Ecotoxicological profiling of selected cyanobacterial strains using multi-endpoint effect-directed analysis.

The main goal of this study was to perform an ecotoxicological profiling of terrestrial and aquatic cyanobacterial strains found in different soils or in toxic cyanobacterial blooms in Vojvodina region, Serbia, using the effect-directed analysis (EDA) approach. The applied procedure was based on a series of in vitro or small-scale bioassays covering multiple endpoints in combination with advanced chemical analytical protocols. Non-selective and non-target preparation techniques were used for the extraction of a broad range of chemical compounds present in three terrestrial (Anabaena C2, Anabaena C5, Nostoc S8) and three aquatic (Nostoc Z1, Phormidium Z2, Oscillatoria K3) strains. Ecotoxicological endpoints addressed included evaluation of the fish cytotoxicity in vitro (acute toxicity), algal growth inhibition (chronic toxicity), and interaction with cellular detoxification mechanisms. All cyanobacterial strains tested in the 1st tier EDA showed significant effects in terms of chronic toxicity and interaction with cellular detoxification. Three major fractions of different polarities were further tested in the 2nd tier, using bioassays which showed the strongest response: induction of CYP1A1 biotransformation enzyme and inhibition of zebrafish organic anion (Oatp1d1) and cation (Oct1) uptake transporters. Oscillatoria K3 strain was selected for a more detailed 3rd tier EDA, and the obtained results revealed that positive sub-fractions possess polar anion and cation compounds that are reactive to both uptake transporters, and compounds responsible for the strongest effects have a pronounced lipophilic character. Apart from lipophilic non-polar compounds that represent typical phase I substrates, sub-fractions that contained polar substances are also shown to significantly induce CYP1A1.

Effect-directed analysis reveals inhibition of zebrafish uptake transporter Oatp1d1 by caulerpenyne, a major secondary metabolite from the invasive marine alga Caulerpa taxifolia.

Caulerpa taxifolia is a marine alga of tropical and subtropical distribution and a well-known invasive species in several temperate regions. Its invasiveness mainly stems from the production of secondary metabolites, some of which are toxic or repellent substances. In this study we investigated the possible inhibitory effects of C. taxifolia secondary metabolites on the activity of two zebrafish (Danio rerio) uptake transporters that transport organic anions (Oatp1d1) and cations (Oct1). Both transporters were transiently transfected and overexpressed in human embryonic kidney HEK293T cells. Transport activity assays using lucifer yellow (LY) and 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as model substrates were applied for the determination of Oatp1d1 and Oct1 interactors. A two-step Effect-Directed Analysis (EDA) procedure was applied for the separation and identification of compounds. We identified caulerpenyne (CYN) as the major metabolite in C. taxifolia and reveal its potent inhibitory effect towards zebrafish Oatp1d1 as well as weak effect on zebrafish Oct1 transport. The observed effect was confirmed by testing CYN purified from C. taxifolia, resulting in an IC50 of 17.97 µM, and a weak CYN interaction was also determined for the zebrafish Oct1 transporter. Finally, using Michaelis-Menten kinetics experiments, we identified CYN as a non-competitive inhibitor of the zebrafish Oatp1d1. In conclusion, this study describes a novel mechanism of biological activity in C. taxifolia, shows that CYN was a potent non-competitive inhibitor of zebrafish Oatp1d1, and demonstrates that EDA can be reliably used for characterization of environmentally relevant complex biological samples.

International round-robin study on the Ames fluctuation test.

An international round-robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate version of the classic plate-incorporation method for the detection of mutagenicity in water, wastewater and chemicals was performed by 18 laboratories from seven countries. Such a round-robin study is a precondition for both the finalization of the ISO standardization process and a possible regulatory implementation in water legislation. The laboratories tested four water samples (spiked/nonspiked) and two chemical mixtures with and without supplementation of a S9-mix. Validity criteria (acceptable spontaneous and positive control-induced mutation counts) were fulfilled by 92-100%, depending on the test conditions. A two-step method for statistical evaluation of the test results is proposed and assessed in terms of specificity and sensitivity. The data were first subjected to powerful analysis of variance (ANOVA) after an arcsine-square-root transformation to detect significant differences between the test samples and the negative control (NC). A threshold (TH) value based on a pooled NC was then calculated to exclude false positive test results. Statistically, positive effects observed by the William's test were considered negative, if the mean of all replicates of a sample did not exceed the calculated TH. By making use of this approach, the overall test sensitivity was 100%, and the test specificity ranged from 80 to 100%.

Evaluation of the genotoxic and cytochrome P450 monooxygenase-inhibitory potential of Dicuran on procaryotic and eucaryotic test systems.

The effect of the herbicide Dicuran 500 FL (formulated product) on the phenotypical and genotypical changes in procaryotic and eucaryotic organisms was investigated using short-term tests for detecting genotoxins. Since pesticides discharged in the water environment can modulate the mixed-function monooxygenases (MFO) detoxification system of water organisms, the in vivo and in vitro effects of Dicuran on hepatic cytochrome P450 (cyt P450) monooxygenase activities were also examined in juvenile carp (Cyprinus carpio L.). By measuring the activities of MFO in experimental carp exposed to Dicuran an attempt was made to establish whether Dicuran could be bioactivated by MFO into ultimate mutagens. Our results on the bacterial strains Salmonella typhimurium TA100 and TA98 show that Dicuran does not possess either mutagenic or premutagenic characteristics. The micronucleus test on the erythrocytes of experimental carp did not establish any clastogenic effect either. However, Dicuran significantly inhibited the MFO activity of 7-ethoxyresorufin-O-deethylase (EROD) and benzo[a]pyrene monooxygenase (BaPMO) in the liver of experimental carp in vitro, as well as in in vivo conditions. These findings demonstrate the potentially damaging effect of Dicuran on the xenobiotic metabolizing enzyme systems of fish, and suggest the applicability of described methods for the prediction of the ecotoxicological significance of the presence of pesticides in the water environment.

Interspecies differences in P-glycoprotein mediated activity of multixenobiotic resistance mechanism in several marine and freshwater invertebrates.

The presence and function of the P-glycoprotein mediated multixenobiotic resistance (MXR) mechanism was demonstrated in numerous aquatic organisms. The aim of this study was to investigate whether in aquatic organisms exists the inherent, species-specific basal level of MXR activity. Here the results of the direct comparison of the basal (noninduced) level of MXR activity measured in several marine (Mytilus galloprovincialis, Monodonta turbinata, Patella lusitanica) and freshwater (Dreissena polymorpha, Viviparus viviparus, Anodonta cygnea) molluscs species are presented. The primary criterion for the assessment and quantification of the basal level of MXR activity was the ratio (R) between the accumulation or efflux of the fluorescent model MXR substrates (rhodamine B or rhodamine 123) in or from the gills, measured with and in the absence of model MXR inhibitors verapamil or cyclosporin A. Significantly different levels of MXR activity were found in the species investigated. These levels generally show a relatively good correlation with the level of pollution present in their natural habitats. Considering these results a conclusion was reached that in aquatic organisms indeed exist the different inherent, species-specific levels of MXR activity. The identified levels might be, at least partly, responsible either for the resistance to, or for the sensitivity of a particular species to organic pollution.

The chemosensitizers of multixenobiotic resistance mechanism in aquatic invertebrates: a new class of pollutants.

Mechanism of multixenobiotic resistance (MXR), identical to multidrug resistance (MDR) in tumor cells, has been found in aquatic invertebrates. The presence of this ATP-dependent membrane P-glycoprotein (Pgp) pump was confirmed by biochemical ('binding'), molecular (immunohistochemical, Western, Northern), physiological (verapamil-sensitivity) and toxicological (modulation of toxicity) methods. The inducibility of MXR in the presence of xenobiotics and its wide taxonomic distribution suggests its role as a general biological defense mechanism that rescues organisms by pumping potentially toxic xenobiotics out of the cells. Some xenobiotics, the chemosensitizers, can inhibit this defense mechanism. The presence of these MXR-inhibitors has important implications on environmental parameters like exposure, uptake, internal dose, bioaccumulation, response, synergism and toxicity. Such MXR-inhibitors, for example, enhance the accumulation of carcinogenic aromatic amines in mussel, with subsequent enhancement in production of their mutagenic metabolites, in induction of single strand breaks in DNA, and in induction of DNA-adducts. The property to inhibit defense mechanism of organisms classifies MXR-inhibitors among top-hazardous environmental chemicals. Therefore, we measured the concentration of chemosensitizers in water concentrates or sediment extracts as their potential to modulate the accumulation of fluorescent dyes in a cell-culture of NIH 3T3 mouse fibroblasts stable transfected with human MDR1 gene, or as the potential of native waters to decrease the efflux-rate of Rhodamine B from gills of mussels. We found significantly higher concentrations of MXR-inhibitors in samples from polluted marine sites or from polluted rivers than in samples from corresponding unpolluted sites. These concentrations were able to enhance the accumulation of fluorescent dyes or carcinogenic aromatic amines in clams, mussels, snails and sponges exposed to these xenobiotics, demonstrating the ecotoxicological relevance of MXR-inhibitors present in polluted waters.

Inhibition of apoptosis is the cause of resistance to doxorubicin in human breast adenocarcinoma cells.

In our previous paper we have described the isolation and characterization of a doxorubicin (DOX) resistant subline of breast adenocarcinoma SC6 cells. These cells were obtained after the treatment with low, clinically relevant doses of doxorubicin. They became cross-resistant to different wide used cytostatics. The expression of several genes involved in mitotic signal transduction, as well as cathepsins D and L, was similar in both parental and doxorubicin treated cells. The aim of this study was to examine the molecular mechanisms involved in resistance of these cells to doxorubicin. Activity of plasma membrane Pgp was examined in parental and resistant cells due to rhodamine-accumulation assay. The involvement of glutathione (GSH) and glutathione S-transferase (GST) in resistance to doxorubicin was determined in MTT modified assay due to the addition of specific inhibitors: buthionine sulfoximine (for GSH) or ethacrynic acid (for GST). The kinetic of apoptosis was followed after the treatment with DOX in control and SC6 cells by fluorescent microscope. The occurrence of apoptosis was confirmed by analysing DNA fragmentation in agarose gel. Our results indicate that P-glycoprotein, glutathione or glutathione transferases were not involved in resistance of SC6 cells to doxorubicin. However, the apoptosis was inhibited in doxorubicin-resistant cells. Therefore, even low doses of doxorubicin can induce the resistance to this drug due to inhibition of apoptosis.