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•Bisphenols exhibited estrogenic activities in OECD validated transactivation assay.•Estrogenic activities of mixtures with BPA followed a concentration addition model.•‘Something ...from nothing’ phenomenon was confirmed for mixture of the six bisphenols.•Mixture effects should be considered in the risk assessment of chemicals.
Bisphenol A and its analogs are environmental contaminants with well known estrogenic and anti-androgenic activities. In studies of human biomonitoring, simultaneous exposure to multiple bisphenols was shown in different biological samples, at picomolar to low nanomolar concentrations. Evaluation of their combined toxicities will therefore be a more realistic and reliable predictor for estimation of health risks than evaluation of only the single chemicals. In the present study, estrogenic activities of individual bisphenols were evaluated, along with their binary and multicomponent mixtures including three- and four-component mixtures, using the Organisation for Economic Co-operation and Development validated transactivation assay with the hERα-Hela9903 cell line. Concentration-dependent estrogenic activity was confirmed for all of the tested bisphenols, in the nanomolar to micromolar range. Estrogenic activities of binary and multicomponent mixtures followed a concentration addition model. Although exposure to individual bisphenols remains below their effective doses, we demonstrate that as a mixture, they can contribute additively to toxicity. This study thus emphasizes the importance of mixture toxicity evaluation for risk assessment of compounds that act like the bisphenols.
Endocrine disrupting chemicals are long suspected to impair reproductive health. Bisphenol A (BPA) has estrogenic activity and therefore the capacity of interfering with endocrine pathways. No ...studies dissected its short-term effects on pregnancy and possible underlying mechanisms. Here, we studied how BPA exposure around implantation affects pregnancy, particularly concentrating on placentation and uterine remodeling. We exposed pregnant female mice to 50 µg/kg BPA/day or 0.1% ethanol by oral gavage from day 1 to 7 of gestation. High frequency ultrasound was employed to document the presence and size of implantations, placentas and fetuses throughout pregnancy. Blood velocity in the arteria uterina was analyzed by Doppler measurements. The progeny of mothers exposed to BPA was growth-restricted compared to the controls; this was evident in vivo as early as at day 12 as analyzed by ultrasound and confirmed by diminished fetal and placenta weights observed after sacrificing the animals at day 14 of gestation. The remodeling of uterine spiral arteries (SAs) was considerably impaired. We show that short-term exposure to a so-called "safe" BPA dose around implantation has severe consequences. The intrauterine growth restriction observed in more than half of the fetuses from BPA-treated mothers may owe to the direct negative effect of BPA on the remodeling of uterine SAs that limits the blood supply to the fetus. Our work reveals unsuspected short-term effects of BPA on pregnancy and urges to more studies dissecting the mechanisms behind the negative actions of BPA during early pregnancy.
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.
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•Estrogenic and retinoid-like activities commonly co-occur in stagnant freshwaters.•Freshwater phytoplankton contribute mainly to the retinoid-like activity.•Retinoid-like effects found in most biomass samples irrespective of dominant species•Cyanobacterial blooms are not major producers of estrogenic compounds.•Phytoplankton samples affected neural differentiation in vitro.
Reaction with soluble Mn(II) has been considered as a main decay pathway for superoxide in natural waters, accompanied by an important Mn redox cycling. In this study, the interaction of Mn(II) and ...humic acid (HA) was investigated in visible light irradiated water. Our results indicate that HA may play a dual role to act as a photosensitizer to produce superoxide anions (O2 –) and as a strong ligand to stabilize the Mn(III), forming soluble Mn(III)L species for substrate transformation. Furthermore, the reaction kinetics, products, and mechanisms of chlorophene (CP) and estradiol (E2) mixture in the Mn(II)/HA/visible light reaction systems were assessed. The removal of CP and E2 was enhanced by 24.3% and 13.2%, respectively, in mixture solution at initial concentration of 1.0 μM for each target contaminant, as compared to the case of single-compound degradation. Product identification and density functional theory calculations indicated that cross-coupling reaction of CP and E2 radicals was more likely to occur than the self-coupling reaction in mixture solution. In addition, estrogenic activities of initial reaction solution were also effectively decreased during the transformation process. These findings provide new insights into Mn(III)-mediated reactions to better understand the environmental fate of organic contaminant mixture in waters.
Endocrine active substances, including naturally occurring hormones and various synthetic chemicals have received much concern owing to their endocrine disrupting potencies. It is essential to ...monitor their environmental occurrence since these compounds may pose potential threats to biota and human health. In this study, yeast-based reporter assays were carried out to investigate the presence of (anti-)androgenic, (anti-)estrogenic, and (anti-)thyroid compounds in the aquatic environment in southern Taiwan. Liquid chromatography tandem mass spectrometry (LC–MS/MS) was also used to measure the environmental concentrations of selected endocrine active substances for assessing potential ecological risks and characterizing contributions to the endocrine disrupting activities. Bioassay results showed that anti-androgenic (ND–7489 μg L−1 flutamide equivalent), estrogenic (ND–347 ng L−1 17β-estradiol equivalent), and anti-thyroid activities were detected in the dissolved and particulate phases of river water samples, while anti-estrogenic activities (ND–10 μg L−1 4-hydroxytamoxifen equivalent) were less often found. LC–MS/MS analysis revealed that anti-androgenic and estrogenic contaminants, such as bisphenol A, triclosan, and estrone were frequently detected in Taiwanese rivers. In addition, their risk quotient values were often higher than 1, suggesting that they may pose an ecological risk to the aquatic biota. Further identification of unknown anti-androgenic and estrogenic contaminants in Taiwanese rivers may be necessary to protect Taiwan's aquatic environment.
•Anti-androgenic activities were firstly detected in Taiwan's surface waters.•Estrogenic and anti-thyroid activities were often found in Taiwanese rivers.•Estrone was the most contributing compounds to the estrogenic activities.•Estrone, triclosan, and bisphenol A in Taiwanese rivers may pose ecological risks.•Bioassays combined with LC–MS/MS can better assess endocrine disrupting activities.
Biological assays can evaluate the cumulative effect of a mixture, considering synergistic/antagonistic interactions and effects of unknown/unconsidered compounds. Therefore, their application could ...increase in the next years also to analyse biological samples.
The aim of this review is to discuss the methodological approach and the application of estrogenic activity assays in human biological samples. 75 research articles were analysed and divided according to whether they used these assays: i) to quantify the level of estrogens and/or as a biomarker of estrogenic status ii) as a biomarker of exposure to endocrine disrupting chemicals (EDCs).
For the first purpose, some authors extracted biological samples while others tested them directly without any treatment. The study of these methodologies outlined that the methodology applied influenced the specificity of analysis. The estrogenic activity biomarker was used to analyse physiological variations of estrogens, pediatric diseases, hormone-dependent diseases and estrogen suppression/enhancement after pharmaceutical treatments.
For the second purpose, some authors extracted samples while others tested them directly, some authors divided endogenous estrogens from xenoestrogens while others tested samples without separation. The analysis of these methodologies outlined some limitations related to the efficiency of extraction and the incorrect separation of some compounds. The studies which applied this EDC biomarker showed that it was correlated with some EDCs, it varied according to the exposure of the population and it allowed the identification of some relationships between EDC exposure and breast cancer, type 1 diabetes and adverse health effects on children.
In conclusion, the estrogenic activity of biological samples can be a useful tool: to quantify low levels of 17β-estradiol, to assess the combined effect of endogenous estrogens and xenoestrogens, to estimate the estrogenic status providing considerable insight into physiological or pathological conditions, to evaluate EDC presence implementing the existing knowledge about EDC exposure and adverse health effects.
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•Estrogenic activity assays can be used to quantify estrogens in biological samples.•Estrogenic activity assays can be used as a estrogenic status biomarker.•Estrogenic activity assays can be used as a EDC exposure biomarker.•Hormone-dependent diseases can be deepened by means of estrogenic activity assays.•Evaluation of human EDC exposure can be implemented by estrogenic activity assays.
Bisphenols are endocrine-disrupting chemicals ubiquitously present in the environment, with the consequent exposure to humans. In humans, bisphenols are metabolized to glucuronide and sulfate ...conjugates. Recent studies indicate that sulfation represents an important bisphenol metabolic pathway for the most vulnerable humans, such as the growing fetus. Our aim was to evaluate sulfation kinetics of commonly detected bisphenols in biological samples: bisphenol A (BPA), bisphenol S (BPS), and bisphenol F (BPF). Furthermore, we evaluated estrogenic agonist potencies and long-term stability of these bisphenol sulfates. BPS and BPF sulfates were prepared by chemical synthesis. Sulfation kinetics of the selected bisphenols were tested in pooled human liver cytosol, as a source for soluble phase II enzymes, including liver sulfotransferases, with quantification by LC-MS/MS. A validated transactivation assay using the hERα-Hela 9903 cell line was used to determine estrogenic agonist potencies. Moreover, BPA, BPS, and BPF sulfate stabilities were examined under various conditions and during storage. In vitro sulfation of BPA and BPS followed Michaelis–Menten kinetics; BPF sulfation followed a substrate inhibition model. Sulfation rates were comparable for these bisphenols, although their KM values indicated some large differences in affinities. BPA and BPS sulfates are not hERα agonists. The bisphenol sulfates can be considered stable for at least 2 days under these tested media conditions. These data indicate that bisphenol sulfation is an important route in their biotransformation. Compared to glucuronidation, these bisphenols show slower sulfation rates but similar KM values. BPA and BPS metabolic biotransformation by sulfation provides their detoxification as they are without estrogenic activities.
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•In vitro sulfation of BPA and BPS followed Michaelis–Menten kinetics.•BPF sulfation followed a substrate inhibition model.•BPA and BPS sulfates are not hERα agonists.•BPA, BPS, and BPF sulfates can be considered stable for at least 2 days.
Bisphenol A (BPA) is a widely used chemical that has been extensively studied as an endocrine-disrupting chemical (EDC). Other bisphenols sharing close structural features with BPA, are increasingly ...being used as alternatives, increasing the need to assess associated hazards to the endocrine system. In the present study, the estrogenic activity of BPA, bisphenol S (BPS) and bisphenol F (BPF) was assessed by using a combination of zebrafish-specific mechanism-based in vitro and in vivo assays. The three bisphenols were found to efficiently transactivate all zebrafish estrogen receptor (zfER) subtypes in zebrafish hepatic reporter cell lines (ZELH-zfERs). BPA was selective for zfERα while BPS and BPF were slightly more potent on zfERβ subtypes. We further documented the estrogenic effect in vivo by quantifying the expression of brain aromatase using a transgenic cyp19a1b-GFP zebrafish embryo assay. All three bisphenols induced GFP in a concentration-dependent manner. BPS only partially induced brain aromatase at the highest tested concentrations (>30µM) while BPA and BPF strongly induced GFP, in an ER-dependent manner, at 1–10µM. Furthermore, we show that BPF strongly induced vitellogenin synthesis in adult male zebrafish. Overall, this study demonstrates the estrogenic activity of BPA, BPF and BPS in different cell- and tissue-contexts and at different stages of development. Differences between in vitro and in vivo responses are discussed in light of selective ER activation and the fate of the compounds in the models. This study confirms the relevance of combining cellular and whole-organism bioassays in a unique model species for the hazard assessment of candidate EDCs.
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•Selective zfER activation by bisphenols in ZELH-zfER cell lines.•Induction of brain aromatase in transgenic cyp19a1b-GFP zebrafish embryos.•Different estrogenic potency in vitro and in vivo.•BPS could act through a distinct mode of action in vivo on brain aromatase.•Relevance of an integrated strategy to assess EDCs and their substitutes.
Organic-diffusive gradients in thin-film samplers (o-DGT), were developed and applied for accumulation of estrogen and estrogen-like compounds on a XAD18 resin and deployed in situ in the effluents ...of Beijing Gaobeidian Wastewater Treatment Plant (GWWTP) and Brussels North Wastewater Treatment Plant as well as in several aquatic systems in Belgium, including the Zenne River, the Belgian Oostende Harbor and the North Sea. Estrogenic compounds accumulate on the XAD18 resin and the estrogenic activity of the resin extract was measured with the Estrogen Responsive Elements-Chemically Activated LUciferase gene eXpression (ERE-CALUX) bioassay. With this result and by applying Fick's diffusion law, it is possible to calculate the estrogenic activity in the aquatic system, if the diffusion boundary layer (DBL) is known or negligible compared to the hydrogel diffusive layer thickness. The DBL thickness in our study varied from 0.010 to 0.023 cm and ignoring the DBL thickness would for instance, underestimate the estrogenic activity by 10–20%. Estrogenic activities in the secondary effluent of GWWTP were the highest (29 ± 4 ng E2-equivalents L−1), while the lowest level was found at the Belgian Oostende Harbor (0.05 ± 0.01 ng E2-equivalents L−1). Comparable estrogenic activities in water samples measured by o-DGT and grab sampling were obtained, confirming that o-DGT can be efficiently used in various aquatic systems. The advantage of our sampling and measuring method is that very low, time averaged estrogenic activities can be determined, with a minimum of sample treatment. The risk of sample contamination is very low as well as the cost of the whole analytical procedure.
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•DGT combined with ERE-CALUX was used to measure in situ water estrogenic activity.•Neglecting the DBL underestimates the estrogenic activity by 10–20%.•DGT and grab sampling give comparable results for the estrogenic activity assessment.•DGT is better suited for a time-weighted average (TWA) estrogenic activity.
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.
The CXCL‐test is a sensitive in vitro assay developed in T47D cell line to identify estrogenic properties of anthropogenic chemicals and to monitor pollution caused by endocrine disruptors. As the test sensitivity can be modified by several factors, multivariable linear model was employed as an innovative statistical approach for fast evaluation of contributing factors. It revealed that cell lines, media, sera, cell number, and cell density significantly affected CXCL12 secretion. This knowledge enables new interpretation of the test results.
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