Potential risk of estrogenic compounds produced by water blooms to aquatic environment.

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 17ß-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.

From fish to cells: Establishment of continuous cell lines from embryos of annual killifish Nothobranchius furzeri and N. kadleci.

There is a growing need of alternative experimental models that avoid or minimize the use of animals due to ethical, economical, and scientific reasons. Surprisingly, the stable embryonic cell lines representing Nothobranchius spp., emerging vertebrate models in aging research, regenerative medicine, ecotoxicology, or genomics, have been not derived so far. This paper reports establishment and deep characterization of ten continuous cell lines from annual killifish embryos of N. furzeri and N. kadleci. The established cell lines exhibited mostly fibroblast- and epithelial-like morphology and steady growth rates with cell doubling time ranging from 27 to 40 h. All cell lines retained very similar characteristics even after continuous subcultivation (more than 100 passages) and extended storage in liquid nitrogen (∼3 years). The cytogenetic analysis of the cell lines revealed a diploid chromosome number mostly equal to 38 elements (i.e., the native chromosome count for both killifish species), with minor but diverse line/passage-specific karyotype changes compared to the patterns observed in non-cultured N. furzeri and N. kadleci somatic cells. Based on transcriptional analysis of marker genes, the cell lines displayed features of an undifferentiated state without signs of senescence even in advanced passages. We confirmed that the cell lines are transfectable and can form viable 3-D spheroids. The applicability of the cell lines for (eco)toxicological surveys was confirmed by assessing the effect of cytotoxic and growth inhibitory agents. Properties of established Nothobranchius embryonic cell lines open new possibilities for the application of this model in various fields of life sciences including molecular mechanisms of aging, karyotype (in)stability or differences in lifespan.

Cyanobacterial Harmful Bloom Lipopolysaccharides Induce Pro-Inflammatory Effects in Immune and Intestinal Epithelial Cells In Vitro.

Freshwater cyanobacterial harmful blooms (CyanoHABs) produce a variety of toxic and bioactive compounds including lipopolysaccharides (LPSs). The gastrointestinal tract can be exposed to them via contaminated water even during recreational activities. However, there is no evidence of an effect of CyanoHAB LPSs on intestinal cells. We isolated LPSs of four CyanoHABs dominated by different cyanobacterial species and LPSs of four laboratory cultures representing the respective dominant cyanobacterial genera. Two intestinal and one macrophage cell lines were used to detect in vitro pro-inflammatory activity of the LPS. All LPSs isolated from CyanoHABs and laboratory cultures induced cytokines production in at least one in vitro model, except for LPSs from the Microcystis PCC7806 culture. LPSs isolated from cyanobacteria showed unique migration patterns in SDS-PAGE that were qualitatively distinct from those of endotoxins from Gram-negative bacteria. There was no clear relationship between the biological activity of the LPS and the share of genomic DNA of Gram-negative bacteria in the respective biomass. Thus, the total share of Gram-negative bacteria, or the presence of Escherichia coli-like LPSs, did not explain the observed pro-inflammatory activities. The pro-inflammatory properties of environmental mixtures of LPSs from CyanoHABs indicate their human health hazards, and further attention should be given to their assessment and monitoring.

Cyanobacteria, cyanotoxins and lipopolysaccharides in aerosols from inland freshwater bodies and their effects on human bronchial cells.

Components of cyanobacterial water blooms were quantified in aerosols above agitated water surfaces of five freshwater bodies. The thoracic and respirable aerosol fraction (0.1-10 µm) was sampled using a high-volume sampler. Cyanotoxins microcystins were detected by LC-MS/MS at levels 0.3-13.5 ng/mL (water) and < 35-415 fg/m3 (aerosol). Lipopolysaccharides (endotoxins) were quantified by Pyrogene rFC assay at levels < 10-119 EU/mL (water) and 0.13-0.64 EU/m3 (aerosol). Cyanobacterial DNA was detected by qPCR at concentrations corresponding to 104-105 cells eq./mL (water) and 101-103 cells eq./m3 (aerosol). Lipopolysaccharides isolated from bloom samples induced IL-6 and IL-8 cytokine release in human bronchial epithelial cells Beas-2B, while extracted cyanobacterial metabolites induced both pro-inflammatory and cytotoxic effects. Bloom components detected in aerosols and their bioactivities observed in upper respiratory airway epithelial cells together indicate that aerosols formed during cyanobacterial water blooms could induce respiratory irritation and inflammatory injuries, and thus present an inhalation health risk.

Estrogenic and retinoid-like activity in stagnant waters with mass occurrence of water blooms.

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.

In vitro testicular toxicity of environmentally relevant endocrine-disrupting chemicals: 2D vs. 3D models of prepubertal Leydig TM3 cells.

The testis is a priority organ for developing alternative models to assess male reproductive health hazards of chemicals. This study characterized a 3D in vitro model of murine prepubertal Leydig TM3 cells with improved expression of steroidogenesis markers suitable for image-based screening of testicular toxicity. This 3D scaffold-free spheroid model was applied to explore the impact of prototypical endocrine-disrupting chemicals (EDCs) and environmental reprotoxicants (benzo[a]pyrene, 2- and 9-methylanthracenes, fluoranthene, triclosan, triclocarban, methoxychlor) on male reproductive health. The results were compared to the male reprotoxicity potential of EDCs assessed in a traditional monolayer (2D) culture. The testicular toxicity was dependent not only on the type of culture (2D vs. 3D models) but also on the duration of exposure. Benzo[a]pyrene and triclocarban were the most active compounds, eliciting cytotoxic effects in prepubertal Leydig cells at low micromolar concentrations, which might be a mechanism contributing to their male reprotoxicity.

Potential estrogenic background in aquatic laboratory cultivations.

Aquatic biotests are important tools targeting various effects in ecotoxicology, including endocrine disruption. Unintentional exposure of bioassay organisms to endocrine disruptors during cultivation or testing may interfere with assessed endpoints. We illustrate this issue on the example of laboratory phytoplankton cultivation, where possible sources of estrogenic compounds have been revealed. Fifty-four blank samples (water and fresh or cultivated growth media) were assessed by in vitro biotests for their estrogenicity, and major known estrogens originating from plastic materials, bisphenol A and alkylphenols, were analyzed in selected samples. The samples of freshly prepared growth medium elicited weak estrogenic response in bioassays and some samples of the aerated media caused responses even above the 50% of maximum of the reference compound (17ß-estradiol, E2), while the samples from diverse laboratory water sources did not show significant estrogenic activity. The results identified substances contained in the growth medium as minor but reproducible contributors to estrogenicity in the cultivations. Sporadic but significant effects (up to 4.9 ng E2 equivalent/L) can be ascribed to compounds released from the used plastic materials during aeration of the cultivations. The potential sources of unintentional exposure to estrogenic compounds need to be considered in aquatic cultivations and biotests, since they could impact their outcomes, especially in arrangements assessing reproduction or whole life cycle biotests, or production of bioactive compounds by phytoplankton. The findings emphasize the necessity to assess all relevant blanks, ideally by sensitive high throughput in vitro assays that reflect also unknown pollutants and minimize all potential sources of background contamination. In vitro assays show very good applicability for this purpose since they enable to screen for any background estrogenicity of the used media and materials without the need of analyzing individual compounds, which often might not be known.

Endocrine-disrupting chemicals affect Sertoli TM4 cell functionality through dysregulation of gap junctional intercellular communication in vitro.

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.

Endocrine-disrupting chemicals rapidly affect intercellular signaling in Leydig cells.

A decline in male fertility possibly caused by environmental contaminants, namely endocrine-disrupting chemicals (EDCs), is a topic of public concern and scientific interest. This study addresses a specific role of testicular gap junctional intercellular communication (GJIC) between adjacent prepubertal Leydig cells in endocrine disruption and male reproductive toxicity. Organochlorine pesticides (lindane, methoxychlor, DDT), industrial chemicals (PCB153, bisphenol A, nonylphenol and octylphenol) as well as personal care product components (triclosan, triclocarban) rapidly dysregulated GJIC in murine Leydig TM3 cells. The selected GJIC-inhibiting EDCs (methoxychlor, triclosan, triclocarban, lindane, DDT) caused the immediate GJIC disruption by the relocation of gap junctional protein connexin 43 (Cx43) from the plasma membrane and the alternation of Cx43 phosphorylation pattern (Ser368, Ser279, Ser282) of its full-length and two N-truncated isoforms. After more prolonged exposure (24 h), EDCs decreased steady-state levels of full-length Cx43 protein and its two N-truncated isoforms, and eventually (triclosan, triclocarban) also tight junction protein Tjp-1. The disturbance of GJIC was accompanied by altered activity of mitogen-activated protein kinases MAPK-Erk1/2 and MAPK-p38, and a decrease in stimulated progesterone production. Our results indicate that EDCs might disrupt testicular homeostasis and development via disruption of testicular GJIC, a dysregulation of junctional and non-junctional functions of Cx43, activation of MAPKs, and disruption of an early stage of steroidogenesis in prepubertal Leydig cells. These critical disturbances of Leydig cell development and functions during a prepubertal period might be contributing to impaired male reproduction health later on.

Polycyclic Aromatic Hydrocarbons and Endocrine Disruption: Role of Testicular Gap Junctional Intercellular Communication and Connexins.

Ambient air pollution and smoking are well-documented risk factors for male infertility. Prevalent air pollutants and cigarette smoke components, polycyclic aromatic hydrocarbons (PAHs), are environmental and occupational toxicants that act as chemicals disrupting endocrine regulation and reproductive potential in males. Testicular gap junctional intercellular communication (GJIC) is critical for normal development and function of testicular tissue, thus we assessed GJIC as a process potentially targeted by PAHs in testes. Lower MW PAHs with a bay or bay-like region rapidly dysregulated GJIC in Leydig TM3 cells by relocalization of major testicular gap junctional protein connexin 43 (Cx43) from plasma membrane to cytoplasm. This was associated with colocalization between Cx43 and ubiquitin in intracellular compartments, but without any effect on Cx43 degradation rate or steady-state Cx43 mRNA levels. A longer exposure to active PAHs decreased steady-state levels of full-length Cx43 protein and its 2 N-truncated isoforms. Inhibition of GJIC by PAHs, similarly to a prototypic GJIC-inhibitor TPA, was mediated via the MAP kinase-Erk1/2 and PKC pathways. Polycyclic aromatic hydrocarbon-induced GJIC dysregulation in testes was cell-type-specific because neither PAH dysregulated GJIC in Sertoli TM4 cells, despite PAHs were rapidly taken up by both Leydig TM3 as well as Sertoli TM4 cells. Because TPA effectively dysregulated GJIC in both testicular cell types, a unique regulator of GJIC targeted by PAHs might exist in Leydig TM3 cells. Our results indicate that PAHs could be a potential etiological agent contributing to reproductive dysfunctions in males through an impairment of testicular GJIC and junctional and/or nonjunctional functions of Cx43.

Intracellular and extracellular retinoid-like activity of widespread cyanobacterial species.

Cyanobacterial species produce wide range of bioactive compounds. This study characterized production of retinoid-like compounds with embryotoxic and teratogenic potential by commonly occurring cyanobacterial species with tendency to form massive water blooms. The major goal was to simultaneously assess the intracellular and extracellular retinoid-like activity from several independent cultivations of one coccal (Microcystis aeruginosa) and four filamentous cyanobacteria (Aphanizomenon gracile, Cylindrospermopsis raciborskii, Limnothrix redekeii, and Planktothrix agardhii) and characterize the variability in its production among cultivations. The retinoid-like activity was evaluated by in vitro assay along with chemical analyses of nine retinoids: all-trans retinoic acid (ATRA), 9-cis retinoic acid (9cis-RA), 13cis-RA, 13cis-RA methyl ester, 5,6 epoxy-RA, 4keto-ATRA, 4keto-retinal, 4hydoxy-retinoic acid (4OH-ATRA), retinal and retinol. The production of retinoid-like compounds was recalculated per volume, per biomass dry weight and per cell to provide relevant data for risk assessment in relation to occurrence of massive water blooms in the environment. Total produced retinoid-like activity of five selected species ranged from 170 to 25,600ng ATRA-equivalents (REQ)/g dm corresponding to 0.001-0.392ng REQ/106 cyanobacterial cells. Results from chemical analyses showed that all tested extracts contained 4keto-ATRA and retinal. All-trans retinoic acid, 9/13cis-retinoic acid and 5,6 epoxy-retinoic acid were detected in most exudate and extract samples. The reported results of recalculated total retinoid-like activity enable potential predictions of its production by the studied species in water blooms of known cell densities relevant for risk assessment.

Phytoestrogens and sterols in waters with cyanobacterial blooms - Analytical methods and estrogenic potencies.

Compounds with estrogenic potencies and their adverse effects in surface waters have received much attention. Both anthropogenic and natural compounds contribute to overall estrogenic activity in freshwaters. Recently, estrogenic potencies were also found to be associated with cyanobacteria and their blooms in surface waters. The present study developed and compared the solid phase extraction and LC-MS/MS analytical approaches for determination of phytoestrogens (8 flavonoids - biochanin A, coumestrol, daidzein, equol, formononetin, genistein, naringenin, apigenin - and 5 sterols - ergosterol, ß-sitosterol, stigmasterol, campesterol, brassicasterol) and cholesterol in water. The method was used for analyses of samples collected in stagnant water bodies dominated by different cyanobacterial species. Concentrations of individual flavonoids ranged from below the limit of detection to 3.58 ng/L. Sterols were present in higher amounts up to 2.25 µg/L. Biological potencies of these phytoestrogens in vitro were characterized using the hERα-HeLa-9903 cell line. The relative estrogenic potencies (compared to model estrogen - 17ß-estradiol) of flavonoids ranged from 2.25E-05 to 1.26E-03 with coumestrol being the most potent. None of the sterols elicited estrogenic response in the used bioassay. Estrogenic activity was detected in collected field water samples (maximum effect corresponding to 2.07 ng/L of 17ß-estradiol equivalents, transcriptional assay). At maximum phytoestrogens accounted for only 1.56 pg/L of 17ß-estradiol equivalents, contributing maximally 8.5% of the total estrogenicity of the water samples. Other compounds therefore, most likely of anthropogenic origin such as steroid estrogens, are probably the major drivers of total estrogenic effects in these surface waters.

Characterization of total retinoid-like activity of compounds produced by three common phytoplankton species.

Phytoplankton can produce various bioactive metabolites, which may affect other organisms in the aquatic environment. This study provides the first information on the total retinoid-like activity associated with both intracellular and extracellular metabolites produced by selected phytoplankton species that could play a role in teratogenic effects and developmental disruption in exposed organisms. The studied species included a coccoid cyanobacteria (Microcystis aeruginosa), a filamentous cyanobacteria (Aphanizomenon gracile) and a green alga (Desmodesmus quadricauda), all of which commonly occur in freshwater bodies in Europe. Methanolic extracts from cellular material and extracellular exudates were prepared from cultures cultivated in two light-intensity variants with five replicates for each species. The retinoid-like activity was evaluated by in vitro assays along with chemical analyses of two potent retinoic acids (all-trans retinoic acid (ATRA) and 9cis-RA). The mean total retinoid-like activity of metabolites produced by the three studied species representing different phytoplankton taxonomic groups ranged from 705 to 5572ng ATRA equivalent/g dry matter corresponding to 0.064-0.234ng ATRA/106 cells. Retinoid-like activity was found in the cellular extracts of all species, while only the extracellular exudates of cyanobacteria exhibited detectable activity (41-1081ng ATRA/L). The greatest extracellular as well as total (extra- and intra- cellular together) retinoid-like activity was detected for Microcystis aeruginosa. The two potent retinoic acids studied were more frequently detected in cellular extracts than in extracellular exudates of all species. Their contribution to observed in vitro effects was relatively low for all tested samples (<10%), indicating a substantial contribution of other retinoid-like compounds to the overall activity. The results indicate possible influence of light intensity and cell density on the production of metabolites with retinoid-like activity and the cyanotoxin microcystin by the studied species. The recalculation of the results per dry weight, water volume, per 106 cells and biovolume enables a direct comparison of the retinoid-like activity distribution between extracts and exudates and the use of the data for risk assessment in water bodies.

Endocrine, teratogenic and neurotoxic effects of cyanobacteria detected by cellular in vitro and zebrafish embryos assays.

Cyanobacteria contain various types of bioactive compounds, which could cause adverse effects on organisms. They are released into surface waters during cyanobacterial blooms, but there is little information on their potential relevance for effects in vivo. In this study presence of bioactive compounds was characterized in cyanobacteria Microcystis aeruginosa (Chroococcales), Planktothrix agardhii (Oscillatoriales) and Aphanizomenon gracile (Nostocales) with selected in vitro assays. The in vivo relevance of detected bioactivities was analysed using transgenic zebrafish embryos tg(cyp19a1b-GFP). Teratogenic potency was assessed by analysis of developmental disorders and effects on functions of the neuromuscular system by video tracking of locomotion. Estrogenicity in vitro corresponded to 0.95-54.6 ng estradiol equivalent(g dry weight (dw))(-1). In zebrafish embryos, estrogenic effects could not be detected potentially because they were masked by high toxicity. There was no detectable (anti)androgenic/glucocorticoid activity in any sample. Retinoid-like activity was determined at 1-1.3 µg all-trans-retinoic acid equivalent(g dw)(-1). Corresponding to the retinoid-like activity A. gracile extract also caused teratogenic effects in zebrafish embryos. Furthermore, exposure to biomass extracts at 0.3 gd wL(-1) caused increase of body length in embryos. There were minor effects on locomotion caused by 0.3 gd wL(-1)M. aeruginosa and P. agardhii extracts. The traditionally measured cyanotoxins microcystins did not seem to play significant role in observed effects. This indicates importance of other cyanobacterial compounds at least towards some species or their developmental phases. More attention should be paid to activity of retinoids, estrogens and other bioactive substances in phytoplankton using in vitro and in vivo bioassays.