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
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.
- Klíčová slova
- Cyanobacteria, Exudates, Retinoic acid receptor, Retinoid-like activity, Retinoids,
- MeSH
- Aphanizomenon * MeSH
- fytoplankton MeSH
- Microcystis * MeSH
- retinoidy MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- retinoidy MeSH
Cyanobacteria are known for their ability to produce and release mixtures of up to thousands of compounds into the environment. Recently, the production of novel metabolites, retinoids, was reported for some cyanobacterial species along with teratogenic effects of samples containing these compounds. Retinoids are natural endogenous substances derived from vitamin A that play a crucial role in early vertebrate development. Disruption of retinoid signalling- especially during the early development of the nervous system- might lead to major malfunctions and malformations. In this study, the toxicity of cyanobacterial biomass samples from the field containing retinoids was characterized by in vivo and in vitro bioassays with a focus on the potential hazards towards nervous system development and function. Additionally, in order to identify the compounds responsible for the observed in vitro and in vivo effects the complex cyanobacterial extracts were fractionated (C18 column, water-methanol gradient) and the twelve obtained fractions were tested in bioassays. In all bioassays, all-trans retinoic acid (ATRA) was tested along with the environmental samples as a positive control. Retinoid-like activity (mediated via the retinoic acid receptor, RAR) was measured in the transgenic cell line p19/A15. The in vitro assay showed retinoid-like activity by specific interaction with RAR for the biomass samples. Neurotoxic effects of selected samples were studied on zebrafish (Danio rerio) embryos using the light/dark transition test (Viewpoint, ZebraLab system) with 120 hpf larvae. In the behavioural assay, the cyanobacterial extracts caused significant hyperactivity in zebrafish at 120 hpf after acute exposure (3 h prior to the measurement) at concentrations below the teratogenicity LOEC (0.2 g dw L-1). Similar effect was observed after exposure to fractions of the extracts with detected retinoid-like activity and additive effect was observed after combining the fractions. However, the effect on behaviour was not observed after exposure to ATRA only. To provide additional insight into the behavioural effects and describe the underlying mechanism gene expression of selected biomarkers was measured. We evaluated an array of 28 genes related to general toxicity, neurodevelopment, retinoid and thyroid signalling. We detected several affected genes, most notably, the Cyp26 enzymes that control endogenous ATRA concentration, which documents an effect on retinoid signalling.
- Klíčová slova
- ATRA, Biomarkers, Cyanobacteria, Locomotor response, Retinoids, Zebrafish,
- MeSH
- biomasa MeSH
- biotest MeSH
- chemické látky znečišťující vodu metabolismus toxicita MeSH
- chování zvířat účinky léků MeSH
- dánio pruhované růst a vývoj metabolismus MeSH
- embryo nesavčí účinky léků metabolismus MeSH
- exprese genu účinky léků MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- receptory kyseliny retinové genetika metabolismus MeSH
- sinice růst a vývoj metabolismus MeSH
- tretinoin metabolismus toxicita MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chemické látky znečišťující vodu MeSH
- receptory kyseliny retinové MeSH
- tretinoin 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.
- Klíčová slova
- All-trans retinoic acid, Cyanobacteria, Retinoid-like activity, Retinoids, Teratogenicity, Zebrafish,
- MeSH
- Aphanizomenon patogenita MeSH
- dánio pruhované embryologie genetika MeSH
- embryo nesavčí účinky léků MeSH
- Microcystis patogenita MeSH
- reportérové geny MeSH
- retinoidy biosyntéza toxicita MeSH
- sinice chemie patogenita MeSH
- teratogeny toxicita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
- Názvy látek
- retinoidy MeSH
- teratogeny MeSH
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.
- Klíčová slova
- ATRA, Cyanobacteria, Extract, Exudates, Retinoid-like activity, Retinoids,
- MeSH
- druhová specificita MeSH
- látky znečišťující vodu analýza MeSH
- monitorování životního prostředí metody MeSH
- retinoidy analýza MeSH
- sinice chemie růst a vývoj MeSH
- škodlivý vodní květ MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- látky znečišťující vodu MeSH
- retinoidy MeSH
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.
- Klíčová slova
- 9-Cis retinoic acid, All-trans-retinoic acid, Biomass extracts, Cyanobacteria, Exudates, Light conditions, Microcystin, Retinoic acid receptor, Retinoid-like activity,
- MeSH
- buněčné linie MeSH
- Chlorophyta chemie MeSH
- fytoplankton chemie metabolismus mikrobiologie MeSH
- Microcystis MeSH
- mikrocystiny MeSH
- retinoidy farmakologie MeSH
- rostlinné extrakty farmakologie MeSH
- sinice chemie MeSH
- tretinoin farmakologie MeSH
- viabilita buněk účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
- Názvy látek
- mikrocystiny MeSH
- retinoidy MeSH
- rostlinné extrakty MeSH
- tretinoin MeSH
