Endocrine Disruptors and Estrogens in Human Prostatic Tissue.

Endocrine disruptors (EDs) are ubiquitous substances both in the environment and everyday products that interfere with the hormonal system. Growing evidence demonstrates their adverse effects on the organism, including the reproductive system and the prostate, owing to their (anti)estrogenic or antiandrogenic effects. Since EDs can interact with steroid hormone actions on-site, understanding the levels of intraprostatic EDs in conjunction with steroids may hold particular significance. The aim of this study was to develop and validate a method for determining estrogens, various groups of EDs (bisphenols, parabens, oxybenzone and nonylphenol) and phytoestrogens in their unconjugated and conjugated forms in prostate tissue by liquid chromatography-tandem mass spectrometry, and subsequently analyze 20 human prostate tissue samples. The method enabled 20 compounds to be analyzed: estrogens (estrone, estradiol, estriol), bisphenols (bisphenol A- BPA, BPS, BPF, BPAF, BPAP, BPZ, BPP), parabens (methyl-, ethyl-, propyl-, butyl-, benzyl- paraben), oxybenzone, nonylphenol and phytoestrogens (daidzein, genistein, equol) with LLOQs between 0.017-2.86 pg/mg of tissue. The most frequently detected EDs in prostate tissues were propylparaben (conjugated and unconjugated forms in 100 % of tissues), methylparaben (unconjugated in 45 % and conjugated in 100 %), ethylparaben (unconjugated in 25 % and conjugated in 100 % BPA (unconjugated in 35 % and conjugated in 60 % and oxybenzone (both forms in 45 % To the best of our knowledge, this is the first study detecting EDs, phytoestrogens and estriol conjugate (E3C) in the prostate. E3C was the most abundant estrogen in prostatic tissue. This highlights the need for further explorations into estrogen metabolism within the prostate.

Derivatized versus non-derivatized LC-MS/MS techniques for the analysis of estrogens and estrogen-like endocrine disruptors in human plasma.

Bisphenols, parabens, alkylphenols and triclosan are anthropogenic substances with a phenolic group that have been introduced to the environment in recent decades. As they possess hormone-like effects, they have been termed endocrine disruptors (EDs), and can interfere with steroid pathways in organisms. To evaluate the potential impact of EDs on steroid biosynthesis and metabolism, sensitive and robust methods enabling the concurrent measurement of EDs and steroids in plasma are needed. Of crucial importance is the analysis of unconjugated EDs, which possess biological activity. The aim of the study was to develop and validate LC-MS/MS methods with and without a derivatization step for the analysis of unconjugated steroids (estrone-E1, estradiol-E2, estriol-E3, aldosterone-ALDO) and different groups of EDs (bisphenols, parabens, nonylphenol-NP and triclosan-TCS), and compare these methods on a set of 24 human plasma samples using Passing-Bablok regression analysis. Both methods were validated according to FDA and EMA guidelines. The method with dansyl chloride derivatization allowed 17 compounds to be measured: estrogens (E1, E2, E3), bisphenols (bisphenol A-BPA, BPS, BPF, BPAF, BPAP, BPZ, BPP), parabens (methylparaben-MP, ethylparaben-EP, propylparaben-PP, butylparaben-BP, benzylparaben-BenzylP), TCS and NP, with lower limits of quantification (LLOQs) between 4 and 125 pg/mL. The method without derivatization enabled 15 compounds to be analyzed: estrogens (E1, E2, E3), ALDO, bisphenols (BPA, BPS, BPF, BPAF, BPAP, BPZ), parabens (MP, EP, PP, BP, BenzylP) with LLOQs between 2 and 63 pg/mL, and NP and BPP in semiquantitative mode. Adding 6 mM ammonium fluoride post column into mobile phases in the method without derivatization achieved similar or even better LLOQs than the method with the derivatization step. The uniqueness of the methods lies in the simultaneous determination of different classes of unconjugated (bioactive) fraction of EDs together with selected steroids (estrogens + ALDO in the method without derivatization), which provides a useful tool for evaluating the relationships between EDs and steroid metabolism.

11-Keto-testosterone and other androgens of adrenal origin.

The adrenal glands produce significant amounts of steroid hormones and their metabolites, with various levels of androgenic activities. Until recently, the androgenic potency of these adrenal-derived compounds were not well known, but some recent studies have shown that the production of 11-oxo- and 11beta-hydroxy-derived testosterone and dihydrotestosterone evidently have high androgenic activity. This fact has clinical importance, for instance, in various types of congenital adrenal hyperplasia with androgenization or polycystic ovarian syndrome, and laboratory determinations of these substances could help to better evaluate the total androgen pressure in patients with these disorders. Another area of concern is the treatment of prostate cancer with androgen deprivation, which loses effectiveness after a certain time. The concurrent blocking of the secretion of adrenal C(19)-steroids, whether using corticoids or adrenostatics, could increase the effectiveness of androgen-deprivation therapy.

Steroid diagnostics of 21st century in the light of their new roles and analytical tools.

The determination of steroid hormones and subsequent interpretation of results is accompanied by a range of difficulties. The amount of information that current technology can provide on the circulating concentrations of more than a hundred various steroid compounds can lead to problems with interpretation. The aim of this study is to help provide orientation in this maze of data on steroid hormones. First we focus on specific aspects arising from the pre-analytical phase of steroid determination that need to be considered when planning sampling, whether for diagnostics or research. Then, we provide a brief summary of the characteristics and diagnostic relevance of several steroid hormones and/or their metabolites: pregnenolone, 17alpha-hydroxy-pregnenolone, dehydroepiandrosterone, hydroxyderivatives of dehydroepiandrosterone, androstenedione, testosterone, estrone, estradiol, estriol, cortisol, cortisone, which in our institute are determined with validated LC-MS/MS methods. For these steroids, we also provide newly calculated reference values in fertile women according to the phase of their menstrual cycle.

Endocrine disruptors, obesity, and cytokines - how relevant are they to PCOS?

As environmental and genetic components contribute to the PCOS expression, we compared levels of endocrine disruptors, steroid hormones, cytokines, and metabolic parameters in twenty healthy, nine normal-weight PCOS women, and ten obese PCOS women. Steroid hormones, bisphenols (BPA, BPS, BPF, BPAF) and parabens (methyl-, ethyl-, propyl-, butyl-, benzyl-parabens) were measured by liquid chromatography-tandem mass spectrometry. Differences between the groups were assessed using the Mann-Whitney U test. Spearman correlation coefficients were calculated for the individual parameters relationship. Significantly higher levels of BPA, anti-Müllerain hormone, lutropine, lutropine/folitropine ratio, testosterone, androstenedione, 7beta-OH-epiandrosterone, and cytokines (IL-6, VEGF, PDGF-bb), were found in normal-weight PCOS women compared to controls. Between normal-weight and obese PCOS women, there were no differences in hormonal, but in metabolic parameters. Obese PCOS women had significantly higher insulin resistance, fatty-liver index, triglycerides, cytokines (IL-2, IL-13, IFN-gamma). In healthy, but not in PCOS, women, there was a positive correlation of BPA with testosterone, SHBG with lutropine, and folitropine, while testosterone negatively correlated with SHBG. In obese women with PCOS, insulin resistance negatively correlated with SHBG and estradiol. No differences were observed in the paraben exposure. Levels of BPA were higher in PCOS women, indicating its role in the etiology. Obesity significantly worsens the symptoms.

A method for determination of one hundred endogenous steroids in human serum by gas chromatography-tandem mass spectrometry.

Steroid profiling helps various pathologies to be rapidly diagnosed. Results from analyses investigating steroidogenic pathways may be used as a tool for uncovering pathology causations and proposals of new therapeutic approaches. The purpose of this study was to address still underutilized application of the advanced GC-MS/MS platform for the multicomponent quantification of endogenous steroids. We developed and validated a GC-MS/MS method for the quantification of 58 unconjugated steroids and 42 polar conjugates of steroids (after hydrolysis) in human blood. The present method was validated not only for blood of men and non-pregnant women but also for blood of pregnant women and for mixed umbilical cord blood. The spectrum of analytes includes common hormones operating via nuclear receptors as well as other bioactive substances like immunomodulatory and neuroactive steroids. Our present results are comparable with those from our previously published GC-MS method as well as the results of others. The present method was extended for corticoids and 17alpha-hydroxylated 5alpha/ß-reduced pregnanes, which are useful for the investigation of alternative "backdoor" pathway. When comparing the analytical characteristics of the present and previous method, the first exhibit by far higher selectivity, and generally higher sensitivity and better precision particularly for 17alpha-hydroxysteroids.

Differences in bisphenol A and estrogen levels in the plasma and seminal plasma of men with different degrees of infertility.

The general population is potentially exposed to many chemicals that can affect the endocrine system. These substances are called endocrine disruptors (EDs), and among them bisphenol A (BPA) is one of the most widely used and well studied. Nonetheless, there are still no data on simultaneous measurements of various EDs along with steroids directly in the seminal fluid, where deleterious effects of EDs on spermatogenesis and steroidogenesis are assumed. We determined levels of BPA and 3 estrogens using LC-MS/MS in the plasma and seminal plasma of 174 men with different degrees of infertility. These men were divided according their spermiogram values into 4 groups: (1) healthy men, and (2) slightly, (3) moderate, and (4) severely infertile men. Estradiol levels differed across the groups and body fluids. Slightly infertile men have significantly higher BPA plasma and seminal plasma levels in comparison with healthy men (p<0.05 and p<0.01, respectively). Furthermore, seminal BPA, but not plasma BPA, was negatively associated with sperm concentration and total sperm count (-0.27; p<0.001 and -0.24; p<0.01, respectively). These findings point to the importance of seminal plasma in BPA research. Overall, a disruption of estrogen metabolism was observed together with a weak but significant impact of BPA on sperm count and concentration.