Phenylethanoid Glycosides of Cistanche on menopausal syndrome model in mice.

Cistanche is the traditional and precious Chinese herbal, with two thousand years of use history in China. It has the effect on tonifying kidney, strong supplement to the liver and kidney, and replenishing essence and blood, known as the "desert ginseng". Here, we explored the mechanism of Phenylethanoid Glycosides of Cistanche (PGC) to the model mice of menopausal syndrome, as well as the therapeutic effect and characteristics of PGC to the menopausal syndrome. In this study, KM mice were reproduced by the complete resection of the ovaries on both sides of the back to establish the model mice of menopausal syndrome (MPS), and received distilled water or drugs, respectively. Model mice received distilled water. Mice received 200 mg/(kg day) high doses of Phenylethanoid Glycosides of Cistanche (HPGC), and 100 mg/(kg day) medium doses of Phenylethanoid Glycosides of Cistanche (MPGC), and 50 mg/(kg day) low doses of Phenylethanoid Glycosides of Cistanche (LPGC). After 21 days, it could determine the number of independent activities and the number of standing, the latent period of first entering the dark room, and the electric number. It also calculated the viscera index of uterus, thymus, spleen, measured the levels of estradiol (E2), testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in the serum. Furthermore, it observed the pathological changes of uterus, thymus, spleen and pituitary of mice. The results showed that behavioral indicators: Compared with the model group (MG), HPGC, MPGC, LPGC could increase the independent activities (P < 0.01); HPGC, MPGC could increase the number of standing, the latent period of first entering the dark room, and reduce the electric number (P < 0.01); LPGC could increase the number of standing (P < 0.05); Viscera index: Compared with MG, HPGC, MPGC could increase the viscera index of uterus, thymus, spleen (P < 0.01); LPGC could increase the viscera index of uterus (P < 0.05); Serum index: Compared with MG, all groups could decrease the levels of LH in the serum (P < 0.01); HPGC, MPGC could improve the levels of E2, T and decrease the levels of LH, FSH in the serum (P < 0.01); LPGC could improve the levels of E2 and decrease the levels of FSH in the serum (P < 0.05). Meanwhile, it had the trend to improve the levels of T in the serum. Pathological changes: Compared with MG, HPGC could significant improve the pathological changes of uterus, thymus, spleen and pituitary of mice; other groups also has a certain effect. The results indicated that PGC could improve the sex hormone disorder of MPS, and restore the function of uterus, thymus and spleen, with better therapeutic effect on MPS.

A novel fully-automated method to measure steroids in serum by liquid chromatography-tandem mass spectrometry.

Background: Steroids play a key role in numerous physiological processes. Steroid determination is a useful tool to explore various endocrine diseases. Because of its specificity, mass spectrometry is considered to be a reference method for the determination of steroids in serum compared to radioimmunoassay. This technology could progress towards more automation for the optimal organization of clinical laboratories and ultimately for the benefit of patients. Methods: A fully automated ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and fully validated to determine five steroids in serum. Sample preparation was based on protein precipitation with filtration followed by online solid phase extraction. Chromatographic separation was performed using a biphenyl stationary phase. Results: The method was successfully validated according to European Medicine Agency guidelines. Coefficients of variation did not exceed, respectively, 8.4% and 8.1% for intra- and inter-assay precision. Method comparison with radioimmunoassay showed a proportional bias for all compounds, except for testosterone in men. Comparison with another LC-MS/MS method demonstrated acceptable concordance for all steroids, although a small bias was observed for androstenedione. Conclusion: The novelty of this method is that it has been fully automated. Automation provides benefits in traceability and allows significant savings in cost and time.

Low free testosterone is associated with increased mortality in frail surgical patients.

BACKGROUND: Preoperative frailty has been associated with adverse postoperative outcomes. Additionally, low testosterone has been associated with physical frailty and cognitive decline. However, the impact of simultaneous frailty and low testosterone on surgical outcomes is understudied. METHODS: Preoperative frailty status and testosterone levels were obtained in patients undergoing a diverse range of surgical procedures. Preoperative frailty was evaluated independently and in combination with testosterone through the creation of composite risk groups. Relationships between preoperative frailty and composite risk groups with overall survival were determined using Kaplan-Meier and logistic regression analyses. Bivariate analysis was used to determine the associations between frailty and testosterone status on postoperative complications, length of hospital stay, and readmission rates. RESULTS: Median age of the cohort was 63 years, and the median follow-up time was 105 weeks. Thirty-one patients (23%) were frail, and 36 (27%) had low free testosterone. Bivariate analysis demonstrated a statistically significant relationship between preoperative frailty and overall survival (P = .044). In multivariate analysis, coexisting frailty and low free testosterone were significantly associated with decreased overall survival (hazard ratio 4.93, 95% confidence interval, 1.68-14.46, P = .004). CONCLUSION: We observed preoperative frailty, both independently and in combination with low free testosterone levels, to be significantly associated with decreased overall survival across various surgical procedures. Personalizing the surgical risk assessment through the incorporation of preoperative frailty and testosterone status may serve to improve the prognostication of patients undergoing major surgery.

Role of soy isoflavone in preventing aging changes in rat testis: Biochemical and histological studies.

Testicular function and structure harmed by ageing. Goal of this research was to assess preventive actions of soy isoflavone oral administration for 8 weeks on testes of old male albino rats, and potential mechanisms of action. Adult control (N = 10) and elderly control (N = 10) rats were fed usual diet, while aged treatment group (N = 10) gave oral 100 mg/kg soy isoflavone daily for 8 weeks. ELISA kits were used to measure testosterone levels and oxidative stress indicators [malonaldehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD)] in serum. Aging produced functional and structural testicular changes and decreased ki67 proliferative marker immunoexpression versus adult control rats due to enhancement of oxidative stress. Soy isoflavone exerted protective effect on testicular function and structure as assessed by increase serum levels of testosterone and preserved histological structure and immune-expression features. These protected effects due to isoflavone antioxidant properties proved by decrease in serum values of MDA, while GSH and SOD were elevated after treatment. These data demonstrated protective effects of isoflavone against age changes in rat testes, by reducing oxidative stress and increasing antioxidants and testicular ki67 proliferative marker immunoexpression.

Continuous reference intervals for pediatric testosterone, sex hormone binding globulin and free testosterone using quantile regression.

Testosterone (T), sex hormone binding globulin (SHBG), free testosterone (FT) and bioavailable testosterone (BAT) are commonly employed tests in pediatric endocrinology and all require age-dependent reference intervals for interpretation. The common methods used to derive these reference intervals require decisions about data shape and/or age partition thresholds, which can result in sharp differences between age groups, particularly for pubescent children. Partitioning also results in a form of data loss, where data from one age-bin is completed disconnected from the adjacent age-bins. Non-parametric continuous reference intervals methods have previously been developed to avoid some of these drawbacks. These strategies use all the available data and smooth transitions between ages avoiding partitioning. However, the fitting process involves selection and adjustment of many parameters and it can be difficult to maintain a reproducible approach. Here we provide a workflow for non-parametric continuous reference intervals applied to T, FT, BAT, and SHBG using the R language quantregGrowth package. T measurements were determined by LC-MS/MS, FT and BAT were calculated, and SHBG was measured on the Roche Cobas e601. The continuous interval methodology is described in detail with code examples and illustrations for reproducibility.

Inhibitory and inductive effects of 4- or 5-methyl-2-mercaptobenzimidazole, thyrotoxic and hepatotoxic rubber antioxidants, on several forms of cytochrome P450 in primary cultured rat and human hepatocytes.

Effects of 4-methyl-2-mercaptobenzimidazole (4-MeMBI) and 5-methyl-2- mercaptobenzimidazole (5-MeMBI) on cytochrome P450 (CYP) activity were examined in primary cultured rat hepatocytes. Hepatocytes from male Wistar rats were cultured in the presence of 4-MeMBI or 5-MeMBI (0-400 µM), and the activity of CYPs 3A2/4 (48 and 96 h) and 1A1/2 (48 h) was determined by measuring the activity of testosterone 6ß-hydroxylation and 7-ethoxyresorufin O-deethylation, respectively. As a result, 4-MeMBI and 5-MeMBI (≥12.5 µM) inhibited CYP3A2 activity. On the other hand, 4-MeMBI (≥25 µM) and 5-MeMBI (≥100 µM) induced CYP1A1/2 activity, being consistent with the previous in vivo results. In a comparative metabolism study using primary cultured human hepatocytes from two Caucasian donors, 4-MeMBI and 5-MeMBI induced the activity of CYPs 3A4 and 1A1/2 with individual variability. It was concluded from these results that 4-MeMBI, 5-MeMBI and MBI caused inhibition of CYP3A2 activity in primary cultured rat hepatocytes, suggesting their potential for metabolic drug-drug interactions. Primary cultured rat and human hepatocytes were considered to be useful for the evaluation of effects of the benzimidazole compounds on their inducibility and inhibitory activities of cytochrome P450 forms.

Simultaneous measurement of 18 steroids in human and mouse serum by liquid chromatography-mass spectrometry without derivatization to profile the classical and alternate pathways of androgen synthesis and metabolism.

BACKGROUND: The recently identified alternate, or backdoor, pathway of DHT synthesis provides important novel information on androgen biosynthesis beyond the classical pathway. We report a rapid and versatile liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously and accurately quantify key steroids in human or mouse serum involved in either the classical or backdoor androgen synthesis pathways. METHODS: Serum (200 µL) fortified with isotopically labelled internal standards underwent liquid-liquid extraction (LLE) with MTBE and extracts were analysed on a LC-MS/MS. The targeted steroids for quantification were testosterone (T), dihydrotestosterone (DHT), 5α-androstane-3α,17ß-diol (3α diol), 5α-androstane-3ß,17ß-diol (3ß diol), dehydroepiandrosterone (DHEA), androstenedione (A4), androsterone (AD), estradiol (E2), estrone (E1), progesterone (P4), pregnenolone (P5), androstenediol (Adiol), 17-hydroxyprogesterone (17-OHP4) and 17-hydroxypregnenolone (17-OHP5), corticosterone (B), cortisol (F), allopregnanolone (Allo-P5) and dihydroprogesterone (DHP). RESULTS: The limits of quantification (LOQ) were 5 pg/mL for E2 and E1, 25 pg/mL for T, 50 pg/mL for A4 and 0.10 ng/mL for DHT, 17OHP5, P4, P5, AD, Adiol, DHEA, AlloP5 and 0.20 ng/mL for 17OHP4, 3α diol, 3ß diol, DHP, 0.25 ng/mL for B and 1 ng/mL for F. Accuracy, precision, reproducibility and recovery were within acceptable limits for bioanalytical method validation. The method is illustrated in human and mouse, male and female serum. CONCLUSIONS: The presented method is sufficiently sensitive, specific and reproducible to meet the quality criteria for routine laboratory application for accurate quantitation of 18 steroid concentrations in male and female serum from humans or mice for the purpose of profiling androgen synthesis and metabolism pathways.

Data on spermatogenesis in rat males gestationally exposed to bisphenol A and high fat diets.

This data article contains supporting information regarding the research article entitled "High butter-fat diet and bisphenol A additively impair male rat spermatogenesis" (P. Tarapore, M. Hennessy, D. Song, J. Ying, B. Ouyang, V. Govindarajah, et al.,) [1]. Sprague-Dawley females were fed AIN, high fat butter, 17α-ethinyl estradiol, or high fat butter plus four bisphenol A doses (2500 µg/kg bw-d, 250 µg/kg bw-d, 25 µg/kg bw-d, and 2.5 µg/kg bw-d) before and during pregnancy. All diets were switched to AIN after the pups were born. Male offspring received testosterone (T)- and estradiol-17ß (E2)-filled implants from postnatal day 70-210 for 20 weeks (T+E2 rat model). The testes were weighed, and examined for impairments in spermatogenesis.

Identification of secretaglobin Scgb2a1 as a target for developmental reprogramming by BPA in the rat prostate.

Secretoglobins are a superfamily of secreted proteins thought to participate in inflammation, tissue repair, and tumorigenesis. Secretoglobin family 2A member 1 (Scgb2a1) is a component of prostatein, a major androgen-binding protein secreted by the rat prostate. Using a rat model for developmental reprogramming of susceptibility to prostate carcinogenesis, we identified, by RNA-seq, that Scgb2a1 is significantly upregulated (>100-fold) in the prostate of adult rats neonatally exposed to bisphenol A (BPA), with increased gene expression confirmed by quantitative RT-PCR and chromatin immunoprecipitation for histone H3 lysine 9 acetylation. Bisulfite analysis of both CpG islands located within 10 kb of the Scgb2a1 promoter identified significant hypomethylation of the CpG island upstream of the transcription start site of this gene in the reprogrammed prostate. These data suggest that expression of Scgb2a1 in the adult prostate could be epigenetically reprogrammed by BPA exposure during prostate development, with potential implications for cancer risk and response to chemotherapeutics associated with prostatein binding.

Effects of crude kerosene on testosterone levels, aggression and toxicity in rat.

The use of crude kerosene as a dietary supplement in boarding schools has been a common practice in east Africa and other countries for many years, with the belief of it reducing the sex drive (libido) at the pubertal stage. There is however no scientific basis for this belief. The present study aimed at using a rat animal model to investigate the effects of crude kerosene on serum testosterone levels, aggression and its possible toxic effects. Fifteen male albino rats of approximately similar age and average weights were put into three groups of five animals each; the control group (placebo), low kerosene dose (10 µl/day) group and high kerosene dose (300 µl/day) group. ELISA was used to determine the serum testosterone levels. During treatment, changes in aggression were observed and noted. Liver toxicity was determined using enzyme assays, total protein and albumin while renal toxicity was monitored using serum creatinine levels. A full hemogram was conducted to determine hematological effects. Various tissue biopsies were obtained and examined using histopathological techniques for evidence of toxicity. Contrary to the common belief, our findings showed an overall increase of serum testosterone levels of up to 66% in the low dose and 75% in the high dose groups, with an increasing trend by the end of the study. The high dose group showed significantly increased levels of white blood cells (WBC) (p = 0.036), red blood cells (RBC) (p = 0.025), hematocrit (HCT) (p = 0.03), red cell distribution width (p = 0.028) and platelets (p = 0.017). The histological results of the stomach indicated chronic gastritis.

Conazole fungicides inhibit Leydig cell testosterone secretion and androgen receptor activation in vitro.

Conazole fungicides are widely used in agriculture despite their suspected endocrine disrupting properties. In this study, the potential (anti-)androgenic effects of ten conazoles were assessed and mutually compared with existing data. Effects of cyproconazole (CYPRO), fluconazole (FLUC), flusilazole (FLUS), hexaconazole (HEXA), myconazole (MYC), penconazole (PEN), prochloraz (PRO), tebuconazole (TEBU), triadimefon (TRIA), and triticonazole (TRIT) were examined using murine Leydig (MA-10) cells and human T47D-ARE cells stably transfected with an androgen responsive element and a firefly luciferase reporter gene. Six conazoles caused a decrease in basal testosterone (T) secretion by MA-10 cells varying from 61% up to 12% compared to vehicle-treated control. T secretion was concentration-dependently inhibited after exposure of MA-10 cells to several concentrations of FLUS (IC50 = 12.4 µM) or TEBU (IC50 = 2.4 µM) in combination with LH. The expression of steroidogenic and cholesterol biosynthesis genes was not changed by conazole exposure. Also, there were no changes in reactive oxygen species (ROS) formation that could explain the altered T secretion after exposure to conazoles. Nine conazoles decreased T-induced AR activation (IC50s ranging from 10.7 to 71.5 µM) and effect potencies (REPs) were calculated relative to the known AR antagonist flutamide (FLUT). FLUC had no effect on AR activation by T. FLUS was the most potent (REP = 3.61) and MYC the least potent (REP = 0.03) AR antagonist. All other conazoles had a comparable REP from 0.12 to 0.38. Our results show distinct in vitro anti-androgenic effects of several conazole fungicides arising from two mechanisms: inhibition of T secretion and AR antagonism, suggesting potential testicular toxic effects. These effects warrant further mechanistic investigation and clearly show the need for accurate exposure data in order to perform proper (human) risk assessment of this class of compounds.