Eliška Sychrová

Estrogenic activity in extracts and exudates of cyanobacteria and green algae.

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.

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.

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.

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.