Flutamide Treatment Research Articles

Differential Effects of Exogenous Androgen and an Androgen Receptor Antagonist in the Peri- and Postpubertal Murine Mammary Gland

There is emerging evidence that androgens inhibit proliferation of normal and malignant breast epithelial cells, but the actions of androgens in normal mammary gland morphogenesis are not well understood. In this study, we investigated whether development of the murine mammary gland could be altered by stimulating or suppressing androgen receptor (AR) signaling in vivo. Intact virgin female mice aged 5 wk (midpuberty) or 12 wk (postpuberty) were implanted with slow-release pellets containing either placebo, 5α-dihydrotestosterone (1.5 mg) or the AR antagonist flutamide (60 mg). Treatment with 5α-dihydrotestosterone from midpuberty to 12 wk of age-retarded ductal extension by 40% (P = 0.007), but treatment from 12-21 wk had no significant effect on gland morphology. In contrast, inhibition of AR signaling with flutamide from midpuberty had no effect on the mammary gland, but flutamide treatment from 12-21 wk increased ductal branching (P = 0.004) and proliferation (P = 0.03) of breast epithelial cells. The increased proliferation in flutamide-treated mice was not correlated with serum estradiol levels or estrogen receptor-α (ERα) expression. In control mice, the frequency and intensity of AR immunostaining in mammary epithelial cells was significantly increased in the 12- to 21-wk treatment group compared with the 5- to 12-wk group (P < 0.001). In contrast, no change in ERα occurred, resulting in a marked increase in the AR to ERα ratio from 0.56 (±0.12) to 1.47 (±0.10). Our findings indicate that androgen signaling influences development and structure of the adult mammary gland and that homeostasis between estrogen and androgen signaling in mature glands is critical to constrain the proliferative effects of estradiol.

The effects of testosterone on craniosynostotic calvarial cells: a test of the gene/environmental model of craniofacial anomalies

The gene-environmental interaction model for craniofacial development proposes that if a genetic predisposition for an anomaly is coupled with an environmental factor that can exacerbate this predisposition, more severe phenotypes will result. Here, we utilize cells derived from our non-syndromic rabbit model of craniosynostosis to test the hypothesis that an insult, testosterone (TP) administration (exogenous source) will alter the osteogenic activity of these cells. Calvarial cells from wild-type (WT) (N=13) or craniosynostotic (CS) rabbits (N=11) were stimulated with TP, an androgen receptor blocker, flutamide, and combined treatments. Proliferation and differentiation assays were conducted after 7 days. anova and t-tests were used to determine differences in stimulation and cell type. The CS cells had significantly greater proliferation after TP administration compared to WT. There were no appreciable changes in differentiation after TP stimulation. Flutamide administration or combined TP and flutamide administration decreased both proliferation and differentiation for both cell types similarly. Testosterone exposure caused an increase in cell proliferation for CS osteoblast cells. However, a therapy targeted to mitigate this response (flutamide therapy) similarly affected CS and WT cells, suggesting that the administration of flutamide or TP in the presence of flutamide decreases osteogenesis of these cells. Thus, although our data support a mechanism of gene-environmental interaction, these results would not support a therapeutic intervention based on this interaction.

Epigenetic and Phenotypic Transgenerational Changes Induced by Vinclozolin in the Mouse Germ Line.

Endocrine disrupting chemicals are known for their ability to alter development and reproduction in mammals. One of the periods most sensitive to endocrine disruptor s is embryonic gonadal sex determination, when the germ line is undergoing epigenetic programming and DNA re-methylation. These environmental compounds can induce physiological changes in mammalian mothers, leading to epigenetic alterations in the developing fetus and the emergence of disease conditions such as cancers, reproductive defects or obesity. Interestingly, in addition to one-generation effects, the incidence of epigenetic changes or diseases resulting from exposure to endocrine disruptors can be transgenerationally transmitted. The mechanism behind this transmission would be the alteration of epigenetic DNA features in the male germ line. Previous work in rats have shown that the exposure of pregnant F0 mothers to intra-peritoneal injections of vinclozolin (fungicide), while the F1 undergoes early development, is capable of inducing adult onset disease states in the F1 generation. These phenotypes can be transmitted through the male germ line and maintained across the following generations. Vinclozolin early embryonic exposure also induces a number of transgenerational epigenetic changes in DNA methylation of promoter regions, detected in the sperm epigenome of F3-generation male rats. The present study replicates these results previously obtained with the rat model now using the mouse model and also expands the analysis to interrogate changes in females. The effects of transient exposure to the endocrine disruptors vinclozolin and flutamide during germ line development were analyzed in two strains of mice, one inbred (129) and one outbred (CD-1), in F1, F2 and F3 generations. Transgenerational increases in apoptosis with vinclozolin in the two mouse strains were observed with the vinclozolin but not the flutamide treatment. Increases in the incidence of diseased animals were also observed across generations, when analyzing testes, prostate and kidney. Also, a transgenerational disease in females was observed, which corresponds to an increase in the number of ovarian cysts following vinclozolin exposure observed from the F1 to F3 generations in a dose dependent manner. In addition to phenotypic effects, a number of epigenetic changes were also detected in the F3 generation when analyzing the epigenome in promoter regions of the sperm DNA. These changes were detected using methylated DNA immunoprecipitation follow by chip comparative arrays (MeDip-chip) and then confirmed regionally with quantification of the changed regions through qPCR of this immunoprecipitated DNA (qChIP). These findings show that the transgenerational transmission of these disease states is accompanied by transgenerational changes in the sperm epigenome. Further studies are required to identify if specific epigenetic changes may associate with particular disease states. Both phenotypic and epigenetic results presented here can have profound implications for the study of endocrine disrupting chemicals on toxicological or epidemiological research. (platform)

Female tail wagging enhances sexual performance in male goats

Preference testing has shown that sexually experienced male goats choose females that are tail wagging, a behavior that may function as both attractivity and proceptivity, over those that are not. We hypothesized that exposure to females expressing high rates of tail wagging would arouse males, increasing sexual performance. Tail wagging rate could be manipulated because we have shown previously that flutamide treatment increases the frequency of tail wagging in estrous goats. Sexually experienced males observed different stimuli for 10min before a 20min sexual performance test (SPT). The stimuli were an empty pen (MT), or groups of three females that were all estrous (E), non-estrous (NE), estrous+flutamide (EF) or non-estrous+flutamide (NEF). During the stimulus observation period, tail wagging was recorded. During SPT, frequencies and latencies of sexual behaviors were recorded. EF females displayed the most tail wagging. Viewing EF females before SPT increased the number of ejaculations attained by males and decreased the latencies to first and second ejaculation, as well as the inter-ejaculatory interval. Viewing estrous females (E and EF) before SPT decreased the latency to first mount, as compared to non-estrous females (NE and NEF). We conclude that male goats are sexually aroused by tail wagging. This study and previous work demonstrate that tail wagging functions as both attractivity and proceptivity in goats.

Differential Expression of Connexin 43 in Adult Pig Testes During Normal Spermatogenic Cycle and After Flutamide Treatment

Evidence is mounting that the foetal and neonatal period of reproductive tract development is highly sensitive to hormonal disruption induced by various endocrine active compounds. Thus, we asked whether androgen withdrawal caused by prenatal (GD20, GD80) or neonatal (PD2) exposure to an anti-androgen flutamide alters Cx43 gene expression and may induce delayed effects on morphology and function of adult pig testes. Flutamide was given in five doses (50 mg/kg bw). Our histological analysis and TUNEL staining revealed varying degrees of seminiferous tubules abnormalities in all experimental pigs. Testes of pigs exposed to flutamide in utero exhibited moderate alterations of the spermatogenic process, whereas those of exposed neonatally were severely impaired. The most striking effects were spermatogenic arrest, germ cell detachment and a statistically significant increase in the frequency of germ cell apoptosis (p<0.01). Moreover, all pigs exposed to flutamide displayed Leydig cell hyperplasia. Because the network of cell-cell communication provided by gap junction channels plays an essential role in the regulation and maintenance of spermatogenesis, the physiological significance of Cx43-based gap junctions with regards to the gonadal impairment was evaluated by analysis of its expression using immunohistochemical, Western blot and qRT-PCR approaches. Significantly, lower Cx43 expression was found when flutamide was administered neonatally, which has coincided with severe disruption of spermatogenesis. Our data suggest that neonatal exposure to flutamide induces long-term effects on the spermatogenic capacity of the pig testis through alterations of Cx43-mediated intercellular communication and permanent alteration of both Sertoli and Leydig cell functions.

Prospective Cohort Study on the Effects and Tolerability of Flutamide in Patients with Female Pattern Hair Loss

Flutamide has been rarely used for the treatment of female pattern hair loss (FPHL). Flutamide treatment for FPHL has not been evaluated in long-term studies with sufficiently large numbers of women. To evaluate long-term effects, safety, and tolerability of flutamide in women with FPHL. A prospective cohort study was conducted in our tertiary care university hospital. The cohort included 101 women diagnosed with FPHL from January 1991 to January 2006. These women received yearly reducing doses (250, 125, and 62.5 mg/day) of flutamide for 4 years. The cohort included 33 patients treated with flutamide alone and 68 treated with flutamide combined with an oral contraceptive. Clinical and endocrine evaluations were assessed semiannually and annually, respectively, in the first 3 years of the study, and once in the following year. Liver function was evaluated quarterly. Both groups showed a marked decrease in alopecia scores after 12 months of flutamide therapy, compared with baseline values. The maximum drug effect occurred after 2 years and was maintained during the following 2 years of treatment. Androgens were strongly suppressed. During the first year of treatment, 4% of patients abandoned the study due to hepatic disorders related to the drug. During the following years, with the lower treatment regimen, no patient abandoned the study because of hepatic alterations. Flutamide is a satisfactory therapeutic regimen for treatment of FPHL in the long run. Moreover, the use of very low doses (62.5 mg/day) of flutamide is associated with complete hepatic tolerability and high adherence.

The effect of prenatal androgen receptor blockade on breathing development in neonatal male and female rats

There are sex biases in the occurrence of respiratory disorders including Obstructive Sleep Apnea (OSA) and Sudden Infant Death Syndrome (SIDS). In neonatal rats, there are changes in respiratory control during the early neonatal period. Postnatal day 12–13 (P12–13) appears to be a “sensitive period” in breathing development in rats. However, little is known about the sex differences at this age and the effects of testosterone in breathing development. The purpose of our study was to eliminate the pre and perinatal testosterone surges and measure eupneic ventilation and the hypoxic ventilatory response (HVR) in awake neonatal rats from P10–15, at puberty (~P30) and in young adult (P90) male and female rats. Pregnant dams were given daily subcutaneous injections of the androgen antagonist flutamide (20mg/kg) from G14 until delivery. At P10-P15, P30 and P90 rats were studied in a whole-body flow-through plethysmograph and breathing and blood O2 saturation were continuously monitored in room-air and in hypoxia (12% O2). Following plethysmography, each rat was anesthetized and arterial blood collected for hormone measurement. Animals were perfused and the carotid bodies and brainstems harvested for immunohistochemical analysis. Flutamide treatment significantly changed O2 saturation in P30 and P90 female but not male rats. Supported by NIH AG18760 and the Parker B Francis Foundation.

Enhanced Proinflammatory Cytokine Response to Bacterial Lipopolysaccharide in the Adult Male Rat after either Neonatal or Prepubertal Ablation of Biological Testosterone Activity

A sex steroid-dependent modulation of the immune function in mammals is accepted, and evidence suggests that while estrogens enhance, androgens inhibit the immune response. The aim of this study was to explore in the adult male rat the effect of either neonatal flutamide (FTM) treatment or prepubertal orchidectomy (ODX) on endocrine markers in the basal condition and peripheral tumor necrosis factor alpha (TNFα) levels during inflammatory stress. For these purposes, (1) 5-day-old male rats were subcutaneously injected with either sterile vehicle alone or containing 1.75 mg FTM, and (2) 25-day-old male rats were sham operated or had ODX. Rats were sacrificed (at 100 days of age) in the basal condition for determination of peripheral metabolite levels. Additional rats were intravenously injected with bacterial lipopolysaccharide (LPS; 25 µg/kg body weight, i.v.) and bled for up to 4 h. Data indicate that (1) ODX increased peripheral glucocorticoid levels and reduced those of testosterone, whereas FTM-treated rats displayed low circulating leptin concentrations, and (2) LPS-induced TNFα secretion in plasma was significantly enhanced in the FTM and ODX groups. Our study supports that neonatal FTM treatment affected adiposity function, and adds data maintaining that androgens have a suppressive role in proinflammatory cytokine release in plasma during inflammation.

Copulatory efficiency and fertility in male rats exposed perinatally to flutamide

This study investigated the effects of perinatal treatment with flutamide on male sexual behavior, semen parameters, and fertility in adult male rats. Pregnant rats received 15 mg/kg of flutamide or peanut oil, s.c., at days 19 and 22 of pregnancy and for the first five postnatal days. Treated male offspring showed increases in latency to copulatory behavior, number of mounts without penis intromission, number of intromissions until ejaculation, latency to ejaculation, and reduced number of ejaculations. Flutamide treated rats presented reductions in weight of testes and prostate, percentage of normal spermatozoa, spermatozoa concentration, testicular sperm production, and testosterone level. Normal females mated with treated males presented more pre-implantation losses, reduced implantation rates, and consequently reduced offspring size. The results indicated that perinatal flutamide treatment damaged organizational processes of sexual differentiation, which led to inefficiency in copulatory behavior and reductions in sperm quality and count, resulting in low capacity for producing descendants.

Prenatal and neonatal exposure to the antiandrogen flutamide alters connexin 43 gene expression in adult porcine ovary

Connexin 43 (Cx43) is the predominant gap junction protein within porcine ovary and is required for proper follicle and corpus luteum (CL) development. Recent research suggests maternally or neonatally mediated effects of antiandrogens on reproductive function during adulthood, notably those dependent on gap junctional communication. The current study was conducted to determine whether late gestational or neonatal exposure to the antiandrogen flutamide influences Cx43 gene expression in the adult porcine ovary. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation and into female piglets between days 2 and 10 posnatally. After animals reached sexual maturity, the ovaries were collected from treated and nontreated (control) pigs. Expression of Cx43 mRNA and protein was determined for preantral and antral follicles and for CLs. In addition, 3β-hydroxysteroid dehydrogenase (3β-HSD) expression and progesterone concentration were determined for luteal tissues. In preantral follicles, Cx43 mRNA was down-regulated ( P < 0.01) following maternal and neonatal flutamide exposure. In large antral follicles, Cx43 mRNA was up-regulated ( P < 0.01) after neonatal flutamide administration. Immunofluorescence showed that Cx43 expression decreased ( P < 0.001) in preantral follicles and increased ( P < 0.001) in large antral follicles following flutamide exposure. In luteal tissues, Cx43 and 3β-HSD expression and progesterone concentration decreased ( P < 0.01) after postnatal flutamide treatment. Overall, these results suggest the involvement of androgens in the regulation of Cx43 expression in pig ovary. Moreover, alteration of Cx43 expression by the administration of flutamide during particular prenatal and neonatal time periods may affect porcine follicle development, as well as CL formation and function.

Testosterone-Down-Regulated Akt Pathway During Cardiac Ischemia/Reperfusion: A Mechanism Involving BAD, Bcl-2 and FOXO3a

Lower levels of myocardial Akt activity in males are associated with a higher incidence of heart failure and worsened cardiac function after ischemia/reperfusion (I/R). While Akt activation by estrogen provides cardioprotection in females, no information exists regarding the effect of testosterone on the myocardial Akt pathway following I/R. We hypothesized that following I/R: (1) endogenous testosterone will decrease myocardial Akt activation in male hearts; (2) endogenous testosterone will mediate downstream signals of Akt, including Bad, Bcl-2, and FOXO3a; (3) administration of exogenous testosterone will recapitulate negative effects on the Akt pathway in castrated male hearts. Rat hearts from age-matched adult males, females, castrated males, males with androgen receptor blocker-flutamide, castrated males with chronic 5α-dihydrotestosterone (DHT) implantation, or acute testosterone infusion (ATI) (n = 9/group) were subjected to I/R (Langendorff). Castration or flutamide treatment significantly up-regulated myocardial Akt activation, increased downstream apoptosis-regulatory molecules p-Bad, Bcl-2, p-FOXO3a, but reduced Fas-L, consistent with decreased myocardial injury in male hearts following I/R. ATI administration, but not chronic DHT, reversed these effects on Akt signaling associated with further exacerbated cardiac dysfunction in castrated males. Notably, lower levels of MnSOD were observed in male hearts, and castration or flutamide treatment restored myocardial MnSOD expression to the levels of females in male hearts after I/R. Our study represents the initial evidence of testosterone-induced down-regulation of the Akt pathway in male hearts following I/R, thereby mediating cardiac injury through decreased p-Bad, reduced ratio of Bcl-2/Bax in the cytoplasm, and increased FOXO3a in the nucleus.

The lack of estrogen and excess luteinizing hormone are responsible for the female ArKO mouse phenotype

It remains to be established as to whether the absence of estrogen (direct) or the elevated levels of gonadotrophins and androgens (indirect) are responsible for the ArKO (aromatase knockout) ovarian phenotype. The aim of this study was to determine the effects of E 2 (17β-estradiol) replacement, acyline (GnRH antagonist) and flutamide (anti-androgen) treatment on the ovarian phenotype of ArKO mice. E 2 replacement and acyline treatment but not flutamide treatment, reduced serum gonadotrophin levels of ArKO mice to within normal ranges. E 2 replacement improved uterine and ovarian follicular phenotypes and reduced the number of Sertoli-like filled cords by 62%. Acyline treatment reduced the number of hemorrhagic cysts and the number of Sertoli-like filled cords within ArKO ovaries. The data indicate that the absence of estrogen in concert with elevated levels of circulating gonadotrophins, principally LH, is responsible for the abnormal reproductive phenotype of the female ArKO mouse.

Short‐Term Antiandrogen Flutamide Treatment Causes Structural Alterations in Somatic Cells Associated with Premature Detachment of Spermatids in the Testis of Pubertal and Adult Guinea Pigs

In spite of widespread application of flutamide in the endocrine therapies of young and adult patients, the side effects of this antiandrogen on spermatogenesis and germ-cell morphology remain unclear. This study evaluates the short-term androgen blockage effect induced by the administration of flutamide to the testes of pubertal (30-day old) and adult (65- and 135-day old) guinea pigs, with an emphasis on ultrastructural alterations of main cell types. The testes removed after 10 days of treatment with either a non-steroidal antiandrogen, flutamide (10 mg/kg of body weight) or a pharmacological vehicle alone were processed for histological, quantitative and ultrastructural analysis. In pubertal animals, flutamide androgenic blockage induces spermatogonial differentiation and accelerates testes maturation, causing degeneration and detachment of primary spermatocytes and round spermatids, which are subsequently found in great quantities in the epididymis caput. In post-pubertal and adult guinea pigs, in addition to causing germ-cell degeneration, especially in primary spermatocytes, and leading to the premature detachment of spherical spermatids, the antiandrogen treatment increased the relative volume of Leydig cells. In addition, ultrastructural evaluation indicated that irrespective of age antiandrogen treatment causes an increase in frequency of organelles involved with steroid hormone synthesis in the Leydig cells and a dramatic accumulation of myelin figures in their cytoplasm and, to a larger degree, in Sertoli cells. In conclusion, the transient exposition of the guinea pigs to flutamide, at all postnatal ages causes some degenerative lesions including severe premature detachment of spermatids and accumulation of myelin bodies in Leydig and Sertoli cells, compromising, at least temporarily, the spermatogenesis.

Effects of neonatal flutamide treatment on hippocampal neurogenesis and synaptogenesis correlate with depression-like behaviors in preadolescent male rats

The prevalence of major depressive disorder (MDD) in adult men is roughly half that of women. Clinical evidence supports a protective effect of androgens against depressive disorders in men. The developing brain is subject to androgen exposure but a potential role for this in depression during adulthood has not been considered. In order to explore this question we treated newborn male rat pups with the androgen receptor antagonist flutamide to block endogenous androgen action and then conducted behavioral tests prior to puberty. Depression-like behaviors were assessed with the Forced Swim Test (FST) and the Sucrose Preference Test (SPT), and anxiety-like behaviors were assessed with the Open Field Test (OFT) and the Novelty-Suppressed Feeding Test (NSFT). Compared to the vehicle-treated controls, neonatal-flutamide treatment caused a significant increase in depression-like behaviors in preadolescent male rats but did not cause any significant difference in anxiety-like behaviors. In separate experiments, male pups with and without flutamide treatment were injected with 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdU) from postnatal day (PND) 1 to 4 to label newly produced cells or the hippocampi were Golgi-Cox imbedded and pyramidal neurons visualized. Three lines of evidence indicate neonatal flutamide treatment inhibits hippocampal neurogenesis and neuronal dendritic spine formation in preadolescent male rats. Compared to vehicle controls, flutamide treatment significantly decreased (1) the number of microtubal associated protein-2+ (MAP-2) neurons in the CA1 region, (2) the number of MAP-2+ neurons in the dentate gyrus (DG) region of the hippocampus, and (3) the density of dendritic spines of pyramidal neurons in the CA1 region. However, there was no effect of flutamide treatment on the number of glial fibrillary acidic protein (GFAP)+ or GFAP+/BrdU+ cells in the hippocampus. This study suggests that the organizational effect of androgen-induced hippocampal neurogenesis is antidepressant.

The role of androgen receptors in regulating territorial aggression in male song sparrows

This paper examines the role that androgen receptors (ARs) play in modulating aggressive behavior in male song sparrows, Melospiza melodia morphna. Song sparrows are seasonally breeding, territorial birds that maintain year-round territories with male–female pair bonds formed during the spring breeding season. Plasma testosterone levels peak as territories are established and mates acquired. In late summer, testosterone levels fall and remain basal during the non-breeding season. We examined the role of ARs in regulating territorial aggression in captive song sparrows under short- and long-day conditions as well as just prior to, and at the start of the breading season in freely living birds using the nonsteroidal antiandrogen flutamide to block AR function. Birds were implanted with either empty or drug filled silastic implants for 18 to 42 days and then challenged with a novel male decoy to assess the individual birds level of male–male aggression. Freely living birds remained on their home territory and underwent a simulated territorial intrusion, whereas laboratory-held birds were assessed using a laboratory simulated territorial intrusion and remained in their home cage. Experimental treatment of male song sparrows decreased aggressive behavior during the pre-breeding life history substage (March–April) in freely living birds as well as in laboratory-held birds under long-day (16L:8D) conditions. During the early breeding substage (April–May) there was no measurable effect of flutamide treatment on aggressive behavior, nor was there a difference in behavior in the (8L:16D) laboratory birds. This demonstrates that ARs are an important component of the neuroendocrine control of aggressive behavior. Given that flutamide only affected aggression during the pre-breeding substage and in LD birds, the results suggest that AR dependent control of aggressive behavior changes as song sparrow life history states change.

The aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) reduces disinhibitory behavior in intact adult male rats treated with a high dose of testosterone

Anabolic androgenic steroids and high testosterone doses have been reported to induce impulsive behavior in man and behavioral disinhibition in rats. The purpose of the present study was to investigate whether aromatization of testosterone to estradiol is of importance for the behavioral disinhibiting effect of a high testosterone dose in adult male rats. Testosterone administered via five testosterone-filled silastic capsules implanted subcutaneously (s.c.) to non-castrated, group-housed rats for six days induced behavioral disinhibition in a modified Vogel's drinking conflict model and yielded supraphysiological serum levels of testosterone and increased accessory sex organ weights. Moreover, concurrent administration of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD; 60mg/kg/day s.c.) decreased behavioral disinhibition in testosterone-treated rats (without affecting accessory sex organ weights) while behavior was not significantly affected in sham-treated animals. Since some reports indicate that ATD, in addition to inhibit aromatase, also may affect the binding of testosterone to the androgen receptor, the effect of the non-steroidal androgen receptor antagonist flutamide was investigated. Flutamide treatment did not affect disinhibited behavior in testosterone-treated rats. However, in sham-treated animals, flutamide (50mg/kg/day) produced behavioral disinhibition. These results suggest that estradiol is of importance in the mechanisms underlying behavioral disinhibition in non-castrated rats treated with a high testosterone dose. Speculatively, aromatization may be involved in pro-impulsive effects of high testosterone doses in humans.

Long-Term Safety and Tolerability of Flutamide for the Treatment of Hirsutism

Hirsutism in young women is not uncommon. Several drugs have been investigated as treatment and have been largely ineffective or inconsistent although cosmetic measures have been achieved in some patients. Previous studies have suggested that flutamide and other nonestrogenic antiandrogen agents may be effective in patients with hirsutism. However, severe adverse effects such as liver toxicity limit their clinical usefulness. Reports addressing this issue have been either short-term studies (6-12 months) or had a small sample size. This prospective study assessed the safety and tolerability of long-term flutamide treatment in hirsute women. The participants were 93 women with moderate to severe hirsutism. Women who fulfilled inclusion criteria (n = 83) received either flutamide 250 mg/d alone (n = 27) or combined with a fixed dose of an oral contraceptive containing 0.020 mg of ethinyl estradiol and 0.15 mg of desogestrel (n = 56) for 84 months. Baseline and treatment data for both groups was very similar and were pooled in the final analysis. Each patient was evaluated for efficacy, safety and tolerability at 6 and 12 months follow-up, and at yearly intervals until the end of the study. Self-perception of therapeutic effect was rated by the patients using a visual analog scale. Tolerability and patient satisfaction were determined by use of questionnaires. Compared with basal values, treatment with flutamide produced a significant progressive decrease in hirsutism after 6 months of treatment (P < 0.001). Improvement in hirsutism scores was maximal at 12 months and maintained at this level throughout the 84-month study period. At follow-up, 34 patients (40.96%, 34/83) had one or more adverse events and 28 (33.73%, 28/83) presented with at least one adverse effect possibly related to flutamide treatment. Twenty patients (24.09%, 20/83) left the study due to intolerance or adverse effects requiring discontinuation of the drug. In total, 39 women (47%, 39/83) withdrew from the study during follow-up. Among those remaining in the study, self-perception of therapeutic efficacy and satisfaction with effects of therapy was high at both 6 and 84 months and none reported dissatisfaction of any type. These findings showing the long-term effectiveness of flutamide for hirsutism are consistent with data from short-term studies but further demonstrate the high incidence of adverse effects and poor treatment compliance.

Up-regulation of circadian clock gene Period 2 in the prostate mesenchymal cells during flutamide-induced apoptosis

Androgen regulates the proper development and physiological function of the prostate. Here, we investigated the modulation of androgen and androgen receptor (AR) antagonist on circadian oscillations of a clock core gene Period 2 (Per2) in rat prostate mesenchymal cells (PMCs). Circadian oscillations were analyzed with the real-time monitoring system of gene expression using transgenic rats introduced with mouse Per2 promoter fused to a destabilized luciferase (Per2-dLuc) reporter gene. Analyses of circadian oscillations, immunofluorescence, and androgen response element (ARE)-luciferase reporter assay revealed that circadian clocks are operative and the AR protein is functional in PMCs in vitro. Androgen such as testosterone (T) and dihydrotestosterone (DHT) did not cause any changes in circadian Per2-dLuc oscillations of confluent cells. Conversely, flutamide (FL) up-regulated the amplitude of circadian Per2-dLuc oscillations in a dose-dependent manner, whereas T antagonized the action of FL. The PER2 protein was markedly accumulated by FL treatment and localized in both the nucleus and cytoplasm during the first peak period of circadian Per2-dLuc oscillations. Simultaneously, FL treatment increased apoptotic cell death. Collectively, the present study demonstrates that a clock gene Per2 is up-regulated in PMCs during FL-induced apoptotic cell death. Thus, circadian oscillations of Per2 gene expression may be closely linked to the cellular states of PMCs such as apoptotic cell death.

Calcitonin gene-related peptide is a survival factor, inhibiting apoptosis in neonatal rat gubernaculum in vitro

Background Testicular descent is proposed to occur in 2 stages. During the second stage, calcitonin gene–related peptide (CGRP) released from the genitofemoral nerve (GFN) causes maximal mitosis in the gubernacular bulb. As normal development requires a balance between cell proliferation and apoptosis, this study explored the effect of CGRP on apoptosis in the rat gubernacular bulb. Methods Gubernacula were collected from male Sprague-Dawley rats at birth (D0) or 2 days post birth (D2), and placed in organ culture for 24 hours with or without CGRP (0.001 mol/L). The D2 rats were pretreated with capsaicin (sensory nerve toxin) or flutamide (antiandrogen) or untreated. D0 rats were untreated (n = 64). Sections of the bulb were stained using the TUNEL method to identify apoptotic cells. Apoptosis was calculated as the percentage of positive cells per hundred cells. Results Normal Sprague-Dawley rat gubernacula showed reduced apoptosis when cultured with CGRP, in D0 (7.0% vs 4.8%, P < .05) and D2 (4.9% vs 2.3%, P < .001). Greater apoptosis occurred at D0 compared to D2, without CGRP added (7.0% vs 4.9%, P < .05) and with CGRP (4.8% vs 2.3%, P < .001). For D2 gubernacula, capsaicin treatment increased apoptosis compared to controls, without CGRP added (4.9% vs 7.3%, P < .05) and with CGRP (2.3% vs 6.7%, P < .001). There was no difference in apoptosis when cultured with or without CGRP (7.3% vs 6.7%, nonsignificant) after capsaicin treatment. Flutamide treatment increased apoptosis compared to controls, but only with CGRP (2.3% vs 7.3%, P < .001). There was no difference in apoptosis when cultured with or without CGRP (7.1% vs 7.3%, nonsignificant) after flutamide. Conclusions Calcitonin gene–related peptide (CGRP) acts as a survival factor in the rat gubernaculum, possibly to steer cells away from a defined apoptotic pathway. Greater apoptosis occurs earlier in development. However, in vivo CGRP released from the genitofemoral nerve may be required to prevent apoptosis, as shown by pretreatment with the sensory nerve toxin capsaicin. Androgen is also involved in the pathway controlling apoptosis, as androgen blockade with flutamide inhibited the action of CGRP.

Both endogenous and exogenous testosterone decrease myocardial STAT3 activation and SOCS3 expression after acute ischemia and reperfusion

Signal transducer and activator of transduction 3 (STAT3) pathway has been shown to be cardioprotective. We observed decreased STAT3/suppressor of cytokine signaling 3 (SOCS3) in male hearts, which was associated with worse postischemic myocardial function compared with females. However, it is unclear whether this downregulation of myocardial STAT3/SOCS3 is due to testosterone in males. We hypothesized that after ischemia/reperfusion (I/R), (1) endogenous testosterone decreases myocardial STAT3 and SOCS3 in males, and (2) administration of exogenous testosterone reduces myocardial STAT3/SOCS3 in female and castrated male hearts. To study this, hearts from I/R injury (Langendorff) were homogenized and assessed for phosphorylated-STAT3 (p-STAT3), total-STAT3 (T-STAT3), SOCS3, and GAPDH by Western blot. We grouped age-matched adult males, females, castrated males, males with androgen receptor blocker-flutamide implantation, females, and castrated males with chronic (3-week) 5alpha-dihydrotestosterone (DHT) release pellet implantation or acute (5-minute) testosterone infusion (ATI) before ischemia (n = 5-9 per group). Castration or flutamide treatment significantly increased SOCS3 expression in male hearts after I/R. However, only castration increased myocardial STAT3 activation. Notably, DHT replacement or ATI decreased markedly myocardial STAT3/SOCS3 in castrated males and females subjected to I/R. These results suggest that endogenous and exogenous testosterone decrease myocardial STAT3 activation and SOCS3 expression after I/R. This represents the initial demonstration of testosterone-downregulated STAT3/SOCS3 signaling in myocardium.