Anna Sierosławska

Microcystin-LR modulates selected immune parameters and induces necrosis/apoptosis of carp leucocytes.

Microcystins (MCs) are potent hepatotoxins acting by the inhibition of protein phosphatase 1 and 2A, and may promote liver tumors. Moreover, studies also suggest they are nephrotoxic. The aim of the present study was to assess possible in vitro effects of microcystin-LR (which contains the amino acids leucine and arginine, the most widely studied and distributed variant of all microcystins) on the selected immune functions of the cells isolated from the head kidney of carp. In the experiments, pure microcystin-LR (MC-LR), was used at concentrations of 0.01, 0.1, 0.5, and 1 microg/ml RPMI-1640 medium. Leucocytes (lymphocytes and phagocytes) were isolated by centrifugation on a density gradient. Lymphocyte proliferation, intracellular production of reactive oxygen species by phagocytes, and the presence of apoptotic and/or necrotic cells were assessed. The respiratory burst activity of phagocytic cells was increased at the lowest toxin concentration used in the study, but it was decreased at higher concentrations. Using a sensitive luminescent immunoassay, MC-LR was observed to have no influence on the T-cell proliferation but decreased the proliferation of B lymphocytes. Moreover, it was noted that MC-LR induced necrosis to a higher degree than apoptosis in fish leucocytes. The results of the present study suggest the modulatory potency of microcystin-LR on fish leucocytes.

Toxicity of cyanobacterial bloom in the eutrophic dam reservoir (Southeast Poland).

Cyanobacterial bloom was observed in a highly eutrophic dam reservoir, Zemborzycki, near Lublin (SE Poland) over a warm period in the year 2007. The water bloom consisted of several cyanobacterial taxa: Anabaena circinalis, Anabaena spiroides, Anabaena flos-aquae, Planktothrix agardhii, Aphanizomenon flos-aquae, Aphanizomenon gracile, and Microcystis flos-aquae. Anabaena spp., and Aphanizomenon spp., potential producers of neurotoxic anatoxin-a, quantitatively predominated in the studied bloom. High-performance liquid chromatography (HPLC) analysis of surface scum sampled during Anabaena circinalis domination revealed the presence of anatoxin-a at a high concentration (1,035.59 microg per liter of surface scum). At the same time, neither gas chromatography/mass spectrometry (GC/MS) nor microcystin enzyme-linked immunosorbent assay (ELISA) test showed the presence of other frequently found cyanotoxins, microcystins. Toxicity of cyanobacterial bloom was assessed by the crustacean acute toxicity test Daphtoxkit F pulex using Daphnia pulex, and by the chronic toxicity test Protoxkit F with a ciliate protozoan Tetrahymena thermophila. The crude extract of cyanobacterial scum showed high toxicity for Daphnia pulex, with 24-h median effective concentration (EC50) value of 90.3 microg/L of anatoxin-a, which corresponded to the cyanobacterial density in the scum of 1.01 g dry weight/L. For Tetrahymena thermophila, 24-h EC50 was lower, evaluated to be 60.48 microg/L of anatoxin-a, which corresponded to a cyanobacterial density of 0.68 g dry weight/L of the scum. On the basis of evaluated toxicity units, the cyanobacterial extract was classified at class IV toxicity, which means high toxic hazard.

Effects of cylindrospermopsin on a common carp leucocyte cell line

Cylindrospermopsin (CYN) is a cytotoxin produced by different cyanobacterial species, increasingly detected in water reservoirs worldwide. There is very little information available concerning the effects of the toxin on fish immune cells. The aim of the study was to elucidate the potential impact of cylindrospermopsin on the selected parameters of a common carp (Cyprinus carpio L.) leucocyte cell line (CLC). The cells were incubated with the cyanotoxin at concentrations of 10, 1 or 0.1 µg ml–1 for up to 48 h. Cell viability and proliferation, apoptosis/necrosis induction, cell morphology and phagocytic activity were determined. The two higher toxin concentrations occurred to be evidently cytotoxic in a time‐dependent manner and influenced all studied parameters. The lowest used concentration had no effects on cell viability and cell number; however, a strong reduction of bacteria uptake after 24‐h exposure was detected. The obtained results indicate that cylindrospermopsin may interfere with the basic functions of fish phagocytic cells and as a consequence influence the fish immunity. Copyright

Cylindrospermopsin induces oxidative stress and genotoxic effects in the fish CLC cell line

Cylindrospermopsin (CYN) is a cyanotoxin detected in water reservoirs worldwide. The toxin is a potent protein synthesis inhibitor capable of adversely influencing a wide range of cell functions. While data on the prooxidative potency of CYN are inconsistent, genotoxic effects towards certain mammalian cell types have been described. However, such a potential on fish cells has not yet been investigated. Hence, the aim of the study was to elucidate the prooxidative and genotoxic impact of CYN on a common carp (Cyprinus carpio L.) leucocyte cell line (CLC). The cells were incubated with the cyanotoxin at concentrations of 0.1, 0.5 or 1 µg ml–1. After 24 h, cytotoxic activity of CYN at the highest used concentration was confirmed by decreased cell membrane integrity and inhibited cell proliferation. Additionally, CYN at 0.5 and 1 µg ml–1 increased intracellular ATP levels and decreased the reduced to oxidized glutathione ratio. Furthermore, a significant increase in the reactive oxygen species (ROS) production with concomitant changes in superoxide dismutase activity was observed after a 3.5‐h exposure of the cells to the toxin. Genotoxic activity of CYN, manifested as oxidative DNA damage and elevated number of micronuclei, was also detected in exposed cells. The obtained results indicate that CYN is able to exert a wide range of adverse effects, including oxidative stress and genotoxicity in fish leucocytes. Copyright

Effects of cylindrospermopsin on the phagocytic cells of the common carp (Cyprinus carpio L.).

Cylindrospermopsin is a cyanotoxin with cytotoxic activity. It is released into water during and after cyanobacterial water blooms and thus poses a threat to the health of fish. There is very little information available concerning the effects of the toxin on fish immune cells. In this study, we assessed the potential impact of cylindrospermopsin on the basic functions of phagocytic cells from common carp (Cyprinus carpio L.), including phagocytosis, reactive oxygen and nitrogen species production, and the structure of microfilaments and selected cytokine expression. Phagocytic cells, isolated from fish head kidneys, were exposed to the toxin at concentrations of 0.05, 0.1, 0.5 or 1 µg ml−1, for up to 24 h. Cytotoxicity, detected by lactate dehydrogenase release, was observed at the highest studied concentration. A decrease in phagocytic activity and changes in actin cytoskeletal structures were observed after the cell exposure to the toxin at 0.5 and 1 µg ml−1. Moreover, at all tested concentrations, cylindrospermopsin increased the production of reactive oxygen and nitrogen species. It also evidently influenced the expression of genes of proinflammatory cytokines interleukin‐1β and tumour necrosis factor‐α and, to a minor extent, anti‐inflammatory transforming growth factor‐β, but had no effects on interleukin‐10. The results indicated that the cyanotoxin cylindrospermopsin is able to modify basic features of carp phagocytic cells, which might result in adverse consequences for fish health. Copyright

Comparative studies on the cytotoxic effects induced by nodularin in primary carp leukocytes and the cells of the fish CLC line

ABSTRACT Nodularin, one of the toxins produced by cyanobacteria commonly occurring in brackish waters, is a cyclic pentapeptide, that may exert adverse effects on animal and human health. The aim of the present study was to evaluate the cytotoxicity of nodularin on different fish cellular models. The leukocytes isolated from carp head kidney and common carp (Cyprinus carpio L.) leukocyte cell line (CLC) were incubated with the cyanotoxin at the concentrations of 0.001, 0.01, 0.05 or 0.1&mgr;g/mL. Treatment of cells with the higher concentrations of the toxin resulted in a dramatic decrease in cell viability. Simultaneously, we observed an increase in the percentage of necrotic cells. Nodularin also induced apoptosis as demonstrated by increased caspase‐3/7 activity, and DNA fragmentation. We found, that primary cell cultures were more sensitive to the toxin than CLC cells. Our study also demonstrated significant increases in intracellular ROS production and decreases in GSH levels, observed in both primary leukocytes and CLC cells exposed to nodularin at 0.01–0.1&mgr;g/mL. These cells exhibited similar GSH/GSSG ratios and similar level of oxidized glutathione. HIGHLIGHTSPrimary cells were more sensitive to the nodularin than CLC cells.This cyanotoxin decreased cell viability and increased apoptosis in fish leukocytes in a concentration‐dependent manner.The toxin induced oxidative stress and dysregulated the glutathione‐redox state of the immune cells.

A representative of arylcyanomethylenequinone oximes effectively inhibits growth and formation of hyphae in Candida albicans and influences the activity of protein kinases in vitro

In this study, we applied various assays to reveal new activities of phenylcyanomethylenequinone oxime-4-(hydroxyimino) cyclohexa-2,5-dien-1-ylidene](phenyl)ethanenitrile (4-AN) for potential anti-microbial applications. These assays demonstrated (a) the antimicrobial effect on bacterial and fungal cultures, (b) the effect on the in vitro activity of the kinase CK2, (c) toxicity towards human erythrocytes, the Caco-2 cancer cell line, and embryonic development of Zebrafish. We demonstrated the activity of 4-AN against selected bacteria and Candida spp. The MIC ranging from 4 µg/ml to 125 µg/ml proved effective in inhibition of formation of hyphae and cell aggregation in Candida, which was demonstrated at the cytological level. Noteworthy, 4-AN was found to inhibit the CK2 kinase with moderate potency. Moreover, at low concentrations, it did not exert any evident toxic effects on human erythrocytes, Caco-2 cells, or Zebrafish embryos. 4-AN can be a potential candidate as a novel drug against Candida infections.

Application of Biotests in Cyanobacterial Extract Toxicity Assessment

Abstract The aim of the study was to determine the toxicity of the extract obtained from the cyanobacterial cells derived from the waters of Zemborzycki dam reservoir with use of a battery of biotests. The taxonomic identification of the bloom-forming cyanobacteria revealed high abundance of Aphanizomenon flos-aquae and Dolichospermum spp. (Anabaena spp.) and in a lower degree of Microcystis aeruginosa and Planktothrix agardhii. In the extract obtained from concentrated cyanobacterial cells, hepatotoxin microcystin-LR at a concentration of 22.89 ± 3.74 μg/L and neurotoxin Antx-a at 13.02 ± 0.01 μg/L have been detected. Toxicity of the extract was evaluated with the following assays: Daphtoxkit F magna with the crustacean Daphnia magna, Thamnotoxkit F with the crustacean Thamnocephalus platyurus, Rotoxkit F with the rotifer Brachionus calyciflorus and Protoxkit F with ciliate Tetrahymena thermophila. The most sensitive organism among all studied was T. platyurus for which EC50 was estimated to be 1.2% of the initial extract concentration. On the basis of the highest obtained value of the toxicity unit (TU = 83) the studied sample was classified to the IV class, which is of high acute toxicity. Additionally, it was found that reactivity on cyanobacterial products differs greatly among organisms used in bioassays, which indicate the need for using a set of biotests.