Estrogens in aquatic environments pose significant ecological and health risks due to their cumulative effects rather than individual impacts. This study investigates the voltammetric behavior of estrone (E1), 17β-estradiol (E2), estriol (E3), and 17α-ethinylestradiol (EE2), presenting a cost-effective and straightforward method for their simultaneous determination. Using differential pulse voltammetry (DPV) with a boron-doped diamond electrode, the method demonstrates high precision (deviations under 4%) and a linear dynamic range of 15.35-134.55 µmol·L-1. Integration of a vacuum evaporation step reduced detection limits to 10-8 mol·L-1, enabling effective analysis of real water samples. This optimized approach ensures practical applicability for monitoring total estrogen content in aquatic systems, providing an accessible and reliable alternative to conventional methods.

• Klíčová slova
• boron-doped diamond electrode, differential pulse voltammetry, estradiol, estriol, estrone, ethinylestradiol,
• MeSH
• chemické látky znečišťující vodu * analýza   MeSH
• elektrochemické techniky * metody   MeSH
• elektrody MeSH
• estradiol analýza   MeSH
• estriol analýza   MeSH
• estrogeny * analýza   MeSH
• estron analýza   MeSH
• ethinylestradiol analýza   MeSH
• limita detekce MeSH
• voda chemie   analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chemické látky znečišťující vodu * MeSH
• estradiol MeSH
• estriol MeSH
• estrogeny * MeSH
• estron MeSH
• ethinylestradiol MeSH
• voda MeSH

Some freshwater phytoplankton species have been suggested to produce estrogenic compounds in concentrations which could cause adverse effects to aquatic biota, while other studies showed no estrogenic effects after exposure to phytoplankton extracts or pointed out possible sources of the overestimation of the estrogenic activity. This study aimed to clarify these research inconsistencies by investigating estrogenicity of biomass extracts from both environmental freshwater blooms and laboratory cyanobacterial and algae cultures by in vitro reporter bioassay. Biomasses of 8 cyanobacterial and 3 algal species from 7 taxonomic orders were extracted and tested. Next to this, samples of environmental water blooms collected from 8 independent water bodies dominated by phytoplankton species previously assessed as laboratory cultures were tested. The results showed undetectable or low estrogenicity of both freshwater blooms and laboratory cultures with E2 equivalent concentration (EEQ) in a range from LOQ up to 4.5 ng EEQ/g of dry mass. Moreover, the co-exposure of biomass extracts with environmentally relevant concentration of model estrogen (steroid hormone 17β-estradiol; E2), commonly occurring in surface waters, showed simple additive interaction. However, some of the biomass extracts elicited partially anti-estrogenic effects in co-exposure with higher E2 concentration. In conclusion, our study documents undetectable or relatively low estrogenic potential of biomass extracts from both environmental freshwater blooms and studied laboratory cultured cyanobacterial and algae species. Nevertheless, in case of very high-density water blooms, even this low estrogenicity (detected for two cyanobacterial species) could lead to EEQ content in biomass reaching effect-based trigger values indicating potential risk, if recalculated per water volume at field sites. However, these levels would not occur in water under realistic environmental scenarios and the potential estrogenic effects would be most probably minor compared to other toxic effects caused by massive freshwater blooms of such high densities.

• Klíčová slova
• Anti-Estrogenicity, Aquatic ecosystem, Cyanobacteria, Estrogen, Freshwater bloom, Phytoplankton,
• MeSH
• estradiol toxicita   MeSH
• estrogeny * toxicita   analýza   MeSH
• estron MeSH
• fytoplankton MeSH
• sinice * MeSH
• voda MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• estradiol MeSH
• estrogeny * MeSH
• estron MeSH
• voda MeSH

Estrogen receptor alpha (ER) is a key biomarker for breast cancer, and the presence or absence of ER in breast and other hormone-dependent cancers decides treatment regimens and patient prognosis. ER is activated after ligand binding - typically by steroid. 2682 steroid compounds were used in a molecular docking study to identify novel ligands for ER and to predict compounds that may show anticancer activity. The effect of the most promising compounds was determined by a novel luciferase reporter assay. Two compounds, 7 and 12, showing ER inhibitory activity comparable to clinical inhibitors such as tamoxifen or fulvestrant were selected. We propose that the inhibitory effect of compounds 7 and 12 on ER is related to the presence of a double bond in their D-ring, which may protect against ER activation by reducing the electron density of the keto group, or may undergo metabolism leading to an active compound. Western blotting revealed that compound 12 decreased the level of ER in the breast cancer cell line MCF7, which was associated with reduced expression of both isoforms of the progesterone receptor, a well-known downstream target of ER. However, compound 12 has a different mechanism of action from fulvestrant. Furthermore, we found that compound 12 interferes with mitochondrial functions, probably by disrupting the electron transport chain, leading to induction of the intrinsic apoptotic pathway even in ER-negative breast cancer cells. In conclusion, the combination of computational and experimental methods shown here represents a rapid approach to determine the activity of compounds towards ER. Our data will not only contribute to research focused on the regulation of ER activity but may also be useful for the further development of novel steroid receptor-targeted drugs applicable in clinical practice.

• Klíčová slova
• Apoptosis, Docking of steroid library, Estrogen receptor alpha, Luciferase assay, Mitochondrial membrane potential,
• MeSH
• alfa receptor estrogenů genetika   metabolismus   MeSH
• estradiol farmakologie   terapeutické užití   MeSH
• estron * farmakologie   MeSH
• fulvestrant farmakologie   terapeutické užití   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prsu * farmakoterapie   metabolismus   MeSH
• receptory pro estrogeny metabolismus   MeSH
• simulace molekulového dockingu MeSH
• tamoxifen farmakologie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alfa receptor estrogenů MeSH
• estradiol MeSH
• estron * MeSH
• fulvestrant MeSH
• receptory pro estrogeny MeSH
• tamoxifen MeSH

Monitoring methodologies reflecting the long-term quality and contamination of surface waters are needed to obtain a representative picture of pollution and identify risk drivers. This study sets a baseline for characterizing chemical pollution in the Danube River using an innovative approach, combining continuous three-months use of passive sampling technology with comprehensive chemical (747 chemicals) and bioanalytical (seven in vitro bioassays) assessment during the Joint Danube Survey (JDS4). This is one of the world's largest investigative surface-water monitoring efforts in the longest river in the European Union, which water after riverbank filtration is broadly used for drinking water production. Two types of passive samplers, silicone rubber (SR) sheets for hydrophobic compounds and AttractSPETM HLB disks for hydrophilic compounds, were deployed at nine sites for approximately 100 days. The Danube River pollution was dominated by industrial compounds in SR samplers and by industrial compounds together with pharmaceuticals and personal care products in HLB samplers. Comparison of the Estimated Environmental Concentrations with Predicted No-Effect Concentrations revealed that at the studied sites, at least one (SR) and 4-7 (HLB) compound(s) exceeded the risk quotient of 1. We also detected AhR-mediated activity, oxidative stress response, peroxisome proliferator-activated receptor gamma-mediated activity, estrogenic, androgenic, and anti-androgenic activities using in vitro bioassays. A significant portion of the AhR-mediated and estrogenic activities could be explained by detected analytes at several sites, while for the other bioassays and other sites, much of the activity remained unexplained. The effect-based trigger values for estrogenic and anti-androgenic activities were exceeded at some sites. The identified drivers of mixture in vitro effects deserve further attention in ecotoxicological and environmental pollution research. This novel approach using long-term passive sampling provides a representative benchmark of pollution and effect potentials of chemical mixtures for future water quality monitoring of the Danube River and other large water bodies.

• Klíčová slova
• Bioanalytical equivalent, Concentration addition, Iceberg modelling, Mixture effects, Passive sampling, Risk prioritization,
• MeSH
• antagonisté androgenů MeSH
• chemické látky znečišťující vodu * toxicita   analýza   MeSH
• ekotoxikologie MeSH
• estron MeSH
• kvalita vody * MeSH
• monitorování životního prostředí metody   MeSH
• řeky chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antagonisté androgenů MeSH
• chemické látky znečišťující vodu * MeSH
• estron MeSH

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.

• Klíčová slova
• Aldosterone, Bisphenol, Estrogen, Nonylphenol, Paraben, Triclosan,
• MeSH
• benzhydrylové sloučeniny analýza   MeSH
• chromatografie kapalinová MeSH
• endokrinní disruptory * analýza   MeSH
• estrogeny analýza   MeSH
• estron analýza   MeSH
• lidé MeSH
• parabeny analýza   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• triclosan * analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• benzhydrylové sloučeniny MeSH
• endokrinní disruptory * MeSH
• estrogeny MeSH
• estron MeSH
• parabeny MeSH
• triclosan * MeSH

Stagnant freshwaters can be affected by anthropogenic pollution and eutrophication that leads to massive growth of cyanobacteria and microalgae forming complex water blooms. These can produce various types of bioactive compounds, some of which may cause embryotoxicity, teratogenicity, endocrine disruption and impair animal or human health. This study focused on potential co-occurrence of estrogenic and retinoid-like activities in diverse stagnant freshwaters affected by phytoplankton blooms with varying taxonomic composition. Samples of phytoplankton bloom biomass and its surrounding water were collected from 17 independent stagnant water bodies in the Czech Republic and Hungary. Total estrogenic equivalents (EEQ) of the most potent samples reached up to 4.9 ng·g-1 dry mass (dm) of biomass extract and 2.99 ng·L-1 in surrounding water. Retinoic acid equivalent (REQ) measured by in vitro assay reached up to 3043 ng·g-1 dm in phytoplankton biomass and 1202 ng·L-1in surrounding water. Retinoid-like and estrogenic activities at some sites exceeded their PNEC and effect-based trigger values, respectively. The observed effects were not associated with any particular species of cyanobacteria or algae dominating the water blooms nor related to phytoplankton density. We found that taxonomically diverse phytoplankton communities can produce and release retinoid-like compounds to surrounding water, while estrogenic potency is likely related to estrogens of anthropogenic origin adsorbed to phytoplankton biomass. Retinoids occurring in water blooms are ubiquitous signalling molecules, which can affect development and neurogenesis. Selected water bloom samples (both water and biomass extracts) with retinoid-like activity caused effects on neurodifferentiation in vitro corresponding to those of equivalent all-trans-retinoic acid concentrations. Co-occurrence of estrogenic and retinoid-like activities in stagnant water bodies as well as the potential of compounds produced by water blooms to interfere with neural differentiation should be considered in the assessment of risks associated with water blooms, which can comprise complex mixtures of natural and anthropogenic bioactive compounds.

• Klíčová slova
• Endocrine disruption, Estrogenic activity, Neural differentiation, Phytoplankton water bloom, Retinoid-like activity,
• MeSH
• estrogeny analýza   MeSH
• estron MeSH
• eutrofizace MeSH
• fytoplankton MeSH
• komplexní směsi MeSH
• lidé MeSH
• retinoidy * MeSH
• rostlinné extrakty MeSH
• sinice * MeSH
• tretinoin MeSH
• voda MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• estrogeny MeSH
• estron MeSH
• komplexní směsi MeSH
• retinoidy * MeSH
• rostlinné extrakty MeSH
• tretinoin MeSH
• voda MeSH

The fast-track process to approve vaccines against COVID-19 has raised questions about their safety, especially in relation to fertility. Over the last 2 years, studies have appeared monitoring female fertility, especially from assisted reproduction centers or in animal experiments. However, studies monitoring healthy populations are still limited. The aim of our study was to monitor the relevant parameters of female fertility (sex and other steroids, LH, FSH, SHBG, Antimüllerian hormone and antral follicle count) before and then 2-4 months after the third dose of vaccination against COVID-19 in a group of 25 healthy fertile woman. In addition, anti-SARS-CoV-2 and anti-SARS-CoV-2S antibodies were determined. We did not observe significant changes in the measured parameters before and after the third dose of vaccination. By comparing levels of the analytes with antibodies indicating a prior COVID-19 infection, we found that women who had experienced the disease had statistically lower levels of estrone, estradiol, SHBG and 5α-dihydroprogesterone, and conversely, higher levels of androgen active dehydroepiandrosterone and dihydrotestosterone. Our results confirm that vaccination does not affect female fertility, and that what fertile women should be worried about is not vaccination, but rather COVID-19 infection itself.

• Klíčová slova
• AMH, COVID-19, antral follicle count, infection, safety, steroids, vaccination, woman fertility,
• MeSH
• 20-alfa-dihydroprogesteron MeSH
• androgeny MeSH
• antimülleriánský hormon * MeSH
• COVID-19 * prevence a kontrola   MeSH
• dehydroepiandrosteron MeSH
• dihydrotestosteron MeSH
• estradiol MeSH
• estron MeSH
• fertilita MeSH
• folikuly stimulující hormon MeSH
• lidé MeSH
• vakcíny proti COVID-19 MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• 20-alfa-dihydroprogesteron MeSH
• androgeny MeSH
• antimülleriánský hormon * MeSH
• dehydroepiandrosteron MeSH
• dihydrotestosteron MeSH
• estradiol MeSH
• estron MeSH
• folikuly stimulující hormon MeSH
• vakcíny proti COVID-19 MeSH

In comparison with analytical tools, bioassays provide higher sensitivity and more complex evaluation of environmental samples and are indispensable tools for monitoring increasing in anthropogenic pollution. Nevertheless, the disadvantage in cellular assays stems from the material variability used within the assays, and an interlaboratory adaptation does not usually lead to satisfactory test sensitivities. The aim of this study was to evaluate the influence of material variability on CXCL12 secretion by T47D cells, the outcome of the CXCL-test (estrogenic activity assay). For this purpose, the cell line sources, sera suppliers, experimental and seeding media, and the amount of cell/well were tested. The multivariable linear model (MLM), employed as an innovative approach in this field for parameter evaluation, identified that all the tested parameters had significant effects. Knowledge of the contributions of each parameter has permitted step-by-step optimization. The most beneficial approach was seeding 20,000 cells/well directly in treatment medium and using DMEM for the treatment. Great differences in both basal and maximal cytokine secretions among the three tested cell lines and different impacts of each serum were also observed. Altogether, both these biologically based and highly variable inputs were additionally assessed by MLM and a subsequent two-step evaluation, which revealed a lower variability and satisfactory reproducibility of the test. This analysis showed that not only parameter and procedure optimization but also the evaluation methodology must be considered from the perspective of interlaboratory method adaptation. This overall methodology could be applied to all bioanalytical methods for fast multiparameter and accurate analysis.

• Klíčová slova
• T47D, cytokine CXCL12/SDF1, estrogenic activity cellular assay, material variability, multivariate linear model (MLM),
• MeSH
• biotest MeSH
• buněčné linie MeSH
• chemické látky znečišťující vodu * MeSH
• estrogeny * toxicita   MeSH
• estron MeSH
• lineární modely MeSH
• monitorování životního prostředí metody   MeSH
• reprodukovatelnost výsledků MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chemické látky znečišťující vodu * MeSH
• estrogeny * MeSH
• estron MeSH

A development of robust and rapid method with simple sample preparation for the analysis of steroids of C18-, C19-, C21- families is of interest of many research groups. Here we present a novel LC-MS/MS method for the simultaneous quantification of 32 steroid hormones in human plasma. Twenty-two of them were analyzed directly without the need for derivatization, while ten were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate. The steroids were separated on a C18 column with a gradient elution consisting of methanol and water with the addition of 0.1% formic acid. The mass spectrometer was operated in positive ESI mode. Validation demonstrated that the method was applicable for the quantitative analysis of two C18- steroids (estrone, estradiol), nineteen C19- steroids (testosterone, epitestosterone, dihydrotestosterone, 11-ketodihydrotestosterone, 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketotestosterone, dehydroepiandrosterone, 7α-hydroxydehydroepiandrosterone, 7β-hydroxydehydroepiandrosterone, 7-ketodehydroepiandrosterone, androsterone, epiandrosterone, androstenedione, androstenediol, 5α-androstane-3α,17β-diol, 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol, 5β-androstane-3β,17β-diol), and eleven C21- steroids (cortisol, 21-deoxycortisol, 11-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 5α-dihydroprogesterone). The lower limits of quantification are appropriate for analyses in both physiological and various pathophysiological conditions. The accuracy, intra- and inter-day precision values as well as stability tests were in accordance with FDA Guidelines. The method will be a useful tool in investigating the mechanisms of steroid-related diseases and will serve as a steppingstone for the development of other methods for steroid analyses in various biological matrices such as prostate tissue, cerebrospinal fluid, urine, seminal fluid, and saliva.

• Klíčová slova
• Human plasma, LC-MS/MS, Liquid chromatography, Mass spectrometry, Steroids,
• MeSH
• androgeny MeSH
• androstendion * MeSH
• chromatografie kapalinová metody   MeSH
• estron MeSH
• lidé MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• androgeny MeSH
• androstendion * MeSH
• estron MeSH

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17β-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17β-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

• Klíčová slova
• Bioassay, Chemical mixture, Endocrine disrupting compound (EDC), Environmental quality standard (EQS), Estrogenicity, Hormone mixture,
• MeSH
• biotest MeSH
• chemické látky znečišťující vodu * analýza   toxicita   MeSH
• endokrinní disruptory * analýza   MeSH
• estrogeny analýza   toxicita   MeSH
• estron MeSH
• monitorování životního prostředí MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chemické látky znečišťující vodu * MeSH
• endokrinní disruptory * MeSH
• estrogeny MeSH
• estron MeSH