Toxic potencies of metabolite(s) of non-cylindrospermopsin producing Cylindrospermopsis raciborskii isolated from temperate zone in human white cells

Chemosphere 120 (2015) 608–614

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Toxic potencies of metabolite(s) of non-cylindrospermopsin producing Cylindrospermopsis raciborskii isolated from temperate zone in human white cells Barbara Poniedziałek a,⇑, Piotr Rzymski a, Mikołaj Kokocin´ski b,c, Jacek Karczewski a a b c

´ , Poland Department of Biology and Environmental Protection, Poznan University of Medical Sciences, Poznan ´ , Poland Department of Hydrobiology, Faculty of Biology, Adam Mickiewicz University, Poznan Collegium Polonicum, Adam Mickiewicz University, Słubice, Poland

h i g h l i g h t s  The effect of non-CYN C. raciborskii extract & CYN was studied in human white cells.  Extract induced apoptosis and necrosis in white cells during 1 h exposure.  Pure CYN did not alter the viability of white cells during 1 h exposure.  Pure CYN revealed higher antiproliferative properties in lymphocytes culture.  C. raciborskii from temperate zone produce metabolite(s) toxic in human cells.

a r t i c l e

i n f o

Article history: Received 11 July 2014 Received in revised form 8 September 2014 Accepted 22 September 2014

Handling Editor: A. Gies Keywords: Cylindrospermopsis raciborskii Cylindrospermopsin Lymphocytes Neutrophils Whole blood

a b s t r a c t Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) has worldwide distribution and is well known for producing the toxic alkaloid, cylindrospermopsin (CYN). Strains unable to synthesize this compound but potentially toxic were recently identified in Europe. Here, for the first time the effect of cell-free extracts of a non-CYN-producing strain of C. raciborskii was studied in human cells (neutrophils and lymphocytes) isolated from healthy donors. The observed effects were compared to those induced by CYN (1.0–0.01 lg mL 1). Short-term (1 h) extract treatments resulted in altered viability of cells demonstrated by increased necrosis and apoptosis in neutrophils and elevated apoptosis in lymphocytes. CYN did not induce similar effects, regardless of the toxin concentration. Exposure of T-lymphocytes to 100% C. raciborskii extract in isolated and whole-blood 72 h cultures resulted in decrease of proliferation by 20.6% and 32.5%, respectively. In comparison, exposure to 1.0 lg mL 1 of CYN caused lymphocytes proliferation to be inhibited by 91.0% in isolated cultures and 56.5% in whole-blood assay. Significant antiproliferative properties were also found for 0.1 lg mL 1 of CYN in whole-blood culture. From the results we conclude that strains occurring in temperate zones may pose a threat to human health through the production of hitherto unknown metabolites that reveal a toxic pattern different to that of CYN. At the same time our study demonstrates that CYN is a powerful but slowly-acting toxin in human immune cells. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) has a wide geographical distribution, inhabits different climate zones and it has been identified in freshwater of South and North Amer⇑ Corresponding author at: Department of Biology and Environmental Protection, Poznan University of Medical Sciences, Rokietnicka 8, 60-806 Poznan´, Poland. Tel./ fax: +48 61 854 91 78. E-mail addresses: [email protected] (B. Poniedziałek), [email protected] (P. Rzymski). http://dx.doi.org/10.1016/j.chemosphere.2014.09.067 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

ica, Africa, Australia, Asia and Europe (Sukenik et al., 2012; Rzymski and Poniedziałek, 2014). It is predicted to spread to new environments as a result of a variety of reasons, including its biological plasticity (Soares et al., 2012), compensation of nitrogen deficiency through N2-fixing (Moisander et al., 2012), allelopathic potencies (Rzymski et al., 2014; Rzymski and Poniedziałek, 2014) cultural eutrophication (O’Neil et al., 2012) and climate changes (Sinha et al., 2012). C. raciborskii is best known for the production of cylindrospermopsin (CYN), a water-soluble alkaloid which demonstrates relatively high chemical stability, poor degradation (Chiswell et al., 1999; Wörmer et al., 2008; Klitzke and Fastner,

B. Poniedziałek et al. / Chemosphere 120 (2015) 608–614

2012), and a high share of extracellular form in the total CYN quota determined in water bodies (Rücker et al., 2007). CYN production has also been associated with other cyanobacterial species, members of genera Aphanizomenon, Anabaena, Raphidopsis, Umezakia and Oscillatoria (Rzymski and Poniedziałek, 2014). CYN exhibits high toxicity potencies in different types of eukaryotic cells (Poniedziałek et al., 2012a). It was found to induce DNA damage in various cell lines, including lymphoblastoid WIL2-NS (Humpage et al., 2000), differentiated HepaRG and colon-derivated Caco-2 (Bazin et al., 2010). It was also found to disrupt endocrine function by inhibiting basal and hCG-stimulated progesterone in human granulosa cells (Young et al., 2008). Moreover, CYN demonstrates immunomodulatory potencies and can suppress functions of human T-lymphocytes (Poniedziałek et al., 2012b, 2014a) and neutrophils (Poniedziałek et al., 2014b). Experiments involving rodent models demonstrated that CYN can affect various organs, including adrenal glands (Hawkins et al., 1985), thymus, heart (Terao et al., 1994), and lungs (Oliveira et al., 2012) when administrated orally or intraperitoneally. A skin-sensitizing action of CYN was also found (Torokne et al., 2001; Stewart et al., 2006). The toxin has been also demonstrated to be potentially toxic to aquatic organisms. It was found to disrupt the function of common carp leucocyte line (Sierosławska and Rymuszka, 2014) and induce oxidative stress in fish after single dose (Gutiérrez-Praena et al., 2011) and subchronic exposures (Guzmán-Guillén et al., 2014). There is evidence that CYN-producing C. raciborskii and pure CYN can be toxic to aquatic invertebrates (Fabbro et al., 2001; Metcalf et al., 2002; Nogueira et al., 2004). Finally, two documented epidemic cases of human poisoning revealed its high degree of hepatotoxicity and nephrotoxicity (Carmichael et al., 2001; Griffiths and Saker, 2003). A provisional safety level for CYN in freshwater was set at 1.0 lg L 1 (Humpage and Falconer, 2003) but the concentrations observed in the environment can largely exceed 100 lg L 1 (Rzymski and Poniedziałek, 2014; Messineo et al., 2010; Saker and Eaglesham, 1999). Despite the relatively large contribution of C. raciborskii in global CYN production, African and European strains of this species were not found to synthesize this compound; the phenomenon resulting from a lack of sulfotransferase cyrJ gene in cyr cluster (Haande et al., 2008; Poniedziałek et al., 2012a; Rzymski and Poniedziałek, 2014). Nevertheless, some non-CYN-producing strains have been shown to exhibit harmful effects in different eukaryotic systems. For example, Hungarian strains isolated from Lake Balaton and not producing any known cyanotoxin, demonstrated significant toxicities in crustaceans (Acs et al., 2013) and induced membrane damage in hamster CHO-K1 cell line (Antal et al., 2011). Crude extracts of other Hungarian strains modulated the acetylcholine receptors of the Helix pomata neurons, indicating neuroactive potencies of its metabolites (Vehovszky et al., 2013). Furthermore, the ability of non-CYN-producing C. raciborskii exudates to inhibit gap junctional intercellular communication (Nováková et al., 2012), an important non-genotoxic mechanism associated with tumour promotion and cancer (Trosko, 2007), was also demonstrated. Toxicological assessment of strains isolated from Portuguese freshwater revealed hepatotoxic (hepatocellular necrosis and eosinophilic deposits) and neurotoxic (lethargy, piloerection, and difficulty breathing) action of crude extracts in the mouse assay despite the absence of CYN production (Saker et al., 2003). French and German non-CYN-producing strains of C. raciborskii were also found to reveal hepatotoxicity in rodents (Bernard et al., 2003; Fastner et al., 2003). Finally, the bioactive potencies of the non-CYN-producing strain were also demonstrated in experiments involving other cyanobacteria species, Microcystis aeruginosa. As found, spent C. raciborskii medium decreased growth and increased the activity of alkaline phosphates

609

in tested organisms; the effects were also found to be induced by purified CYN (Rzymski et al., 2014). These findings altogether strongly indicate that non-CYN-producing C. raciborskii strains found in the temperate zone may produce and release hitherto unknown compounds posing a relevant ecotoxicological risk. The following study was undertaken to evaluate the toxic potencies of aqueous extracts obtained from a non-CYN-producing C. raciborskii strain isolated in Poland. As an in vitro experimental model we used human white cells, lymphocytes and neutrophils, isolated from healthy donors. Investigations were conducted on isolated cultures and whole-blood and compared with the effects induced by purified CYN. Our study demonstrates that non-CYNproducing C. raciborskii strain in Poland contains unknown metabolite(s) with potential toxicities in human cells. 2. Material and methods 2.1. Culture conditions The C. raciborskii strain (Fig. 1) was previously isolated under a Carl Zeiss light microscope (magnification 4000) from the environmental samples collected from Lake Bytyn´skie (MankiewiczBoczek et al., 2012). Single filaments were collected using a Pasteur glass pipette and transferred to culture flasks containing sterile BG-11 media (Sigma–Aldrich, USA). This procedure was repeated until monocultures of the cyanobacteria were obtained. The isolates were incubated in 250 mL Erlenmeyer flasks containing 150 mL of sterile BG-11 medium in an incubation chamber (POLEKO, Poland) at 21 °C under 80 lmol m 2 s 2 irradiance using cool white fluorescent light with a photoperiod regime of 12 h dark and 12 h light. Previous study had demonstrated the C. raciborskii strain as unable to produce CYN (Kokocin´ski et al., 2013). 2.2. Extract preparation Cell-free extracts of C. raciborskii were prepared by subjecting cell suspensions (325 600 trichomes per mL) to ultrasonic treatment (UD-20, Techpan, Poland) on ice (2 min in 2 cycles with 60 s break). Complete cell lyses was confirmed by microscopic examination. The broken cell suspensions were centrifuged (12 000 g, 10 min). The supernatants were sterilized by filtration on 0.22 lm syringeless filter devices (Roth) and stored at 20 °C until use.

Fig. 1. Micrograph of the Polish C. raciborskii strain investigated in this study.

610

B. Poniedziałek et al. / Chemosphere 120 (2015) 608–614

2.3. Blood collection Heparinized blood samples (6.0 mL) were collected in lithium heparin tubes from 10 healthy (screened by physical examination, medical history and initial blood tests), non-smoking and normal weighted (BMI 18.5–24.9) donors (aged 21–28, 5 female, 5 male) at the Regional Center of Blood and Blood Treatment in Poznan, Poland, according to accepted safeguard standards and legal requirements. 2.4. Experimental design To assess the toxic potencies of aqueous C. raciborskii extracts in human white cells, two independent sets of experiments (each using blood collected from 10 different healthy donors) were conducted: (i) Samples of human whole-blood (45 lL) were exposed for 1 h to three extract concentrations: 100% (corresponding to 325 600 trichomes per mL), 10% (corresponding to 32 560 trichomes per mL) and 1% (corresponding to 3256 trichomes per mL) and three concentrations 1.0, 0.1 and 0.01 lg mL 1) of purified CYN (>95%, HPLC. Alexis Biochemicals, USA) isolated from an Australian C. raciborskii strain for 1 h. Incubation was carried out in a CO2 incubator under controlled conditions (5% CO2, 37 °C, 95% humidity). Following the exposure, viability of lymphocytes and neutrophils was evaluated. (ii) Human lymphocytes were exposed to 100%, 10% and 1% C. raciborskii cell-free extract concentrations added at the beginning of a 72 h culture of isolated cells or whole-blood. Additionally, using a whole-blood assay, lymphocytes were exposed to 1.0, 0.1 and 0.01 lg mL 1 of CYN. The effects of CYN in these concentrations in isolated culture have already been reported in our previous study (Poniedziałek et al., 2014a) and were used here only for comparison. After 72 h of cultures, the rate of lymphocyte proliferation was evaluated. 2.5. Lymphocyte isolation and culture Mononuclear cells (lymphocytes and monocytes) were isolated from blood under sterile conditions by centrifugation (30 min, 1750 rpm, g = 420) on Gradisol-L (Aqua-Med, Poland) and washed twice in Eagle’s medium (Biomed, Poland). The isolated cells suspension (1  106 cells per mL 1) was supplemented with 10% fetal bovine serum (Sigma Chemicals, USA) and antibiotic (gentamycine at 50 lg mL 1, Sigma Chemicals, USA). Purity of lymphocytes cultures was verified by counting under a light microscope, and by flow cytometry using CD3- and CD19-specific antibodies conjugated with fluorescein isothiocyanate and R-phycoerythrin, respectively (Loken et al., 2000), and exceeded 90% (n = 10). Lymphocyte cultures were established in a 96-well microplate (200 lL aliquots per well) and were incubated in a CO2 incubator under controlled conditions (5% CO2, temp. 37 °C, humidity 95%). Each culture was prepared in triplicate for further experimental procedures. 2.6. Whole-blood culture For whole-blood assay, 1:10 dilutions were made in sterile Eagle’s medium (Biomed, Poland) supplemented with 10% fetal bovine serum (Sigma Chemicals, USA) and gentamycine (concentration of 50 lg mL 1, Sigma Chemicals, USA), and were then seeded in 96-well microplates at 200 lL/well and incubated in a CO2 incubator under controlled conditions (5% CO2, temp. 37 °C,

humidity 95%). Each culture was prepared in triplicate for further experimental procedures. 2.7. Lymphocytes and neutrophils viability assay After 1 h of incubation, samples were labelled fluorescently for detection of apoptotic and necrotic cells by adding 20 lL of binding buffer, 5 lL of Annexin V-FITC and 5 lL of propidium iodine (Pharmingen, San Diego, CA). Samples were mixed gently and incubated at 25 °C in the dark for 15 min. Next, 1 mL of red blood cell lysis solution (Miltenyi Biotec, USA) was added and after 10 min, all samples were analysed by flow cytometry (CyFlow Space flow cytometer with 488 nm excitation, Partec GmbH, Germany) using FL1 (Annexin V-FITC) and FL3 (propidium iodine) channels. Using a log FL-1 versus log FL-3 quadrant dot-plot, three cell subpopulations were identified: alive, apoptotic and necrotic. The position of sub-populations was first determined using campothecin, a known apoptosis inducer. Three technical replicates were performed for each sample, the obtained values were averaged. 2.8. Lymphocyte proliferation assay Lymphocyte proliferation was measured after 72 h culture of isolated cells or whole-blood, using an assay with tritiated thymidine (Amersham, UK) which was added in 1 lCi per well concentration for the last 24 h of cultivation. Cultures were then transferred using the harvester (Skatron Instruments, Norway) on glass fiber filters (Perkin Elmer, USA) and placed in a scintillation cocktail (Perkin Elmer, USA). Measurement of thymidine incorporation was determined using a scintillation counter (Perkin Elmer, USA). Proliferation rate was expressed as counts per minute (CPM). 2.9. Statistical analyses Statistical analyses were performed on raw data using the Statistica 10.0 software package (StatSoft, USA). Because the data did not meet the assumption of Gaussian distribution (tested with Shapiro–Wilk method), the non-parametric Wilcoxon signed-rank test was employed to compare the studied samples with the control; p < 0.05 was considered as significant. 3. Results 3.1. Viability of lymphocytes and neutrophils after 1h exposure The population of neutrophils and lymphocytes exhibited different responses to cell-free C. raciborskii extract but both indicated its toxic action (Fig. 2). After 1 h of exposure, a significant decrease of alive neutrophils, followed by an increase of necrosis and apoptosis was observed in samples treated with 100% and 10% concentration of extract, and apoptosis in samples exposed to 1% concentration. The observed effects revealed a concentrationdependent manner (Fig. 2). In lymphocytes, 100% and 10% concentrations of C. raciborskii extracts revealed pro-apoptotic action and a significantly decreased percentage of alive cells (Fig. 2). Contrary to the extracts, no increase in necrosis or apoptosis was observed in neutrophils and lymphocytes treated with CYN at any assayed concentration of the toxin. 3.2. Proliferation of lymphocytes after 72 h culture As measured by incorporation of thymidine, cell-free extract of C. raciborskii had a significant effect on T-lymphocyte proliferation after 72 h in both isolated and whole-blood cultures (Fig. 3A). In isolated and whole-blood cultures the highest assayed CYN

B. Poniedziałek et al. / Chemosphere 120 (2015) 608–614

611

Fig. 2. The viability (measured by annexin V-FITC/propidium iodide activities using flow cytometry) of human neutrophils and lymphocytes (n = 10) treated with 100%, 10% or 1% of cell-free C. raciborskii extract expressed as a percentage of live (A), necrotic (B) and apoptotic (C). Column and bar represent mean and standard deviation, respectively. Asterisks indicate statistically significant differences to the control (⁄p < 0.05; ⁄⁄p < 0.01, Wilcoxon signed-rank test).

Fig. 3. Mean proliferation (measured by incorporation of thymidine) of human T-lymphocytes (n = 10) treated with 100%, 10% or 1% of cell-free C. raciborskii extract (A), and 1.0, 0.1 or 0.01 lg mL 1 of purified CYN (B) in isolated (white columns) or whole-blood (black columns) cultures. Bars represent standard deviation, asterisks represent statistically significant differences to the control (⁄⁄⁄p < 0.001, Wilcoxon signed-rank test); a-results published earlier (Poniedziałek et al. 2014a).

concentration decreased it by 20.6% and 32.5%, respectively. Significantly suppressed proliferation was also found in lymphocytes exposed to 1% of extract but only in isolated cell cultures. In general, whole-blood cultures were characterised by higher variability, as revealed by the standard deviation (Fig. 3A). CYN also had an effect on T-lymphocyte proliferation (Fig. 3B). In isolated and whole-blood cultures the highest assayed CYN concentration decreased it by 91.0% and 56.5%, respectively. Significantly suppressed proliferation was also found in lymphocytes exposed to 0.1 lg L 1 but only in the whole-blood culture. 4. Discussion Cyanobacteria are known to produce a variety of bioactive compounds including cyclic and non-cyclic peptides, polyketides, alkaloids, phenols and chlorinated aromatic compounds (Leao et al., 2009; Rzymski et al. 2011). The toxin profile of C. raciborskii appears to be diversified in relation to the geographical zone (Rzymski and Poniedziałek, 2014). In general, CYN-producing strains occur in Australia and Asia (Jiang et al., 2014), whereas strains capable of saxitoxins (neurotoxic alkaloids) production are found in South America (Hoff-Risseti et al, 2013). African and European strains have not been shown to produce any known cyanotoxin (Haande et al., 2008; Rzymski and Poniedziałek, 2014) although the crude extracts of the latter were demonstrated to exhibit potentially toxic properties (Antal et al., 2011). In our study, we have confirmed that extracts of C. raciborskii contained compound(s) which are capable of altering the function of one of the most abundant cells of human immune system, lymphocytes and neutrophils. This is the first report to show directly that metabolite(s) of the European strain can potentially affect the function of cells derived from healthy donors through hitherto unknown and yet to be analytically identified compounds.

Previously, toxic effects of German non-CYN-producing C. raciborskii were investigated in a cell line model and found to be slightly toxic in human hepatoblastoma (HEP-G2) but not in human colon adenocarcinoma (CACO-2) cell lines (Fastner et al., 2003). European C. raciborskii isolates have also been demonstrated to be toxic in bioassays employing rodents (Fastner et al., 2003; Saker et al., 2003), molluscs (Kiss et al., 2002; Vehovszky et al., 2009) and insects (Hiripi et al., 1998). Considering that C. raciborskii is expected to increasingly inhabit European lakes within the next 10–20 years and has already formed dense blooms in some locations within the continent (Padisák, 1997; Burford et al., 2006; Sinha et al., 2012), its occurrence may represent a threat to human and animal health and this should be taken into account in risk assessments. In Poland, C. raciborskii can constitute up to 13.9% of the total phytoplankton biomass, particularly in shallower lakes (Kokocin´ski and Soininen, 2012). In other European countries, it was already reported to reach higher densities, including total domination of phytoplankton (Briand et al. 2002; Karadzˇic´ et al. 2013). As found in our study, extracts of tested C. raciborskii revealed different toxic patterns than purified CYN. It is worth noting that the effects were observed in the whole-blood model in which specific responses can be measured in the presence of autologous cellular and serum components that may be physiologically relevant. The whole-blood model has also been shown to be a convenient assay to study immune cell responses as it requires low blood volumes and shorter procedures (Deenadayalan et al., 2013). The use of blood samples collected from healthy donors ensured that individual variability and susceptibility was included in our study, conditions not found when using cell line models. Our results may therefore closely reflect a possible in vivo situation of exposure to non-CYN-producing C. raciborskii metabolite(s) and CYN. On the other hand, the isolation and culturing of lymphocytes performed

612

B. Poniedziałek et al. / Chemosphere 120 (2015) 608–614

in our study, allowed T-cell specific analysis to be conducted and so it was possible to clarify whether the observed alterations of proliferation are the results of direct action of cyanobacterial metabolites on these cells. In general, lymphocytes appeared to be more resistant to cellfree extracts of the tested C. raciborskii strain than neutrophils. Nevertheless, their short-term (1 h) exposure to 100% and 10% concentrations of extract resulted in a significantly increased rate of apoptotic cells. Interestingly, this effect was not observed when cells were treated with CYN, regardless of the concentration of toxin. As revealed in previous studies, in eukaryotic cells CYN can induce oxidative stress (Runnegar et al., 1995; Humpage et al., 2005; Gutiérrez-Praena et al., 2012), which was postulated as the potential mechanism underlying toxin-induced DNA strain breaks in human lymphocytes (Zˇegura et al., 2011) and consequently, G0/ G1 arrest followed by apoptotic cell death (Poniedziałek et al., 2014a). All of these effects were, however, found after significantly longer exposure than 1 h. On the other hand, CYN exhibited very high anti-proliferative potencies against T-lymphocytes in 72 h of cultures of isolated cells (Poniedziałek et al., 2012b, 2014a) and whole-blood. These findings indicate that CYN is a slow acting but powerful toxin which is in line with the study of Froscio et al. (2009) demonstrating a relatively slow but progressive uptake of CYN in the Vero cell line. Contradictory to this, metabolite(s) present in the Polish strain of C. raciborskii revealed rapid action in lymphocytes by inducing apoptosis and consequently, decreasing their viability in a time as short as 1 h. However, the effect of C. raciborskii extracts on T-lymphocyte proliferation was not as pronounced as for 1.0 lg L 1 CYN. This suggests that these cells can partially overcome the toxic effects induced by C. raciborskii exudates or the metabolite(s) are degraded due to their lower stability than CYN. Interestingly, we observed some differences between lymphocyte responses in isolated and whole-blood cultures. The results obtained using whole-blood assay were characterised by higher values of standard deviation, which may indicate the role of other cellular components in response to CYN and C. raciborskii extract. In fact, it was shown that lymphocyte proliferation requires cellular cooperation with macrophages and monocytes (Passwell et al., 1982; Bloemena et al., 1989). In our study, the differences in magnitude of the effects between whole-blood and isolated cultures were specifically found for experiments involving CYN. In the whole-blood model, exposure to the highest assayed concentration of CYN resulted in the less pronounced suppression of T-lymphocyte proliferation compared to the isolated culture, indicating that the effect of the toxin was spread over the other cellular population. On the other hand, proliferation also remained significantly decreased in lymphocytes treated with 0.1 lg L 1 CYN; an effect not observed in isolated cultures. Therefore, it may indicate that the toxin at a lower and environmentally-relevant concentration may alter the function of other cellular components essential in lymphocyte viability. In most abundant human leukocytes, neutrophils, short-term exposure (1 h) induced a relatively high rate of necrosis and apoptosis. It is worth noting that these effects were found for every studied concentration of extract, including those as low as 1%. This on one hand, highlights the susceptibility of neutrophils to metabolite(s) produced by the tested C. raciborskii strain while on the other, indicates the high toxicity of this (these) unknown compound(s). Decreased viability of neutrophils, short-lived cells with a circulating half life of 6–8 h, can cause a particular susceptibility to bacterial and fungal infections (Nathan, 2006). Unlike C. raciborskii extract, CYN at any assayed concentration revealed neither a pro-necrotic nor pro-apoptotic mode of action. This finding was already reported in our previous study in which neutrophil viability was studied with carboxyfluorescein-labelled

fluoromethyl ketone peptide inhibitor of caspases (Poniedziałek et al., 2014b). Interestingly, CYN demonstrated the ability to decrease ROS production in both stimulated and unstimulated neutrophils through, what is most likely to be, altered activity of NADPH oxidase (Poniedziałek et al., 2014b). In this study ROS production was not investigated but different patterns in cell viability strongly suggest that tested C. raciborskii metabolite(s) may differ from CYN in chemical structure and mode of action. It was, however, reported that the same strain of C. raciborskii can produce compound(s) mimicking postulated allelopathic CYN action in sympatric cyanobacteria (Rzymski et al., 2014) but since this effect was observed in prokaryotic organisms it cannot be extrapolated to eukaryotic systems. Apart from humans, our results may have indirect implications for aquatic organisms. It was previously demonstrated in vitro and in vivo that cyanotoxins, such as microcystins, anatoxin-a and CYN, can have immunomodulatory potencies in fish (Bownik et al., 2012; Rymuszka and Adaszek, 2013; Sieroslawska and Rymuszka, 2014). The effects posed by cyanobacterial metabolites may be even greater than they are for humans because cyanotoxins can be continuously released during exponential growth of cyanobacteria or at the time of heavy bloom events (Chiswell et al., 1999; Shaw et al., 1999). This can lead to sub-chronic or chronic exposures to fish as well as other aquatic organisms and alterations to their functioning. In turn, humans and terrestrial animals are more likely to be subject to single but acute poisonings. Nevertheless, further studies should address the potential toxicity of European C. raciborskii strains in various freshwater organisms. So far, only one study employing a battery of ecotoxicological assays has been performed and demonstrated that non-CYN-producing Hungarian strains can affect aquatic crustaceans and disrupt fish embryogenesis (Acs et al., 2013). In summary, this and other studies investigating the bioactive potencies of European C. raciborskii exudates in in vitro and in vivo models suggest that these strains can produce powerful but hitherto unknown compound(s), toxic to eukaryotes but also potentially possessing therapeutical properties as biological reagents or drug candidates. Further studies are required to analytically identify all metabolites produced by C. raciborskii, clarify the molecular and biochemical bases of their synthesis and transport, specify the route of their release to the extracellular environment, determine their stability to chemical, physical and microbial factors and finally, fully assess their toxicity in different organisms – including humans, terrestrial and aquatic animals. Nevertheless, the present study demonstrated that both, CYN and non-CYN-producing C. raciborskii extract exhibited immunomodulatory potencies. Their presence in freshwater, particularly at high concentrations/densities may represent a serious health threat and a challenge to water treatment. Acknowledgments We would like to thank Ms. Anna S´wiejkowska (Poznan University of Medical Sciences) for her help with the cell cultures and food supply, and Mr. Jan Kazimierz Poniedziałek-Rzymski for keeping us awake and working. References } , N., Kiss, G., Gyo }ri, J., Vehovszky, A., Kováts, Acs, A., Kovács, A.W., Csepregi, J.Z., Töro N., Farkas, A., 2013. The ecotoxicological evaluation of Cylindrospermopsis raciborskii from Lake Balaton (Hungary) employing a battery of bioassays and chemical screening. Toxicon 70, 98–106. http://dx.doi.org/10.1016/ j.toxicon.2013.04.019. Antal, O., Karisztl-Gácsi, M., Farkas, A., Kovács, A., Acs, A., Töro, N., Kiss, G., Saker, M.L., Gyori, J., Bánfalvi, G., Vehovszky, A., 2011. Screening the toxic potential of Cylindrospermopsis raciborskii strains isolated from Lake Balaton, Hungary. Toxicon 57, 831–840. http://dx.doi.org/10.1016/j.toxicon.2011.02.007.

B. Poniedziałek et al. / Chemosphere 120 (2015) 608–614 Bazin, E., Mourot, A., Humpage, A.R., Fessard, V., 2010. Genotoxicity of a freshwater cyanotoxin cylindrospermopsin in two human cell lines: Caco-2 and HepaRG. Environ. Mol. Mutagen. 51, 251–259. http://dx.doi.org/10.1002/em.20539. Bernard, C., Harvey, M., Briand, J.F., Biré, R., Krys, S., Fontaine, J.J., 2003. Toxicological comparison of diverse Cylindrospermopsis raciborskii strains: evidence of liver damage caused by a French C raciborskii strain. Environ. Toxicol. 18, 176–186. http://dx.doi.org/10.1002/tox.10112. Bloemena, E., Roos, M.T.L., Van Heijst, J.L.A.M., Vossen, J.M.J.J., Schellekens, P.T.A., 1989. Whole-blood lymphocyte cultures. J. Immunol. Methods 122, 161–167. http://dx.doi.org/10.1016/0022-1759(89)90260-3. Bownik, A., Rymuszka, A., Sierosławska, A., Skowron´ski, T., 2012. Anatoxin-a induces apoptosis of leukocytes and decreases the proliferative ability of lymphocytes of common carp (Cyprinus carpio L.) in vitro. Pol. J. Vet. Sci. 15, 531–535. http:// dx.doi.org/10.2478/v10181-012-0082-7. Briand, J.F., Robillot, C., Quiblier-Llobéras, C., Humbert, J.F., Couté, A., Bernard, C., 2002. Environmental context of Cylindrospermopsis raciborskii (Cyanobacteria) blooms in a shallow pond in France. Water Res. 36, 3183–3192. http:// dx.doi.org/10.1016/S0043-1354(02)00016-7. Burford, M.A., Mcneale, K.L., Mckenzie-Smith, F.J., 2006. The role of nitrogen in promoting the toxic cyanophyte Cylindrospermopsis raciborskii in a subtropical water reservoir. Freshwater Biol. 51, 2143–2153. http://dx.doi.org/10.1111/ j.1365-2427.2006.01630.x. Carmichael, W.W., Azevedo, S.M., An, J.S., Molica, R.J., Jochimsen, E.M., Lau, S., Rinehart, K.L., Shaw, G.R., Eaglesham, G.K., 2001. Human fatalities from cyanobacteria: chemical and biological evidence for cyanotoxins. Environ. Health Perspect. 109, 663–668. Chiswell, R.K., Shaw, G.R., Eaglesham, G., Smith, M.J., Norris, R.L., Seawright, A.A., Moore, M.R., 1999. Stability of cylindrospermopsin the toxin from the cyanobacterium Cylindrospermopsis raciborskii: effect of pH temperature and sunlight on decomposition. Environ. Toxicol. 14, 155–161. http://dx.doi.org/ 10.1002/(SICI)1522-7278(199902)14:1<155::AID-TOX20>3.0.CO;2-Z. Deenadayalan, A., Maddineni, P., Raja, A., 2013. Comparison of whole blood and PBMC assays for T-cell functional analysis. BMC Research Notes 6, 120. http:// dx.doi.org/10.1186/1756-0500-6-120. Fabbro, L., Baker, M., Dilvenvoorden, L., Pegg, G., Shiel, R., 2001. The effects of the ciliate Paramecium cf. caudatum Ehrenberg on toxin producing Cylindrospermopsis isolated from the Fitzroy River. Australia. Environ. Toxicol. 16, 489–497. http://dx.doi.org/10.1002/tox.10007. Fastner, J., Heinze, R., Humpage, A.R., Mischke, U., Eaglesham, G.K., Chorus, I., 2003. Cylindrospermopsin occurrence in two German lakes and preliminary assessment of toxicity and toxin production of Cylindrospermopsis raciborskii (Cyanobacteria) isolates. Toxicon 42, 313–321. http://dx.doi.org/10.1016/ S0041-0101(03)00150-8. Froscio, S.M., Cannon, E., Lau, H.M., Humpage, A.R., 2009. Limited uptake of the cyanobacterial toxin cylindrospermopsin by Vero cells. Toxicon 54, 862–868. http://dx.doi.org/10.1016/j.toxicon.2009.06.019. Griffiths, D.J., Saker, M.L., 2003. The Palm Island mystery disease 20 years on: a review of research on the cyanotoxin cylindrospermopsin. Environ. Toxicol. 18, 78–93. http://dx.doi.org/10.1002/tox.10103. Gutiérrez-Praena, D., Jos, A., Pichardo, S., Cameán, A.M., 2011. Oxidative stress responses in tilapia (Oreochromis niloticus) exposed to a single dose of pure cylindrospermopsin under laboratory conditions: influence of exposure route and time of sacrifice. Aquat. Toxicol. 105, 100–106. http://dx.doi.org/10.1016/ j.aquatox.2011.05.015. Gutiérrez-Praena, D., Pichardo, S., Jos, A., Moreno, F.J., Cameán, A.M., 2012. Alterations observed in the endothelial HUVEC cell line exposed to pure Cylindrospermopsin. Chemosphere 89, 1151–1160. http://dx.doi.org/10.1016/ j.chemosphere.2012.06.023. Guzmán-Guillén, R., Prieto, A.I., Moreno, I., Ríos, V., Vasconcelos, V.M., Cameán, A.M., 2014. Effects of depuration on oxidative biomarkers in tilapia (Oreochromis niloticus) after subchronic exposure to cyanobacterium producing cylindrospermopsin. Aquat. Toxicol. 149, 40–49. http://dx.doi.org/10.1016/ j.aquatox.2014.01.026. Haande, S., Rohrlack, T., Ballot, A., Roberg, K., Skulberg, R., Beck, M., Wiedner, C., 2008. Genetic characterization of Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) isolates from Africa and Europe. Harmful Algae 7, 692–701. http://dx.doi.org/10.1016/j.hal.2008.02.010. Hawkins, P.R., Runnegar, M.T., Jackson, A.R., Falconer, I.R., 1985. Severe hepatotoxicity caused by the tropical cyanobacterium (blue-green alga) Cylindrospermopsis raciborskii (Woloszynska) Seenaya and Subba Raju isolated from a domestic water supply reservoir. Appl. Environ. Microbiol. 50, 1292– 1295. Hiripi, L., Nagy, L., Kalmár, T., Kovács, A., Vörös, L., 1998. Insect (Locusta migratoria migratorioides) test monitoring the toxicity of cyanobacteria. Neurotoxicology 19, 605–608. Hoff-Risseti, C., Dorr, F.A., Schaker, P.D.C., Pinto, E., Werner, V.R., Fiore, M.F., 2013. Cylindrospermopsin and saxitoxin synthetase genes in Cylindrospermopsis raciborskii strains from Brazilian freshwater. PLoS ONE 8 (8), 74238. Humpage, A.R., Falconer, I.R., 2003. Oral toxicity of the cyanobacterial toxin cylindrospermopsin in male Swiss albino mice: determination of no observed adverse effect level for deriving a drinking water guideline value. Environ. Toxicol. 18, 94–103. http://dx.doi.org/10.1002/tox.10104. Humpage, A.R., Fenech, M., Thomas, P., Falconer, I.R., 2000. Micronucleus induction and chromosome loss in transformed human white cells indicate clastogenic and aneugenic action of the cyanobacterial toxin cylindrospermopsin. Mutat. Res. 472, 155–161. http://dx.doi.org/10.1016/S1383-5718(00)00144-3.

613

Humpage, A.R., Fontaine, F., Froscio, S., Burcham, P., Falconer, I.R., 2005. Cylindrospermopsin genotoxicity and cytotoxicity: role of cytochrome P-450 and oxidative stress. J. Toxicol. Environ. Health A 68, 739–753. http://dx.doi.org/ 10.1080/15287390590925465. Jiang, Y., Xiao, P., Yu G, G., Shao, J., Liu, D., Azevedo, S.M., Li R, R., 2014. Sporadic distribution and distinctive variations of cylindrospermopsin genes in cyanobacterial strains and environmental samples from Chinese freshwater bodies. Appl. Environ. Microbiol. http://dx.doi.org/10.1128/AEM.00551-14. Karadzˇic´, V., Simic´, G.S., Natic´, D., Rzˇanicˇanin, A., C´iric´, M., Gacˇic´, Z., 2013. Changes in the phytoplankton community and dominance of Cylindrospermopsis raciborskii (Wolosz.) Subba Raju in a temperate lowland river (Ponjavica, Serbia). Hydrobiologia 711, 43–60. http://dx.doi.org/10.1007/s10750-013-1460-6. Kiss, T., Vehovszky, Á., Hiripi, L., Kovács, A., Vörös, L., 2002. Membrane effects of toxins isolated from a cyanobacterium, Cylindrospermopsis raciborskii, on identified molluscan neurones. Comp. Biochem. Physiol. Part C: Toxicol. Pharmacol. 131, 167–176. http://dx.doi.org/10.1016/S1532-0456(01)00290-3. Klitzke, S., Fastner, J., 2012. Cylindrospermopsin degradation in sediments – the role of temperature, redox conditions and dissolved organic carbon. Water Res. 46, 1549–1555. http://dx.doi.org/10.1016/j.watres.2011.12.014. Kokocin´ski, M., Soininen, J., 2012. Environmental factors related to the occurrence of Cylindrospermopsis raciborskii (Nostocales, Cyanophyta) at the north-eastern limit of its geographical range. Eur. J. Phycol. 47, 12–21. Kokocin´ski, M., Mankiewicz-Boczek, J., Jurczak, T., Spoof, L., Meriluoto, J., Rejmonczyk, E., Hautala, H., Vehniäinen, M., Pawełczyk, J., Soininen, J., 2013. Aphanizomenon gracile (Nostocales), a cylindrospermopsin-producing cyanobacterium in Polish lakes. Environ. Sci. Pollut. Res. Int. 20, 5243–5264. http://dx.doi.org/10.1007/s11356-012-1426-7. Leao, P.N., Vasconcelos, M., Vasconcelos, V.M., 2009. Allelopathy in freshwater cyanobacteria. Crit. Rev. Microbiol. 35, 271–282. http://dx.doi.org/10.3109/ 10408410902823705. Loken, M.R., Green, C.L., Wells, D.A., 2000. Immunofluorescence of surface markers. In: Ormerod, M.G. (Ed.), Flow cytometry: a practical approach. Oxford University Press, Oxford (UK), pp. 61–82. Mankiewicz-Boczek, J., Kokocin´ski, M., Gagała, I., Pawełczyk, J., Jurczak, T., Dziadek, J., 2012. Preliminary molecular identification of cylindrospermopsin-producing Cyanobacteria in two Polish lakes (Central Europe). FEMS Microbiol. Lett. 326, 173–179. http://dx.doi.org/10.1111/j.1574-6968.2011.02451.x. Messineo, V., Melchiorre, S., Di Corcia, A., Gallo, P., Bruno, M., 2010. Seasonal succession of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum blooms with cylindrospermopsin occurrence in the volcanic Lake Albano, Central Italy. Environ. Toxicol. 25, 18–27. http://dx.doi.org/10.1002/tox.20469. Metcalf, J.S., Lindsay, J., Beattie, K.A., Birmingham, S., Saker, M.L., Törökné, A.K., Codd, G.A., 2002. Toxicity of cylindrospermopsin to the brine shrimp Artemia salina: comparisons with protein synthesis inhibitors and microcystins. Toxicon 40, 1115–1120. http://dx.doi.org/10.1016/S0041-0101(02)00105-8. Moisander, P.H., Cheshire, L.A., Braddy, J., Calandrino, E.S., Hoffman, M., Piehler, M.F., Paerl, H.W., 2012. Facultative diazotrophy increases Cylindrospermopsis raciborskii competitiveness under fluctuating nitrogen availability. FEMS Microbiol. Ecol. 79, 800–811. http://dx.doi.org/10.1111/j.1574-6941.2011. 01264.x. Nathan, C., 2006. Neutrophils and immunity: challenges and opportunities. Nat. Rev. Immunol. 3, 173–182. http://dx.doi.org/10.1038/nri1785. Nogueira, I.C., Saker, M.L., Pflugmacher, S., Wiegand, C., Vasconcelos, V.M., 2004. Toxicity of the cyanobacterium Cylindrospermopsis raciborskii to Daphnia magna. Environ. Toxicol. 19, 453–459. http://dx.doi.org/10.1002/tox.20050. Nováková, K., Bláha, L., Babica, P., 2012. Tumor promoting effects of cyanobacterial extracts are potentiated by anthropogenic contaminants–evidence from in vitro study. Chemosphere 89, 30–37. http://dx.doi.org/10.1016/j.chemosphere.2012. 04.008. Oliveira, V.R., Carvalho, G.M., Avila, M.B., Soares, R.M., Azevedo, S.M., Ferreira, T.S., Valença, S.S., Faffe, D.S., Zin, W.A., 2012. Time-dependence of lung injury in mice acutely exposed to cylindrospermopsin. Toxicon 60, 764–772. http://dx.doi.org/ 10.1016/j.toxicon.2012.06.009. O’Neil, J.M., Davis, T.W., Burford, M.A., Gobler, C.J., 2012. The rise of harmful cyanobacteria blooms: the potential roles of eutrophication and climate change. Harmful Algae 14, 313–334. http://dx.doi.org/10.1016/j.hal.2011.10.027. Padisák, J., 1997. Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju, an expanding, highly adaptive cyanobacterium: worldwide distribution and review of its ecology. Arch. Hydrobiol. 107 (suppl.), 563–593. Passwell, J.H., Levanon, M., Davidsohn, J., Kohen, F., Ramot, B., 1982. The effect of human monocytes and macrophages on lymphocyte proliferation. Immunology 47, 175–181. Poniedziałek, B., Rzymski, P., Kokocin´ski, M., 2012a. Cylindrospermopsin: waterlinked potential threat to human health in Europe. Environ. Toxicol. Pharmacol. 34, 651–660. http://dx.doi.org/10.1016/j.etap.2012.08.005. Poniedziałek, B., Rzymski, P., Wiktorowicz, K., 2012b. First report of cylindrospermopsin effect on human peripheral blood lymphocytes proliferation in vitro. Centr. Eur. J. Immunol. 37, 314–317. http://dx.doi.org/ 10.5114/ceji.2012.32717. Poniedziałek, B., Rzymski, P., Wiktorowicz, K., 2014a. Toxicity of cylindrospermopsin in human lymphocytes: proliferation, viability and cell cycle studies. Toxicol. Vitro 28, 968–974. http://dx.doi.org/10.1016/ j.tiv.2014.04.015. Poniedziałek, B., Rzymski, P., Karczewski, J., 2014b. Cylindrospermopsin decreases the oxidative burst capacity of human neutrophils. Toxicon 87, 113–199. http:// dx.doi.org/10.1016/j.toxicon.2014.05.004.

614

B. Poniedziałek et al. / Chemosphere 120 (2015) 608–614

Rücker, J., Stuken, A., Nixdorf, B., Fastner, J., Chorus, I., Wiedner, C., 2007. Concentrations of particulate and dissolved cylindrospermopsin in 21 Aphanizomenon-dominated temperate lakes. Toxicon 50, 800–809. http:// dx.doi.org/10.1016/j.toxicon.2007.06.019. Runnegar, M.T., Kong, S.M., Zhong, Y.Z., Lu, S.C., 1995. Inhibition of reduced glutathione synthesis by cyanobacterial alkaloid cylindrospermopsin in cultured rat hepatocytes. Biochem. Pharmacol. 49, 219–225. http://dx.doi.org/ 10.1016/S0006-2952(94)00466-8. Rymuszka, A., Adaszek, Ł., 2013. Cytotoxic effects and changes in cytokine gene expression induced by microcystin-containing extract in fish immune cells–an in vitro and in vivo study. Fish Shellfish Immunol. 34, 1524–1532. http:// dx.doi.org/10.1016/j.fsi.2013.03.364. Rzymski, P., Poniedziałek, B., Karczewski, J., 2011. Gastroenteritis and liver carcinogenesis induced by cyanobacterial toxins. Gastroenetrol Pol. 18, 159– 162. Rzymski, P., Poniedziałek, B., 2014. In search of environmental role of cylindrospermopsin: a review on global distribution and ecology of its producers. Water Res. http://dx.doi.org/10.1016/j.watres.2014.08.029. Rzymski, P., Poniedziałek, B., Kokocin´ski, M., Jurczak, T., Lipski, D., Wiktorowicz, K., 2014. Interspecific allelopathy in cyanobacteria: cylindrospermopsin and Cylindrospermopsis raciborskii effect on the growth and metabolism of Microcystis aeruginosa. Harmful Algae 35, 1–8. http://dx.doi.org/10.1016/ j.hal.2014.03.002. Saker, M.L., Eaglesham, G.K., 1999. The accumulation of cylindrospermopsin from the cyanobacterium Cylindrospermopsis raciborskii in tissues of the Redclaw crayfish Cherax quadricarinatus. Toxicon 37, 1065–1077. http://dx.doi.org/ 10.1016/S0041-0101(98)00240-2. Saker, M.L., Nogueira, I.C., Vasconcelos, V.M., Neilan, B.A., Eaglesham, G.K., Pereira, P., 2003. First report and toxicological assessment of the cyanobacterium Cylindrospermopsis raciborskii from Portuguese freshwaters. Ecotoxicol. Environ. Saf. 55, 243–250. http://dx.doi.org/10.1016/S0147-6513(02)00043-X. Shaw, G.R., Sukenik, A., Livine, A., Chiswell, R.K., Smith, M.J., Seawright, A.A., Norris, R.L., Eaglesham, G.K., Moore, M.R., 1999. Blooms of the cylindrospermopsin containing cyanobacterium, Aphanizomenon ovalisporum (Forti), in newly constructed lakes, Queensland. Australia. Environ. Toxicol. 14, 167–177. Sieroslawska, A., Rymuszka, A., 2014. Effects of cylindrospermopsin on a common carp leucocyte cell line. J. Appl. Toxicol. http://dx.doi.org/10.1002/jat.2990. Sinha, R., Pearson, L.A., Davis, T.W., Burford, M.A., Orr, P.T., Neilan, B.A., 2012. Increased incidence of Cylindrospermopsis raciborskii in temperate zones–is climate change responsible? Water Res. 46, 1408–1419. http://dx.doi.org/ 10.1016/j.watres.2011.12.019.

Soares, M.C.S., Lürling, M., Huszar, V.L.M., 2012. Growth and temperature-related phenotypic plasticity in the cyanobacterium Cylindrospermopsis raciborskii. Phycol. Res. 61, 61–67. http://dx.doi.org/10.1111/pre.12001. Stewart, I., Seawright, A.A., Schluter, P.J., Shaw, G.R., 2006. Primary irritant and delayed-contact hypersensitivity reactions to the freshwater cyanobacterium Cylindrospermopsis raciborskii and its associated toxin cylindrospermopsin. BMC Dermatology. http://dx.doi.org/10.1186/1471-5945-6-5. Sukenik, A., Hadas, O., Kaplan, A., Quesada, A., 2012. Invasion of Nostocales (cyanobacteria) to subtropical and temperate freshwater lakes – physiological regional and global driving forces. Front. Microbiol. http://dx.doi.org/10.3389/ fmicb.2012.00086. Terao, K., Ohmori, S., Igarashi, K., Ohtani, I., Watanabe, M.F., Harada, K.I., Ito, E., Watanabe, M., 1994. Electron microscopic studies on experimental poisoning in mice induced by cylindrospermopsin isolated from blue-green alga Umezakia natans. Toxicon 32, 833–843. Torokne, A., Palovics, A., Bankine, M., 2001. Allergenic (sensitization, skin and eye irritation) effects of freshwater cyanobacteria- experimental evidence. Environ. Toxicol. 16, 512–516. http://dx.doi.org/10.1002/tox.10011. Trosko, J.E., 2007. Gap junctional intercellular communication as a biological ‘‘Rosetta stone’’ in understanding, in a systems biological manner, stem cell behavior, mechanisms of epigenetic toxicology, chemoprevention and chemotherapy. J. Membr. Biol. 218, 93–100. http://dx.doi.org/10.1007/s00232007-9072-6. Vehovszky, A., Ács, A., Kovács, W.A., Szabó, H., GyTri, J., Farkas, A., 2009. Isolated strains of Cylindrospermopsis raciborskii from Lake Balaton (Hungary) produce anatoxin-a like neurotoxins. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol 153, S88. http://dx.doi.org/10.1016/j.cbpa.2009.04.083. } ri, J., Szabó, H., Vasas, G., 2013. Vehovszky, A., Kovács, A.W., Farkas, A., Gyo Pharmacological studies confirm neurotoxic metabolite(s) produced by the bloom-forming Cylindrospermopsis raciborskii in Hungary. Environ. Toxicol. http://dx.doi.org/10.1002/tox.21927. Wörmer, L., Cires, S., Carrasco, D., Quesada, A., 2008. Cylindrospermopsin is not degraded by co-occurring natural bacterial communities during a 40-day study. Harmful Algae 7, 206–213. http://dx.doi.org/10.1016/j.hal.2007.07.004. Young, F.M., Micklem, J., Humpage, A.R., 2008. Effects of blue-green algal toxin cylindrospermopsin (CYN) on human granulosa cells in vitro. Reprod. Toxicol. 25, 374–380. http://dx.doi.org/10.1016/j.reprotox.2008.02.006. Zˇegura, B., Gajski, G., Štraser, A., Garaj-Vrhovac, V., 2011. Cylindrospermopsin induced DNA damage and alteration in the expression of genes involved in the response to DNA damage, apoptosis and oxidative stress. Toxicon 58, 471–479. http://dx.doi.org/10.1016/j.toxicon.2011.08.005.