The frequencies of adverse outcomes associated with male reproductive health, including infertility and testicular cancer, are increasing. These adverse trends are partially attributed to increased exposure to environmental agents such as endocrine-disrupting chemicals (EDCs). This study addresses effects on EDCs on adjacent prepubertal Sertoli TM4 cells, specifically on 1) testicular gap junctional intercellular communication (GJIC), one of the hallmarks of non-genotoxic carcinogenicity, 2) GJIC building blocks connexins (Cx), and 3) mitogen-activated protein kinases MAPKs. We selected eight representatives of EDCs: organochlorine chemicals such as pesticides dichlorodiphenyltrichloroethane, lindane, methoxychlor, and vinclozolin, industrial chemicals bisphenol A and 2,2',4,4',5,5'-hexachlorobiphenyl, and components of personal care products, triclocarban and triclosan. EDCs rapidly dysregulated GJIC in Sertoli TM4 cells mainly via MAPK p38 and/or Erk1/2 pathways by the intermediate hyper- or de-phosphorylation of Cx43 (Ser368, Ser282) and translocation of Cx43 from the plasma membrane, suggesting disturbed intracellular trafficking of Cx43 protein. Surprisingly, EDCs did not rapidly activate MAPK Erk1/2 or p38; on the contrary, TCC and TCS decreased their activity (phosphorylation). Our results indicate that EDCs might disrupt testicular homeostasis and development via testicular GJIC, junctional and non-junctional functions of Cx43 and MAPK-signaling pathways in Sertoli cells.
- MeSH
- časové faktory MeSH
- endokrinní disruptory aplikace a dávkování metabolismus toxicita MeSH
- lidé MeSH
- metabolické nemoci chemicky indukované epidemiologie metabolismus MeSH
- rozmnožování účinky léků fyziologie MeSH
- vystavení vlivu životního prostředí škodlivé účinky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- úvodní články MeSH
- úvodníky MeSH
- MeSH
- antibakteriální látky metabolismus škodlivé účinky MeSH
- antiflogistika nesteroidní metabolismus škodlivé účinky MeSH
- Basidiomycota enzymologie metabolismus MeSH
- biodegradace MeSH
- celulosa metabolismus MeSH
- endokrinní disruptory metabolismus škodlivé účinky MeSH
- látky znečišťující půdu chemie MeSH
- látky znečišťující životní prostředí škodlivé účinky MeSH
- lignin metabolismus MeSH
- očkovadla agrotechnická MeSH
- perzistentní organické znečišťující látky * škodlivé účinky MeSH
- Pleurotus enzymologie metabolismus MeSH
- polychlorované bifenyly metabolismus škodlivé účinky MeSH
- polycyklické aromatické uhlovodíky metabolismus škodlivé účinky MeSH
- regenerace a remediace životního prostředí metody MeSH
- Publikační typ
- přehledy MeSH
The equilibrium dissociation constant of competitive antagonists represents the affinity of the receptor-ligand interaction, and it is a key characteristic of many therapeutic drugs or toxic compounds. Two commonly used methods by which the affinity of the antagonist can be estimated are Schild analysis and the Cheng-Prusoff method. However, both methods yield different results when applied to systems with slopes not equal to one. The Gaddum equation, which is fundamental for both methods, should be extended to incorporate the slope parameter of the dose-response curves and this extension should diminish the differences between the Schild and Cheng-Prusoff methods. In this study, we derived a novel form of the Gaddum equation with a slope parameter (Hill coefficient) of agonist dose-response curve. We also derived the subsequent equations for Schild and Cheng-Prusoff analysis and we validated the proposed model by the measurement of several known estrogen receptor competitive antagonists. Standardized in vitro yeast reporter gene assay (BMAEREluc/ERα) has been used for the measurements and the range of used antagonist concentrations was 1.37-46.03 μM. By applying our mathematical model, both Schild and Cheng-Prusoff methods provide more similar values of antagonist affinity than the original mathematical approach. The correctness of the model has also been demonstrated by the measurement of a partial agonist with a known receptor affinity. The presented mathematical model significantly reduces the differences in values calculated by the Cheng-Prusoff and Schild methods and yields more accurate estimations of antagonist affinity.
- MeSH
- alfa receptor estrogenů antagonisté a inhibitory metabolismus MeSH
- antagonisté estrogenového receptoru metabolismus farmakologie MeSH
- benzhydrylové sloučeniny metabolismus toxicita MeSH
- biologické modely * MeSH
- endokrinní disruptory metabolismus toxicita MeSH
- estradiol metabolismus farmakologie MeSH
- estrogeny metabolismus farmakologie MeSH
- fenoly metabolismus toxicita MeSH
- kompetitivní vazba MeSH
- lidé MeSH
- ligandy MeSH
- parciální agonismus léků MeSH
- reprodukovatelnost výsledků MeSH
- vazba proteinů MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Klíčová slova
- bisfenol A, obezogeny, tributyltin,
- MeSH
- diabetes mellitus 1. typu MeSH
- diabetes mellitus 2. typu MeSH
- diethylstilbestrol škodlivé účinky MeSH
- endokrinní disruptory * dějiny metabolismus škodlivé účinky toxicita MeSH
- fytoestrogeny škodlivé účinky MeSH
- kostní dřeň metabolismus patofyziologie MeSH
- kyseliny ftalové analýza škodlivé účinky MeSH
- lidé MeSH
- mužská infertilita chemicky indukované MeSH
- obezita MeSH
- parabeny škodlivé účinky MeSH
- sledování ekologických parametrů MeSH
- zákonodárství jako téma MeSH
- ženská infertilita chemicky indukované MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
OBJECTIVES: The aim of this study was to detect endocrine disruption potential of selected bisphenols and phthalates, compare in silico prediction with results from two in vitro methods and bring up-to-date information on development of EU legislation, available in vitro methods and biomechanisms involved in endocrine disruption. MATERIAL AND METHODS: In silico approach based on the OECD QSAR Toolbox was used for prediction of estrogen receptor α binding. OECD TG 455 assay and a yeast-based YES/YAS assay was used to determine the interactions with human estrogen (ERα) and androgen receptors. RESULTS: In silico results predicted the screened phthalates as non binders and bisphenols as very strong binders of the ERα. In vitro results differed from in silico prediction in several cases but exhibited concordance mainly for strong binders of ERα. Most of the substances exhibited parallel activity (agonist-antagonist) on both estrogen and androgen receptors. Agonistic studies showed the effective concentration of 10% activity (EC10) from 5.0E-07 for strong agonists (e.g. BPC, BPTMC). Cytotoxicity was observed after 48 h exposure of S. cerevisiae to BPFL, BPG, BPM, BPTMC in concentrations starting at 3.6E-05 mol/l. CONCLUSION: Our results suggest multiple parallel interactions of tested compounds and emphasize the importance of determination of an appropriate battery of in vitro methods that will include more receptors and will be appropriate to target specific molecular mechanisms involved in endocrine disruption. Results in agonistic studies indicate agonistic potential and are supported by results of antagonistic studies with consideration of possible multiple interactions.
- MeSH
- alfa receptor estrogenů metabolismus MeSH
- androgenní receptory metabolismus MeSH
- androgeny metabolismus MeSH
- biotest metody MeSH
- endokrinní disruptory metabolismus MeSH
- estrogeny metabolismus MeSH
- lidé MeSH
- receptory pro estrogeny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that interact in a complex manner with both the aryl hydrocarbon receptor (AhR) and estrogen receptors (ER). Their potential endocrine-disrupting activities may depend on both inhibitory AhR-ER cross-talk and on AhR-dependent metabolic production of estrogenic PAH metabolites. Here, we analyzed the impact of AhR on estrogen-like effects of PAHs, such as benzo[a]pyrene (BaP), in particular, on control of cell cycle progression/cell proliferation. Using AhR knockout variant of estrogen-sensitive human breast cancer MCF-7 cells (MCF-7 AhRKO cells), we observed that the AhR-dependent control of cytochrome P450 family 1 (CYP1) expression played a major role in formation of estrogenic BaP metabolites, most notably 3-OH-BaP, which contributed to the ER-dependent induction of cell cycle progression/cell proliferation. Both BaP metabolism and the BaP-induced S-phase transition/cell proliferation were inhibited in MCF-7 AhRKO cells, whereas these cells remained sensitive towards both endogenous estrogen 17β-estradiol or hydroxylated BaP metabolites. BaP was found to increase the activity of ER-dependent luciferase reporter gene in wild-type MCF-7 cells; however, unlike its hydroxylated metabolite, BaP failed to stimulate luciferase activity in MCF-7 AhRKO cells. Similarly, estrogen-like effects of other known estrogenic PAHs, such as benz[a]anthracene or 3-methylcholanthrene, were diminished in MCF-7 AhRKO cells. Ectopic expression of human CYP1A1 and CYP1B1 enzymes partly restored both BaP metabolism and its effects on cell proliferation. Taken together, our data suggest that the AhR-dependent metabolism of PAHs contributes significantly to the impact of PAHs on cell proliferation in estrogen-sensitive cells.
- MeSH
- buněčné kultury MeSH
- buněčný cyklus účinky léků genetika MeSH
- cytochrom P-450 CYP1A1 genetika metabolismus MeSH
- cytochrom P450 CYP1B1 genetika metabolismus MeSH
- endokrinní disruptory metabolismus toxicita MeSH
- exprese genu účinky léků MeSH
- genetické vektory MeSH
- genový knockdown MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- plazmidy MeSH
- polycyklické aromatické uhlovodíky metabolismus toxicita MeSH
- proliferace buněk účinky léků genetika MeSH
- receptory aromatických uhlovodíků genetika metabolismus MeSH
- receptory pro estrogeny genetika metabolismus MeSH
- reportérové geny MeSH
- transfekce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The white rot fungus Pleurotus ostreatus HK 35, which is also an edible industrial mushroom commonly cultivated in farms, was tested in the degradation of typical representatives of endocrine disrupters (EDCs; bisphenol A, estrone, 17β-estradiol, estriol, 17α-ethinylestradiol, triclosan and 4-n-nonylphenol); its degradation efficiency under model laboratory conditions was greater than 90% within 12 days and better than that of another published strain P. ostreatus 3004. A spent mushroom substrate from a local farm was tested for its applicability in various batch and trickle-bed reactors in degrading EDCs in model fortified and real communal wastewater. The reactors were tested under various regimes including a pilot-scale trickle-bed reactor, which was finally tested at a wastewater treatment plant. The result revealed that the spent substrate is an efficient biodegradation agent, where the fungus was usually able to remove about 95% of EDCs together with suppression of the estrogenic activity of the sample. The results showed the fungus was able to operate in the presence of bacterial microflora in wastewater without any substantial negative effects on the degradation abilities. Finally, a pilot-scale trickle-bed reactor was installed in a wastewater treatment plant and successfully operated for 10days, where the bioreactor was able to remove more than 76% of EDCs present in the wastewater.
The mechanisms contributing to toxic effects of airborne lower-chlorinated PCB congeners (LC-PCBs) remain poorly characterized. We evaluated in vitro toxicities of environmental LC-PCBs found in both indoor and outdoor air (PCB 4, 8, 11, 18, 28 and 31), and selected hydroxylated metabolites of PCB 8, 11 and 18, using reporter gene assays, as well as other functional cellular bioassays. We focused on processes linked with endocrine disruption, tumor promotion and/or regulation of transcription factors controlling metabolism of both endogenous compounds and xenobiotics. The tested LC-PCBs were found to be mostly efficient anti-androgenic (within nanomolar - micromolar range) and estrogenic (at micromolar concentrations) compounds, as well as inhibitors of gap junctional intercellular communication (GJIC) at micromolar concentrations. PCB 8, 28 and 31 were found to partially inhibit the aryl hydrocarbon receptor (AhR)-mediated activity. The tested LC-PCBs were also partial constitutive androstane receptor (CAR) and pregnane X receptor (PXR) agonists, with PCB 4, 8 and 18 being the most active compounds. They were inactive towards other nuclear receptors, such as vitamin D receptor, thyroid receptor α, glucocorticoid receptor or peroxisome proliferator-activated receptor γ. We found that only PCB 8 contributed to generation of oxidative stress, while all tested LC-PCBs induced arachidonic acid release (albeit without further modulations of arachidonic acid metabolism) in human lung epithelial cells. Importantly, estrogenic effects of hydroxylated (OH-PCB) metabolites of LC-PCBs (4-OH-PCB 8, 4-OH-PCB 11 and 4'-OH-PCB 18) were higher than those of the parent PCBs, while their other toxic effects were only slightly altered or suppressed. This suggested that metabolism may alter toxicity profiles of LC-PCBs in a receptor-specific manner. In summary, anti-androgenic and estrogenic activities, acute inhibition of GJIC and suppression of the AhR-mediated activity were found to be the most relevant modes of action of airborne LC-PCBs, although they partially affected also additional cellular targets.
- MeSH
- buněčné linie MeSH
- endokrinní disruptory metabolismus toxicita MeSH
- epitelové buňky účinky léků MeSH
- hydroxylace MeSH
- látky znečišťující vzduch toxicita MeSH
- lidé MeSH
- nádory metabolismus MeSH
- polychlorované bifenyly metabolismus toxicita MeSH
- receptory cytoplazmatické a nukleární metabolismus MeSH
- signální transdukce účinky léků MeSH
- steroidní receptory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Stagnant water bodies have generally received little attention regarding the presence of endocrine disruptive compounds, although they can integrate diverse pollutants from multiple different sources. Many compounds of anthropogenic as well as natural origin can contribute to the overall estrogenicity of surface waters and some of them can exhibit adverse effects on aquatic biota even in very low concentrations. This study focused on freshwater ponds and reservoirs affected by water blooms and determined the estrogenic activity of water by in vitro bioassay as well as concentrations of several important groups of estrogenic compounds (estrogenic hormones, alkylphenols, and phytoestrogens) by LC-MS/MS analyses. Estrogenic hormones were found at concentrations up to 7.1 ng.L-1, similarly to flavonoids, whose concentrations did not exceed 12.5 ng.L-1. Among alkylphenols, only bisphenol A and 4-tert-octylphenol were detected in levels reaching 100 ng.L-1 at maximum. Estrogenic activity of water samples varied from below the quantification limit to 1.95 ng.L-1. There does not seem to be any general causal link of the massive phytoplankton occurrence with the estrogenicity of water or concentration of phytoestrogens, since they showed no direct relationship with the phytoplankton abundance or composition across sites. The contribution of the analysed compounds to the estrogenic activity was calculated in three scenarios. In minimum scenario, just the compounds above quantification limit (LOQ) were taken into account and for most samples, only minor part (<6%) of the biological activity could be explained. In the mean and maximum scenarios, we included also compounds below LOQ into the calculations at the level of LOQ/2 and LOQ, respectively. In these cases, a considerable part of the estrogenic activity could be attributed to the possible presence of steroid estrogens below LOQ. However, for the samples with estrogenic activity greater than 1 ng.L-1, more than 50% of the estrogenic activity remained unexplained even in the maximum scenario. Probably other compounds or possible interactions between individual substances cause the estrogenic activity in these types of water bodies and in this case, the results of LC-MS/MS analyses cannot sufficiently predict the biological effects. A complex approach including bioassays is needed when assessing the estrogenicity of these types of surface waters.
- MeSH
- chemické látky znečišťující vodu analýza metabolismus MeSH
- chromatografie kapalinová MeSH
- endokrinní disruptory analýza metabolismus MeSH
- estrogeny analýza metabolismus MeSH
- fytoplankton chemie metabolismus MeSH
- sladká voda chemie MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
