Kateřina Nováková

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.

Novel metabolites in cyanobacterium Cylindrospermopsis raciborskii with potencies to inhibit gap junctional intercellular communication

Despite intensive research into toxic bloom-forming cyanobacteria, the majority of their metabolites remain unknown. The present study explored in detail a novel bioactivity identified in cyanobacteria, i.e. inhibition of gap junctional intercellular communication (GJIC), a marker of tumor promotion. The extracellular mixture (exudate) of the cyanobacterial strain Cylindrospermopsis raciborskii (SAG 1.97) was fractionated by semi-preparative reversed phase HPLC, and the fractions assessed for their potencies to inhibit GJIC. Two non-polar fractions that significantly inhibited GJIC were further fractionated, tested and analyzed using multiple mass spectrometric methods. Investigations led to the identification of a putative chemical compound (molecular formula C18H34O3, m/z 299.2581 for the [M+H](+) ion) responsible for observed bioactivities. Specific inhibitors of signaling pathways were used to screen for biochemical mechanisms beyond GJIC inhibition, and the results indicate the involvement of ERK1/2 kinases via a mechanism related to the action of epidermal growth factor EGF but clearly distinct from other anthropogenic tumor promoters like polychlorinated biphenyls or polycyclic aromatic hydrocarbons. The chemical and in vitro toxicological characterizations of the newly described metabolite provide important insights into the still poorly understood health impacts of complex toxic cyanobacterial blooms and indicate that currently applied monitoring practices may underestimate actual risks.

A combined approach for the study of histone deacetylase inhibitors

Overexpression of histone deacetylases (HDACs), with consequent hypoacetylation of histones, is reportedly associated with transcriptional repression of tumour suppressor genes. Thus, inhibition of HDACs has emerged as a promising strategy in cancer therapy. In order to monitor the effects of potential HDAC inhibitors, a multi-level approach consisting of preliminary screening (measurement of HDAC activity and semi-quantitative evaluation of histone H4 modification profile by MALDI-TOF MS) and detailed analysis of histone modification forms (using 2-D AUT/AU PAGE and LC-ESI-IT MS) has been used in this study. The data obtained provide a global insight into the effects of HDAC inhibitors on the histone acetylation status that participates in gene transcription control. Using two example inhibitors, valproic acid sodium salt and entinostat, we show that similar levels of HDAC inhibition induced by different agents can lead to distinct rates of histone hyperacetylation, suggesting that except for the direct inhibition of HDACs, additional molecular mechanisms amplifying the response are likely to be involved in the inhibitory process. The approach used in our study makes it possible not only to follow the dynamics of individual histone modification forms, but also of their combined occurrence in the N-terminal fragment.

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.