Kolaviron modulates angiogenesis, apoptosis and inflammatory signaling in rat model of testosterone propionate-induced benign prostate hyperplasia.

Benign prostatic hyperplasia (BPH) is a non-malignant disease of the prostate characterized by uncontrolled proliferation of the prostate gland. Inflammation and oxidative stress have been reported to play a role in the development of BPH. Kolaviron, a bioflavonoid complex of Garcinia kola seed, has been shown to possess anti-inflammatory effect. In this study, we investigated the effect of Kolaviron on testosterone propionate (TP)-induced BPH in rats. Fifty male rats were assigned in 5 groups. Groups 1 and 2 were orally exposed to corn oil (2 ml/kg) and Kolaviron (200 mg/kg/day, p.o) for 28 days. Group 3 rats received TP (3 mg/kg/day, s.c) for 14 days while Groups 4 and 6 were treated with Kolaviron (200 mg/kg/day, p.o) and Finasteride (5 mg/kg/day, p.o), respectively, for 14 days prior to TP (3 mg/kg, s.c) co-exposure for the remaining 14 days. Administration of Kolaviron to TP-treated rats reverted histological alteration and significantly decreased prostate weight, prostate index, 5α-reductase, dihydrotestosterone, androgen receptor expression, tumor necrosis factor α, interleukin-1ß, cyclooxygenase-2, prostaglandin E2, 5-lipoxygenase leukotriene B4, inducible nitric oxide synthase and nitric oxide concentration. In addition, Kolaviron alleviated TP-induced oxidative stress and reduced the expression of Ki-67, VEGF, and FGF to almost control levels. Furthermore, Kolaviron promoted apoptosis in TP-treated rats through downregulation of BCL-2 and upregulation of P53 and Caspase 3 expressions. Overall, Kolaviron prevented BPH via regulation of androgen/androgen receptor signaling, anti-oxidative and anti-inflammatory mechanisms.

A metabolomics study of Qianliexin capsule treatment of benign prostatic hyperplasia induced by testosterone propionate in the rat model.

A metabolomics investigation of the treatment effect of Qianliexin (QLX) capsules was conducted on rats with benign prostatic hyperplasia (BPH) induced by testosterone propionate. Establishment of the BPH model was confirmed using the prostatic index. Hematoxylin and eosin (HE) staining for TGF-ß, EGFR, collagen, IL-1 ß, TNF-α was performed and changes in urine volume were measured. Urine and serum samples were collected from three groups, including a control group, a BPH model group and a QLX-treated group and subjected to metabolomics profiling based on ultrahigh-performance liquid chromatography-mass spectrometry. Pharmacodynamics analysis showed that the QLX group had significantly lower histopathological damage, fibrosis damage, and inflammation and higher urine output compared with the model group. Twenty-two potential biomarkers were identified in urine samples and 23 metabolites were identified in plasma samples. Alterations in metabolic patterns were evident in all sample types. The treatment effects of QLX appear to involve various metabolic pathways including lipid metabolism, fatty acid metabolism and purine generation and significantly reduced the pathological symptoms and related biochemical indicators of BPH and improved the level of potential marker metabolites. This comprehensive study suggested that differential markers provided insights into the metabolic pathways involved in BPH and the treatment effects of QLX.

Androgenic regulation of beta-defensins in the mouse epididymis.

BACKGROUND: The majority of beta-defensin family members are exclusively expressed in the epididymis, and some members have been shown to play essential roles in sperm maturation and fertility in rats, mice and humans. Therefore, beta-defensins are hypothesized to be potential targets for contraception and infertility diagnosis and treatment. Clarifying the regulatory mechanisms for the expression of these genes is necessary. Androgen/androgen receptor (AR) signaling plays an important regulatory role in epididymal structure and function. However, very little is known about the androgenic regulation on the production and secretion of the epididymal beta-defensins. METHODS: The expression of beta-defensins was detected by quantitative RT-PCR. The androgen dependence of beta-defensins was determined by bilateral orchiectomy and androgen supplementation. The androgen response elements (AREs) in the promoters of beta-defensins were identified using the MatInspector software. The binding of AR to AREs was assayed by ChIP-PCR/qPCR. RESULTS: We demonstrated that 23 mouse caput epididymal beta-defensins were differentially regulated by androgen/androgen receptor. Six genes, Defb18, 19, 20, 39, 41, and 42, showed full regulation by androgens. Ten genes, Defb15, 30, 34, 37, 40, 45, 51, 52, 22 and Spag11a, were partially regulated by androgens. Defb15, 18, 19, 20, 30, 34, 37, 39, 41, 42, 22 and Spag11a were associated with androgen receptor binding sites in their promoter or intronic regions, indicating direct regulation of AR. Six genes, Defb1, 12, 13, 29, 35, and spag11b/c, exhibited an androgen-independent expression pattern. One gene, Defb25, was highly dependent on testicular factors rather on androgens. CONCLUSIONS: The present study provides novel insights into the mechanisms of androgen regulation on epididymal beta-defensins, enabling a better understanding of the function of beta-defensins in sperm maturation and fertility.

Testosterone Propionate Promotes Proliferation and Viability of Bone Marrow Mesenchymal Stem Cells while Preserving Their Characteristics and Inducing Their Anti-Cancer Efficacy

Background: Various studies have reported the effects of testosterone on different cell types, yet bone marrow-derived mesenchymal stem cells' cellular responses to testosterone remain unknown. Aims: To investigate the effects of testosterone propionate, an oil-soluble short-acting form of testosterone, on human bone marrow-derived mesenchymal stem cells' proliferation and viability after 24 hours of incubation. We also investigated the impact of testosterone propionate on bone marrow-derived mesenchymal stem cell's polarization and cytotoxicity on K562 leukemia cell line. Study Design: In vitro study. Methods: We expanded commercially available bone marrow derived mesenchymal stem cells in vitro and treated them with testosterone propionate at concentrations ranging from 10-6-10-10 M for 24 hours. Ideal concentration was determined by evaluating cellular viability and proliferation with Annexin V/Propidium Iodide assay and carboxyfluorescein succinimidyl ester staining. The characteristic features of bone marrow-derived mesenchymal stem cells were evaluated by immunophenotyping and investigating their differentiation capacities. Bone marrow-derived mesenchymal stem cells' cytotoxic properties upon testosterone propionate treatment were determined by co-culturing the cells with K562 cells and with confocal imaging investigating polarization. Results: Testosterone propionate promoted proliferation and maintained the viability of bone marrow-derived mesenchymal stem at 10-8 M concentration. Further evaluations were conducted with the determined dose. The results showed that, apart from promoting mesenchymal stem cells' polarization and increasing their cytotoxicity on K562 cells, testosterone propionate did not alter differentiation capacities of bone marrow-derived mesenchymal stem cells and certain cell surface markers, but led to a significant increase in HLA-DR expression. Conclusion: The findings reveal that testosterone propionate promotes the proliferation and survival of bone marrow-derived mesenchymal stem cells in a dose-dependent manner without hampering their differentiation capacities, induces their polarization to the pro-inflammatory phenotype, and increases their cytotoxicity on the K562 cell line.

Masculinization of the distal tubular and external genitalia in female sheep with prenatal androgen exposure.

Prenatal exposure to endogenous or exogenous androgens alters the development of the female reproductive tract. Although lesions in ovaries and external genitalia of androgenized female sheep have been reported, lesions of the tubular genitalia have not. Testosterone propionate (TP) or dihydrotestosterone (DHT) was administered by intramuscular injection twice weekly to 32 ewes from 30 to 90 days of pregnancy. The ewes lambed normally. The reproductive tracts from 24 treated and 13 control postpubertal female offspring were examined at 10 months of age. The ovaries, oviducts, and uteri were grossly and histologically normal in both TP- and DHT-exposed sheep. However, in the DHT-treated sheep, the uterus connected to a misshapen, saccular vagina that opened into the urethra; in the TP-treated sheep, it ended in a blind sac. In both TP- and DHT-treated sheep, the urethra was approximately 5 times longer than that of control sheep, and it resembled a male urethra with bilateral male accessory genital glands. The urethra terminated in a fully developed penis in both TP- and DHT-treated sheep, and a scrotal sac was present (without testes). These results show that prenatal exposure of female sheep to exogenous androgens results in masculinization of the tubular and external genitalia.

Developmental programming: impact of excess prenatal testosterone on intrauterine fetal endocrine milieu and growth in sheep.

Prenatal testosterone excess in sheep leads to reproductive and metabolic disruptions that mimic those seen in women with polycystic ovary syndrome. Comparison of prenatal testosterone-treated sheep with prenatal dihydrotestosterone-treated sheep suggests facilitation of defects by androgenic as well as androgen-independent effects of testosterone. We hypothesized that the disruptive impact of prenatal testosterone on adult pathology may partially depend on its conversion to estrogen and consequent changes in maternal and fetal endocrine environments. Pregnant Suffolk sheep were administered either cottonseed oil (control) or testosterone propionate in cottonseed oil (100 mg, i.m. twice weekly), from Day 30 to Day 90 of gestation (term is ~147 d). Maternal (uterine) and fetal (umbilical) arterial samples were collected at Days 64-66, 87-90, and 139-140 (range; referred to as D65, D90, and D140, respectively) of gestation. Concentrations of gonadal and metabolic hormones, as well as differentiation factors, were measured using liquid chromatography/mass spectrometer, radioimmunoassay, or ELISA. Findings indicate that testosterone treatment produced maternal and fetal testosterone levels comparable to adult males and D65 control male fetuses, respectively. Testosterone treatment increased fetal estradiol and estrone levels during the treatment period in both sexes, supportive of placental aromatization of testosterone. These steroidal changes were followed by a reduction in maternal estradiol levels at term, a reduction in activin A availability, and induction of intrauterine growth restriction in D140 female fetuses. Overall, our findings provide the first direct evidence in support of the potential for both androgenic as well as estrogenic contribution in the development of adult reproductive and metabolic pathology in prenatal testosterone-treated sheep.

Androgenic profile and genotoxicity evaluation of testosterone propionate and novel synthesized heterocyclic steroids.

In this study, we tested the androgenic activity of three structurally promising novel synthesized heterocyclic steroids compared with testosterone propionate in male mice. Additionally, the possible genotoxic effects of the novel synthesized heterocyclic steroids in comparison with testosterone propionate on male mice using chromosomal analysis of somatic and germ cells as well as RAPD-PCR were investigated. Male mice were administered with two doses of testosterone propionate, pyridoandrostene derivative 4b, pyrimidinoandrostene derivative 9a and thienoandrostene derivative 12 (200 and 400mg/kg b.w.) daily for 2 weeks. Results indicated that compounds 4b and 12 have androgenic activity as well as testosterone propionate. There were no significant differences in the frequencies of total chromosomal aberrations in both somatic and germ cells as well as no alteration in the DNA bands patterns between control, testosterone propionate and pyridoandrostene 4b treated animals. However, the pyrimidinoandrostene derivative 9a caused significant increase in the mean value of total chromosomal aberrations of both somatic and germ cells (P< or =0.01) as well as enhanced the polymorphic bands patterns as compared to the control and the other tested compounds. On the other hand, thienoandrostene derivative 12 induced significant decrease in the mean values of chromosomal aberrations in both somatic and germ cells, decreased sperm morphological abnormalities, increased the sperm count and motility than control. Our data indicate that testosterone propionate; pyridoandrostene 4b and thienoandrostene derivative 12 have no genotoxic activity. However, pyrimidinoandrostene derivative 9a has genotoxic activity possibly due to a modulation of the different expression of the catalyzing enzyme systems which will be investigated in the nearly future.

Transformations of steroid esters by Fusarium culmorum.

The course of transformations of the pharmacological steroids: testosterone propionate, 4-chlorotestosterone acetate, 17beta-estradiol diacetate and their parent alcohols in Fusarium culmorum AM282 culture was compared. The results show that this microorganism is capable of regioselective hydrolysis of ester bonds. Only 4-ene-3-oxo steroid esters were hydrolyzed at C-17. 17beta-Estradiol diacetate underwent regioselective hydrolysis at C-3 and as a result, estrone--the main metabolite of estradiol--was absent in the reaction mixture. The alcohols resulting from the hydrolysis underwent oxidation at C-17 and hydroxylation. The same products (6beta- and 15alpha-hydroxy derivatives) as from testosterone were formed by transformation of testosterone propionate, but the quantitative composition of the mixtures obtained after transformations of both substrates showed differences. The 15alpha-hydroxy derivatives were obtained from the ester in considerably higher yield than from the parent alcohol. The presence of the chlorine atom at C-4 markedly reduced 17beta-saponification in 4-chlorotestosterone acetate. Only 3beta,15alpha-dihydroxy-4alpha-chloro-5alpha-androstan-17-one (the main product of transformation of 4-chlorotestosterone) was identified in the reaction mixture. 6beta-Hydroxy-4-chloroandrostenedione, which was formed from 4-chlorotestosterone, was not detected in the extract obtained after conversion of its ester.

Dose-dependent regulation of steroid receptor coactivator-1 and steroid receptors by testosterone propionate in the hippocampus of adult male mice.

Androgens have been proposed to play important roles in the regulation of hippocampus function either directly, through the androgen receptor (AR), or indirectly, through estrogen receptors (ERs), after aromatization into estradiol. Steroid receptor coactivator-1 (SRC-1) is present in the hippocampus of several species, and its expression is regulated by development and aging, as well as by orchidectomy and aromatase inhibitor letrozole administration, while ovariectomy only transiently downregulated hippocampal SRC-1. However, whether the expression of hippocampal SRC-1 can be directly regulated by testosterone, the principal male sex hormone, remains unclear. In the present study, we investigated the expression of hippocampal SRC-1 after orchidectomy and testosterone treatment using immunohistochemistry and Western blot analysis. We found that while hippocampal SRC-1 was significantly downregulated by orchidectomy (ORX), its expression was rescued by treatment with testosterone in a dose-dependent manner. Furthermore, we noticed that the decreased expression of hippocampal AR, ERs and the synaptic proteins GluR-1 and PSD-95 induced by ORX was also rescued by testosterone treatment in a dose-dependent manner. However, we found that hippocampal membrane estrogen receptor GPR30 and dendritic spine marker spinophilin were not altered by ORX or testosterone treatment. Together, the above results provided the first direct evidence for the androgenic regulation on hippocampal SRC-1, indicating that SRC-1 may be a direct target of androgenic regulation on the hippocampus. Furthermore, because AR and ERs can be differentially regulated by testosterone, and the transcriptional activity requires the involvement of local SRC-1, and considering the complicated regulatory pathway of each individual receptor, the converged hub regulator SRC-1 of these nuclear receptor networks is worthy of further investigation.

Evaluation of estrogenic and androgenic activity of butylated hydroxyanisole in immature female and castrated rats.

We evaluated the estrogenic and androgenic activity of butylated hydroxyanisole (BHA) using immature rat uterotrophic assay and Hershberger assay. To investigate (anti-) estrogenicity, BHA alone or with 17beta-estradiol was administered to 20-days-old immature female rats for three consecutive days. Absolute and relative uterine weights were significantly decreased by BHA (50, 100, 250, 500 mg/kg) alone and 17beta-estradiol-stimulated weights of uterine and vagina were also decreased by BHA (500 mg/kg), while uterine epithelial cell height was not affected. In Hershberger assay, BHA alone or with testosterone propionate (TP) was administered daily to 51-days-old castrated male rats for 10 days. BHA alone or with testosterone propionate (TP) caused no significant effect on androgen-dependent accessory sex organ weights; seminal vesicle/coagulative glands, glans penis, Cowper's gland, ventral prostate gland and levator ani plus bulbocarvernosus muscle. Although, the relative weight of ventral prostate gland was increased by the co-treatment of BHA 250 mg/kg with TP 0.4 mg/kg compared to that of TP alone, the relative and absolute weights of other androgen-dependent organs and absolute and formalin-fixed ventral prostate gland weight showed no changes. Our studies suggest that BHA have anti-estrogenic activity for the decrease of uterine weight in immature female rat but have negligible effect on the androgenic activity in castrated male rats.

Interactive effects of vinclozolin and testosterone propionate on pregnancy and sexual differentiation of the male and female SD rat.

In mammals, androgens are essential in directing mammalian sexual differentiation of the male phenotype. Administration of testosterone during this period alters female development in a male-like direction, whereas exposure to an androgen receptor antagonist like vinclozolin (V) demasculinizes and feminizes the male offspring. In the current study, we administered V (gavage at 200 mg/kg/day) and/or testosterone propionate (TP, sc, at 1 mg/rat/day), alone and in combination to Sprague-Dawley (SD) rats on days 14 through 19 of pregnancy, to determine if V would antagonize the effects of TP in the female and, conversely, if TP would antagonize the effects of V in the male offspring. These doses of TP and V were selected because they significantly alter sexual differentiation in the majority of female and male rat offspring, respectively, without producing severe toxicity in the dam or offspring. The study design is a 2 x 2 factorial (7 dams per group) including vehicle control, V, TP, and V + TP groups. As expected, individually, both V and TP reduced maternal weight gain and the V + TP group was affected in a cumulative fashion. Litter size on postnatal day (PND) 2 was reduced only by V + TP, whereas pup body weight was reduced in all three treated groups, the effect of V + TP again being cumulative. In female offspring, TP-induced alterations (i.e., increased anogenital distance [AGD] and fewer nipples, vaginal agenesis, hydrometrocolpos, induced prostate and bulbourethral glands, and levator ani muscle tissues) were all reversed by coadministration of V. In male offspring, V-induced alterations were only modestly antagonized by TP. At the dosage levels used herein, V + TP-treated male offspring had less well-developed nipples as infants and adults and a lower incidence of ectopic testis than did the V group. However, V-induced changes in reproductive organ weights, AGD, atrophic testes, vaginal pouch, and agenesis of the sex accessory tissues were not antagonized by concurrent TP treatment in male offspring. We observed that the combination of V and TP, two chemicals with opposing endocrine action, antagonized one another during sexual differentiation, especially in the female offspring and induced cumulative effects on maternal and neonatal toxicity. We suspect that antagonism of V by TP would be enhanced in the male if lower dose levels of V were used, but then the antagonism of TP by V in the female would likely be attenuated.

Atomistic description of the solubilisation of testosterone propionate in a sodium dodecyl sulfate micelle.

Large-scale molecular dynamics simulations were used to study the structural and dynamic properties of the solubilization process of testosterone propionate (TSTP) within sodium dodecyl sulfate (SDS) micelles. We observed that the TSTP spontaneously adsorb onto the SDS micelles and preferentially reside among the polar head groups of the SDS molecules. We found that the SDS micelle is slightly aspherical in size and has a surface area of ∼170 Å(2)/molecule, while the SDS+TSTP micelle is more aspherical and has a surface area of ∼156 Å(2)/molecule. The structural properties of the interior of the SDS micelle and the hydration of the SDS headgroup are largely undisturbed by the presence of the TSTP. However, there seems to be a correlation between the location of the TSTP molecules and the location of Na(+) counterions on the surface of the SDS micelle. Additionally, we also observe that the TSTP molecules diffuse on the surface of the SDS micelle and try to organize themselves such that they are approximately equidistant from one another.

Gonadectomy and hormone replacement exert region- and enzyme isoform-specific effects on monoamine oxidase and catechol-O-methyltransferase activity in prefrontal cortex and neostriatum of adult male rats.

Sex differences and gonadal hormone influences are well known for diverse aspects of forebrain amine and indolamine neurotransmitter systems, the cognitive and affective functions they govern and their malfunction in mental illness. This study explored whether hormone regulation/dysregulation of these systems could be related to gonadal steroid effects on catechol-O-methyltransferase and monoamine oxidase which are principal enzymatic controllers of forebrain dopamine, serotonin and norepinephrine levels. Driven by male over female differences in cortical enzyme activities, by male-specific associations between monoamine oxidase and catechol-O-methyltransferase gene polymorphisms and cognitive and dysfunction in disease and by male-specific consequences of gene knockouts in mice, the question of hormone sensitivity was addressed here using a male rat model where prefrontal dopamine levels and related behaviors are also known to be affected. Specifically, quantitative O-methylation and oxidative deamination assays were used to compare the activities of catechol-O-methyltransferase's soluble and membrane-bound isoforms and of monoamine oxidase's A and B isoforms in the pregenual medial prefrontal cortex and dorsal striatum of male rats that were sham operated, gonadectomized or gonadectomized and supplemented with testosterone propionate or with estradiol for 28 days. These studies revealed significant effects of hormone replacement but not gonadectomy on the soluble but not the membrane-bound isorfom of catechol-O-methyltransferase in both striatum and cortex. A significant, cortex-specific testosterone-but not estradiol-attenuated effect (increase) of gonadectomy on monoamine oxidase's A but not B isoform was also observed. Although none of these actions suggest potential roles in the regulation/dysregulation of prefrontal dopamine, the suppressive effects of testosterone on cortical monoamine oxidase-A that were observed could have bearing on the increased incidence of cognitive deficits and symptoms of depression and anxiety that are repeatedly observed in males in conditions of hypogonadalism related to aging, other biological factors or in prostate cancer where androgen deprivation is used as a neoadjuvant treatment.

Parametrial adipose tissue and metabolic dysfunctions induced by fructose-rich diet in normal and neonatal-androgenized adult female rats.

Hyperandrogenemia predisposes an organism toward developing impaired insulin sensitivity. The aim of our study was to evaluate endocrine and metabolic effects during early allostasis induced by a fructose-rich diet (FRD) in normal (control; CT) and neonatal-androgenized (testosterone propionate; TP) female adult rats. CT and TP rats were fed either a normal diet (ND) or an FRD for 3 weeks immediately before the day of study, which was at age 100 days. Energy intake, body weight (BW), parametrial (PM) fat characteristics, and endocrine/metabolic biomarkers were then evaluated. Daily energy intake was similar in CT and TP rats regardless of the differences in diet. When compared with CT-ND rats, the TP-ND rats were heavier, had larger PM fat, and were characterized by basal hypoadiponectinemia and enhanced plasma levels of non-esterified fatty acid (NEFA), plasminogen activator inhibitor-1 (PAI-1), and leptin. FRD-fed CT rats, when compared with CT-ND rats, had high plasma levels of NEFA, triglyceride (TG), PAI-1, leptin, and adiponectin. The TP-FRD rats, when compared with TP-ND rats, displayed enhanced leptinemia and triglyceridemia, and were hyperinsulinemic, with glucose intolerance. The PM fat taken from TP rats displayed increase in the size of adipocytes, decrease in adiponectin (protein/gene), and a greater abundance of the leptin gene. PM adipocyte response to insulin was impaired in CT-FRD, TP-ND, and TP-FRD rats. A very short duration of isocaloric FRD intake in TP rats induced severe metabolic dysfunction at the reproductive age. Our study supports the hypothesis that the early-androgenized female rat phenotype is highly susceptible to developing endocrine/metabolic dysfunction. In turn, these abnormalities enhance the risk of metabolic syndrome, obesity, type 2 diabetes, and cardiovascular disease.

Organizational influence of the postnatal testosterone surge on the circadian rhythm of core body temperature of adult male rats.

The suprachiasmatic nucleus (SCN) of the hypothalamus coordinates physiological and behavioral circadian rhythms such as activity, body temperature, and hormone secretion. Circadian rhythms coordinated by the SCN often show sex differences arising from both organizational and activational effects of gonadal hormones. In males, little is known about the organizational role of testosterone on the circadian regulation of core body temperature (CBT) in adulthood. To explore this, we castrated or sham-operated male rats on the day of birth, and at 4 months of age, implanted them with transmitters that measured CBT rhythms under a 12:12 light/dark cycle. This study revealed a significantly earlier rise in CBT during the light phase in neonatally castrated males. Subsequently, we found that treating neonatally castrated males with testosterone propionate (TP) in adulthood did not reverse the effect of neonatal castration, thus indicating an organizational role for testosterone. In contrast, a single injection of TP at the time of neonatal surgery, to mimic the postnatal surge of testosterone, coupled with TP treatment in adulthood, normalized the circadian rise in CBT. In a final study we examined CBT circadian rhythms in intact adult male and female rats and detected no differences in the rise of CBT during the light phase, although there was a greater overall elevation in female CBT. Together, results of these studies reveal an early organizational role of testosterone in males on the timing of the circadian rise of CBT, a difference that does not appear to reflect "defeminization".