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.

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.

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.

Low female stress hormone levels are predicted by same- or opposite-sex sociality depending on season in wild Assamese macaques.

The social environment can have a powerful impact on an individual's stress response and thus affect health and biological fitness. Positive social interactions are particularly important for females of species living in complex societies, e.g. humans and non-human primates. Existing studies have mainly focussed on the effect of same-sex social interaction on the stress response, rather than both same- and opposite-sex social interaction simultaneously. However, consideration of both may be crucial since females may have different 'social needs' across different life-history stages. Applying the conceptual framework of allostasis, we tested the hypothesis that female allostatic load (measured through faecal glucocorticoid levels [fGCs]), of wild seasonally breeding Assamese macaques (Macaca assamensis), would increase if their social needs were not maintained in accordance with season. We found significant seasonal differences in same- and opposite-sex sociality which, depending on season, predicted female fGCs. In the mating season, females which spent more time close to males and more frequently groomed with them exhibited lower fGCs. In the non-mating season, when female-male interaction was infrequent, positive female-female sociality predicted lower fGCs. Our results support the hypothesis that same- and opposite-sex sociopositive interactions, specific to certain life-history stages, can mediate fGCs. We interpret this as a consequence of the positive direct and/or indirect effects of social contact in accordance with interactions pertaining to a given life-history stage, which are likely to impact positively upon fitness.

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.

Increased activation of the alternative "backdoor" pathway in patients with 21-hydroxylase deficiency: evidence from urinary steroid hormone analysis.

BACKGROUND: 17-Hydroxyprogesterone (17-OHP) can be converted to dihydrotestosterone (DHT) via an alternative "backdoor" route that bypasses the conventional intermediates androstenedione and testosterone. In this backdoor pathway, 17-OHP is converted to 5α-pregnane-3α,17α-diol-20-one (pdiol), which is an excellent substrate for the 17,20 lyase activity of CYP17A1 to produce androsterone. OBJECTIVE AND HYPOTHESES: The objective of this study was to obtain evidence for the presence of the backdoor pathway in patients with 21-hydroxylase deficiency (21-OHD). METHODS: We compared urinary steroid hormone profiles determined by gas chromatography-mass spectrometry of 142 untreated 21-OHD patients (age range, 1 d to 25.4 yr; 51 males) with 138 control subjects. The activity of the backdoor pathway was assessed using the ratios of the urinary concentrations of pdiol to those of the metabolites of the classic Δ4 and Δ5 pathways. In contrast to etiocholanolone, which originates almost exclusively from the classic pathways, androsterone may be derived additionally from the backdoor pathway. Therefore, the androsterone to etiocholanolone ratio can be used as an indicator for the presence of the backdoor pathway. RESULTS: Untreated 21-OHD subjects showed increased urinary ratios of pdiol to the Δ4 and Δ5 pathway metabolites and a higher androsterone to etiocholanolone ratio. CONCLUSIONS: The elevated ratios of pdiol to the Δ4 and Δ5 pathway metabolites as well as the higher androsterone to etiocholanolone ratio in patients with 21-OHD indicate postnatal activity of the backdoor pathway with maximum activity during early infancy. Our data provide new insights into the pathophysiology of androgen biosynthesis of 21-OHD.

The 5α-androstanedione pathway to dihydrotestosterone in castration-resistant prostate cancer.

The survival and progression of prostate cancer are generally dependent on expression of the androgen receptor (AR), as well as the availability of endogenous AR agonists. Originating from the gonads, testosterone is released into circulation and is converted by steroid-5α-reductase in prostate cancer to 5α-dihydrotestosterone (DHT), potently activating AR and driving tumor progression. Advanced prostate cancer is initially treated with gonadal testosterone depletion, which suppresses this cascade of events and typically leads to a treatment response. Eventually, resistance to testosterone deprivation occurs with "castration-resistant" prostate cancer (CRPC) and is driven by the intratumoral synthesis of DHT. The generation of DHT occurs in large part from adrenal 19-carbon precursor steroids, which are dependent on expression of CYP17A1. Although the path from adrenal precursor steroids to DHT was generally thought to require 5α-reduction of testosterone, recent data suggest that it instead involves conversion from Δ-androstenedione by steroid-5α-reductase isoenzyme-1 to 5α-androstanedione, followed by subsequent conversion to DHT. The 5α-androstanedione pathway to DHT therefore bypasses testosterone entirely. Abiraterone acetate effectively inhibits CYP17A1, blocks the synthesis of androgens, and extends the survival of men with CRPC. Further progress in the hormonal treatment of CRPC is dependent on an understanding of the mechanisms that underlie CRPC and resistance to abiraterone acetate.

Glucuronidation of the steroid enantiomers ent-17ß-estradiol, ent-androsterone and ent-etiocholanolone by the human UDP-glucuronosyltransferases.

Steroids enantiomers are interesting compounds for detailed exploration of drug metabolizing enzymes, such as the UDP-glucuronosyltransferases (UGTs). We have now studied the glucuronidation of the enantiomers of estradiol, androsterone and etiocholanolone by the 19 human UGTs of subfamilies 1A, 2A and 2B. The results reveal that the pattern of human UGTs of subfamily 2B that glucuronidate ent-17ß-estradiol, particularly 2B15 and 2B17, resembles the glucuronidation of epiestradiol (17α-estradiol) rather than 17ß-estradiol, the main physiological estrogen. The UGTs of subfamilies 1A and 2A exhibit higher degree of regioselectivity than enantioselectivity in the conjugation of these estradiols, regardless of whether the activity is primarily toward the non-chiral site, 3-OH (UGT1A1, UGT1A3, UGT1A7, UGT1A8 and, above all, UGT1A10), or the 17-OH (UGT1A4). In the cases of etiocholanolone and androsterone, glucuronidation of the ent-androgens, like the conjugation of the natural androgens, is mainly catalyzed by UGTs of subfamilies 2A and 2B. Nevertheless, the glucuronidation of ent-etiocholanolone and ent-androsterone by both UGT2B7 and UGT2B17 differs considerably from their respective activity toward the corresponding endogenous androgens, whereas UGT2A1-catalyzed conjugation is much less affected by the stereochemistry differences. Kinetic analyses reveal that the K(m) value of UGT2A1 for ent-estradiol is much higher than the corresponding value in the other two high activity enzymes, UGT1A10 and UGT2B7. Taken together, the results highlight large enantioselectivity differences between individual UGTs, particularly those of subfamily 2B.

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.

Simplified method to measure glucocorticoid metabolites in faeces of horses.

Glucocorticoids or their metabolites can be measured in several body fluids or excreta, including plasma, saliva, urine and faeces. In recent years the measurement of glucocorticoid metabolites (GCMs) in faeces has gained increasing attention, because of its suitability for wild populations. In horses, however, the group-specific enzyme immunoassay described so far has a limited practicability due to its complex extraction procedure. Therefore, we tested the applicability of other enzyme immunoassays for glucocorticoid metabolites. The present study clearly proved that an enzyme immunoassay (EIA) for 11-oxoaetiocholanolone using 11-oxoaetiocholanolone-17-CMO: BSA (3alpha,11-oxo-A EIA) as antigen showed high amounts of immunoreactive substances. Therefore it was possible to use just a small amount of the supernatant of a methanolic suspension of faeces. The results correlated well with the already described method for measuring GCMs in horse faeces, i.e. analysing the samples with an EIA after a two step clean up procedure of the samples (Merl et al. 2000). In addition, the 3alpha,11-oxo-A EIA has the advantage of providing a bigger difference between baseline values and peak values after ACTH stimulation. The new assay increased the accuracy of the test, lowered the expenses per sample, and storing samples at room temperature after collection was less critical than with other assays investigated in our study. This is a big advantage both in the field of wildlife management of equids and in the field of equestrian sports and it shows the importance of choosing an assay which is in good accordance with the metabolites excreted in a given species.

Aldo-keto reductase 1C3 expression in MCF-7 cells reveals roles in steroid hormone and prostaglandin metabolism that may explain its over-expression in breast cancer.

Aldo-keto reductase (AKR) 1C3 (type 5 17beta-hydroxysteroid dehydrogenase and prostaglandin F synthase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast. Purified recombinant AKR1C3 reduces PGD(2) to 9alpha,11beta-PGF(2), Delta(4)-androstenedione to testosterone, progesterone to 20alpha-hydroxyprogesterone, and to a lesser extent, estrone to 17beta-estradiol. We established MCF-7 cells that stably express AKR1C3 (MCF-7-AKR1C3 cells) to model its over-expression in breast cancer. AKR1C3 expression increased steroid conversion by MCF-7 cells, leading to a pro-estrogenic state. Unexpectedly, estrone was reduced fastest by MCF-7-AKR1C3 cells when compared to other substrates at 0.1muM. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response to estrone and 17beta-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen receptor alpha induced proliferation. MCF-7-AKR1C3 cells also reduced PGD(2), limiting its dehydration to form PGJ(2) products. The AKR1C3 product was confirmed as 9alpha,11beta-PGF(2) and quantified with a stereospecific stable isotope dilution liquid chromatography-mass spectrometry method. This method will allow the examination of the role of AKR1C3 in endogenous prostaglandin formation in response to inflammatory stimuli. Expression of AKR1C3 reduced the anti-proliferative effects of PGD(2) on MCF-7 cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor gamma (PPARgamma) signaling by reducing formation of 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)).

A randomized placebo-controlled study on the effects of pioglitazone on cortisol metabolism in polycystic ovary syndrome.

OBJECTIVE: To investigate possible effects of insulin-sensitizing treatment on cortisol metabolism in insulin-resistant patients with polycystic ovary syndrome (PCOS). DESIGN: Randomized placebo-controlled study. SETTING: Academic tertiary care medical center. PATIENT(S): Thirty insulin-resistant PCOS patients. INTERVENTION(S): Sixteen weeks of pioglitazone (30 mg/day) or placebo treatment. MAIN OUTCOME MEASURE(S): Twenty-four-hour 20 min integrated blood sampling for measurement of cortisol and 24 h urinary excretion of steroid metabolites. Relative 5alpha-reductase activity was evaluated by allotetrahydrocortisol (alloTHF)/THF and androsterone/etiocholanolone (A/E) ratios. Delta values denoted changes during the treatment period (16 weeks--basal). Pyridostigmine growth hormone (GH) stimulation tests were performed, and testosterone (T), dihydrotestosterone (DHT), DHEA, DHEAS, adiponectin, and insulin-like growth factor I (IGF-I) were measured before and after intervention. RESULT(S): Insulin sensitivity, GH, adiponectin, and IGF-I significantly increased during pioglitazone treatment, whereas alloTHF/THF levels significantly decreased. Delta alloTHF/THF levels inversely correlated with Delta adiponectin levels. Delta A/E ratio inversely correlated with Delta IGF-I and Delta peak GH during GH stimulation tests. No significant changes were measured in T, DHT, DHEA, DHEAS, 24 h mean cortisol, or urinary excretion of steroid metabolites. CONCLUSION(S): Pioglitazone decreased relative 5alpha-reductase activity, whereas no significant changes were measured in cortisol levels or urinary cortisol excretion.

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.

Enzyme kinetics of zearalenone biotransformation: pH and cofactor effects.

The aim of the present study was to investigate the hepatic biotransformation of the mycotoxin zearalenone (ZEA) in vitro using subcellular fractions of pig livers. The dependencies of the enzymatic reactions involved on the enzyme velocity, on the cofactor and on pH were analysed in both the microsomal fraction and the post-mitochondrial cell fraction. Finally, the inhibitory effects of various endogenous substrates on the enzymes involved (3alpha- and 3beta-hydroxysteroid dehydrogenase) were examined. Significant differences were observed between the individual subcellular fractions in terms of prevailing metabolites and absolute amounts of the metabolites produced. Moreover, this study also demonstrated that the reactions for both subcellular fractions of porcine liver are dependent on the cofactor, as alpha-zearalenol (alpha-ZOL) formation increased in the presence of NADPH, whereas beta-zearalenol (beta-ZOL) production only increased in the presence of NADH (P<0.001). The optimal pH for alpha-ZOL production was pH 5.6 and that for beta-ZOL formation pH 7.4. Subsequent inhibition studies showed significant inhibitory effects for 5alpha-androstanedione>androstanedione>pregnenolone on alpha-ZOL formation, whereas beta-ZOL production was only inhibited by pregnenolone. Finally, the contributions of 3alpha- and 3beta-hydroxysteroid dehydrogenase during the bioconversion of ZEA are discussed in the context of these experiments.

In vitro biosynthesis of novel 5beta-reduced steroids by the testis of the round goby, Neogobius melanostomus.

Previous studies indicate that, in the round goby Neogobius melanostomus, the reproductively mature male releases a pheromone that attracts ripe females. Furthermore, studies suggest that the pheromone may be a steroid (more specifically a 5beta-reduced androgen) produced by specialized glandular tissue in the testes. In the present study, it is shown that the testis of the male round goby contains such specialized glandular tissue. In vitro, the testes convert [3H]androstenedione into 3alpha-hydroxy-5beta-androstane-11,17-dione (i.e., 11-oxo-etiocholanolone, 11-oxo-ETIO); 11-oxo-ETIO sulfate (11-oxo-ETIO-s); 11-oxo-testosterone (i.e., 11-ketotestosterone), 3alpha-hydroxy-5beta-androstan-17-one (etiocholanolone, ETIO); 11beta-hydroxy-androstenedione; ETIO sulfate and testosterone. Glucuronidated steroids were not identified. Neither 11-oxo-ETIO nor 11-oxo-ETIO-s has previously been identified in teleost gonads. Both these steroids are formed in the round goby testis even when [3H]17-hydroxyprogesterone is used as a precursor. The fact that, for both steroids, the carbon A ring has a 5beta-configuration (already linked with olfactory sensitivity and behavior induction in two other species of gobies) makes them likely candidate pheromones in the round goby. However, their in vivo production and pheromonal activity remain to be proved.

Effects of androgens on aromatase activity and 11C-vorozole binding in granulosa cells in vitro.

BACKGROUND: Locally produced androgens and estrogens are important in the hormonal regulation of follicular development. The present study aimed to further elucidate the mechanism through which androgens exert their ambivalent effects on aromatization. METHODS: Non-cultured human granulosa-luteal cells (GC) were treated with different concentrations of androstenedione (A4), testosterone (T), 5alpha-androstane-3,17-dione (5alpha-A) and dihydrotestosterone (DHT). The effects on aromatase activity were evaluated in a tritiated water assay (incubation time 2 h) and the availability of aromatase active sites was measured in a radiotracer-binding assay using the non-steroidal competitive aromatase inhibitor [11C]-vorozole (incubation time 15 min). RESULTS: A4, T and 5alpha-A caused dose-dependent inhibition of both aromatase activity and [11C]-vorozole binding; IC50-values for both inhibition processes were calculated for these three steroids, revealing A4 as the most potent inhibitor and T and 5alpha-A as moderate inhibitors. At low concentrations (0.01 and 0.1 micro M), DHT stimulated aromatase activity but did not affect [11C]-vorozole binding. At the higher concentrations tested (1 and 10 micro M) DHT suppressed both processes thus weakly binding the aromatase active site. CONCLUSION: Because the incubation time in the tritiated water assay was short, the stimulation by DHT at low concentrations might therefore most likely include mechanisms other than new synthesis of aromatase protein such as allosteric action of DHT upon aromatase or liganded androgen receptor-aromatase interaction.