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.

The Endocannabinoid System - The Prediction of Spontaneous Preterm Birth in High-Risk Women: Protocol of a Study.

Spontaneous preterm birth (sPTB) is a major cause of perinatal morbidity and mortality, even in developed countries. Prediction of sPTB is therefore a valuable tool to reduce the associated risks. The current standard for the prediction of sPTB consists, in addition to anamnestic data, of previous sPTB and previous second trimester miscarriage, measurement of cervical length by transvaginal ultrasound (TVU CL) together with assessment of fetal fibronectin levels in cervicovaginal fluid. Other evaluation parameters, such as the level of endocannabinoids in the pregnant woman's blood, could increase the sensitivity of this management. Endocannabinoids (eCBs) are a part of the endocannabinoid system (ECS); out of them anandamide (arachidonoyl-ethanolamide, AEA), in particular, plays an important role in the regulation of pregnancy and childbirth. We present the protocol for an open, non-randomized study to evaluate concentrations of AEA and other endocannabinoids: 2 linoleoylglycerol (2-AG), 2 linoleoylglycerol (2-LG), 2 oleoylglycerol (2-OG), and 2 arachidonoyldopamine (2-ADOPA or also NADA) in the blood of pregnant women as potential predictors of sPTB. In a total of 230 women with a history of sPTB or miscarriage, eCBs levels between 22 and 28 weeks of gestation will be assessed from maternal blood, in addition to the standard procedure. The aim of the study is to determine the relationship between blood concentrations of the endocannabinoids tested and the risk of sPTB. The results of this study will describe the prognostic significance of maternal blood eCBs levels for sPTB, and could subsequently enable improved screening programs for early identification of sPTB.

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.

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.

Are there sex differences in the reaction of undercarboxylated osteocalcin to hypoglycemia?

There has been increasing evidence in recent years for the hypothesis of bones as endocrine organs. Osteocalcin, long considered just a marker of new bone formation, is now seen as the first hormone produced by bones, and seems to be associated with regulating glucose metabolism and reproduction. The aim of this work was to monitor changes of osteocalcin in reaction to hypoglycemia, and determine if there are differences in such reactions between the sexes. The study included 61 healthy probands with physiological calciophosphate metabolism (30 men and 31 women). We applied to each of them an insulin tolerance test, and then monitored levels of undercarboxylated osteocalcin and reactions to hypoglycemia at regular time intervals. We found differences in the reaction to hypoglycemia between the sexes. In men there was a significant decline in undercarboxylated osteocalcin between the 30 and 40 min (p<0.0015), which reflects a reaction to a glycemic decline between 25-30 min, followed by reversal. Low undercarboxylated osteocalcin in men lasted up to 90 min, after which they returned to levels before the test. In women we did not find any significant changes in undercarboxylated osteocalcin levels. Changes in undercarboxylated osteocalcin induced by hypoglycemia indicate a relationship between bones and glucose metabolism. There was an interesting difference between the sexes. However, a definitive conclusion about the role of osteocalcin in human metabolism will require numerous future studies.

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.

Androgens in women - critical evaluation of the methods for their determination in diagnostics of endocrine disorders.

The androgens dehydroepiandrosterone sulfate, dehydro-epiandrosterone, androstenedione and testosterone are routinely assessed in women, and circulating levels of these androgens reflect their production. These androgens are measured in most laboratories using various immuno-analytical methods. Recently, however, androgen assays have begun to be performed using gas or liquid chromatography combined with mass spectrometry. To better understand the difficulties and issues of androgen laboratory diagnostics, it is important to assess each of the methods used, how and why they were introduced into practice, and their advantages, limits, historic milestones and current status. It is also necessary to understand how reference ranges are determined and specifics arising from the physiology of individual androgens. Here we present a summary and discussion of these issues.

Phytoestrogens and the intestinal microbiome.

The microflora of the digestive tract is composed of a unique set of bacteria, yeasts, viruses and other microorganisms, generally known as the microbiome. The microbiome exhibits considerable inter-individual variability, with up to two-thirds of the microflora differing between individuals. Because of this, the variable intestinal microflora is responsible for many differences in metabolic, hormonal and immunological processes in humans and animals. Significant differences have been observed in the metabolism of phytoestrogens, naturally occurring substances that possess estrogenic or anti-estrogenic activity. These substances occur predominately in legumes, especially in soy and many soy products. Because of their effects, phytoestrogens are used as an alternative therapy for menopausal disorders and benign prostate hyperplasia. In connection with the worldwide expansion of soy products as part of healthy lifestyles including vegetarianism and veganism, phytoestrogens have become a regular part of everyday life. The activity of phytoestrogens is strongly dependent on the microbiome. Their metabolites have stronger estrogenic activity than the natural substances themselves, and because of the variability in microbiomes, there are large differences in the effects of phytoestrogens among individuals.

Parabens and their relation to obesity.

Parabens are a group of chemicals used as preservatives in the food, cosmetic and pharmaceutical industries. They are known to possess estrogenic effects, and therefore have been classified as endocrine disruptors. In addition to the classical endocrine organs, other tissues have endocrine activity, including adipose tissue. Several chemicals are known to cause obesogenic effects, and parabens are currently being studied in this context. The aim of this study was to investigate the possible connections of paraben exposure and obesity. Blood plasma from 27 healthy women was collected during their menstrual cycle. Basal anthropometric measures, levels of parabens (methylparaben, ethylparaben and propylparaben), adipokines (adiponectin, adipsin, leptin, resistin and visfatin) and hormones affecting energy balance and metabolic health (c-peptide, ghreline, GIP, GLP-1, glucagon, insulin, PAI-1) were measured. A Kolmogorov-Smirnov test showed higher methylparaben and propylparaben levels in women with BMI 25-34.9 compared to those with BMI 18.5-24.9. Plasma levels of methylparaben as well as the sum of parabens were positively associated with the plasma adipsin levels. Negative associations for methylparaben were found for glucagon, leptin and PAI-1. In accordance with other experimental studies we observed important associations of methylparaben and hormones affecting energy balance and metabolic health, indicating its obesogenic potential.

Endocrine disruptors of the bisphenol and paraben families and bone metabolism.

After menopause, when estrogen levels decrease, there is room for the activity of anthropogenic substances with estrogenic properties - endocrine disruptors (EDs) - that can interfere with bone remodeling and changes in calcium-phosphate metabolism. Selected unconjugated EDs of the bisphenol group - BPA, BPS, BPF, BPAF, and the paraben family - methyl-, ethyl-, propyl-, butyl-, and benzyl-parabens - were measured by high performance liquid chromatography-tandem mass spectrometry in the plasma of 24 postmenopausal women. Parameters of calcium-phosphate metabolism and bone mineral density were assessed. Osteoporosis was classified in 14 women, and 10 women were put into the control group. The impact of EDs on calcium-phosphate metabolism was evaluated by multiple linear regressions. In women with osteoporosis, concentrations of BPA ranged from the lower limit of quantification (LLOQ) - 104 pg/ml and methyl paraben (MP) from LLOQ - 1120 pg/ml. The alternative bisphenols BPS, BPF and BPAF were all under the LLOQ. Except for MP, no further parabens were detected in the majority of samples. The multiple linear regression model found a positive association of BPA (beta=0.07, p<0.05) on calcium (Ca) concentrations. Furthermore, MP (beta=-0.232, p<0.05) was negatively associated with C-terminal telopeptide. These preliminary results suggest that these EDs may have effects on calcium-phosphate metabolism.

Phthalate metabolites in maternal and cord plasma and their relations to other selected endocrine disruptors and steroids.

Endocrine disruptors (EDs) are known to have harmful effects on the human endocrine system; special effort is actually given to the exposure during pregnancy. Humans are usually exposed to a mixture of EDs, which may potentiate or antagonize each other, and the combined effect may be difficult to estimate. The main phthalate monoesters monoethyl-, mono-n-butyl-, monoisobutyl-, monobenzyl-, mono-(2-ethylhexyl)-, mono-(2-ethyl-5-hydroxyhexyl)- and mono-(2-ethyl-5-oxohexyl) phthalate were determined in 18 maternal (37th week of pregnancy) and cord plasma samples using liquid chromatography-tandem mass spectrometry. Previously determined levels of selected bisphenols, parabens and steroids were also considered in this study. In cord blood, there were significantly higher mono-n-butyl phthalate levels than in maternal blood (p=0.043). The results of multiple regression models showed that maternal plasma phthalates were negatively associated with cord plasma androstenedione, testosterone and dehydroepiandrosterone and positively associated with estradiol and estriol. For estriol, a cumulative association was also observed for sumabisphenols. To the best of our knowledge, this is the first pilot study evaluating the effect of prenatal exposure by multiple EDs on newborn steroidogenesis. Our results confirmed phthalate accumulation in the fetal area and disruption of fetal steroidogenesis. This preliminary study highlights the negative impacts of in utero EDs exposure on fetal steroidogenesis.

Steroid hormone levels in the peripartum period - differences caused by fetal sex and delivery type.

Progesterone, estrogens, androgens and glucocorticoids all play important roles during pregnancy, from implantation to delivery. Focusing on selected steroid hormones in the peripartum period, we defined reference ranges measured using LS-MS/MS, and assessed relationships with maternal age, pregnancy weight gain, delivery type, and fetal sex. Samples were taken from 142 healthy women with physiological gravidity at the 37th week, during the first period of labor, and from newborn mixed cord blood. We found higher cortisol and 17-OH-pregnenolone plasma levels in mothers at the 37th week that carried male fetuses (p=0.03), but no significant differences in any studied hormones in newborns of different sex. Neither maternal age nor weight gain nor newborn birth weight had any relationships to any of the studied hormones. However, there were differences depending on vaginal versus planned cesarean section deliveries. In women carrying a male fetus we found significantly higher levels of 17-OH-pregnenolone, progesterone, cortisol, corticosterone and significantly lower levels of estradiol in those undergoing spontaneous vaginal delivery. However, we found no significant differences in the cord blood of newborn males from either delivery type. We established reference ranges for our analysis methods, which should be useful for further studies as well as in standard clinical practice.

The quantitation of 7beta-hydroxy-epiandrosterone in the plasma and seminal plasma of men with different degrees of fertility.

7beta-hydroxy-epiandrosterone (7beta-OH-EpiA) is an endogenous androgen metabolite that has been shown to exert neuroprotective, anti-inflammatory and anti-estrogenic effects. However, to the best of our knowledge no information is available about this androgen steroid in relation to sperm quality. We analyzed 7beta-OH-EpiA in plasma and seminal plasma using a newly developed isotope dilution ultra-high performance liquid chromatography - mass spectrometry method. Validation met the requirements of FDA guidelines. Levels of 7beta-OH-EpiA were measured in 191 men with different degrees of infertility. One-way analysis of variance followed by multiple comparison and correlation analysis adjusted for age, BMI and abstinence time were performed to evaluate the relationships between this steroid and sperm quality. Concentrations of 7beta-OH-EpiA in seminal plasma were significantly higher in severely infertile men in comparison with healthy men and slightly infertile men. The same trend was found when blood plasma was evaluated. Furthermore, plasma 7beta-OH-EpiA negatively correlated with sperm concentration (-0.215; p<0.01) and total count (-0.15; p<0.05). Seminal 7beta-OH-EpiA was negatively associated with motility (-0.26; p<0.01), progressively motile spermatozoa (-0.233; p<0.01) and nonprogressively motile spermatozoa (-0.188; p<0.05). 7beta-OH-EpiA is associated with lower sperm quality and deserves more research in that respect.

Serum and intratesticular sex steroids in azoospermic men: how do they correlate?

Five intratesticular sex steroids (testosterone, dihydrotestosterone, androstenedione, estradiol and epitestosterone) along with six serum hormones (LH, FSH, prolactin, SHBG, testosterone and estradiol) were determined in 84 non-obstructive azoospermic men, in order to evaluate to what extent serum and testicular tissue as well as individual hormones in the same material mutually correlate. With exception of androstenedione, tight correlations were found among tissue content of sex steroids, while only weak correlation was recorded between serum and testicular concentrations of major sex steroids testosterone and estradiol. It points to importance of measurement of intratesticular steroids in combination with examination of sperm parameters for assessment of testicular function and spermatogenesis.

Physiological changes after spa treatment - a focus on endocrinology.

The paper presents the results of our effort to reveal objective parameters for evaluation of the spa treatment for patients with anxiety-depressive disorders. The study was based on our previous experience with neuroactive steroids and neurosteroids, which play a crucial role in the psychological well-being of patients by maintaining balance of the organism. A total number of 94 steroids were determinated in a group of 70 female patients diagnosed with anxiety-depressive disorders. Patients underwent a month spa treatment while maintaining unchanged medication dosing with SSRI (selective serotonin reuptake inhibitors). The other investigated factors contributing to improving the health of treated subjects were amino-acid homocysteine and serotonin. The blood samples were collected at the beginning and the end of the spa treatment. Serotonin in all patients increased by a relative 23 % (results given as relative differences in percent), while homocysteine decreased by 17.1 %. Statistically significant increases were found in 21 steroids, which indicate activation of the adrenal cortex. It can be assumed, that the overall improvement in the mental condition of patients, which was proved by questionnaire from Knobloch and Hausner, the increase in immune suppressive substances and anti-autoimmune responses, will maintain for a longer time after the spa treatment.

Prenatal exposure to bisphenols and parabens and impacts on human physiology.

In modern societies, living organisms are exposed daily to multiform pollution from industrial chemical products. Some of these substances have been shown to affect the endocrine system, and have been termed endocrine disruptors (EDs). Bisphenol A (BPA), which can leach from plastics, and parabens, used in cosmetic products, are among the most well-studied. Prenatal development is a vulnerable phase of human life, and disruptions during this period may have lifelong consequences. Since EDs are known to cross the placental barrier and BPA may accumulate in the fetus, "BPA-free" products have been introduced to the market. However, such products often contain alternative bisphenols (e.g. BPS, BPF) that have not yet been extensively examined or regulated. Moreover, alternative bisphenols often occur together with BPA. The human organism is thus exposed to a mixture of EDs, some of which can have additive or synergic effects. Recent findings have also shown that paraben exposure can alter bisphenol pharmacokinetics. Taking into account the widespread occurrence of various EDs and the potential multiplicity of their effects, doses of EDs currently considered safe may not actually be as safe as they appear, especially during pregnancy.

Sex differences in the effect of prenatal testosterone exposure on steroid hormone production in adult rats.

Maternal hyperandrogenism during pregnancy might have metabolic and endocrine consequences on the offspring as shown for the polycystic ovary syndrome. Despite numerous experiments, the impact of prenatal hyperandrogenic environment on postnatal sex steroid milieu is not yet clear. In this study, we investigated the effect of prenatal testosterone excess on postnatal concentrations of luteinizing hormone, corticosterone and steroid hormones including testosterone, pregnenolone, progesterone, estradiol and 7beta-hydroxy-epiandrosterone in the offspring of both sexes. Pregnant rats were injected daily with either testosterone propionate or vehicle from gestational day 14 until parturition. The hormones were evaluated in plasma of the adult offspring. As expected, females had lower testosterone and higher pregnenolone, progesterone and estradiol in comparison to males. In addition, corticosterone was higher in females than in males, and it was further elevated by prenatal testosterone treatment. In males, prenatal testosterone exposure resulted in higher 7beta-hydroxy-epiandrosterone in comparison to control group. None of the other analyzed hormones were affected by prenatal testosterone. In conclusion, our results did not show major effects on sex hormone production or luteinizing hormone release in adult rats resulting from testosterone excess during their fetal development. However, maternal hyperandrogenism seems to partially affect steroid biosynthesis in sex-specific manner.

Changes to fetal steroidogenesis caused by maternal smoking.

Smoking during pregnancy presents health risks for both the mother and her child. In this study we followed changes in the production of steroid hormones in pregnant smokers. We focused on changes in steroidogenesis in the blood of mothers in their 37(th) week of pregnancy and in mixed cord blood from their newborns. The study included 88 healthy women with physiological pregnancies (17 active smokers and 71 non-smokers). We separately analyzed hormonal changes associated with smoking according to the sex of newborns. In women with male fetuses, we found higher levels of serum cortisone, dehydroepiandrosterone (DHEA), 7alpha-OH-DHEA, 17-OH pregnenolone, testosterone, and androstenedione in smokers at the 37(th) week compared to non-smokers. In women with female fetuses, we found lower serum levels of 7beta-OH-DHEA and higher androstenedione in smokers at the 37(th) week. We found significantly higher levels of testosterone in newborn males of smokers and higher levels of 7alpha-OH-DHEA in female newborns of smokers. Smoking during pregnancy induces changes in the production of steroids in both the mother and her child. These changes are different for different fetal sexes, with more pronounced changes in mothers carrying male newborns as well as in the newborn males themselves.