles. ConclusionsYeast-expressed zebrafish CYP1s will be useful in determining further functionality with endogenous and xenobiotic compounds. General significanceDetermining the roles of zebrafish CYP1s in physiology and toxicology depends on knowing the substrate selectivity of these enzymes.