Flutamide Treatment Research Articles

The effect of flutamide on the physical working capacity and activity of some of the key enzymes for the energy supply in adult rats.

The aim of the study was to assess the effects of androgen receptor antagonists on the physical working capacity and activity of some of the key muscle enzymes for the energy supply in rats. Young adult male Wistar rats were divided into two groups. One group received 15 mg kg−1 of flutamide daily for 6 days a week and the other group served as control for 8 weeks. At the beginning and at the end of the experiment, all rats were subjected to submaximal running endurance (SRE), maximum time to exhaustion (MTE), and maximal sprinting speed (MSS) tests. At the end of the trial, maximum oxygen consumption (VO2max) test was performed and the levels of testosterone, erythrocytes, hemoglobin as well as enzyme activity of succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), and NAD.H2-cytochrome-c reductase (NAD.H2) of the gastrocnemius muscle were measured. Serum testosterone of the flutamide-treated rats was higher than that of the controls, which verifies the effectiveness of the dose chosen. MTE and SRE of the anti-androgen-treated group were lower compared with the initial values. Flutamide treatment decreased the activity of SDH and NAD.H2 compared with the controls. We found no effect of the anti-androgen treatment on MSS, VO2max, running economy, LDH activity, and hematological variables. Our findings indicate that the maintenance of the submaximal and maximal running endurance as well as the activity of some of the key enzymes associated with muscle oxidative capacity is connected with androgen effects mediated by androgen receptors.

SBR among targets for biotech firm Anellotech

Water contamination is one of the factors influencing the decline of amphibians. Flutamide is an antiandrogenic medicine that occurs as water contaminant. This compound especially affects the reproductive organs, but it can also show hepatotoxic effects. The Bufonidae family has a peculiar organ named Bidder’s organ, considered by some authors as a rudimentary ovary, but capable to respond to some external stimuli. This study investigated flutamide effects on testes and Bidder’s organ germ cells, liver pigmentation, and sexual hormones levels in Rhinella schneideri males. We randomly divided 15 males in three groups (N = 5): two groups were injected with flutamide, at 1 and 5 mg/kg, while the control group received only mineral oil, for 7 days. After euthanasia, blood samples were collected and the organs were sent to histological routine. In the testes, both treatments caused an increase in spermatogonia and spermatocytes, and a decrease in spermatozoa and locular area. In the Bidder’s organ, the final diplotene oocytes increased, but the initial diplotene, degrading and atresic oocytes reduced in both treatments. The lipofuscin in the Bidder’s organ was not affected. In the liver, melanin and lipofuscin increased only for the 1 mg/kg flutamide treatment. The 5 mg/kg treatment did not affect the liver. Serum testosterone and estradiol levels did not vary compared with the control group. This compound has antiandrogenic activity, which can affect the spermatogenetic process. The decrease in degrading and atresic Bidderian oocytes indicated that flutamide could stimulate the organ, retarding the degradation processes. The increase in liver melanin, which has protective role, and lipofuscin, a sign of degradation, indicates that flutamide cause hepatotoxic effects. So we conclude that flutamide negatively affects the testes, especially by reducing the sperm area, and the liver, inducing cell degradation and producing protective responses. Furthermore, the compound encourages lower degradation rates of the Bidder’s organ germ cells.

Biomarkers of Flutamide-Bioactivation and Oxidative Stress In Vitro and In Vivo.

The nonsteroidal androgen-receptor antagonist flutamide is associated with hepatic injury. Oxidative stress and reactive metabolite formation are considered contributing factors to liver toxicity. Here we have used flutamide as a model drug to study the generation of reactive drug metabolites that undergo redox cycling to induce oxidative stress (OS) in vitro and in vivo. Lipid peroxidation (LPO) markers, as well as genes regulated by the redox-sensitive Nrf2 pathway, have been identified as surrogates for the characterization of OS. These markers and metabolism biomarkers for drug bioactivation have been investigated to characterize drug-induced hepatic damage. Rat hepatocytes and in vivo studies showed that several LPO markers, namely the isoprostanes 15R-PD2, dihydro keto PE2, and iPF(2α)-VI, as well as hydroxynonenal mercapturic acid metabolites, had increased significantly by 24 hours after flutamide treatment from 4.9 to 15.3-fold in hepatocytes and from 2.6 to 31.0-fold in rat plasma. Induction of mRNA expression levels for Nrf2-regulated genes was evident as well, with heme oxygenase 1, glutathione-S-transferase π1 and NAD(P)H dehydrogenase showing a 3.6-, 4.1-, and 1.9-fold increase in hepatocytes and 5.6-, 7.5-, and 94.1-fold in rat liver. All effects were observed at drug concentrations that did not show overt liver toxicity. Addition of an in situ hydrogen peroxide-generating system to in vitro experiments demonstrated the formation of a reactive di-imine intermediate as the responsible metabolic pathway for the generation of OS. The dataset suggests that hepatic oxidative stress conditions can be mediated via metabolic activation and can be monitored with suitable biomarkers preceding the terminal damage.

Pharmacologic blockade and genetic deletion of androgen receptor attenuates aortic aneurysm formation

Testosterone is theorized to play a major role in the pathophysiology of abdominal aortic aneurysms (AAAs) because this disease occurs primarily in men. The role of the androgen receptor (AR) in the formation of AAAs has not been well elucidated, and therefore, it is hypothesized that androgen blockade will attenuate experimental aortic aneurysm formation. Aortas of 8- to 12-week-old male C57Bl/6 wild-type (WT) mice or male AR knockout (AR(-/-)) mice were perfused with purified porcine pancreatic elastase (0.35 U/mL) to induce AAA formation. Two groups of WT male mice were treated with the AR blockers flutamide (50mg/kg) or ketoconazole (150mg/kg) twice daily by intraperitoneal injection. Aortas were harvested on day 14 after video micrometry was used to measure AAA diameter. Cytokine arrays and histologic analysis were performed on aortic tissue. Groups were compared using an analysis of variance and a Tukey post hoc test. Flutamide and ketoconazole treatment (mean± standard error of the mean) attenuated AAA formation in WT mice (84.2%± 22.8% [P= .009] and 91.5%± 18.2% [P= .037]) compared with WT elastase (121%± 5.23%). In addition, AR(-/-) mice showed attenuation of AAA growth (64.4%± 22.7%; P< .0001) compared with WT elastase. Cytokine arrays of aortic tissue revealed decreased levels of proinflammatory cytokines interleukin (IL)-α, IL-6, and IL-17 in flutamide-treated and AR(-/-) groups compared with controls. Pharmacologic and genetic AR blockade cause attenuation of AAA formation. Therapies for AR blockade used in prostate cancer may provide medical treatment to halt progression of AAAs in humans.

Effect of Prenatal and Neonatal Anti-Androgen Flutamide Treatment on Aquaporin 5 Expression in the Adult Porcine Ovary.

The growth of ovarian follicles is accompanied by fluid-filled antrum formation. Water movement within the follicular wall is predominantly transcellular via membranous water channels named aquaporins (AQPs). Androgens are important regulators of mammalian folliculogenesis, and their prenatal and/or neonatal deficiency affects female fertility in adulthood. Therefore, this study was performed to determine whether gestational or neonatal exposure to the anti-androgen flutamide influences androgen-dependent AQP5 expression in pre-antral and large antral follicles of adult pigs. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation and into female piglets between days 2 and 10 post-natally. The ovaries were collected from flutamide-treated and non-treated (control) sexually mature pigs. In pre-antral follicles, AQP5 mRNA and protein levels were both downregulated following maternal (p < 0.01 and p < 0.01, respectively) and neonatal (p < 0.01 and p < 0.01, respectively) flutamide exposure. Likewise, the expression of mRNA (p < 0.01 and p < 0.001, respectively) and protein (p < 0.05 and p < 0.01, respectively) for AQP5 were diminished in large antral follicles in both groups. Immunohistochemistry showed decreased intensity of AQP5 immunoreaction in pre-antral (p < 0.01) and large antral (p < 0.001) follicles following flutamide treatment. Moreover, radioimmunological analysis revealed that changes observed in AQP5 expression corresponded with diminished follicular androgens production after both maternal (p < 0.05 and p < 0.05, respectively) and neonatal (p < 0.05 and p < 0.01, respectively) flutamide administration. Therefore, AQP5 appears to be a potential regulator of follicular fluid accumulation, under androgen control, and may be a key factor in antral follicle growth.

Beyond aggression: Androgen-receptor blockade modulates social interaction in wild meerkats

In male vertebrates, androgens are inextricably linked to reproduction, social dominance, and aggression, often at the cost of paternal investment or prosociality. Testosterone is invoked to explain rank-related reproductive differences, but its role within a status class, particularly among subordinates, is underappreciated. Recent evidence, especially for monogamous and cooperatively breeding species, suggests broader androgenic mediation of adult social interaction. We explored the actions of androgens in subordinate, male members of a cooperatively breeding species, the meerkat (Suricata suricatta). Although male meerkats show no rank-related testosterone differences, subordinate helpers rarely reproduce. We blocked androgen receptors, in the field, by treating subordinate males with the antiandrogen, flutamide. We monitored androgen concentrations (via baseline serum and time-sequential fecal sampling) and recorded behavior within their groups (via focal observation). Relative to controls, flutamide-treated animals initiated less and received more high-intensity aggression (biting, threatening, feeding competition), engaged in more prosocial behavior (social sniffing, grooming, huddling), and less frequently initiated play or assumed a ‘dominant’ role during play, revealing significant androgenic effects across a broad range of social behavior. By contrast, guarding or vigilance and measures of olfactory and vocal communication in subordinate males appeared unaffected by flutamide treatment. Thus, androgens in male meerkat helpers are aligned with the traditional trade-off between promoting reproductive and aggressive behavior at a cost to affiliation. Our findings, based on rare endocrine manipulation in wild mammals, show a more pervasive role for androgens in adult social behavior than is often recognized, with possible relevance for understanding tradeoffs in cooperative systems.

Germ Cell Nuclear Factor (GCNF/RTR) Regulates Transcription of Gonadotropin-Regulated Testicular RNA Helicase (GRTH/DDX25) in Testicular Germ Cells--The Androgen Connection.

Gonadotropin-regulated testicular RNA helicase (GRTH) (GRTH/DDX25), is a testis-specific protein essential for completion of spermatogenesis. Transgenic mice carrying 5'-flanking regions of the GRTH gene/green fluorescence protein (GFP) reporter revealed a region (-6.4/-3.6 kb) which directs its expression in germ cells (GCs) via androgen action. This study identifies a functional cis-binding element on the GRTH gene for GC nuclear factor (GCNF) (GCNF/RTR) required to regulate GRTH gene expression in postmeiotic testis GCs and explore the action of androgen on GCNF and GRTH transcription/expression. GCNF expression decreased in mice testis upon flutamide (androgen receptor antagonist) treatment, indicating the presence of an androgen/GCNF network to direct GRTH expression in GC. Binding studies and chromatin immunoprecipitation demonstrated specific association of GCNF to a consensus half-site (-5270/-5252) of the GRTH gene in both round spermatids and spermatocytes, which was abolished by flutamide treatment in round spermatids. Moreover, flutamide treatment of wild-type mice caused selective reduction of GCNF and GRTH in round spermatids. GCNF knock-down in seminiferous tubules from GRTH-transgenic mice (dark zone, round spermatid rich) caused decreased GFP expression. Exposure of tubules to flutamide caused decrease in GCNF and GFP expression, whereas androgen exposure induced significant increase. Our studies provide evidence for actions of androgen on GCNF cell-specific regulation of GRTH expression in GC. GRTH associates with GCNF mRNA, its absence caused increase on GCNF expression and mRNA stability indicative of a negative autocrine regulation of GCNF by GRTH. These in vivo/in vitro models link androgen actions to GC through GCNF, as regulated transfactor that controls transcription/expression of GRTH.

Critical role of androgen receptor in the postnatal period in male sexual behavior in rats

Gonadal hormones have a developmental role in organization of the nervous system that regulates sexually dimorphic behavior. It is well known that androgen secreted from testes in the perinatal period is converted to estrogen by aromatase in rodent brain, and that estrogen and its receptor play a pivotal role in masculinization of brain structure and function. Treatment with flutamide, an androgen receptor (AR) antagonist, during the perinatal period inhibits development of malespecific brain structure and function, suggesting that androgen signaling via AR also influences brain masculinization. In this study, we investigated which stage during the postnatal period is critical for androgen signaling in brain masculinization. The postnatal period was designated as postnatal days (PD) 0–22, and divided into stages I (PD 0–7), II (PD 8–14), and III (PD 15–22). Newborn male rats were given flutamide subcutaneously in each stage. After adulthood, the effects of postnatal flutamide treatment on brain masculinization were evaluated byanalysis of male sexual behavior. Continuous inhibition of AR throughout stages I and II caused a robust reduction of the intromission ratio and ejaculation frequency compared with other groups. AR inhibition in stage I, II, or III did not cause any change. AR inhibition had no effect onmount behavior. These results show that stage-specific AR activation in the first two postnatal weeks may contribute to brain masculinization mediating male sexual behavior in adulthood.

Gynecomastia in Patients with Prostate Cancer: A Systematic Review.

IntroductionGynecomastia and/or mastodynia is a common medical problem in patients receiving antiandrogen (bicalutamide or flutamide) treatment for prostate cancer; up to 70% of these patients result to be affected; furthermore, this can jeopardise patients’ quality of life.AimsTo systematically review the quality of evidence of the current literature regarding treatment options for bicalutamide-induced gynecomastia, including efficacy, safety and patients’ quality of life.MethodsThe PubMed, Medline, Scopus, The Cochrane Library and SveMed+ databases were systematically searched between January 1, 2000 and December 31, 2014. All searches were undertaken between January and February 2015. The search phrase used was:”gynecomastia AND treatment AND prostate cancer”. Two reviewers assessed 762 titles and abstracts identified. The search and review process was done in accordance with the PRISMA statement. The PICOS (patients, intervention, comparator, outcomes and study design) process was used to specify inclusion criteria. Quality of evidence was rated according to GRADE.Main Outcome MeasuresPrimary outcomes were: treatment effects, number of complications and side effects. Secondary outcome was: Quality of Life.ResultsEleven studies met the inclusion criteria and are analysed in this review. Five studies reported pharmacological intervention with tamoxifen and/or anastrozole, either as prophylactic or therapeutic treatment. Four studies reported radiotherapy as prophylactic and/or therapeutic treatment. Two studies compared pharmacological treatment to radiotherapy. Most of the studies were randomized with varying risk of bias. According to GRADE, quality of evidence was moderate to high.ConclusionsBicalutamide-induced gynecomastia and/or mastodynia can effectively be managed by oral tamoxifen (10–20 mg daily) or radiotherapy without relevant side effects. Prophylaxis or therapeutic treatment with tamoxifen results to be more effective than radiotherapy.

MP49-16 ENZALUTAMIDE INHIBITS ANDROGEN RECEPTOR-POSITIVE BLADDER CANCER CELL GROWTH

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2015MP49-16 ENZALUTAMIDE INHIBITS ANDROGEN RECEPTOR-POSITIVE BLADDER CANCER CELL GROWTH Takashi Kawahara, Hiroki Ide, Eiji Kashiwagi, Leonardo Reis, Yichun Zheng, and Hiroshi Miyamoto Takashi KawaharaTakashi Kawahara More articles by this author , Hiroki IdeHiroki Ide More articles by this author , Eiji KashiwagiEiji Kashiwagi More articles by this author , Leonardo ReisLeonardo Reis More articles by this author , Yichun ZhengYichun Zheng More articles by this author , and Hiroshi MiyamotoHiroshi Miyamoto More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2811AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Emerging preclinical evidence suggests the involvement of androgen-mediated androgen receptor (AR) signals in bladder cancer progression. However, little is known about the efficacy of enzalutamide, an AR signaling inhibitor that not only blocks androgen binding to AR but also prevents AR nuclear translocation, DNA binding, and coactivator recruitment, in the growth of bladder cancer cells. In this study, we compared the effects of enzalutamide and 2 other anti-androgens, flutamide and bicalutamide, on androgen-induced bladder cancer cell proliferation, migration, and invasion. METHODS MTT assay, colony formation assay, scratch wound healing assay, transwell invasion assay, real-time polymerase chain reaction, and reporter gene assay were performed in AR-positive (e.g. UMUC3, TCCSUP, 5637-AR, 647V-AR) and AR-negative (e.g. UMUC3-AR-shRNA, TCCSUP-AR-shRNA, 5637, 647V) bladder cancer lines treated with dihydrotestosterone and/or each anti-androgen. We also used a mouse xenograft model for bladder cancer. RESULTS Dihydrotestosterone increased cell viability, colony formation, cell migration, and cell invasion, indicating that endogenous or exogenous AR was functional. Enzalutamide, hydroxyflutamide, and bicalutamide showed similar inhibitory effects, without significant agonist activity, on androgen-induced cell proliferation/migration/invasion in AR-positive lines. No significant effects of dihydrotestosterone as well as anti-androgens on the growth were seen in AR-negative cells. Correspondingly, in UMUC3 cells, these anti-androgens down-regulated androgen-induced expression of MMP-2 and IL-6. Androgen-enhanced AR-mediated transcriptional activity was also blocked by each anti-androgen exhibiting marginal agonist activity. In UMUC3 xenograft-bearing mice, oral gavage treatment with each anti-androgen retarded tumor growth, and enzalutamide showed more significant suppressive effects, compared with flutamide or bicalutamide treatment. CONCLUSIONS These findings support recent findings indicating that androgens promote bladder cancer progression via the AR pathway and further suggest that anti-androgens, including enzalutamide, are of therapeutic benefit in AR-positive bladder cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e608-e609 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takashi Kawahara More articles by this author Hiroki Ide More articles by this author Eiji Kashiwagi More articles by this author Leonardo Reis More articles by this author Yichun Zheng More articles by this author Hiroshi Miyamoto More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Postmenopausal Hypertension Is Blunted Following Chronic Flutamide Treatment in Intrauterine Growth Restricted Female Rat

Intrauterine growth restriction (IUGR) programs the development of hypertension associated with increased adiposity and accelerated reproductive senescence by 12 months of age in female IUGR rats. At 12 months of age the estradiol (E) to testosterone (T) ratio is shifted with E levels remaining similar whereas T levels are increased in female IUGR relative to control. Postmenopausal women have a higher prevalence of hypertension associated with increased adiposity. In addition, blood pressure (BP) is increased in association with increased serum T in postmenopausal women. Thus, we tested the hypothesis that chronic blockade of the androgen receptor would attenuate hypertension in female IUGR rats at 12 months of age. Rats were treated with flutamide (8mg/kg/day), an androgen receptor antagonist, or vehicle subcutaneously for two weeks. BP was measured in conscious, chronically catheterized rats. T levels were measured via radioimmunoassay. BP was increased in IUGR relative to control at 12 months of age (Δ15 mmHg, P&lt;0.05); T levels were also elevated in IUGR vs. control (45.33±3.48 vs. 37.56 ±1.72 pg/ml; P&lt;0.05). Flutamide decreased BP in IUGR relative to untreated IUGR (Δ7 mmHg) with no impact in control (Δ0 mmHg). Flutamide did not alter fat mass. Thus, these data indicate that T contributes to the etiology of hypertension that develops with age following IUGR in the female rat. NIH: HL074927, HL51971, P20GM104357, AHA GRNT19900004

TGFβ (transforming growth factor β) superfamily members and their receptors in the fetal porcine ovaries: effect of prenatal flutamide treatment.

We have recently demonstrated that antiandrogen treatment during fetal life resulted in delayed folliculogenesis. The aim of the present study was to investigate the effect of androgen deficiency induced by flutamide on the expression of TGFβ superfamily members and their receptors which are involved in follicle formation and its transition to the primary stage. Pregnant gilts were injected with flutamide (for seven days, 50 mg/day/kg b.w.) or corn oil (controls) starting at 43 (GD50), 83 (GD90) or 101 (GD108) gestational day. The expression in fetal ovaries of selected TGFβ superfamily members (AMH, BMP4, GDF9), their receptors (AMHR-II, BMPR-IB, BMPR-II), and Smad1 and Smad3 proteins involved in signal transduction were investigated by real-time PCR and/or immunohistochemistry. Flutamide treatment increased the expression of BMP4 mRNA on GD50 and GD108 and BMPR-IB mRNA on GD50. The expression of BMPR-II was decreased at mRNA level and lower immunostaining intensity was observed after flutamide administration only on GD50. GDF9 and AMHR-II mRNA expression levels were significantly downregulated in both GD90 and GD108 groups. However, AMHR-II was immunolocalized only on GD108 and less positively stained oocytes were found after flutamide treatment. AMH mRNA level was diminished in the GD90 group, while it was elevated in the GD108 group. Moreover, the higher amounts of positively stained oocytes for phosphorylated form of Smad1 were observed following flutamide administration on GD108. Experimentally-induced androgen deficiency during fetal development deregulates the expression level of some of TGFβ superfamily members and their receptors which may affect primordial follicle assembly. Our findings further underline the role of androgens in the early stages of follicle development.

Enhanced mesenteric arterial responsiveness to angiotensin II is androgen receptor-dependent in prenatally protein-restricted adult female rat offspring.

Gestational protein restriction results in intrauterine growth restriction and hypertension in adult female growth-restricted rats. Enhanced vascular responsiveness to angiotensin II is observed, and blockade of the renin-angiotensin system abolishes hypertension in adult growth-restricted rats, suggesting that the renin-angiotensin system contributes to intrauterine growth restriction-induced hypertension. Moreover, growth-restricted adult rats have higher plasma testosterone levels, and antiandrogen treatment abolishes hypertension, indicating an important role for testosterone. We hypothesized that androgens may play a pivotal role in the enhanced responsiveness to Ang II and hypertension. Female offspring of pregnant rats fed 20% protein (control) or 6% protein diet (protein restricted), at 6 mo of age, were studied. Plasma testosterone and mean arterial pressure in protein-restricted offspring were significantly higher compared to controls. Flutamide treatment (10 mg/kg/day subcutaneously for 10 days) reduced mean arterial pressure in protein-restricted offspring but was without significant effect in controls. Vascular Agtr1/Agtr2 ratio was significantly higher in protein-restricted offspring, an effect that was reversed by flutamide. Flutamide treatment did not have any effect on Agtr1/Agtr2 ratio in controls. Enhanced contractile response to angiotensin II in mesenteric arteries was observed in protein-restricted offspring compared with control. Flutamide treatment reversed the enhanced contractile response to angiotensin II in protein-restricted offspring without significant effect in controls. Vascular reactivity to phenylephrine was similar between the control and protein-restricted offspring with and without flutamide treatment, suggesting that enhanced contractile response and flutamide's reversal effect is specific to angiotensin II. These results suggest that prenatally protein-restricted rats exhibit an enhanced responsiveness to angiotensin II that is testosterone-dependent.

Influence of the antiandrogen flutamide on connexin 43 (Cx43) gene and protein expression in the porcine placenta and uterus during pregnancy.

The study focuses on the expression of connexin 43 (Cx43), a gap junctional protein in the porcine placenta and uterus. The aim was to examine Cx43 mRNA and protein expression after antiandrogen flutamide treatment. Flutamide was injected into pregnant gilts at a daily dose of 50 mg/kg body weight at different stages of pregnancy: between days 43-49 (50 dpc), 83-89 (90 dpc) and 101-107 (108 dpc) of gestation. The animals were sacrificed and tissues were collected one day after the last injection. Cx43 immunostaining was observed in epithelial and stromal cells of the fetal part of the placenta; luminal and glandular epithelial cells of maternal part of the placenta and myometrium of the uterus within placentation sites. Cx43 was also found in glandular epithelium and myometrium of non-placental uterus. Flutamide treatment caused fluctuations in Cx43 expression especially before parturition. Although significant changes in Cx43 mRNA expression were observed only in the fetal part of the placenta, Cx43 protein level was affected within the maternal part of the placenta and non-placental uterus. These results suggest the involvement of androgens in the regulation of Cx43 expression within the feto-maternal compartment in pigs. However, androgen deficiency caused pronounced changes during late pregnancy and before parturition. These results are interesting due to the functional changes in the porcine uterus during the preparturient period that is determined by Cx43 protein.

2014 AHA Late-Breaking Basic Science Abstracts

### 24178 ### MAVS Mediates Protection against Myocardial Ischemic Injury with Hydrogen Sulfide David Durrant, Adolfo G Mauro, Juan Valle Raleigh, Anindita Das, Jun He, Khoa Nguyen, Stefano Toldo, Antonio Abbate, Fadi N Salloum; Virginia Commonwealth Univ, Richmond, VA Background : Hydrogen sulfide (H2S) has been shown to protect against myocardial ischemic and inflammatory injury in part by preserving mitochondrial integrity. Since mitochondrial antiviral signaling (MAVS) protein has been implicated in attenuating Bax-mediated cytochrome c release from mitochondria caused by oxidative stress or ischemia, we sought to determine whether MAVS mediates the cardioprotective effects of H2S. Methods and Results : After baseline echocardiography, adult male wild type (WT) or MAVS KO mice underwent myocardial infarction (MI) by coronary artery ligation for 30 min. followed by 24 h reperfusion. Mice were pretreated with Na2S (100 μg/kg; ip ) or saline 1 h before MI. Infarct size, measured with TTC staining, was reduced and LV fractional shortening (FS) was preserved with Na2S at 24h post MI in WT mice as compared to saline-treated mice, but not in MAVS KO mice (Figs. A and B). The risk area was not different between the groups. Western blot analysis revealed a significant decline in myocardial MAVS expression at 24h post MI, which was preserved with Na2S (Fig. C). Another subset of mice was subjected to permanent coronary artery occlusion and treated with Na2S or saline daily for 28 days. LVFS decreased significantly at 28 days post-MI in the saline group, but was significantly preserved with Na2S (Fig. D). Moreover, LV infarct scar size, assessed by trichrome staining, was smaller in Na2S group (22.4 ± 2.7%) as compared to control (33.5 ± 2.1%, P<0.05). Survival rate was 2 fold higher with Na2S compared to saline (P<0.05). Western blot analysis confirmed a significant …

Role of testicular luminal factors on Basal cell elongation and proliferation in the mouse epididymis.

A subset of basal cells (BCs) in the initial segment (IS) of the mouse epididymis has a slender body projection between adjacent epithelial cells. We show here that these projections occasionally cross the apical tight junctions and are in contact with the luminal environment. Luminal testicular factors are critical for the establishment of the IS epithelium, and we investigated their role in the regulation of this luminal sensing property. Efferent duct ligation (EDL) was performed to block luminal flow from the testis without affecting blood flow. Cytokeratin 5 (KRT5) labeling showed a time-dependent reduction of the percentage of BCs with intercellular projections from 1 to 5 days after EDL, compared to controls. Double labeling for caspase-3 and KRT5 showed that a subset of BCs undergoes apoptosis 1 day after EDL. Ki67/KRT5 double labeling showed a low rate of BC proliferation under basal conditions. However, EDL induced a marked increase in the proliferation rate of a subset of BCs 2 days after EDL. A 2-wk treatment with the androgen receptor antagonist flutamide did not affect the number of BCs with intercellular projections, but reduced BC proliferation. Flutamide treatment also reduced the increase in BC proliferation induced 2 days after EDL. We conclude that, in the adult mouse IS, 1) luminal testicular factors play an important role in the ability of BCs to extend their body projection towards the lumen, and are essential for the survival of a subset of BCs; 2) androgens play an important role in the proliferation of some of the BCs that survive the initial insult induced by EDL; and 3) the formation and elongation of BC intercellular projections do not depend on androgens.

Anogenital distance plasticity in adulthood: implications for its use as a biomarker of fetal androgen action.

Androgen action during the fetal masculinization programming window (MPW) determines the maximum potential for growth of androgen-dependent organs (eg, seminal vesicles, prostate, penis, and perineum) and is reflected in anogenital distance (AGD). As such, determining AGD in postnatal life has potential as a lifelong easily accessible biomarker of overall androgen action during the MPW. However, whether the perineum remains androgen responsive in adulthood and thus responds plastically to perturbed androgen drive remains unexplored. To determine this, we treated adult male rats with either the antiandrogen flutamide or the estrogen diethylstilbestrol (DES) for 5 weeks, followed by a 4-week washout period of no treatment. We determined AGD and its correlate anogenital index (AGI) (AGD relative to body weight) at weekly intervals across this period and compared these with normal adult rats (male and female), castrated male rats, and appropriate vehicle controls. These data showed that, in addition to reducing circulating testosterone and seminal vesicle weight, castration significantly reduced AGD (by ∼17%), demonstrating that there is a degree of plasticity in AGD in adulthood. Flutamide treatment increased circulating testosterone yet also reduced seminal vesicle weight due to local antagonism of androgen receptor. Despite this suppression, surprisingly, flutamide treatment had no effect on AGD at any time point. In contrast, although DES treatment suppressed circulating testosterone and reduced seminal vesicle weight, it also induced a significant reduction in AGD (by ∼11%), which returned to normal 1 week after cessation of DES treatment. We conclude that AGD in adult rats exhibits a degree of plasticity, which may be mediated by modulation of local androgen/estrogen action. The implications of these findings regarding the use of AGD as a lifelong clinical biomarker of fetal androgen action are discussed.

Altered expression of 3β-HSD, CYP17 and 17β-HSD in the foetal porcine gonads in response to anti-androgen flutamide exposure.

We investigated whether the limited access to androgens during late prenatal period alters expression of steroidogenic enzymes involved in androgen production: 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD), cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17) and 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) or type 3 (17β-HSD3) in the foetal porcine gonads. Pregnant gilts were injected with anti-androgen flutamide (for seven days, 50mg/day/kg bw) or corn oil (control) starting at 83 (GD90) or 101 (GD108) gestational day. To assess 3β-HSD, CYP17 and 17β-HSD1 or 17β-HSD3 expression, real-time PCR and immunohistochemistry were performed. In testes from flutamide-treated foetuses, increased 3β-HSD and CYP17 mRNA expression was observed in the GD90 group, while decreased 3β-HSD and 17β-HSD3 mRNA expression and increased CYP17 mRNA expression were found in the GD108 group. CYP17 and 17β-HSD3 were localized in Leydig cells. Following flutamide administration, the intensity of CYP17 immunostaining was higher in both treated groups, while 17β-HSD3 intensity was lower in the GD108 group. In ovaries from flutamide-treated foetuses in the GD90 group, mRNA level for 3β-HSD was elevated, but it was diminished for CYP17 and 17β-HSD1. In the GD108 group, flutamide treatment led to lower mRNA level for 3β-HSD but higher for CYP17. 3β-HSD was found in granulosa cells, while CYP17 was localized within egg nests and oocytes of forming follicles. Following flutamide treatment, the intensity of 3β-HSD and CYP17 immunostaining was higher in the GD90 and GD108 groups, respectively. Immunohistochemical staining for 3β-HSD was restricted to the ovary. Concluding, diminished androgen action in the porcine foetal gonads during late gestation induces changes in steroidogenic enzymes expression, which may led to changes in gonadal function. However, it seems that androgens exert diverse biological effects depending on the gestational period.

Alterations in luteal production of androstenedione, testosterone, and estrone, but not estradiol, during mid- and late pregnancy in pigs: Effects of androgen deficiency

Recently, we have found that flutamide-induced androgen deficiency altered progesterone production in the porcine corpus luteum (CL) during mid- and late pregnancy. Herein, we tested whether flutamide administration subsequently influences androgen and estrogen metabolism in the CL of pregnancy. Pregnant gilts were treated with flutamide between Days 43 and 49 (GD50F), 83 and 89 (GD90F), or 101 and 107 (GD108F) of gestation. Corpora lutea (CLs) were collected from treated and nontreated (control) pigs. The concentrations of androstenedione (A4), testosterone (T), estrone (E1), and estradiol (E2) together with the levels of expression of mRNAs and proteins for cytochrome P450 17α-hydroxylase/c17-20 lyase (CYP17A1), 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), cytochrome P450 aromatase (CYP19A1), and 17β-hydroxysteroid dehydrogenase type 7 (17β-HSD7) were measured in the CL of control and flutamide-treated animals. Steroidogenic enzymes were also immunolocalized in luteal tissues. The luteal concentrations of A4 and T were higher in the GD50F (P = 0.006, P = 0.03) and GD108F (P = 0.005, P = 0.035) groups, but lower in the GD90F (P = 0.004, P = 0.014) group. The E1 level was greater only in the GD90F (P = 0.03) and GD108F (P = 0.035) groups, whereas E2 concentration was not affected by flutamide treatment. Increased luteal CYP17A1 mRNA and protein expression was found in the GD50F (P = 0.002, P = 0.03) and GD108F (P = 0.0026, P = 0.03) groups, but reduced in the GD90F (P = 0.002, P = 0.03) group. mRNA of 17β-HSD1 was upregulated in the GD50F (P = 0.0005) group, but downregulated in the GD90F (P = 0.002) and GD108F (P = 0.0005) groups. In contrast, 17β-HSD1 protein expression was higher in the GD50F and GD108F (P = 0.03) groups, but lower in the GD90F (P = 0.03) group. Both CYP19A1 mRNA and protein levels were greater in the GD90F (P = 0.001, P = 0.028) and GD108F (P = 0.005, P = 0.03) groups. Neither 17β-HSD7 mRNA nor protein level were affected by flutamide exposure. Both CYP17A1 and 17β-HSD1 were immunolocalized exclusively in small luteal cells, whereas CYP19A1 and 17β-HSD7 were found in large luteal cells of control and flutamide-treated CLs. Overall, flutamide administration led to the alterations in A4, T, and E1, but not in E2, production in the CL of pregnancy in pigs, probably because of disrupted steroidogenic enzymes expression. These changes suggest that androgens are important modulators of luteal function during pregnancy in pigs.

Androgen Deficiency During Mid- and Late Pregnancy Alters Progesterone Production and Metabolism in the Porcine Corpus Luteum

We determined whether androgen deficiency induced by flutamide treatment during mid- and late pregnancy affects the functions of the porcine corpus luteum (CL). Pregnant gilts were injected with flutamide between days 43 and 49 (gestation day [GD] 50F), days 83 and 89 (GD90F), or days 101 and 107 (GD108F) of gestation. Antiandrogen treatment increased the luteal progesterone concentration in the GD50F group and decreased progesterone content in the GD90F and GD108F groups. Luteal levels of side-chain cleavage cytochrome P450 (CYP11A1) mRNA and protein were significantly downregulated in the GD90F and GD108F groups as compared with the respective controls. The 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (HSD3B) mRNA and protein expression were significantly reduced only in the GD108F group as compared with the control. Decreased luteal 20α-hydroxysteroid dehydrogenase (AKR1C1) mRNA and protein levels were observed in the GD50F group. Thus, androgen deficiency during pregnancy in pigs led to CL dysfunction that is marked by decreased progesterone production. Furthermore, exposure to flutamide during late pregnancy downregulated steroidogenic enzymes (CYP11A1 and HSD3B) in pigs. We conclude that androgens are important regulators of CL function during pregnancy.