Androgen-Dependent Differences in the Amounts of CYP mRNAs in the Pig Kidney.

We previously reported androgen-dependent sex and breed differences in the amounts of mRNAs of CYP isoforms in the pig liver. To clarify whether there are such sex and breed differences in the kidney, we examined the amounts of several CYP mRNAs in the kidney using both sexes of 5-month-old Landrace, Meishan and/or their crossbred F1 (LM and ML) pigs. Significant sex differences in the amounts of several CYP mRNAs were found: male < female for CYP2A19 and CYP3A29; and male > female for CYP4A24/25 in all the breeds. Sex differences in the amount of CYP2B22 mRNA (male < female) and in CYP2C33 and CYP2C49 mRNAs (male > female) were also observed in all the breeds except Landrace pigs. Furthermore, a significant sex difference (male < female) in CYP3A46 mRNA was only found in LM and ML pigs. No significant sex differences were found in either Landrace or Meishan pigs for CYP1A1, CYP1A2 and CYP4B1 mRNAs. The amounts of CYP2C33 and CYP4A24/25 mRNAs in males were higher in Meishan pigs than in Landrace pigs. Additional experiments using pigs treated by castration and/or testosterone propionate indicated that sex and breed differences in the amounts of those CYP mRNAs were, at least in part, dependent on the levels of serum testosterone. Furthermore, the effects of androgen on the amounts of CYP mRNAs in the kidney did not necessarily correlate with those in the liver, suggesting that there is a tissue-selective factor responsible for the androgen-related expression of CYP genes.

Determination of Steroids in Bovine Serum: Validation of a Reliable LC-MS/MS Method and In Vivo Studies with Boldenone Undecylenate and Testosterone Propionate.

Serum analysis has received much attention in regulatory analysis of food-producing animals, especially for anabolic steroids. The possibility of confirming the parent drugs with minimum metabolization enables the detection of intact steroid esters, whose identification represents unequivocal proof of drug administration. This work involved the development and validation of a quantitative LC-MS/MS method to determine 30 steroids and steroid esters in bovine serum. Sensitivity was improved using microwave-assisted chemical derivatization with methoxyamine hydrochloride. The validation was successfully conducted in accordance with the Decision 657/2002/EC guidelines. An in vivo experiment was performed on 12 crossbred steers in which two commercial formulations containing boldenone undecylenate and testosterone propionate were administrated via intramuscular injections. The samples were collected over a period of 120 days, in which both intact esters were identified within 11 days postadministration. 17ß-Boldenone was observed after 92 days for 2 steers and 56 days for the other animals. The applicability of a cut-off level to the ratio between 17ß-testosterone and epitestosterone was evaluated in an attempt to differentiate testosterone abuse from endogenous production. It could be observed that a calculated ratio above this level is strong evidence of drug administration, although a high false-negative rate was obtained.

Determination of testosterone esters and estradiol esters in bovine and porcine blood serum.

Monitoring of steroid esters in blood serum is desirable in order to detect the possible illegal use of natural hormones as growth promoters. A method for the determination of testosterone propionate, testosterone benzoate, testosterone isocaproate, testosterone decanoate and estradiol benzoate in bovine and porcine blood serum was developed. The procedure consists of protein precipitation and removal of phospholipids using a HybridSPE®-Phospholipid column followed by clean-up on a hydrophilic modified styrene polymer SupelTM-Select HLB column and LC-MS/MS measurement. The method was validated according to Commission Decision 2002/657/EC. Decision limits for all analytes were observed in the range 5-30 pg ml-1. The method was shown to be robust for bovine and porcine serum analyses and can be applied for both screening and confirmatory determination in routine residue monitoring.

Flaxseed suppressed prostatic epithelial proliferation in a rat model of benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH), a disease occurring frequently among elderly males, is a slow progressive enlargement of the fibromuscular and epithelial structures of the prostate gland. Dietary factors may influence the prostate and exert an influence on prostatic growth and disease. The current study was undertaken to investigate the protective effect of dietary flaxseed supplementation against testosterone-induced prostatic hyperplasia in male rats. Forty male Wistar rats were divided into 5 groups: (1) untreated control; (2) treatment with testosterone propionate (TP) to induce prostate enlargement; (3) TP-treated group fed a diet containing 5% milled flaxseed; (4) TP-treated group fed a diet containing 10% milled flaxseed; and (5) TP-treated group fed a diet containing 20 ppm finasteride. Treatment with TP significantly increased the absolute and relative weights of different prostatic lobes, serum testosterone (T), and testosterone/estradiol ratio, as well as prostatic vascular endothelial growth factor (VEGF) expression, RNA synthesis per cell, and epithelial cell proliferation, detected as Ki67 labeling. Histopathological examination did not reveal marked differences in acinar morphology in ventral prostate, whereas morphometric analysis showed significantly increased epithelial cell height. Co-administration of flaxseed or finasteride with TP significantly reduced prostatic VEFG, epithelial cell proliferation, and RNA/DNA ratio, along with a significant increase in serum T and testosterone/estradiol ratio compared with TP-only-treated rats. Our results indicate that flaxseed, similar to the 5α-reductase inhibitor finasteride, blocked TP-induced prostate enlargement in a rat model of BPH, likely through suppression of prostatic VEFG and cellular proliferation.

Enhanced transdermal bioavailability of testosterone propionate via surfactant-modified ethosomes.

The current investigation aimed to evaluate the transdermal potential of novel testosterone propionate (TP) ethosomes and liposomes prepared by surfactant modification. The effect of hexadecyl trimethyl ammonium bromide and cremophor EL-35 on the particle size and zeta potential of the prepared vesicles was investigated. The entrapment efficiency and stability, as well as in vitro and in vivo skin permeation, were studied with the various techniques, such as differential scanning calorimetry, confocal laser scanning microscopy, transmission electron microscopy, dynamic light scattering, and so on. The results indicated that the ethosomes were defined as spherical, unilamellar structures with low polydispersity (0.100 ± 0.015) and nanometric size (156.5 ± 3.5 nm). The entrapment efficiency of TP in ethosomal and liposomal carriers was 92.7% ± 3.7% and 64.7% ± 2.1%, respectively. The stability profile of the prepared TP ethosomal system assessed for 120 days revealed very low aggregation and very low growth in vesicular size. TP ethosomes also provided an enhanced transdermal flux of 37.85 ± 2.8 µg/cm(2)/hour and a decreased lag time of 0.18 hours across mouse skin. The skin permeation efficiency of the TP ethosomes as further assessed by confocal laser scanning microscopy revealed enhanced permeation of rhodamine red-loaded formulations to the deeper layers of the skin (260 µm) than that of the liposomal formation (120 µm).

Ultra high performance liquid chromatography/tandem mass spectrometry based identification of steroid esters in serum and plasma: an efficient strategy to detect natural steroids abuse in breeding and racing animals.

During last decades, the use of natural steroids in racing and food producing animals for doping purposes has been flourishing. The endogenous or exogenous origin of these naturally occurring steroids has since remained a challenge for the different anti-doping laboratories. The administration of these substances to animals is usually made through an intra-muscular pathway with the steroid under its ester form for a higher bioavailability and a longer lasting effect. Detecting these steroid esters would provide an unequivocal proof of an exogenous administration of the considered naturally occurring steroids. A quick analytical method able to detect at trace level (below 50 pg/mL) a large panel of more than 20 steroid esters in serum and plasma potentially used for doping purposes in bovine and equine has been developed. Following a pre-treatment step, the sample is submitted to a solid phase extraction (SPE) before analysis with UPLC-MS/MS. The analytical method's efficiency has been probed through three different in vivo experiments involving testosterone propionate intra-muscular administration to three heifers, 17-estradiol benzoate intra-muscular administration to a bull and a heifer and nandrolone laurate intra-muscular administration to a stallion. The results enabled detecting the injected testosterone propionate and 17-estradiol benzoate 2 and 17 days, respectively, post-administration in bovine and nandrolone laurate up to 14 days post-administration in equine. The corresponding elimination profiles in bovine serum and equine plasma have been established. The first bovine experiment exhibited a maximal testosterone propionate concentration of 400 pg/mL in one of the three heifer serum within 5h post-administration. The second bovine experiment reported a maximal 17-estradiol benzoate concentration of 480 pg/mL in the same matrix recorded 9 days after its administration. The last equine experiment resulted in a maximal nandrolone laurate concentration of 440 pg/mL in horse plasma 24h after administration.

Socially modulated cell proliferation is independent of gonadal steroid hormones in the brain of the adult green treefrog (Hyla cinerea).

Gonadal steroid hormones have been shown to influence adult neurogenesis in addition to their well-defined role in regulating social behavior. Adult neurogenesis consists of several processes including cell proliferation, which can be studied via 5-bromo-2'-deoxyuridine (BrdU) labeling. In a previous study we found that social stimulation altered both cell proliferation and levels of circulating gonadal steroids, leaving the issue of cause/effect unclear. In this study, we sought to determine whether socially modulated BrdU-labeling depends on gonadal hormone changes. We investigated this using a gonadectomy-implant paradigm and by exposing male and female green treefrogs (Hyla cinerea) to their conspecific chorus or control stimuli (i.e. random tones). Our results indicate that socially modulated cell proliferation occurred independently of gonadal hormone levels; furthermore, neither androgens in males nor estrogen in females increased cell proliferation in the preoptic area (POA) and infundibular hypothalamus, brain regions involved in endocrine regulation and acoustic communication. In fact, elevated estrogen levels decreased cell proliferation in those brain regions in the implanted female. In male frogs, evoked calling behavior was positively correlated with BrdU-labeling in the POA; however, statistical analysis showed that this behavior did not mediate socially induced cell proliferation. These results show that the social modulation of cell proliferation can occur without gonadal hormone involvement in either male or female adult anuran amphibians, and confirms that it is independent of a behavioral response in males.

High-dose testosterone propionate treatment reverses the effects of endurance training on myocardial antioxidant defenses in adolescent male rats.

This study was aimed at evaluation of changes in activities of selected antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) and contents of key nonenzymatic antioxidants (glutathione, protein thiol groups, and α- and γ-tocopherols) in the left heart ventricle of young male Wistar rats subjected to endurance training (treadmill running, 1 h daily, 5 days a week, for 6 weeks) or/and testosterone propionate treatment (8 or 80 mg/kg body weight, intramuscularly, once a week, for 6 weeks) during adolescence. The training alone increased the activities of key antioxidant enzymes, but lowered the pool of nonenzymatic antioxidants and enhanced myocardial oxidative stress as evidenced by elevation of the lipid peroxidation biomarker malondialdehyde. The lower-dose testosterone treatment showed mixed effects on the individual components of the antioxidant defense system, but markedly enhanced lipid peroxidation. The higher-dose testosterone treatment decreased the activities of the antioxidant enzymes, lowered the contents of the nonenzymatic antioxidants, except for that of γ-tocopherol, reversed the effect of endurance training on the antioxidant enzymes activities, and enhanced lipid peroxidation more than the lower-dose treatment. These data demonstrate the potential risk to cardiac health from exogenous androgen use, either alone or in combination with endurance training, in adolescents.

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.

[Effects of sex hormones on bladder function and structure: experiment with ovariectomized female rats].

OBJECTIVE: To investigate the effects of sex hormones on bladder and mechanism thereof. METHODS: Sixty mature female SD rats underwent bilateral ovariectomy and then randomly divided into 6 groups: low dose estrogen group (OVX + E group) treated with estradiol benzoate 0. 25 mg/kg injected intramuscularly qod, high dose estrogen group (OVX + E 1 mg group) treated with estradiol benzoate 1 mg/kg, progesterone (P) group, treated with P 1 mg, OVX + E + P group treated with estradiol benzoate 0.25 mg/kg + progesterone 1 mg, d androgen group (OVX + T group) treated with testosterone propionate 3 mg/kg, and untreated ovariectomized group (OVX group) receiving no medication. Another 10 rats underwent sham operation. Four weeks later the rats were killed with their bladders and urethras taken out to undergo light and electron microscopy. RESULTS: The thickness of bladder wall of the OVX group was (0.97 +/- 0.11) mm, significant thinner than that of the sham operation group [(1.10 +/- 0.1) mm, P < 0.05)]. The thickness levels of bladder walls of the OVX + E and OVX + T groups were (1.23 +/- 0.12) mm and (1.26 +/- 0.12 mm) respectively, both significantly thicker than that of the OVX group (both P < 0.01). Wider spaces between the detrusor muscle fascicles and degeneration of detrusor muscle cells were seen in the bladder walls of the OVX group, and such phenomena were milder in the OVX + E and OVX + T groups, however, wider spaces between the detrusor muscle fascicles could be seen in the OVX + E + P group too. The hypertrophic effect on the detrusor muscle of the OVX + E 1 mg group was weaker than that of the OVX + E group, a lot of cytoplasmic vacuoles was seen in the 2 groups treated with E, especially the OVX + E 1 mg group. The bladder structure of the OVX + P group was similar to that of the OVX group. CONCLUSION: Endogenous testosterone level as well as estrogen level can significantly affect the bladder structure with a hypertrophic effect on bladder detrusor muscle. But higher doze of estrogen also has some unexpected effects which may destroy bladder structure. In addition, progesterone can compromise the effect of estrogen.

Pioglitazone improves insulin action and normalizes menstrual cycles in a majority of prenatally androgenized female rhesus monkeys.

PURPOSE OF THE STUDY: To determine whether pioglitazone will improve menstrual cyclicity in a fetal programming model for polycystic ovary syndrome. BASIC PROCEDURES: Eight prenatally androgenized (PA) and 5 control female rhesus monkeys of similar age, body weight and body mass index received an oral placebo daily for 6-7 months followed, after at least 90 days, by daily oral dosing with pioglitazone (3mg/kg) for an additional 6-7 months. Blood was sampled thrice weekly to monitor ovulatory function, and a variety of endocrine challenges were performed to quantify changes in ovarian, gonadotropin and glucoregulatory function. MOST IMPORTANT FINDINGS: Pioglitazone normalized menstrual cycles in 5 out of 8 (62%) PA females (pioglitazone responsive; Pio(RESP)). Pioglitazone increased serum 17alpha-hydroxyprogesterone responses to an hCG injection in Pio(RESP) PA females, while diminishing serum progesterone, and increasing DHEA and estradiol responses to hCG in Pio(RESP) PA and all normal females. PRINCIPAL CONCLUSIONS: Insulin resistance plays a mechanistic role in maintaining anovulation in a majority of PA female monkeys.

Differential proliferative response of the ventral prostate and seminal vesicle to testosterone replacement.

We have investigated epithelial cell proliferation and the rate of glandular recovery of the ventral prostate (VP) and seminal vesicle (SV) promoted by testosterone replacement (TR) in castration-induced regressed glands. Adult male Wistar rats were castrated and, after 21 days, they were treated with testosterone propionate (4 mg/kg/day). Intact (CT) and castrated rats without TR (CS) were also analysed. VP and SV were processed for histochemistry, morphometric-stereological analysis and immunocytochemistry to determine the PCNA index (PI). After 10 days of TR, the VP weight reached approximately 72% of the CT values, while the SV weight exceeded approximately 17% of the CT values. By the third day of TR, VP and SV presented a mean PI of 34% and 94% for distal region and 14% and 22% for proximal region, respectively. SV also had more luminal cells PCNA-positive than VP, mainly in the distal region. The PI values fell on days 5, 7 and 10, but were still higher than CT. These findings indicate that epithelial cells from involuted SV are more responsive to TR than those from VP when stimulated to proliferate and replace the luminal cell population, suggesting a different mechanism regulating cell proliferation in response to androgenic stimuli.

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.

Changes in serum alpha2u-globulin levels in castrated male rats treated with testosterone propionate in a Hershberger assay.

The Hershberger assay has been proposed as a candidate screening test method for the detection of androgenic and anti-androgenic chemicals and is being validated presently under the test guideline programme of the Organization for Economic Cooperation and Development (OECD). Rat alpha2u-globulin is male rat-specific protein appearing in their serum and urine, and the protein is known to be induced by androgens. We investigated the usefulness of measuring serum alpha2u-globulin levels as a parameter for the androgenic activity of chemicals tested in the Hershberger assay. The serum alpha2u-globulin level was measured after the administration of testosterone propionate at dosages of 0, 20, 100 or 500 microg kg(-1) day(-1) for ten consecutive days in the castrated male rats. The ventral prostate, balbocavernosus/levator ani muscles, glans penis and Cowper's gland were collected and weighed. Although all the androgen-responsive organ weights were increased significantly at dosages of 100 and 500 microg kg(-1) day(-1), the serum alpha2u-globulin level was increased significantly only at a dosage of 500 microg kg(-1) day(-1). These results show that the serum alpha2u-globulin level may be a useful biomarker for detecting androgenic activity caused by test chemicals, but it is less sensitive than the organ weights of androgen-responsive tissues in the Hershberger assay.