Testosterone-induced Increases Research Articles

Protective Effect of Panicum dichotomiflorum in a Rodent Model of Testosterone-Induced Benign Prostatic Hyperplasia

Benign prostatic hyperplasia (BPH) is a common disease in aging men. Panicum dichotomiflorum (PD) is an annual grass species of Poaceae that is distributed worldwide. The present study examined whether PD has a protective effect against BPH. BPH was generated in rats by daily subcutaneous administration of testosterone for four weeks. During this period, the rats were also given daily oral gavages of an extract of PD (150 mg/kg). After the final treatment, all animals were euthanized and their prostates were collected and weighed. In BPH model rats, the prostate weight and levels of dihydrotestosterone (DHT) and 5α-reductase expression were inhibited following treatment with PD extract. Testosterone-induced increases in prostate gland epithelial thickness and expression of cyclin D1 and proliferating cell nuclear antigen (PCNA) were markedly suppressed in PD-treated rats, whereas cleaved caspase-3 levels were increased. PD administration also decreased the expression of transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF), the phosphorylation of Akt, and inflammatory cytokines levels. Taken together, these results show that PD extract protects against testosterone-induced BPH progression by alleviating prostate cell growth and reducing levels of growth factors and inflammatory cytokines, indicating that PD extract may have potential in protecting against BPH.

Testosterone ameliorates age-related brain mitochondrial dysfunction.

Brain mitochondrial dysfunction and reduced testosterone levels are common features of aging in men. Although evidence suggests that the two phenomena are interrelated, it is unclear whether testosterone supplementation ameliorates mitochondrial dysfunction in the aging male brain. Here, we show that testosterone supplementation significantly alleviates exploratory behavioral deficits and oxidative damage in the substantia nigra and hippocampus of aging male rats. These effects were consistent with improved mitochondrial function, reflected by testosterone-induced increases in mitochondrial membrane potential (MMP), antioxidant enzyme (GSH-PX, catalase, and Mn-SOD) expression/activity, and mitochondrial respiratory complex activities in both brain regions. Furthermore, elevated PGC-1α, NRF-1, and TFAM expression (suggestive of enhanced mitochondrial biogenesis), increased citrate synthase activity, mtDNA copy number, and ND1, COX1, and ATP6 expression (indicative of increased mitochondrial content), as well as increased PINK1/Parkin and decreased P62 expression (suggesting mitophagy activation), were detected in the substantial nigra and hippocampus of aged male rats after testosterone supplementation. These findings suggest that testosterone supplementation may be a viable approach to ameliorating brain mitochondrial dysfunction and thus prevent or treat cognitive-behavioral deficits and neurodegenerative conditions associated with aging.

Testosterone administration increases social discounting in healthy males

Although testosterone is thought to induce antisocial and aggressive behavior, research on social economic interactions has associated it with prosocial and affiliative behavior. Here, we investigated the effects of testosterone on social distance-dependent generosity in an economic discounting task where participants chose between selfish and generous alternatives. We administered testosterone gel or placebo to men in a double-blind, randomized design and measured how willing they were to share rewards with close and distant others. Across two studies (total n = 174), testosterone administration consistently increased social discounting, that is participants became more selfish, particularly with regard to distant others (vs. close others). This effect was not explained by testosterone-induced increases in social distance perception. Our findings provide causal evidence that-testosterone reduces generosity in human economic decision-making. Moreover, they suggest that the valuation and the perception of social distance are independently affected by testosterone.

Novel mechanism of endocrine disruption by fungicides through binding to the membrane androgen receptor, ZIP9 (SLC39A9), and antagonizing rapid testosterone induction of the intrinsic apoptotic pathway

A variety of pesticides including vinclozolin, its metabolite M2 (3′,5′-dichloro-2-hydroxy-2-methylbut-3-enanilide), and prochloraz have been shown to exert antiandrogenic effects in animal models by competing with androgen binding to nuclear androgen receptors (nAR) and decreasing transcription of androgen-responsive genes. However, it is not known whether these pesticide antiandrogens also interfere with rapid (often described as nongenomic, nonclassical) androgen actions mediated by membrane androgen receptors (mARs). We recently discovered that ZIP9, a member of the zinc transporter ZIP (SLC39A) family, is a specific, high-affinity mAR that mediates rapid testosterone-dependent signaling, zinc influx, and apoptosis in breast and prostate cancer cell lines. Possible disruption by prochloraz, vinclozolin, and M2 of androgen actions through this mAR was investigated in vitro in PC-3 prostate cancer cells (nAR-) over expressing human ZIP9 (PC3-ZIP9 cells). Single-point competitive binding assays showed 1 μM and 10 μM concentrations of all three pesticides displaced specific [3H]-testosterone binding to PC3-ZIP9 cell membranes with binding affinities <10% that of testosterone. The pesticides also exerted antiandrogen actions through ZIP9. Co-treatments with 100 nM prochloraz, vinclozolin and M2 blocked or attenuated the 20 nM testosterone-induced increases in apoptosis, intracellular free zinc levels, and expression of the proapoptotic gene, Bax. Prochloraz also attenuated testosterone activation of MAPkinase. The finding that prochloraz, vinclozolin and M2 are effective competitors of [3H]-testosterone binding to ZIP9 and block testosterone actions mediated through ZIP9 in vitro at nanomolar concentrations suggests that androgen functions mediated by ZIP9 are also susceptible to disruption by pesticide antiandrogens with potential adverse effects on human health.

Membrane androgen receptor characteristics of human ZIP9 (SLC39A) zinc transporter in prostate cancer cells: Androgen-specific activation and involvement of an inhibitory G protein in zinc and MAP kinase signaling

Characteristics of novel human membrane androgen receptor (mAR), ZIP9 (SLC39A9), were investigated in ZIP9-transfected PC-3 cells (PC3-ZIP9). Ligand blot analysis showed plasma membrane [3H]-T binding corresponds to the position of ZIP9 on Western blots which suggests ZIP9 can bind [3H]-T alone, without a protein partner. Progesterone antagonized testosterone actions, blocking increases in zinc, Erk phosphorylation and apoptosis, further evidence that ZIP9 is specifically activated by androgens. Pre-treatment with GTPγS and pertussis toxin decreased plasma membrane [3H]-T binding and blocked testosterone-induced increases in Erk phosphorylation and intracellular zinc, indicating ZIP9 is coupled to an inhibitory G protein (Gi) that mediates both MAP kinase and zinc signaling. Testosterone treatment of nuclei and mitochondria which express ZIP9 decreased their zinc contents, suggesting ZIP9 also regulates free zinc through releasing it from these intracellular organelles. The results show ZIP9 is a specific Gi coupled-mAR mediating testosterone-induced MAP kinase and zinc signaling in PC3-ZIP9 cells.

Identification and characterization of membrane androgen receptors in the ZIP9 zinc transporter subfamily: II. Role of human ZIP9 in testosterone-induced prostate and breast cancer cell apoptosis.

Recently, we discovered a cDNA in teleost ovarian follicle cells belonging to the zinc transporter ZIP9 subfamily (SLC39A9) encoding a protein with characteristics of a membrane androgen receptor (mAR). Here, we demonstrate that human ZIP9 expressed in MDA-MB-468 breast cancer cells and stably overexpressed in human prostate cancer PC-3 cells (PC-3-ZIP9) also displays the ligand binding and signaling characteristics of a specific, high-affinity mAR. Testosterone treatment of MDA-MB-468 and PC-3-ZIP9 cells caused activation of G proteins and second messenger pathways as well as increases in intracellular free zinc concentrations that were accompanied by induction of apoptosis. [1,2,6,7-(3)H]-testosterone binding and these responses were abrogated in MDA-MB-468 cells after ZIP9 small interfering RNA (siRNA) treatment and absent in PC-3 cells transfected with empty vector, confirming that ZIP9 functions as an mAR. Testosterone treatment caused up-regulation of proapoptotic genes Bax (Bcl-2-associated X protein), p53 (tumor protein p53), and JNK (c-Jun N-terminal kinases) in both cell lines and increased expression of Bax, Caspase 3, and cytochrome C proteins. Treatment with a zinc chelator or a MAPK inhibitor blocked testosterone-induced increases in Bax, p53, and JNK mRNA expression. The results suggest that both androgen signaling and zinc transporter functions of ZIP9 mediate testosterone promotion of apoptosis. ZIP9 is widely expressed in human tissues and up-regulated in malignant breast and prostate tissues, suggesting that it is a potential therapeutic target for treating breast and prostate cancers. These results provide the first evidence for a mechanism mediated by a single protein through which steroid and zinc signaling pathways interact to regulate physiological functions in mammalian cells.

Testosterone enhances tubuloglomerular feedback by increasing superoxide production in the macula densa

Males have higher prevalence of hypertension and renal injury than females, which may be attributed in part to androgen-mediated effects on renal hemodynamics. Tubuloglomerular feedback (TGF) is an important mechanism in control of renal microcirculation. The present study examines the role of testosterone in the regulation of TGF responses. TGF was measured by micropuncture (change of stop-flow pressure, ΔPsf) in castrated Sprague-Dawley rats. The addition of testosterone (10(-7) mol/l) into the lumen increased the ΔPsf from 10.1 ± 1.2 to 12.2 ± 1.2 mmHg. To determine whether androgen receptors (AR) are involved, mRNA of AR was measured in the macula dense cells isolated by laser capture microdissection from kidneys, and a macula densa-like cell line (MMDD1). AR mRNA was expressed in the macula densa of rats and in MMDD1 cells. We next examined the effects of the AR blocker, flutamide (10(-5) mol/l) on the TGF response. The addition of flutamide blocked the effects of testosterone on TGF. The addition of Tempol (10(-4) mol/l) or polyethylene glycol-superoxide dismutase (100 U/ml) to scavenge superoxide blocked the effect of testosterone to augment TGF. We then applied apocynin to inhibit NAD(P)H oxidase and oxypurinol to inhibit xanthine oxidase and found the testosterone-induced augmentation of TGF was blocked. In additional experiments in MMDD1 cells, we found that testosterone increased O2(-) generation. Apocynin or oxypurinol blocked the testosterone-induced increases of O2(-), while blockade of COX-2 with NS-398 had no effect. These findings suggest that testosterone enhances TGF response by stimulating O2(-) production in macula densa via an AR-dependent pathway.

Testosterone and Secondary Hypertension

See related article, pp 1263–1271 The absence of estrogen, in males or after menopause in women, is independently associated with increased cardiovascular risk.1 A number of factors have been implicated as the underlying cause, including effects at the cell-organ level (eg, inflammatory activation, increased oxidative stress, and increased vasoconstriction),1 as well as changes in cardiovascular physiology (eg, sympathetic activation, increased blood pressure, and insulin resistance). All of these parameters are sensitive to modulation by sex steroid hormones,1 particularly 17β-estradiol, testosterone, and its more active metabolite, 5α-dihydrotestosterone, which is produced from testosterone by 5α-reductase.1,2 Testosterone is the main male sex steroid hormone but is also produced by women.2 Testosterone is converted to 17β-estradiol by aromatase in numerous tissues, including the vasculature and adipose tissue, representing an important local source of estrogen.3 In obesity, aromatase-derived 17β-estradiol formation may represent an important cause of feminization in males and the increased risk of estrogen-sensitive cancers in obese females.3 ### Androgenic Steroids and Cardiovascular Risk In many countries, including the United Kingdom, the United States, Canada, and Australia, anabolic androgenic steroids, such as testosterone, are controlled substances, and their nonmedical use is considered drug abuse.4,5 Testosterone has been used since the 1930s for nonmedical, athletic purposes, especially in male and female body builders and swimmers.6 Because testosterone abuse may increase arterial blood pressure,7 leading to left ventricular hypertrophy,8 it should be included among the differential diagnoses of secondary arterial hypertension. Moreover, testosterone abuse has been associated with myocardial infarction because of coronary vasospasm9 or thrombosis.10–12 Among the mechanisms of how exogenous, as opposed to endogenous, testosterone contributes to increased in cardiovascular risk, coagulatory activation, as well as accelerated progression of coronary artery disease, have been described.10 Sex steroids dilate human coronary …

Effects of exogenous testosterone on the ventral striatal BOLD response during reward anticipation in healthy women

Correlational evidence in humans shows that levels of the androgen hormone testosterone are positively related to reinforcement sensitivity and competitive drive. Structurally similar anabolic–androgenic steroids (AAS) are moreover widely abused, and animal studies show that rodents self-administer testosterone. These observations suggest that testosterone exerts activational effects on mesolimbic dopaminergic pathways involved in incentive processing and reinforcement regulation. However, there are no data on humans supporting this hypothesis. We used functional magnetic resonance imaging (fMRI) to investigate the effects of testosterone administration on neural activity in terminal regions of the mesolimbic pathway. In a placebo-controlled double-blind crossover design, 12 healthy women received a single sublingual administration of .5mg of testosterone. During MRI scanning, participants performed a monetary incentive delay task, which is known to elicit robust activation of the ventral striatum during reward anticipation. Results show a positive main effect of testosterone on the differential response in the ventral striatum to cues signaling potential reward versus nonreward. Notably, this effect interacted with levels self-reported intrinsic appetitive motivation: individuals with low intrinsic appetitive motivation exhibited larger testosterone-induced increases but had smaller differential responses after placebo. Thus, the present study lends support to the hypothesis that testosterone affects activity in terminal regions of the mesolimbic dopamine system but suggests that such effects may be specific to individuals with low intrinsic appetitive motivation. By showing a potential mechanism underlying central reinforcement of androgen use, the present findings may moreover have implications for our understanding of the pathophysiology of AAS dependency.

Effects of Environmental Antiandrogenic Chemicals on Expression of Androgen-Responsive Genes in Rat Prostate Lobes

Rat prostate, which is usually used in the Hershberger assay for evaluating the antiandrogenic activity of environmental chemicals in vivo ,h as ac omplex structure consisting 4 lobes, i.e. ,t he ventral prostate (VP), lateral prostate (LP), dorsal prostate (DP) and anterior prostate (AP). The VP is considered to have no counterpart in primates, while the LP and DP are histologically similar to human prostate. However, the Hershberger assay focuses on the VP, not the other lobes. Moreover, there are few other methods for assessment of antiandrogenic activity in vivo .W et herefore investigated androgen-responsive genes in the DP, as well as VP, following treatment with environmental chemicals reported to be androgen antagonists. Male castrated F344 rats were treated with testosterone (0.5 mg·kg −1 ·day −1 )a lone or together with flutamide (6 mg·kg −1 ·day −1 )a s ar eference antiandrogen or fenthion (25 mg·kg −1 ·day −1 )o r fenitrothion (25 mg·kg −1 ·day −1 )o r 2,4,4 � -trihydroxybenzophenone (2,4,4 � -triOHBP) (300 mg·kg −1 ·day −1 )f or 7d ays. Testosterone significantly increased the expression of kallikrein S3, cystatinrelated protein-1 (CRP-1) and prostatein C3 mRNAs in the VP, and prostate secretory protein of 94 amino acids (PSP94) mRNA, but not stem cell growth factor (SCGF) mRNA, in the DP. Coadministration of flutamide blocked the testosterone-induced increases of all three mRNAs in the VP, but not that of PSP94 mRNA in the DP. Coadministration of fenitrothion significantly reduced the testosterone-induced increase of kallikrein S3 mRNA, while fenthion significantly increased the testosterone-induced increase of PSP94 mRNA. 2,4,4 � -TriOH-BP significantly increased the testosterone-induced increases of CRP-1 and prostatein C3 mRNAs. These results indicate that the effects of environmental chemicals on the prostate are very complex. The Hershberger assay alone appears to be inadequate for risk assessment, and it may be useful to employ androgen-responsive genes as additional markers.

PKCδ Mediates Testosterone-induced Increases in Coronary Smooth Muscle Cav1.2

Sex hormones have emerged as important modulators of cardiovascular physiology and pathophysiology. Our previous studies demonstrated that testosterone increases expression and activity of L-type, voltage-gated calcium channels (Cav1.2) in coronary arteries of males. The purpose of the present study was to determine whether testosterone (T) alters coronary protein kinase C delta (PKCdelta) expression and whether PKCdelta plays a role in coronary Cav1.2 expression. For in vitro studies, porcine right coronary arteries (RCA) and post-confluent (passages 3-6) 5-day, serum-restricted coronary smooth muscle cell cultures (CSMC) were incubated in the presence and absence of T or dihydrotestosterone (10 and 100 nm) for 18 h at 37 degrees C in a humidified chamber. For sex and endogenous testosterone-dependent effects, RCA were obtained from intact males, castrated males, castrated males with T replacement, and intact females. In vitro T and dihydrotestosterone caused an approximately 2-3-fold increase in PKCdelta protein levels, approximately 1.5-2-fold increase in PKCdelta kinase activity, and localization of PKCdelta toward the plasma membrane and nuclear envelope. PKCdelta protein levels were higher in coronary arteries of intact males compared with intact females. Elimination of endogenous testosterone by castration reduced RCA PKCdelta protein levels, an effect partially (approximately 45%) reversed by exogenous T (castrated males with T replacement). In CSMC, PKC inhibition with either the general PKC inhibitor, cheylerythrine, or the putative PKCdelta inhibitor, rottlerin, completely inhibited the T-mediated increase in coronary Cav1.2 protein levels. Conversely, Go6976, a conventional PKC isoform inhibitor, failed to inhibit T-induced increases in coronary Cav1.2 protein levels. PKCdelta short interference RNA completely blocked T-induced increases in Cav1.2 protein levels in CSMC. These results demonstrate for the first time that 1) endogenous T is a primary modulator of coronary PKCdelta protein and activity in males and 2) T increases Cav1.2 protein expression in a PKCdelta-dependent manner.

Regulation of caltrin mRNA expression by androgens in the murine prostate.

Testicular androgens induce the proliferation and differentiation of prostatic epithelial cells by regulating the expression of androgen target genes. The use of subtractive hybridization to isolate genes that are differentially expressed during the early phase of androgen-induced prostatic regrowth in castrated mice resulted in identification of the murine caltrin gene. Caltrin messenger RNA (mRNA) was highly expressed in the prostates of intact mice. Five weeks following castration of mice, steady state caltrin mRNA levels were reduced by 70%. Within 12 hours of administration of pharmacological doses of testosterone enanthate, steady state caltrin mRNA levels were elevated and increased to 90% of levels found in intact mice by 24 hours. Reverse transcriptase-polymerase chain reaction analysis of prostate tissue localized caltrin mRNA transcripts to the dorsal but not the ventral or lateral prostate. Within the dorsal prostate, in situ hybridization always localized caltrin mRNAs to the prostatic epithelial cells. Testosterone-induced increases in caltrin mRNA levels were detected prior to S-phase progression and initiation of proliferation in this cell population. Caltrin has been demonstrated previously to function as a calcium transport inhibitor at the plasma membrane. Findings of this study indicate that caltrin is highly expressed and androgen-regulated in the murine prostate, where it is associated with androgen-induced proliferation and differentiation of epithelial cells.

The Selective Estrogen Receptor Modulator, Raloxifene: An Overview of Nonclinical Pharmacology and Reproductive and Developmental Testing

Raloxifene is a nonsteroidal, selective estrogen receptor modulator being developed by Eli Lilly and Company as a therapeutic agent for postmenopausal osteoporosis. In the ovariectomized (OVX) rat, raloxifene prevents the loss of bone at the distal metaphysis of the femur, proximal tibia, and vertebrae; reduces cancellous bone resorption; and reduces serum cholesterol, but does not cause any significant changes in stromal eosinophilia or uterine epithelium. In estrogen-stimulated OVX rats, raloxifene prevents the morning lowering of serum luteinizing hormone levels, produces a reduction in afternoon serum prolactin levels, antagonizes pituitary weight increase, and antagonizes stimulation of mammary gland development. Raloxifene also has been shown to exhibit antiestrogenic activity in several in vivo and in vitro mammary tumor models. Raloxifene treatment results in regression of endometriosis in both a surgically prepared, rat uterine explant model and in Rhesus macaques diagnosed with spontaneous endometriosis before exposure. Also, uterine leiomyomas in estrogen-stimulated OVX guinea pigs regress after the onset of raloxifene treatment. Raloxifene antagonizes testosterone-induced increases in prostate weight of castrated rats, but does not bind to androgen receptors or affect prostatic 5-α-reductase or testicular steroid 17-α-hydroxylase activity. A series of preclinical toxicology studies was designed to characterize reproductive and developmental outcomes following various schedules of raloxifene treatment in rats or rabbits. Studies of female reproduction and developmental outcome were conducted primarily at pharmacologic doses (0.1, 1, or 10 mg/kg/d); male reproductive studies used higher doses (10, 30, or 100 mg/kg/d). In this series of studies, male reproductive end points were not affected, whereas embryo implantation, fetal rabbit morphology, and several aspects of offspring development were disrupted by the lowest dose of maternal raloxifene treatment, a profile consistent with estrogen antagonist activity.

Effects of steroidal and non-steroidal antiandrogens on the androgen binding properties of the rat ventral prostate androgen receptor

Steroidal (cyproterone acetate) and non-steroidal (RU23908 and hydroxyflutamide) antiandrogens are able to block testosterone-induced increases in nuclear androgen receptor (AR) in the prostate of 1-day orchidectomized rats, but when given alone, RU23908 and hydroxyflutamide increase nuclear AR (RU23908 > hydroxyflutamide) in the same animal model. The increases in nuclear AR induced by antiandrogen alone or with testosterone alone are blocked by cycloheximide 1 h after administration, suggesting that androgen or antiandrogens induce de novo AR synthesis. Concomitant to nuclear AR accumulation, testosterone is able to induce depletion of cytosol and microsomal AR. Blockade of testosterone-induced depletion of microsomal AR, but not of cytosol AR, occurs in the presence of antiandrogens. Cyproterone acetate has a higher relative binding affinity (RBA) for microsomal AR and cytosol AR than RU23908 or hydroxyflutamide. This phenomenon is in good agreement with the degree of inhibition by these compounds of the association rate of androgen for the microsomal AR. This correlation between RBA and inhibition of the initial rate of hormone binding to the receptor is not found for cytosol AR. The results show that antiandrogens are not ‘pure’ antagonists of androgen action and they are potent agonists in the absence of testosterone. Furthermore, testosterone alone or antiandrogens per se regulate AR levels acutely by protein-synthesis dependent mechanisms of action, in rat ventral prostate.

Metabolic control mechanisms in mammalian systems. Hormonal regulation of phosphofructokinase in the rat prostate and seminal vesicles.

1. The hormonal regulation of phosphofructokinase was investigated in the accessory reproductive organs of the orchidectomized rat. 2. Phosphofructokinase activities declined to 51% and 47% in the prostate and 9% and 6% of the normal values in seminal vesicles 4 and 8 weeks after castration respectively. Administration of testosterone (100mug./100g. body wt.) for 3 days reversed substantially the effects of orchidectomy, and phosphofructokinase activity increased to 173% in the prostate and 536% in seminal vesicles as compared with the values of castrated controls. 3. Time-course studies demonstrated that after a single injection of testosterone (5mg./100g. body wt.) phosphofructokinase activity was maximally elevated to 236% in the prostate and 342% in seminal vesicles at 24hr. 4. Dose-response studies revealed that 2.5mg. of testosterone propionate/100g. body wt. was the minimal amount necessary to induce significant increases in enzyme activity in both accessory sex organs; maximal increases were obtained with a dose of 5mg./100g. body wt. 5. The observed enzyme increases induced by testosterone were inhibited by the simultaneous administration of oestradiol-17beta, and phosphofructokinase activity in this group of rats remained at 97% in the prostate and 137% of the control values in seminal vesicles. Oestradiol-17beta by itself failed to produce any significant effect on enzyme activity in either of these secondary sexual tissues. 6. The nature of the testosterone-induced increases in phosphofructokinase activity was studied by using a variety of inhibitors of RNA and protein synthesis. Cycloheximide, 5-fluorouracil and ethionine largely blocked the androgen-stimulated rise in enzyme activity observed 24hr. after steroid injection. The inhibitory effect of ethionine was completely reversed by the simultaneous administration of methionine. 7. Actinomycin, which is known to inhibit the synthesis of messenger RNA as well as the synthesis of other cellular RNA fractions, when given simultaneously with the hormone, also inhibited the testosterone-induced increases in prostatic and seminal-vesicular phosphofructokinase. However, when the antibiotic was given 6 or 12hr. after injection of the steroid, practically no inhibition of phosphofructokinase induction was obtained. This indicates that, once the enzyme-forming machinery is turned on and allowed to operate for a few hours, actinomycin is incapable of reversing the hormone-induced enzyme responses. 8. The results presented suggest that new RNA and protein synthesis may be involved in the observed androgen-induced increases in phosphofructokinase activity in the prostate and seminal vesicles of the orchidectomized rat.