Some phytoplankton species were shown to produce teratogenic retinoids. This study assessed the variability in the extracellular production of compounds with retinoid-like activity for 50 independent cultivations of wide spectra of species including 12 cyanobacteria (15 strains) and 4 algae of different orders. Extracellular retinoid-like activity was detected for repeated cultivations of six cyanobacteria. The results were consistent for some species including Microcystis aeruginosa and Aphanizomenon gracile. The detected retinoid-like activities ranged from below the limit of quantification of 16 ng/L to over 6 µg all-trans retinoic acid (ATRA) equivalent/L. Nontargeted virtual fractionation together with suspect screening approach enabled to identify some retinoid-like compounds in exudates, including ATRA, 9/13-cis retinoic acid, all-trans 5,6-epoxy retinoic acid, 4keto-ATRA, 4keto-retinal, 4hydroxy-ATRA, and retinal. Most of them were for the first time repeatedly detected in exudates of all studied algae (at ng/L levels) and cyanobacteria. Their relative potencies ranged from 0.018 (retinal) to 1 compared to ATRA. They accounted for less than 0.1-50% of total detected retinoid-like activity. The high detected activities and concentrations of retinoids in some samples and their direct accessibility from exudates document potential risk of developmental toxicity for organisms in proximity of massive water blooms.

• MeSH
• Aphanizomenon * MeSH
• Phytoplankton MeSH
• Microcystis * MeSH
• Retinoids MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cyanobacteria routinely release potentially harmful bioactive compounds into the aquatic environment. Several recent studies suggested a potential link between the teratogenicity of effects caused by cyanobacteria and production of retinoids. To investigate this relationship, we analysed the teratogenicity of field-collected cyanobacterial bloom samples by means of an in vivo zebrafish embryo test, an in vitro reporter gene bioassay and by the chemical analysis of retinoids. Extracts of biomass from cyanobacterial blooms with the dominance of Microcystis aeruginosa and Aphanizomenon klebahnii were collected from water bodies in the Czech Republic and showed significant retinoid-like activity in vitro, as well as high degrees of teratogenicity in vivo. Chemical analysis was then used to identify a set of retinoids in ng per gram of dry weight concentration range. Subsequent fractionation and bioassay-based characterization identified two fractions with significant in vitro retinoid-like activity. Moreover, in most of the retinoids eluted from these fractions, teratogenicity with malformations typical for retinoid signalling disruption was observed in zebrafish embryos after exposure to the total extracts and these in vitro effective fractions. The zebrafish embryo test proved to be a sensitive toxicity indicator of the biomass extracts, as the teratogenic effects occurred at even lower concentrations than those expected from the activity detected in vitro. In fact, teratogenicity with retinoid-like activity was detected at concentrations that are commonly found in biomasses and even in bulk water surrounding cyanobacterial blooms. Overall, these results provide evidence of a link between retinoid-like activity, teratogenicity and the retinoids produced by cyanobacterial water blooms in the surrounding environment.

• MeSH
• Aphanizomenon pathogenicity   MeSH
• Zebrafish embryology   genetics   MeSH
• Embryo, Nonmammalian drug effects   MeSH
• Microcystis pathogenicity   MeSH
• Genes, Reporter MeSH
• Retinoids biosynthesis   toxicity   MeSH
• Cyanobacteria chemistry   pathogenicity   MeSH
• Teratogens toxicity   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH

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.

• MeSH
• Aphanizomenon chemistry   MeSH
• Biological Assay MeSH
• Zebrafish embryology   genetics   metabolism   MeSH
• Embryo, Nonmammalian drug effects   MeSH
• Endocrine Disruptors toxicity   MeSH
• Animals, Genetically Modified embryology   genetics   metabolism   MeSH
• Microcystis chemistry   MeSH
• Neurotoxins toxicity   MeSH
• Cyanobacteria chemistry   MeSH
• Teratogens toxicity   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The environmental occurrence and concentrations of cyanobacterial toxins (cyanotoxins) were investigated in the Czech Republic. Concentrations of microcystins (MCs), cylindrospermopsin (CYN) or saxitoxins (STXs) were determined immunochemically by ELISA assays in 30 water samples collected from the surface layers of 19 reservoirs during the summer season of 2010. MCs were detected in 18 reservoirs and 83 % of samples, with median and maximal concentration being 1.5 and 18.6 μg/L, respectively. The high frequency of MC occurrence coincided with prevalence of cyanobacterium Microcystis sp., which was detected in 87 % samples, followed by Dolichospermum (Anabaena) sp. observed in 33 % samples. CYN was detected by ELISA only in one sample at a concentration of 1.2 μg/L. STXs presence was indicated for the first time in Czech water reservoirs when the toxins were found at low concentrations (0.03-0.04 μg/L) in two samples (7 %) collected from two different reservoirs, where STXs co-occurred with MCs and eventually also with CYN. In both STX-positive samples, the phytoplankton community was dominated by Microcystis sp., but Dolichospermum sp. and/or Aphanizomenon sp. were also present as putative producers of STX and/or CYN. Cyanotoxins commonly occurred in Czech water reservoirs, and MCs frequently at concentrations possibly associated with human health risks. MCs were the most prevalent and abundant cyanotoxins, but also other cyanotoxins were detected, though sporadically. Further research and regulatory monitoring of cyanotoxins other than MCs is therefore required.

• MeSH
• Anabaena isolation & purification   MeSH
• Aphanizomenon isolation & purification   MeSH
• Bacterial Toxins analysis   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Phytoplankton isolation & purification   MeSH
• Microcystis isolation & purification   MeSH
• Water Microbiology * MeSH
• Microcystins analysis   MeSH
• Environmental Monitoring methods   statistics & numerical data   MeSH
• Marine Toxins analysis   MeSH
• Neurotoxins analysis   MeSH
• Saxitoxin analysis   MeSH
• Cyanobacteria isolation & purification   MeSH
• Fresh Water chemistry   microbiology   MeSH
• Uracil analogs & derivatives   analysis   MeSH
• Water Supply analysis   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH

Here is presented some of the first information on interactions of compounds produced by cyanobacteria and green algae with estrogen receptor signaling. Estrogenic potency of aqueous extracts and exudates (culture spent media with extracellular products) of seven species of cyanobacteria (10 different laboratory strains) and two algal species were assessed by use of in vitro trans-activation assays. Compounds produced by cyanobacteria and algae, and in particular those excreted from the cells, were estrogenic. Most exudates were estrogenic with potencies expressed at 50% of the maximum response under control of the estrogen receptor ranging from 0.2 to 7.2 ng 17β-estradiol (E(2)) equivalents (EEQ)/L. The greatest estrogenic potency was observed for exudates of Microcystis aerigunosa, a common species that forms water blooms. Aqueous extracts of both green algae, but only one species of cyanobacteria (Aphanizomenon gracile) elicited significant estrogenicity with EEQ ranging from 15 to 280 ng 17β-estradiol (E(2))/g dry weight. Scenedesmus quadricauda exudates and extracts of Aphanizomenon flos-aquae were antagonistic to the ER when coexposed to E(2). The EEQ potency was not correlated with concentrations of cyanotoxins, such as microcystin and cylindrospermopsin, which suggests that the EEQ was comprised of other compounds. The study demonstrates some differences between the estrogenic potency of aqueous extracts prepared from the same species, but of different origin, while the effects of exudates were comparable within species. The observed estrogenic potencies are important namely in relation to the possible mass expansion of cyanobacteria and release of the active compounds into surrounding water.

• MeSH
• Aphanizomenon metabolism   MeSH
• Biological Assay MeSH
• Water Pollutants, Chemical metabolism   pharmacology   MeSH
• Chlorophyta metabolism   MeSH
• Endocrine Disruptors metabolism   pharmacology   MeSH
• Estradiol metabolism   pharmacology   MeSH
• Estrogens metabolism   pharmacology   MeSH
• Eutrophication MeSH
• Exudates and Transudates chemistry   MeSH
• Cell Communication physiology   MeSH
• Microcystis drug effects   MeSH
• Receptors, Estrogen metabolism   MeSH
• Cyanobacteria metabolism   MeSH
• Dose-Response Relationship, Drug MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Inhibition of gap junctional intercellular communication (GJIC) is affiliated with tumor promotion process and it has been employed as an in vitro biomarker for evaluation of tumor promoting effects of chemicals. In the present study we investigated combined effects of anthropogenic environmental contaminants 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) and fluoranthene, cyanotoxins microcystin-LR and cylindrospermopsin, and extracts of laboratory cultures of cyanobacteria Aphanizomenon gracile and Cylindrospermopsis raciborskii, on GJIC in the rat liver epithelial cell line WB-F344. Binary mixtures of PCB 153 with fluoranthene and the mixtures of the two cyanobacterial strains elicited simple additive effects on GJIC after 30 min exposure, whereas microcystin-LR and cylindrospermopsin neither inhibited GJIC nor altered effects of PCB 153 or fluoranthene. However, synergistic effects were observed in the cells exposed to binary mixtures of anthropogenic contaminants (PCB 153 or fluoranthene) and cyanobacterial extracts. The synergistic effects were especially pronounced after prolonged (6-24h) co-exposure to fluoranthene and A. gracile extract, when mixture caused nearly complete GJIC inhibition, while none of the individual components caused any downregulation of GJIC at the same concentration and exposure time. The effects of cyanobacterial extracts were independent of microcystin-LR or cylindrospermopsin, which were not detected in cyanobacterial biomass. It provides further evidence on the presence of unknown tumor promoting metabolites in cyanobacteria. Clear potentiation of the GJIC inhibition observed in the mixtures of two anthropogenic contaminants and cyanobacteria highlight the importance of combined toxic effects of chemicals in complex environmental mixtures.

• MeSH
• Aphanizomenon metabolism   MeSH
• Cell Extracts toxicity   MeSH
• Cell Line MeSH
• Cylindrospermopsis metabolism   MeSH
• Epithelial Cells drug effects   metabolism   MeSH
• Fluorenes toxicity   MeSH
• Carcinogens toxicity   MeSH
• Rats MeSH
• Environmental Pollutants toxicity   MeSH
• Gap Junctions drug effects   metabolism   MeSH
• Cell Communication drug effects   physiology   MeSH
• Microcystins toxicity   MeSH
• Polychlorinated Biphenyls toxicity   MeSH
• Drug Synergism MeSH
• Uracil analogs & derivatives   toxicity   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Toxicity and liver tumor promotion of cyanotoxins microcystins have been extensively studied. However, recent studies document that other metabolites present in the complex cyanobacterial water blooms may also have adverse health effects. In this study we used rat liver epithelial stem-like cells (WB-F344) to examine the effects of cyanobacterial extracts on two established markers of tumor promotion, inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) - ERK1/2. Extracts of cyanobacteria (laboratory cultures of Microcystis aeruginosa and Aphanizomenon flos-aquae and water blooms dominated by these species) inhibited GJIC and activated MAPKs in a dose-dependent manner (effective concentrations ranging 0.5-5mgd.w./mL). Effects were independent of the microcystin content and the strongest responses were elicited by the extracts of Aphanizomenon sp. Neither pure microcystin-LR nor cylindrospermopsin inhibited GJIC or activated MAPKs. Modulations of GJIC and MAPKs appeared to be specific to cyanobacterial extracts since extracts from green alga Chlamydomonas reinhardtii, heterotrophic bacterium Klebsiella terrigena, and isolated bacterial lipopolysaccharides had no comparable effects. Our study provides the first evidence on the existence of unknown cyanobacterial toxic metabolites that affect in vitro biomarkers of tumor promotion, i.e. inhibition of GJIC and activation of MAPKs.

• MeSH
• Enzyme Activation drug effects   MeSH
• Aphanizomenon chemistry   isolation & purification   MeSH
• Cell Line MeSH
• Time Factors MeSH
• Extracellular Signal-Regulated MAP Kinases metabolism   MeSH
• Phosphorylation drug effects   MeSH
• Carcinogens chemistry   toxicity   MeSH
• Complex Mixtures chemistry   toxicity   MeSH
• Rats MeSH
• Gap Junctions drug effects   MeSH
• Cell Communication drug effects   MeSH
• Microcystins analysis   chemistry   isolation & purification   toxicity   MeSH
• Mitogen-Activated Protein Kinases metabolism   MeSH
• Cyanobacteria chemistry   isolation & purification   MeSH
• Fresh Water microbiology   MeSH
• Uracil analogs & derivatives   toxicity   MeSH
• Dose-Response Relationship, Drug MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Comparative Study MeSH
• Geographicals
• Czech Republic MeSH