Assessment of the potential of polyphenols as a CYP17 inhibitor free of adverse corticosteroid elevation.

Inhibition of 17α-hydroxylase/17,20-lyase (CYP17), which dictates the proceeding of androgen biosynthesis, is recommended as an effective treatment for androgen-dependent diseases. However, androgen depletion by selective CYP17 inhibition is accompanied with corticosteroid elevation, which increases risk of cardiovascular diseases. In this study, we evaluated the likelihood of polyphenols as a CYP17 inhibitor without cardiovascular complications. All examined polyphenols significantly inhibited CYP17 in human adrenocortical H295R cells, but their effects on androgen and cortisol biosynthesis were diverse. Resveratrol was the most potent CYP17 inhibitor with an approximate IC50 of 4 µM, and the inhibition might weigh on the 17α-hydroxylase activity more than the 17,20-lyase activity. Resveratrol also inhibited 21α-hydroxylase (CYP21) essential for corticosteroid biosynthesis but to a lesser extent, thus preventing the occurrence of cortisol elevation following CYP17 blockade. Although transcriptional down-regulation was important for α-naphthoflavone-mediated CYP17 inhibition, resveratrol inhibited CYP17 and CYP21 mainly at the level of enzyme activity rather than enzyme abundance and cytochrome P450 electron transfer. Daidzein also inhibited CYP17 and CYP21 although less potent than resveratrol. Daidzein was the only polyphenol showing inhibition of 3ß-hydroxysteroid dehydrogenase type II (3ßHSD2). The exceptional 3ßHSD2 inhibition led to dehydroepiandrosterone accumulation alongside daidzein-caused androgen biosynthetic impairment. In contrast, androgen and cortisol secretion was increased or remained normal under α-naphthoflavone and ß-naphthoflavone treatments, suggesting that CYP17 inhibition was counteracted by increased substrate generation. α-naphthoflavone and ß-naphthoflavone also enhanced the formation of cortisol from 17-hydroxyprogesterone and testosterone from androstenedione. Our findings suggest a potential application of resveratrol in androgen deprivation therapy.

Relevance weighting of tier 1 endocrine screening endpoints by rank order.

Weight of evidence (WoE) approaches are recommended for interpreting various toxicological data, but few systematic and transparent procedures exist. A hypothesis-based WoE framework was recently published focusing on the U.S. EPA's Tier 1 Endocrine Screening Battery (ESB) as an example. The framework recommends weighting each experimental endpoint according to its relevance for deciding eight hypotheses addressed by the ESB. Here we present detailed rationale for weighting the ESB endpoints according to three rank ordered categories and an interpretive process for using the rankings to reach WoE determinations. Rank 1 was assigned to in vivo endpoints that characterize the fundamental physiological actions for androgen, estrogen, and thyroid activities. Rank 1 endpoints are specific and sensitive for the hypothesis, interpretable without ancillary data, and rarely confounded by artifacts or nonspecific activity. Rank 2 endpoints are specific and interpretable for the hypothesis but less informative than Rank 1, often due to oversensitivity, inclusion of narrowly context-dependent components of the hormonal system (e.g., in vitro endpoints), or confounding by nonspecific activity. Rank 3 endpoints are relevant for the hypothesis but only corroborative of Ranks 1 and 2 endpoints. Rank 3 includes many apical in vivo endpoints that can be affected by systemic toxicity and nonhormonal activity. Although these relevance weight rankings (WREL ) necessarily involve professional judgment, their a priori derivation enhances transparency and renders WoE determinations amenable to methodological scrutiny according to basic scientific premises, characteristics that cannot be assured by processes in which the rationale for decisions is provided post hoc.

Frailty, sarcopenia, and hormones.

Frailty is now a definable clinical syndrome with a simple screening test. Age-related changes in hormones play a major role in the development of frailty by reducing muscle mass and strength (sarcopenia). Selective Androgen Receptor Molecules and ghrelin agonists are being developed to treat sarcopenia. The role of Activin Type IIB soluble receptors and Follistatin-like 3 mimetics is less certain because of side effects. Exercise (resistance and aerobic), vitamin D and protein supplementation, and reduction of polypharmacy are keys to the treatment of frailty.

The application of reporter gene assays for the detection of endocrine disruptors in sport supplements.

The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC(50) of 0.01 ng mL(-1) and 0.16 ng mL(-1) respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the combination of biological and physio-chemical techniques is optimal.

Disruption of androgen receptor signaling in males by environmental chemicals.

Androgen-disruptors are environmental chemicals in that interfere with the biosynthesis, metabolism or action of endogenous androgens resulting in a deflection from normal male developmental programming and reproductive tract growth and function. Since male sexual differentiation is entirely androgen-dependent, it is highly susceptible to androgen-disruptors. Animal models and epidemiological evidence link exposure to androgen disrupting chemicals with reduced sperm counts, increased infertility, testicular dysgenesis syndrome, and testicular and prostate cancers. Further, there appears to be increased sensitivity to these agents during critical developmental windows when male differentiation is at its peak. A variety of in vitro and in silico approaches have been used to identify broad classes of androgen disrupting molecules that include organochlorinated pesticides, industrial chemicals, and plasticizers with capacity to ligand the androgen receptor. The vast majority of these synthetic molecules act as anti-androgens. This review will highlight the evidence for androgen disrupting chemicals that act through interference with the androgen receptor, discussing specific compounds for which there is documented in vivo evidence for male reproductive tract perturbations. This article is part of a Special Issue entitled 'Endocrine disruptors'.

OECD validation of phase-3 Hershberger assay using the stimulated weanling male rat in Korea.

The OECD has proposed a new, validated test guideline with the stimulated weanling male Hershberger assay to avoid the surgical castration step. In the present study, we assessed the relevance and reliability of the stimulated weanling Hershberger assay in four stages. All chemicals except for testosterone propionate (TP) were orally administered to sexually immature male rats of 22 days old for 10 days. The weights of four mandatory accessory sex organs, two additional reproductive tissues and optional systemic organs were evaluated. At the first two stages, TP, as reference androgen, significantly increased the weights of epididymides and accessory sex organs (ASO) at 1.0 mg kg(-1) and flutamide (FLU), as a positive anti-androgen control, decreased the TP-stimulated organ weights at 3.0 mg kg(-1). At stage 3, trenbolone (40 mg kg(-1)), an anabolic steroid, significantly increased ASO weights, and weak anti-androgens (DDE and linuron) decreased the TP-stimulated ASO weights at each high dose. The above results were confirmed in a blind test with coded substances provided by OECD. Compared with results from our previous castrated male assay, the intact weanling version is less sensitive than the castrated male version, in terms of a smaller response at the reference dose of TP or FLU. However, this study suggests that the stimulated weanling Hershberger assay can detect the effects of both potent and weak anti-androgens on androgen-producing and androgen-dependent tissues.

OECD validation of phase 3 Hershberger assay in Korea using surgically castrated male rats with coded chemicals.

As a participating laboratory for the OECD Hershberger validation program, we conducted a phase 3 trial to test the reliability of the Hershberger assay using coded substances. Male Sprague-Dawley rats were castrated at 6 weeks of age and allowed to recover for 8 days. All the coded substances were administered orally once daily for 10 consecutive days. In the antagonist version of the assay, 0.4 mg kg(-1) of testosterone propionate (TP), a reference androgen, was co-administered with the coded compounds C, D, H, I or K, by a subcutaneous injection. As anticipated, TP alone produced statistically significant increases in the five mandatory accessory sex organ weights. The coded substance L (trenbolone 40 mg kg(-1)), the test agonist, caused significant increases in the weights of the androgen-dependent tissues. The five coded compounds, p,p'-DDE at two doses (codes C and I), linuron at two doses (codes D and K) and flutamide (code H), all significantly decreased the weights of the TP-stimulated sex organs. These results suggest the OECD Hershberger assay to be a reliable screening method for detecting androgen agonists and antagonists.

A concentration addition model for the activation of the constitutive androstane receptor by xenobiotic mixtures.

The effects of contaminants are typically studied in individual exposures; however, environmental exposures are rarely from a single contaminant. Therefore, the study of chemical mixtures is important in determining the effects of xenobiotics. The constitutive androstane receptor (CAR) responds to endobiotics and xenobiotics, and in turn induces detoxification enzymes involved in their elimination. First, we compared several androgens as inverse agonists, including androgens allegedly used by Bay Area Laboratory Co-operative to enhance athletic performance. CAR inverse agonists ranked in order of potency were dihydroandrosterone (DHA) > tetrahydrogestrinone (THG) > androstanol > norbolethone. Therefore, we used DHA as an inverse agonist during transactivation assays. Next, we examined the effects of several pesticides, plasticizers, steroids, and bile acids on CAR activation. Our data demonstrates that several pesticides and plasticizers, including diethylhexylphthalate, nonylphenol, cypermethrin, and chlorpyrifos activate CAR. Both full and partial CAR activators were discovered, and EC(50) values and Hillslopes were determined for use in the concentration addition models. Concentration addition models with and without restraint values to account for partial activators were developed. Measured results from transactivation assays with a mixture of two to five chemicals indicate that the concentration addition model without restraints correctly predicts activity unless all of the chemicals in the mixture are partial activators, and then restraint values be considered. Overall, our data indicates that it is important to consider that we are exposed to a milieu of chemicals, and the efficacy of each individual chemical is not the sole factor in determining CAR's activity in mixture modeling.

Lack of complete cross-resistance between different aromatase inhibitors; a real finding in search for an explanation?

While third-generation aromatase inhibitors (anastrozole, letrozole and exemestane) are successfully implemented as adjuvant and first-line therapy for hormone-sensitive breast cancer in postmenopausal women, important questions remain to be addressed. An issue of particular interest is the question about lack of complete cross-resistance between steroidal and non-steroidal compounds. Although the studies reporting this phenomenon in general contain a small number of patients, the findings across the different reports seem consistent. While several potential mechanisms have been suggested, so far we lack scientific proof what mechanisms may be responsible for this finding. Finally, we do not know whether lack of cross-resistance actually signals an improved efficacy for certain compounds or may be due to alternative mechanisms of action. Neither do we know whether some tumours are more sensitive to particular drugs. This paper summarizes clinical findings up to now with respect to lack of cross-resistance and discuss potential mechanisms involved.

Androgen-mediated cholesterol metabolism in LNCaP and PC-3 cell lines is regulated through two different isoforms of acyl-coenzyme A:Cholesterol Acyltransferase (ACAT).

BACKGROUND: The objective of this work was to determine the effect of an androgen agonist, R1881, on intracellular cholesterol synthesis and esterification in androgen-sensitive (AS) prostate cancer (LNCaP) cells. METHODS: We investigated the activity and expression of cholesterol metabolism enzymes, HMG-CoA-reductase and ACAT in the LNCaP and PC-3 (androgen-independent control) models. RESULTS: Microsomal PC-3 HMG-CoA-reductase activity was increased with R1881 despite having similar cholesterol levels while increased cholesterol levels in microsomes from LNCaPs treated with R1881 (L+) were associated with increased HMG-CoA reductase activity. Increased intracellular cholesteryl esters (CE) found in (L+) were not associated with an increased ACAT1 activity. There was no effect from androgen treatment on ACAT1 protein expression in theses cells; however, ACAT2 expression was induced upon R1881 treatment. In contrast, we found an increase in the in vitro ACAT1 activity in PC-3 cells treated with androgen (P+). Only ACAT1 expression was induced in P+. We further assessed the expression of STAT1 alpha, a transcriptional activator that modulates ACAT1 expression. STAT1 alpha expression and phosphorylation were induced in P+. To determine the role of the AR on ACAT1 expression and esterification, we treated PC-3 cells overexpressing the androgen receptor with R1881 (PAR+). AR expression was decreased in PAR+ cells; ACAT1 protein expression and cholesterol ester levels were also decreased, however, ACAT2 remained unchanged. STAT1 alpha expression was decreased in PAR+. CONCLUSIONS: Overall, these findings support the importance of cholesterol metabolism regulation within prostate cancer cells and unravel a novel role for STAT1 alpha in prostate cancer metabolism.

Detection and assessment of androgenic potency of endocrine-disrupting chemicals using three-spined stickleback, Gasterosteus aculeatus.

The male three-spined stickleback (Gasterosteus aculeatus) produces a glue protein named "spiggin" that is used as a cementing substance for building its nest. The synthesis of spiggin is under the control of androgenic stimulation. Therefore, spiggin is an effective biomarker of any androgenic activity displayed by environmental chemicals, similarly to the use of vitellogenin as an estrogenic biomarker. The aim of this study was to establish a quantification system for spiggin mRNA to develop a highly sensitive system for evaluating environmental androgens. In this process, two different types of cDNA encoding spiggin (SPG-1 and SPG-2) were isolated. They closely resemble each other in primary structure and features. In addition, the transcriptions of both spiggin gene were induced by only androgenic stimulation in a receptor-mediated manner. These findings suggest the multiplicity albeit specificity of spiggin in the stickleback. The quantification system for spiggin mRNA was established using a real-time RT-PCR technique. This system enables accurate quantification within a wide range of spiggin mRNA from 10(1) to 10(6) copies.

The OECD program to validate the rat Hershberger bioassay to screen compounds for in vivo androgen and antiandrogen responses. Phase 1: use of a potent agonist and a potent antagonist to test the standardized protocol.

The Organisation for Economic Cooperation and Development (OECD) has completed phase 1 of the Hershberger validation intended to identify in vivo activity of suspected androgens and antiandrogens. Seventeen laboratories from 7 countries participated in phase 1, and results were collated and evaluated by the OECD with the support of an international committee of experts. Five androgen-responsive tissues (ventral prostate, paired seminal vesicles and coagulating glands, levator ani and bulbocavernosus muscles, glans penis, and paired Cowper's or bulbourethral glands) were evaluated. The standardized protocols used selected doses of a reference androgen, testosterone propionate (TP), and an antiandrogen, flutamide (FLU). All laboratories successfully detected TP-stimulated increases in androgen-responsive tissue weight and decreases in TP-stimulated tissue weights when FLU was co-administered. The standardized protocols performed well under a variety of conditions (e.g., strain, diet, housing protocol, bedding). There was good agreement among laboratories with regard to the TP doses inducing significant increases in tissue weights and the FLU doses decreasing TP-stimulated tissue weights. Several additional procedures (e.g., weighing of the dorsolateral prostate and fixation of tissues before weighing) and serum component measurements (e.g., luteinizing hormone) were also included by some laboratories to assess their potential utility. The results indicated that the OECD Hershberger protocol was robust, reproducible, and transferable across laboratories. Based on this phase 1 validation study, the protocols have been refined, and the next phase of the OECD validation program will test the protocol with selected doses of weak androgen agonists, androgen antagonists, a 5alpha-reductase inhibitor, and chemicals having no androgenic activity.

Pharmacokinetics and pharmacodynamics of nonsteroidal androgen receptor ligands.

Testosterone and structurally related anabolic steroids have been used to treat hypogonadism, muscle wasting, osteoporosis, male contraception, cancer cachexia, anemia, and hormone replacement therapy in aging men or age-related frailty; while antiandrogens may be useful for treatment of conditions like acne, alopecia (male-pattern baldness), hirsutism, benign prostatic hyperplasia (BPH) and prostate cancer. However, the undesirable physicochemical and pharmacokinetic properties of steroidal androgen receptor (AR) ligands limited their clinical use. Nonsteroidal AR ligands with improved pharmacological and pharmacokinetic properties have been developed to overcome these problems. This review focuses on the pharmacokinetics, metabolism, and pharmacology of clinically used and emerging nonsteroidal AR ligands, including antagonists, agonists, and selective androgen receptor modulators.

Strengths and weaknesses of in vitro assays for estrogenic and androgenic activity.

The endocrine and reproductive effects of xenobiotics are believed to be due to (1) their mimicking the effects of endogenous hormones; (2) their antagonizing the effects of endogenous hormones; (3) their altering the pattern of synthesis and metabolism of natural hormones; and (4) their modifying hormone receptor levels. It has been suggested that endocrine disruptors may play a role in the decrease in human semen quantity and quality, an increase in the anomalies of male genital tract, and an increase in the testicular and breast cancer incidence during the last 50 years. Testing these hypotheses will require: (1) identifying estrogen and androgen agonists and antagonists among the chemicals present in the environment; (2) assessing the interactions among the endocrine disruptors to which humans are exposed; and (3) finding markers of estrogen (and androgen) exposure. The development of fast and sensitive bioassays is central to the achievement of these three goals.

OECD validation of the Hershberger assay in Japan: Phase 3. Blind study using coded chemicals.

The Organization for Economic Co-operation and Development (OECD) has initiated the development of new guidelines for the screening and testing of potential endocrine disrupters. The Hershberger assay is one of the assays selected for validation based on the need for in vivo screening to detect androgen agonists or antagonists by measuring the response of five sex accessory organs and tissues of castrated juvenile male rats: the ventral prostate, the seminal vesicles with coagulating glands, the levator ani and bulbocavernosus muscle complex (LABC), Cowper's glands, and the glans penis. The Phase 1 feasibility demonstration stage of the Hershberger validation program has been successfully completed with a single androgen agonist and a single antagonist as reference substances. The Phase 2 validation study was performed, employing a range of additional androgen agonists and antagonists. Recently, the Phase 3 validation study was conducted and performed in several International laboratories. Three Japanese laboratories have contributed to the blind study using coded materials of Phase 3 validation. Four coded test substances in the agonistic version and seven substances in the antagonistic version were orally administered by gavage for 10 consecutive days, respectively. In the antagonist version of the assay, 0.2mg/kg/day of testosterone propionate (TP) was coadministered by subcutaneous injection. All five accessory sex reproductive organs and tissues consistently responded with statistically significant changes in weight within a narrow window in both versions. Therefore, the Japanese studies support the Hershberger assay as a reliable and reproducible screening assay for the detection of androgen agonistic and antagonistic effects.

Differential expression of the tetracycline-controlled transactivator-driven human CYP1B1 gene in double-transgenic mice is due to androgens: application for detecting androgens and antiandrogens.

Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome p450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i). the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii). castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii). treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv). treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.

Immature rat uterotrophic assay of 18 chemicals and Hershberger assay of 30 chemicals.

We performed an immature rat uterotrophic assay of 18 chemicals and Hershberger assay of 30 chemicals to assess the relationship between the results of two assays. The chemicals tested by the uterotopic assay were 4-n-amylphenol, p-dodecyl-phenol, p-(tert-pentyl)phenol, 4-cyclohexylphenol, 4-(1-adamantyl)phenol, 4,4'-thiobis-phenol, diphenyl-p-phenylenediamine, 4-hydroxyazobenzene, 4-(phenylmethyl)phenol, 4,4'-(hexafluoroisopropylidene)diphenol, 2,2-bis(4-hydroxyphenyl)-4-methyl-n-pentane, 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol, 4,4'-dihydroxybenzophenone, 2,2',4,4'-tetrahydroxybenzophenone, 4-hydroxybenzophenone, 2,4,4'-trihydroxybenzophenone, testosterone enanthate, and methyltestosterone. The chemicals tested by the Hershberger assay were the 18 chemicals tested in the uterotrophic assay plus the following: 17 alpha estradiol, estrone, equilin, norethindrone, norgestrel, ethynyl estradiol, bisphenol A, bisphenol B, bisphenol F, 4-tert-octylphenol, p-cumyl phenol, and nonylphenol. All chemicals examined in this study were positive in a reporter gene assay for ER-alpha. In the immature rat uterotrophic assay, all chemicals induced uterotrophy and p-(tert-pentyl)phenol, 4,4'-thiobis-phenol, 4-(phenylmethyl)phenol, 4,4'-(hexafluoroisopropylidene)diphenol, 2,2-bis(4-hydroxyphenyl)-4-methyl-n-pentane, 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol, 4,4'-dihydroxybenzophenone, 2,2',4,4'-tetrahydroxybenzophenone, 4-hydroxybenzophenone, and 2,4,4'-trihydroxybenzophenone exerted both estrogen agonistic effect and reduced the estrogenic effect of ethynylestradiol. In the Hershberger assay, a clear androgen agonistic effect was detected in the androgen derivatives testosterone enanthate and methyltestosterone.