Androgen Receptor Blocker Flutamide Research Articles

Differential Actions of Eplerenone and Spironolactone on the Protective Effect of Testosterone Against Cardiomyocyte Apoptosis In Vitro

Testosterone deficiency is associated with a poor prognosis in patients with heart failure. It is not clear whether testosterone reduces cardiomyocyte apoptosis or whether the effect of spironolactone, an aldosterone receptor blocker with progestogenic and anti-androgen activity, differs from that of the selective aldosterone blocker eplerenone. Apoptosis induced by hyperosmotic stress in the embryonic rat heart cell line H9c2 was monitored by measuring cell viability, DNA fragmentation and caspase-3, -8 and -9 activation. The effect of testosterone was investigated in the presence or absence of spironolactone and eplerenone. Exposure to sorbitol (0.6 M, 3 h) decreased cell viability and increased DNA fragmentation and caspase-3, -8 and -9 activation. These effects were all significantly reduced by testosterone, 100 nM (P< .01). Pretreatment with spironolactone, 10 .M, blocked the effects of testosterone, decreased cell viability (P< .01) and increased caspase activation (P< .01). In contrast, eplerenone, 10 .M, increased cell viability (P< .001) without altering the effect on caspase activation. These actions were not modified by the androgen receptor blocker flutamide. They were mediated by SAPK/JNK and ERK1/2 signaling pathways (P< .01). Testosterone appears to have a protective effect against cardiomyocyte apoptosis which is antagonized by spironolactone but not by eplerenone. These effects await confirmation in in vivo models, but their presence could have clinical and therapeutic implications.

Sex steroids are necessary in the second postnatal week for the expression of male alloparental behavior in prairie voles (Microtus ochragaster).

Sex steroids play a significant role in organizing male social behavior, which is associated with low levels of pro-social behavior and high levels of aggression. However, the role of steroids in organizing behavior in highly social males is unclear. The authors tested the hypothesis that low levels of sex steroids facilitate the expression of pro-social behavior in male prairie voles (Microtus ochragaster), predicting that inhibition of testosterone and estradiol would reduce spontaneous-alloparental behavior. Treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) or the androgen receptor blocker flutamide, days 8-14, significantly reduced the expression of alloparental behavior in 21-day old males. While both treatments reduced alloparental behavior and increased pup-directed aggression, there were differential treatment effects. Flutamide altered initial response, increasing latency to enter the pup cage and the likelihood of retreat from initial contact. ATD-treated males that were alloparental showed increases in sniffing and latencies to lick and huddle. Results indicate that endogenous steroids play a role in the development of male pro-social behavior and the effects of estrogens and androgens differ.

Dihydrotestosterone stimulates aldosterone secretion by H295R human adrenocortical cells

Men exhibit a higher incidence of cardiovascular diseases than do women. The cardiovascular actions of sex steroids have been suggested as primary factors in mediating this sex difference. The mechanisms by which sex steroids, androgens and estrogens, mediate cardiovascular actions remain unclear. Excess aldosterone secretion has been associated with cardiovascular diseases. The hypothesis tested in this study was that at physiological concentrations, androgens stimulate and estradiol inhibits aldosterone secretion by human adrenal cells. In contrast to our hypothesis, physiological concentrations of sex steroids did not modify aldosterone secretion by H295R human adrenocortical cells. However, supraphysiological concentrations (300–1000 nM) of dihydrotestosterone (DHT) significantly stimulated basal and Angiotensin II-mediated aldosterone secretion. The stimulatory effect of DHT on aldosterone secretion was not blocked by the classical androgen receptor blocker flutamide. The stimulatory effect of DHT on aldosterone secretion was also independent of the intra-adrenal renin–angiotensin system since it was neither modified by treatment with the Angiotensin II receptor type 1 blocker losartan or the angiotensin converting enzyme inhibitor captopril. Inhibitors of the calmodulin/calmodulin-dependent protein kinase (CaMK) and protein kinase C intracellular signaling pathways abolished the DHT stimulatory effect on aldosterone secretion by H295R cells. In conclusion, physiological concentrations of sex steroids did not modify aldosterone secretion by human adrenal cells. However, supraphysiological concentrations of DHT-stimulated aldosterone secretion by human adrenal cells by the calmodulin/CaMK and protein kinase C intracellular signaling pathways but independently of the classical androgen receptor. Supraphysiological doses of androgen may promote cardiovascular diseases via stimulation of aldosterone secretion.

Neuroendocrine Effects of Androgens in Adult Polycystic Ovary Syndrome and Female Puberty

In addition to hyperandrogenism and ovulatory dysfunction, polycystic ovary syndrome (PCOS) is characterized by neuroendocrine abnormalities including a persistently rapid gonadotropin-releasing hormone (GnRH) pulse frequency. Rapid GnRH pulsatility favors pituitary secretion of luteinizing hormone (LH) over that of follicle-stimulating hormone (FSH). Excess LH stimulates ovarian androgen production, whereas relative deficits in FSH impair follicular development. The rapid GnRH pulse frequency is a result of reduced progesterone-mediated feedback inhibition of the GnRH pulse generator secondary to infrequent luteal phase increases in progesterone, as well as reduced hypothalamic sensitivity to progesterone feedback. Progesterone sensitivity is restored by treatment with the androgen receptor blocker flutamide. As such, hyperandrogenemia appears to play an important pathophysiologic role in PCOS. Adolescent hyperandrogenemia is believed to be a precursor to adult PCOS. In addition to increased LH concentrations and pulse frequency, some girls with elevated androgen levels also demonstrate reduced hypothalamic sensitivity to progesterone feedback. We hypothesize that excess peripubertal androgens may reduce the sensitivity of the GnRH pulse generator to sex steroid inhibition in susceptible individuals, resulting in increased GnRH pulse frequency and subsequent abnormalities in gonadotropin secretion, ovarian androgen production, and ovulatory function. Over time, these abnormalities may progress to the clinical hyperandrogenism and chronic oligo-ovulation typical of adult PCOS.

Cognitive performance in rhesus monkeys varies by sex and prenatal androgen exposure

Men and women differ on performance and strategy on several spatial tasks. Rodents display similar sex differences, and manipulations of early hormone exposure alter the direction of these differences. However, most cognitive testing of nonhuman primates has utilized sample sizes too small to investigate sexually differentiated behaviors. This study presents an investigation of sex differences and the effects of prenatal androgen on spatial memory and strategy use in rhesus monkeys. Monkeys prenatally exposed to vehicle, testosterone, or the androgen receptor blocker flutamide performed a search task in which 5 of 12 goal boxes contained food rewards. Spatial consistency and the presence of local landmarks were varied. Performance when both spatial and marker cues were available did not differ by sex or prenatal treatment. Contrary to predictions, females easily solved the task when local markers were removed, and their performance outscored males. Although eliminating spatial consistency and requiring subjects to use local markers impaired performance by all monkeys, females continued to locate correct goal boxes at higher than chance levels and scored better than males. Blocking prenatal androgen exposure in males improved use of local markers. These findings suggest that the tendency to attend to landmarks and to use them in solving spatial problems is typical of females across many species, including rodents, humans, and rhesus monkeys. In rhesus monkeys and rodents, developmental androgen eliminates this specialization. However, these results are the only known example of better performance of females than males when salient markers are removed.

The Effect of Flutamide on Systemic and Renal Hemodynamics in Zucker Diabetic Rats: Paradoxic Renal Vasodilator Response to Endothelin-1 and TXA2 Receptor Activation in Female Sex

There is increasing evidence that endogenous sex hormones regulate vascular reactivity, and testosterone may contribute to the worse prognosis for renal disease in men. Male Zucker diabetic rats exhibit improved renal hemodynamic responses after castration. It is, however, unclear whether endogenous testosterone affects renal and systemic microcirculatory responses in the female sex, especially in type 2 diabetes. To test the hypothesis that endogenous testosterone in the female Zucker diabetic rat exerts a pathophysiologically relevant modulation of endothelial and renal microvascular function. Female Zucker diabetic rats (FZDR) aged 5-6 weeks and from the same litter were divided into 2 groups (n = 6-8 each). The experimental group received the androgen receptor blocker flutamide, dissolved in alcohol and added to their drinking water (500 mL) at 20 mg/rat/week. The control FZDR received only the alcohol vehicle added to the same volume of drinking water. Both FZDR groups were treated for 3 months before undergoing the hemodynamic studies. A sex comparison control group of male Zucker diabetic rats (MZDR), also aged 5-6 weeks, was studied, following same protocol. Mean arterial pressure (MAP) and renal cortical blood flow (RCF) response to phenylephrine, acetylcholine, TXA2-mimetic U46619, endothelin-1 (ET-1), angiotensin II, and L-NG-nitro arginine methyl ester were studied. Furthermore, the role of protein kinase C in the responses was assessed using phorbol-12,13 dibutyrate 10(-4) M. The impact of flutamide on body weights and blood glucose of the rats were also determined. Flutamide-treated FZDR had a significant reduction in body weight/adiposity to 432 +/- 44 g, compared to controls at 553 +/- 37 g (P = 0.045), and random blood glucose concentration of 185 +/- 44 g/dL, compared to the control FZDR at 475 +/- 34 g/dL (P = 0.002). Vehicle-treated FZDR (n = 6-8), exhibited little or no systemic or renal response to any of the agonists. By contrast, flutamide treatment of FZDR (n = 5-7) caused a normalization of the dose-dependent MAP and RCF pressor response to phenylephrine [P < 0.005, analysis of variance (ANOVA)] and the vasodilator response to acetylcholine (P <. 0.01, ANOVA). Flutamide-treated FZDR showed enhanced pressor response to U46619 (P = 0.024, ANOVA), ET-1, and angiotensin II (P < 0.03, ANOVA). Surprisingly, the augmented systemic pressor action of U46619 and ET-1 was accompanied by a renal vasodilator action, with paradoxic RCF increases to U46619 (P < 0.003, ANOVA) and to ET-1 (P < 0.001, ANOVA) only in flutamide-treated FZDR. By contrast, flutamide-treated MZDR exhibited no significant change in body weight and an attenuation of the vasoconstrictor responses and enhanced nitric oxide-mediated dilatation compared with male controls. However, no specific effect on ET-1 or TXA2 receptor-mediated renal perfusion was discernible. Both L-NG-nitro arginine methyl ester and the protein kinase C agonist phorbol-12,13 dibutyrate [10(-4)M] significantly increased MAP and reduced RCF (P < 0.03) in the experimental FZDR compared with their controls. Flutamide administration to FZDR resulted in the reversal of abnormal systemic and renal alpha-1-mediated vasoconstriction and enhanced nitric oxide-mediated vasodilation. Flutamide caused a paradoxic but specific increase in renal perfusion during ET-1 and TXA2 receptor activation, which could be renoprotective in females. The salutary effects of flutamide on vascular reactivity in the FZDR may be mediated by a protein kinase C-dependent mechanism. These results are compatible with the notion that endogenous testosterone may regulate systemic and renal microcirculation in the female sex and in the type 2 diabetic state.

The origins and sequelae of abnormal neuroendocrine function in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common clinical disorder characterized by ovulatory dysfunction and hyperandrogenaemia. A neuroendocrine hallmark of PCOS is persistently rapid LH (GnRH) pulsatility, which favours pituitary synthesis of LH over that of FSH and contributes to the increased LH concentrations and LH : FSH ratios typical of PCOS. Inadequate FSH levels contribute to impaired follicular development, whereas elevated LH levels augment ovarian androgen production. Whereas luteal phase elevations in progesterone normally slow GnRH pulse frequency, women with PCOS do not experience normal progesterone-mediated slowing, due in part to impaired hypothalamic progesterone sensitivity. This reduction in hypothalamic progesterone sensitivity appears to be mediated by elevated androgens because sensitivity can be restored with the androgen receptor blocker flutamide. The ovulatory and hormonal abnormalities associated with PCOS generally present during puberty, typically associated with hyperandrogenaemia. Along with elevated LH concentration and pulsatility, some girls with hyperandrogenaemia have impaired hypothalamic progesterone sensitivity similar to that seen in adult women with PCOS. We propose that peripubertal hyperandrogenaemia may lead to persistently rapid GnRH pulse frequency via impaired hypothalamic feedback inhibition. The subsequent abnormalities in gonadotropin secretion, androgen production and ovulatory function may support progression towards the adult PCOS phenotype.

Prenatal androgen blockade accelerates pubertal development in male rhesus monkeys

Aspects of the prenatal environment, including steroid hormones, modulate the timing of puberty onset in many mammalian species. This study tested whether prenatal androgen manipulations altered pubertal development in male rhesus macaques (Macaca mulatta). Pregnant females received testosterone enanthate (TE), the androgen receptor blocker flutamide, or vehicle during one of two periods of gestation, and their male offspring were observed for morphological, endocrine, and behavioral development from 3 to 4.5 years of age. Males exposed to flutamide early in gestation had a greater response to exogenous GnRH prepubertally, and greater testes volume, elevated testosterone, and elevated LH at age 3.5 than did control subjects. Males exposed to flutamide late in gestation also had greater testes volumes at age 3.5 than did control males. However, these differences between flutamide treated males and control males did not persist postpubertally. By 4.5 years of age, development in control males had reached comparable levels to that of flutamide-treated males. Late gestation treatment with TE had no effect on morphological pubertal development but early TE treatment altered some aspects of endocrine function during puberty. None of the prenatal androgen manipulations affected sexual behavior. These findings suggest that prenatal androgens, in conjunction with social factors, masculinize pubertal timing in rhesus monkey males.

Play, copulation, anatomy, and testosterone in gonadally intact male rats prenatally exposed to flutamide

The role of prenatal androgen on the differentiation of sexually dimorphic juvenile play and adult copulatory patterns was evaluated in male offspring of rats injected with 5 mg of the androgen receptor blocker flutamide (4′-nitro-3′-trifluoromethylisobutyranilide) from Days 11–21 of pregnancy. Rough-and-tumble play was incompletely masculinized in flutamide-exposed males at 31 days of age. The copulatory potential tested at 70 days of age was severely attenuated by prenatal flutamide. There was no ejaculatory behavior, low levels of intromissions, and depressed levels of nonintromittive mounting when the animals were tested while gonadally intact. Adult plasma levels of testosterone (T) were not different in flutamide-exposed males and controls, but testicular and epididymal weight, anogenital (AG) distance, and penile length were reduced. While reductions in intromittive mounting and ejaculatory behavior may be due to the abnormalities in the external genitalia, the incomplete masculinization of play and the reduction in nonintromittive mounting probably resulted from effects the androgen antagonist exerted on sexual differentiation of the central nervous system. These data suggest that androgen released prior to birth is needed for the full masculinization of juvenile play behaviors in the rat, just as it is for the adult copulatory pattern.

Parental Responsiveness Is Feminized after Neonatal Castration in Virgin Male Prairie Voles, but Is Not Masculinized by Perinatal Testosterone in Virgin Females

We previously found a large sex difference in the parental responsiveness of adult virgin prairie voles (Microtus ochrogaster) such that most males are spontaneously parental, whereas most females are not. Because this sex difference is independent of the gonadal hormones normally circulating in adult virgin voles, the present study examined whether perinatal hormones influence the development of this sex difference. Males were treated prenatally (via their pregnant dam) with both the androgen receptor blocker flutamide (5 mg/day/dam) and the aromatase inhibitor ATD (1 mg/day/dam), or oil, for the last 2 weeks of gestation. Half of the subjects from each group were castrated on the day of birth and the other half received a sham surgery. As adults, intact males were castrated and all males received a silastic capsule filled with testosterone. Prenatal treatment with flutamide and ATD had no effect on males' behavior toward pups, but neonatal castration significantly reduced the percentage of males acting parentally. In a second experiment, females were exposed to testosterone propionate (TP; 50 μg/day/dam) or oil via their dam during the last 2 weeks of gestation. For the first neonatal week, half of the females from each group were injected with TP (1 mg/day) and the other half oil. As adults, females were ovariectomized and half from each group received a testosterone-filled capsule and the other half received an empty capsule. None of the perinatal TP treatments increased females' parental responsiveness, although females from all groups that received testosterone capsules as adults were highly parental. Therefore, although postnatal testicular hormones are necessary for high parental responsiveness in males, the behavior of females is not influenced by perinatal exposure to testosterone.

Dehydroepiandrosterone: an inexpensive steroid hormone that decreases the mortality due to sepsis following trauma-induced hemorrhage.

Recent studies suggest that male sex steroids play a role in producing immunodepression following trauma-hemorrhage. This notion is supported by studies showing that castration of male mice before trauma-hemorrhage or the administration of the androgen receptor blocker flutamide following trauma-hemorrhage in noncastrated animals prevents immunodepression and improves the survival rate of animals subjected to subsequent sepsis. However, it remains unknown whether the most abundant steroid hormone, dehydroepiandrosterone (DHEA), protects or depresses immune functions following trauma-hemorrhage. In this regard, DHEA has been reported to have estrogenic and androgenic properties, depending on the hormonal milieu. To determine whether administration of DHEA after trauma-hemorrhage has any salutary or deleterious effects on immune responses, and whether it improves the survival of animals subjected to subsequent sepsis. Male C3H/HeN mice underwent laparotomy (ie, trauma-induced) and hemorrhagic shock (blood pressure, 35+/-5 mm Hg for 90 minutes) followed by fluid resuscitation, or sham operation. The animals then received 100 mg of DHEA (4 mg/kg) or propylene glycol (hereafter referred to as vehicle). At 24 hours after trauma-hemorrhage and resuscitation, the animals were killed and blood, spleens, and peritoneal macrophages were harvested. Splenocyte proliferation and interleukin (IL) 2 release and splenic and peritoneal macrophage IL-1 and IL-6 release were determined. In a separate set of experiments, sepsis was induced by cecal ligation and puncture at 48 hours after trauma-hemorrhage and resuscitation. For those studies, the animals received vehicle, a single 100-microg dose of DHEA, or 100 microg/d DHEA for 3 days following hemorrhage and resuscitation. Survival was monitored for 10 days after the induction of sepsis. Administration of DHEA restored the depressed splenocyte and macrophage functions at 24 hours after trauma-hemorrhage. Moreover, daily administration of DHEA for 3 days significantly increased the survival of animals subjected to subsequent sepsis (P=.01). The finding that DHEA markedly improves the depressed immune functions and survival of animals subjected to subsequent sepsis suggests that short-term treatment with DHEA after trauma-hemorrhage is a safe and novel approach for preventing immunodepression and for decreasing the mortality rate due to subsequent sepsis.

Aromatization mediates aggressive behavior in quail

Although testosterone (T) stimulates aggressive and reproductive behaviors in males of many vertebrate species, it is now known that the full expression of T action in the brain requires aromatization to estradiol (E 2) and subsequent interaction of locally formed E 2 with nuclear estrogen receptors. In experiments reported here, we used a behavioral test which quantifies the response of an individual male Japanese quail ( Coturnix coturnix japonica) to the visual stimulus of a conspecific. We have called this behavior aggression because it shares many features in common with traditional measures of aggression, e.g., predicting dominance and subordinance. Nevertheless, the behavior probably also combines a complex steroid-sensitive masculine behavior. The advantage of this test is that it allows the discrimination of individual differences in masculine behavior but avoids fighting and sexual encounters per se, thereby reducing effects of learning, a problem with previous tests of avian aggression. In addition, this test has been applied usefully to identify neuroendocrine correlates to male behavior. Using this test, the arousal of reproductively inactive males (hereafter referred to as aggression) is activated by administration of T or estradiol benzoate (EB), but not by 5α-dihydrotestosterone (DHT). T-induced aggression was blocked by the aromatase inhibitor 4-hydroxyandrostenedione (OHA), an effect partially reversed by treatment with EB. In addition, OHA or the estrogen receptor blocker CI-628 reduced aggressiveness of reproductively active males whereas the androgen receptor blocker flutamide had no effect. Results with the 5α-reductase inhibitor N, N-diethyl-4-methyl-3-oxo-4-aza-5α-androstane-17α-carboxyamide (4-MA) were equivocal. Additionally, treatment of reproductively inactive quail with T or E 2 but not DHT increased aromatase activity in the hypothalamus-preoptic area (HPOA). We conclude, therefore, that T to E 2 conversion is essential for the activation of aggressiveness in this species. Although locally formed estrogen exerts its effects on aggression in part by increasing activity of aromatase per se, analysis of the time course of behavioral induction or suppression by the various treatments suggests that the response has multiple components, including both short latency, receptor-independent and long latency, receptor-dependent events.