Cent. Eur. J. Biol. • 8(12) • 2013 • 1216-1229 DOI: 10.2478/s11535-013-0239-0

Central European Journal of Biology

In vitro and in vivo toxicity evaluation of the freshwater cyanobacterium Heteroleiblenia kuetzingii Research Article

Ivanka Teneva1, Plamen Stoyanov1, Rumen Mladenov1, Balik Dzhambazov2,* Department of Botany, Faculty of Biology, Plovdiv University, 4000 Plovdiv, Bulgaria

1

2 Department of Developmental Biology, Faculty of Biology, Plovdiv University, 4000 Plovdiv, Bulgaria

Received 13 April 2013; Accepted 11 July 2013

Abstract: Cyanobacteria are prokaryotic organisms characterized by their ability to produce secondary metabolites with different biological activities. The aim of this work was to evaluate the in vitro and in vivo toxicity of the cosmopolitan freshwater cyanobacterium H.  kuetzingii. An extract from H.  kuetzingii and cyanobacterial growth media were assessed for presence of intracellular and extracellular toxins by in vitro tests using primary cell cultures from mouse kidney and fibroblasts, cell lines A549 and 3T3, a fish cell line RTgill-W1 as well as by a traditional in vivo mouse bioassay. The presence of toxicity was compared with the ELISA and HPLC data for corresponding cyanotoxins. In vitro tests showed pronounced cytotoxicity of the cyanobacterium extract and growth medium in which H.  kuetzingii released potential extracellular toxic compounds as the mammalian cells were significantly more sensitive to exposure compared to the fish cells. Histopathological analyses of the liver and kidneys of treated mice showed pathological changes such as leukocyte infiltration and necrosis, changes in the proximal and distal convoluted tubules, lack of differentiation of Bowman’s space, enlarged Bowman’s capsules and massive hemorrhages. ELISA and HPLC analyses confirmed the presence of saxitoxins and microcystins at low concentrations. In addition, the histological analyses suggest that H. kuetzingii produces other, yet unknown toxic metabolites. Monitoring efforts are therefore required to evaluate the potential hazard for the freshwater aquatic systems and possible public health implications associated with this cyanobacterium. Keywords: Cyanobacteria • Toxic effects • Cyanotoxins • Microcystin • Saxitoxin • In vitro tests • In vivo bioassay • Histopathology © Versita Sp. z o.o.

1. Introduction Cyanobacteria (Cyanophyta, Cyanoprokaryota) are evolutionarily ancient prokaryotic organisms, unique with their ability to perform photosynthesis. They are characterized by cosmopolitan distribution, species diversity and ability to produce several secondary metabolites with different biological activity. It is suggested that these secondary metabolites determine the high biological adaptability and cosmopolitanism of cyanobacteria and this is a part of their strategy for survival [1]. In recent decades, more attention has been paid to cyanobacteria as potential sources of pharmaceutical products with different activities, including cytotoxicity,

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immunosuppression, antiproliferative activity and antitumor activity [2-4]. For example, the anticancer agents cryptophycins were isolated from cyanobacteria [5-7]. Cryptophycin-52, which is an analogue of the cryptophycin-1 has successfully completed Phase II clinical trials as an anti-tumor agent [7-10]. Dolastatin-10 is a modified pentapeptide isolated from Dolabella auricularia and Lyngbya majuscula, which is also in clinical trials as an anti-tumor agent [11]. Reddy et al. [12] summarized known information about the use of natural products as anticancer agents showing their advantages over synthetic chemicals that cause non-specific destruction of the cells. Along with the useful secondary metabolites, majority of cyanobacteria are producers of cyanotoxins, * E-mail: [email protected]

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which may be harmful to animals, including humans. In freshwater basins, cyanobacteria are part of the phytoplankton. Under certain conditions in the summer months, such as increasing eutrophication of the water pool, higher water temperatures, long sunshine and low values of the ratio TN/TP [13], cyanobacteria can experience exuberant growth, called “cyanobacterial blooms”. It was found that most cyanobacterial blooms are usually concomitant with release of large amounts of cyanotoxins. Depending on the concentration in the water pool, these natural toxins can cause acute intoxication, can have a chronic effect or can accumulate in the food chain. According to the effect on mammals and other vertebrates, the cyanotoxins are classified as hepatotoxins (affecting the liver), neurotoxins (causing damage to the nervous system), cytotoxins (causing cell damage) and dermatotoxins (causing skin allergic reactions). Cyanobacterial genera Anabaena, Aphanizomenon, Nostoc, Microcystis, Nodularia, Cylindrospermopsis, Planktothrix, Oscillatoria, Raphidiopsis, Phormidium and Lyngbya are of increased interest from a toxicological and pharmacological point of view. Species of the genus Lyngbya C. Agardh ex Gomont 1892 have been increasingly reported as producers of various substances with different structure and biological activity. L. majuscula, L. martensiana, L. aestuarii, and L. wollei are the most frequently reported species as producers of secondary metabolites. Sixty eight substances isolated from L. majuscula with a range of biological effects were reported [14]. Lyngbyatoxin-A and debromoaplysiatoxin appear to have the most widely studied effects on human health and ecosystems. Liu and Rein [15] summarized data about the structure and bioactivity of 50 peptides isolated mainly from marine representatives of the genus Lyngbya. A case of acute contact dermatitis (Lyngbya dermatitis) after swimming in rough surf conditions on the shores of Oahu (Hawai) have been recently reported [16]. This specific type of dermatitis is caused usually by L. majuscula, which is commonly distributed in tropical and temperate waters worldwide. The freshwater cyanobacteria, L. wollei is a source of biologically active substances including toxic analogues of the saxitoxins such as dcSTX, dcGTX2, dcGTX3 and LWTXs, as well as of the hepatotoxin deoxy-CYN [17-21]. L. aerugineo-coerulea has been also defined as a potential producer of extracellular and intracellular toxic substances [22]. Most members of the genus Heteroleiblenia (Geitler) Hoffmann 1985 previously belonged to genus Lyngbya and the present status of this genus is not yet clearly supported and delimited

by molecular analyses [23]. Although in the last decade the efforts have been aimed to study the toxic and pharmaceutical potential of cyanobacteria, information for the freshwater species, including those of the genera Heteroleiblenia/Lyngbya, is still limited. So far, there are not data for toxicity associated with cyanobacterial representatives of the genus Heteroleiblenia. Therefore, the aim of our study was to explore the toxic potential and biological activity of the cosmopolitan freshwater cyanobacterial species H. kuetzingii (Schmidle) Compère 1985 (=Lyngbya kuetzingii Schmidle 1897). This study has revealed for the first time the toxic potential of Heteroleiblenia species.

2. Experimental Procedures 2.1 Cyanobacterial preparation

culture

and

extract

H. kuetzingii (Schmidle) Compère 1985, kept in Plovdiv Algal Culture Collection (PACC) under No 5420, has been grown intensively for 21 days under sterile conditions in Cyanobacteria BG-11 freshwater solution (Sigma-Aldrich, Steinheim, Germany). The culture was maintained at 22°C by altering light/dark periods of 14/10 h (light intensity 224 mmol photon s-1 m-2). The culture medium was continuously aerated with 100 liters of air per hour per one liter of medium, adding 1% CO2 during the light period. Cyanobacterial extract was obtained according to the method of Krishnamurthy et al. [24] with slight modifications. Briefly, the cyanobacterial mass was removed from the BG-11 medium and weighed, then frozen and thawed, and extracted twice (3 h and overnight) with water-methanol-butanol solution (15:4:1, v:v:v, analytical grade) at 22°C under magnetic stirring. The extracts were centrifuged at 9,500 xg for 30 min and the supernatants were pooled. Organic solvents were removed via speed-vac centrifugation (SAVANT, Instruments Inc. Farmingdate, NY, USA) at 37°C. The resulting extract was sterilized by filtration through a 0.22 mm Millipore GS filter and prepared to give equivalent final concentrations of 150 mg mL-1 (wet weight/volume) suspended cyanobacterial matter. To study whether H. kuetzingii release extracellular toxic metabolites, the BG-11 freshwater solution in which the cyanobacterium was cultivated during the 21 days was filtered through a 0.22 mm Millipore filter. The final equivalent concentration of suspended cyanobacterial matter was 20 mg mL-1 (wet weight/volume). This cyanobacterial growth medium was tested for toxicity and presence of cyanotoxins. 1217

Toxicity of Heteroleiblenia kuetzingii

2.2 In vitro cell cultures and exposure conditions Primary cell cultures from mouse kidney and mouse skin fibroblasts were prepared as previously described [22]. In addition, two commercially available mammalian cell lines were used for the in vitro tests: A549 (human lung carcinoma, ATCC CCL-185) and 3T3 (mouse embryonic fibroblasts, ATCC CCL-92). Cells were maintained in 75 cm2 flasks (Nunc, Roskilde, Denmark) in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibcoä, Paisley, Scotland, UK), supplemented with 10% heat inactivated fetal calf serum (FCS, PAA Laboratories GmbH, Linz, Austria), 100 IU mL-1) penicillin and 100 mg mL-1 streptomycin (Sigma-Aldrich, Steinheim, Germany), at 37°C in a 5% CO2 humidified incubator. Rainbow trout gill cells (RTgill-W1, ATCC CRL-2523) were cultured in 75 cm2 Nunc culture flasks at 19°C in Leibovitz’s L-15 medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% FCS and penicillin– streptomycin (20 IU mL-1 – 20 μg mL-1). Subcultivation procedures were performed as previously described [25]. Prior to exposure, cells were plated in 96-well tissue culture plates at a density of 5x104. After 24 h (to allow attachment of the cells) the cultures were exposed to three concentrations of the Heteroleiblenia extract or growth medium – 100 mg mL-1, 500 mg mL-1 and 1,000 mg mL-1 equivalent concentration of suspended cyanobacterial matter. Millipore water and BG-11 medium were used as appropriate controls. Mammalian cell cultures and the fish cell line RTgill-W1 were exposed for 24 h and 48 h prior to analysis.

2.3 Cytotoxicity assays 2.3.1 MTT test

The MTT test is based on the capacity of mitochondrial dehydrogenases to convert the yellow 3-(4,5-dimethyl2-thizaolyl)-2,5-diphenyl-2H-tetrazolium bromide into an insoluble and impermeable purple-blue formazan product, which accumulate in healthy cells. The staining was performed according to Edmondson et al. [26]. After the desired time of exposure to the cyanobacterial extract or growth medium (24 or 48 h), 20 mL of a 0.5% (w/v) solution of MTT (Sigma, St. Louis, MO, USA) in PBS were added directly to each well and incubated at 37°C for 3 h in dark. After incubation, the medium with the dye was aspirated and plates inverted to drain unreduced MTT, and 100 mL of DMSO was added to each well in order to facilitate solubilization of the formazan product. The plates were shaken, and absorbance was measured at 570 nm using a microplate reader (SpectraMax M5, Molecular Devices). 1218

2.3.2 alamarBlue™ and CFDA-AM test

The fluorescent dyes alamarBlue™ (BioSource, Solingen, Germany) and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM, Molecular Probes, Eugene, OR, USA) were used in combination, as previously described, using L-15/ex as a simplified culture medium [27]. The alamarBlue™ dye measures the redox potential of a cell, and 5-carboxyfluorescein diacetate acetoxymethyl ester measures cell membrane integrity. After exposure of the cells to the cyanobacterial extract or medium, the wells were emptied and filled with 100 mL of a mixuture of 5% (v/v) alamarBlue™ and 4 mmol L-1 CFDA-AM in L-15/ex and incubated in the dark for 30 min prior to fluorescent measurement. Fluorescence was analyzed using a microplate reader (SpectraMax M5, Molecular Devices) at optimized excitation/emission wavelengths for alamarBlue™ and CFDA-AM of 530/595 nm and 493/541 nm, respectively.

2.3.3 Neutral Red (NR) test

The neutral red working solution was prepared prior to each cytotoxicity test by diluting the stock solution 1:100 in L-15/ex to yield 50 μg neutral red in 1 mL L-15/ex. This working solution was filter-sterilized with a 0.2 μm Millipore filter to remove fine precipitates of the dye. For immediate measurements, aliquots of 100 μL of this working solution were added to the culture plates. After an incubation period of 1 h, which allowed the dye to be taken up by cells with intact lysosomes, the dye solution was removed and the cells were washed with a formolcalcium solution (1% anhydrous CaCl2 w/v in 0.4% formaldehyde) to remove the dead cells. The dye was then extracted from the intact cells with an acetic acid–ethanol solution (1% glacial acetic acid in 50% ethanol). The absorbance of the solution was read at 540 nm using a microplate reader (SpectraMax M5, Molecular Devices).

2.4 Proliferation assay

A549 and 3T3 cells were plated and exposed to 1,000 mg mL-1 of either Heteroleiblenia extract or Heteroleiblenia growth media as described above. During the last 18 h of exposure, cells were pulsed with 1 μCi [3H]-thymidine per well (Amersham Labs, Buckinghamshire, England). After the completion of 48 h of exposure, the cultures were harvested in a Filtermate™ cell harvester (Packard Instrument, Meriden, CT, USA). Incorporation of [3H]-thymidine was measured in a Matrix 96 Direct beta counter (Packard). The mean cpm (counts per minute) values of triplicates were determined.

2.5 In vivo mouse bioassay and histology

Nine male 8 weeks old BALB/c mice (average weight 21 g) were used for the in vivo experiment (three mice

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per group). Animals were kept under standard conditions (24ºC, 12/12 h light/dark cycles) in polystyrene cages containing wood shavings. Mice were fed standard rodent chow and water ad libitum in a specific pathogenfree environment. Experimental mice (group 1 and group 2) were injected i.p. with 500 µL test solution (cyanobacterial extract or growth medium, respectively) containing equivalent final concentrations per mouse of 15 mg suspended cyanobacterial matter. In order to obtain this test solution, the cyanobacterial extract was diluted 1:4 with Dulbecco’s phosphate-buffered saline (DPBS, Gibco®, Paisley, Scotland, UK). Control mice (group 3) were injected with 500 µL DPBS. The animals were observed for 24 h after treatment. Behavioral symptoms, mouse body weights and survival times were recorded. In addition, weights of the liver, kidneys and spleen were also recorded at autopsy in the end of the experiment. The experiment was conducted in accordance with the ‘Bulgarian National Guidelines for the Care and Use of Laboratory Animals’ (Decree No. 14/19.07.2000) and approved by the local ethics committee at Plovdiv University. All mice were subjected to histological examination of the liver and kidneys for pathology. After termination of the experiment, the liver and kidney slices were processed for light microscopy according to standard procedures. Briefly, the tissue samples were fixed in 4% buffered formalin for 24 h, dehydrated in a graded series of alcohol, cleared in xylene, and embedded in paraffin wax. Multiple sections from each block were prepared at 5 mm thickness and stained with hematoxylin and eosin [28].

2.6 High performance liquid chromatography (HPLC) and ELISA analyses HPLC analysis of the cyanobacterial extract and growth medium was performed with an ÄKTA™ explorer 100 Air system (Amersham Pharmacia Biotech AB, Uppsala, Sweden) using an UNICORN V5.11 software. The analytical column was a Discovery® C18 (5x4 mm I.D., 5 µm) from Supelco (Bellefonte, PA, USA). The mobile phase consisted of a mixture of solvent A (10 mmol L-1 ammonium acetate, pH 5.5) and solvent B (10 mmol L-1 ammonium acetate-acetonitrile, 80:20, v/v) as follows: 0% of B at 0 min, 100% of B at 45 min to 65 min using a linear gradient. Flow-rate was 0.8 mL min-1 and UV detection was performed at 238 nm. All runs were carried out at room temperature. The column was reequilibrated with 8 mL of the solvent A between runs. Each standard was run separately (AnTx-a 5 µg mL-1), MC-LR 5 µg mL-1), (STX 40.5 pg mL-1), 200 µL injection volume) and thereafter a mixture of all standards with the

same concentrations in 200 µL was run again. 200 µL of the sample were injected for HPLC analysis. Toxins and their concentrations in the sample were determined by comparing retention times and peak areas for each toxin with those of the standards. The extract of H. kuetzingii and the cyanobacterial growth medium were analyzed for presence of cyanotoxins by the Microcystin Plate kit (EnviroLogix Inc., Portland, USA) and the Ridascreen™ saxitoxin ELISA kit (R-Biopharm, Darmstadt, Germany). These are competitive immunosorbent assays for a quantitative analysis of microcystins/nodularins and saxitoxins with other related toxins based on the competition between the free toxins from samples or standards and an enzyme-conjugated cyanotoxin for the same antibody. The limit of detection of the EnviroLogix Microcystin Plate kit is 0.05 ppb and for the Ridascreen™ saxitoxin assay is about 0.010 ppb.

2.7 Statistics

Results are reported as mean ± SE from individual determinations with at least four replicates. Statistical differences were analyzed by one-way analysis of variance (ANOVA) or Mann–Whitney U-test using the StatView (SAS Institute, Inc.) programme. Values of P<0.05 were considered to be statistically significant.

3. Results 3.1 Toxicity of the H. kuetzingii growth medium and extract in vitro

Cyanobacteria, which produce toxins, emit these substances extracellularly in their habitat and/or accumulate them in the thallus as intracellular ingredients that are released after the death of the cyanobacterium. The toxic potential of each cyanobacterial species is determined from its ability to produce intracellular and/or extracellular cyanotoxins as well as other biologically active substances with a similar mode of action. To assess the toxic potential of H. kuetzingii, both, an extract (for intracellular toxins) and the nutrient solution (growth medium) in which the cyanobacterium was cultured (for extracellular toxins), were tested for toxicity. For the in vitro cytotoxicity tests primary cell cultures from mouse kidney and mouse skin fibroblasts, two commercially available mammalian cell lines as well as a fish cell line RTgill-W1 were used. The strength of the toxic effect exerted by the growth medium in which H. kuetzingii was cultivated, depended on the applied concentration, exposure time and the type of used cells. Kidney cells showed highest sensitivity to exposure. Twenty four hours after treatment with 1219

Toxicity of Heteroleiblenia kuetzingii

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1,000 mg mL-1 growth medium, the toxicity was 23% and after 48 h it has been increased two and half times, reaching 60% cell death (Figure 1A). Cytotoxic effects were not observed or were less pronounced during the first 24 h of treatment of primary fibroblasts, while after 48 h of exposure to 1,000 mg mL-1 growth medium, the toxicity reached 56% (Figure 1B). In both types of primary animal cell cultures, the toxic effects of the released in the growth medium compounds were doseand time-dependent. The potential of H. kuetzingii to produce intracellular cyanotoxins or other substances with toxic effects was tested by using an extract from the cyanobacterium and the same cells and methods described above. Mouse kidney cells treated with increased concentrations of 100 mg mL-1, 500 mg mL-1 and 1,000 mg mL-1 Heteroleiblenia extract for 24 and 48 h showed a clear

cytotoxic dose-dependent effect (Figure 2), reaching 28% toxicity at the highest concentration of extract after 24 h of exposure, and 44% toxicity after 48 h of exposure. Comparable results were obtained for the primary mouse fibroblasts after exposure to the extract (Figure 2B). Weak cytotoxic effects were observed after 24 h of exposure, whereas after 48 h, cytotoxicity was more than 25% at concentration of the extract 100 mg mL-1, reaching 50% at the highest concentration of the extract 1,000 mg mL-1. In order to elucidate if the cytotoxic effects could be reproducible using not primary cell cultures but permanent cell lines, additionally we have treated A549 and 3T3 cells with the same concentrations of Heteroleiblenia growth medium and extract for 24 and 48 h at the conditions described for the primary cell cultures. Results of the cytotoxicity evaluation are shown in Figure 3. The cytotoxic activity against both

Figure 1.

Figure 2.

In vitro cytotoxicity of the Heteroleiblenia growth medium on mammalian primary cell cultures after exposure for 24 and 48 h. (A) – kidney cells; (B) – fibroblasts. Data are reported as mean values ± SE from individual determinations with at least four replicates. Asterisks indicate significant differences in cell viability compared to the control (*P<0.05; **P<0.01; ***P<0.001).

In vitro cytotoxicity of the Heteroleiblenia extract on mammalian primary cell cultures after exposure for 24 and 48 h. (A) – kidney cells; (B) – fibroblasts. Data are reported as mean values ± SE from individual determinations with at least four replicates. Asterisks indicate significant differences in cell viability compared to the control (*P<0.05; **P<0.01).

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Figure 3.

Dose-dependent in vitro cytotoxicity of the Heteroleiblenia growth medium and Heteroleiblenia extract on permanent cell lines after exposure for 24 and 48 h. (A,B) – A549 cells; (C,D) – 3T3 cells. Data are presented as mean values ± SE from individual determinations with at least three replicates. Asterisks indicate significant differences in cell viability compared to the control (0 mg mL-1) (*P<0.05; **P<0.01).

cell lines increased by increasing the concentration of the cyanobacterial extract respectively growth medium from 0 to 1,000 mg mL-1. At the highest dose tested (1,000 mg mL-1), both Heteroleiblenia growth medium and extract showed pronounced cytotoxic effects against A549 cells after 48 h of treatment where the number of viable cells was reduced to 48.4% with an IC50 value of 970.7 mg mL-1 (Figure 3A) and 27.88% with an IC50 value of 504 mg mL-1 (Figure 3B), respectively. The Heteroleiblenia extract was cytotoxic also against 3T3 cells (Figure 3D) giving IC50=951.2 mg mL-1. However, Heteroleiblenia extract was more toxic against the lung cancer cell line A549 than the normal fibroblast cells (3T3). The antiproliferative activity of the Heteroleiblenia extract and growth medium against these two cell lines was also assessed. As shown in Figure 4, the most prominent inhibition of the cell proliferation had the Heteroleiblenia extract against the cancer cells A549. The cyanobacterial growth medium exhibited lower antiproliferative capacity. Fish cells RTgill-W1 were also exposed to 100 mg mL-1, 500 mg mL-1 and 1,000 mg mL-1 of the nutrient solution in which H. kuetzingii was grown and then the cell viability was assessed by using three different dyes: alamarBlue™, CFDA-AM and Neutral red. The obtained results in this experiment showed similar toxic profiles

Figure 4.

Antiproliferative effects of the Heteroleiblenia extract and Heteroleiblenia growth medium on permanent cell lines A549 and 3T3 after exposure to 1,000 mg  mL-1 cyanobacterial matter for 48 h. Data are presented as mean values ± SE from individual determinations with three replicates. Asterisks indicate significant differences in the cell proliferation compared to the control (*P<0.05; **P<0.01).

(Figure 5) with well-defined toxicity of 16% (CFDA-AM), 40% (alamarBlue™) and 43% (Neutral red) at a concentration of 1,000 mg mL-1 of the growth medium. Overall, all animal cell cultures used for the in vitro tests 1221

Toxicity of Heteroleiblenia kuetzingii

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showed pronounced toxicity of the growth medium in which H. kuetzingii released potential toxic compounds, as the mammalian cells were significantly more sensitive to exposure compared to the fish cells. Also, compared to the mammalian cell cultures, fish cells were less sensitive to exposure to the H. kuetzingii extract. Applying CFDA-AM and alamarBlue™ assays, a slight stimulatory effect was observed at the lowest

concentration of the extract (100 mg mL-1), whereas at the highest concentration (1,000 mg mL-1), an increase in cytotoxicity to 16% and 23% was detected (Figure 6A,B). By using Neutral red, a stimulation of lysosomal activity was observed with increasing values of 2%, 19% and 33% corresponding to the increasing concentration of the extract of 100 mg mL-1, 500 mg mL-1 and 1,000 mg mL-1 (Figure 6C).

Figure 5.

Figure 6.

In vitro cytotoxicity of the Heteroleiblenia growth medium on fish cells RTgill-W1 after exposure for 24 and 48 h. (A) – CFDA-AM test; (B) – alamarBlue™ test; (C) Neutral red test. Data are reported as mean values ± SE from individual determinations with at least four replicates. Asterisks indicate significant differences in cell viability compared to the control (*P<0.05; **P<0.01).

In vitro cytotoxicity of the Heteroleiblenia extract on fish cells RTgill-W1 after exposure for 24 and 48 h. (A) – CFDA-AM test; (B) – alamarBlue™ test; (C) Neutral red test. Data are reported as mean values ± SE from individual determinations with at least four replicates. Asterisks indicate significant differences in cell viability compared to the control (*P<0.05).

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3.2 Toxicity of the H. kuetzingii growth medium and extract in vivo The toxicity of the Heteroleiblenia extract and the growth medium in which H. kuetzingii was grown was assessed in BALB/c mice. Neurological symptoms such as reduced activity, respiratory difficulties, convulsions or spasms, as well as survival of the mice were recorded for a period of 24 h after intraperitoneal injection of the test solutions. No signs of neurotoxicity nor lethality was observed either for the experimental or control mice (Table 1). Mice injected with Heteroleiblenia extract showed an average weight loss of 0.7 g, which is 3% of their initial weight, while those treated with growth medium showed an average weight loss of 0.3 g or 1% of their weight recorded at the beginning of the experiment (Table 1). At the same time, the control group mice gained an average of 0.5 g (2%). Collected kidneys and spleens at the end of the experiment from the three groups of mice were normal in size and color. A slight increase of the liver size was observed in the two experimental groups of mice (Table 1).

3.3 Histopathology of liver and kidney

The distribution of toxicants in the body is most pronounced in highly vascularized organs such as heart, liver, kidney, lung or brain. So far, most frequently reported targets for intoxication with cyanotoxins are the liver (microcystins, nodularins) and/or kidneys (cylindrospermopsin). This was the reason to choose exactly the liver and kidneys as organs for histopathological analysis after the in vivo experiment. Despite the lack of macroscopically visible changes in the morphology and size of the collected organs after treatment, the histopathological analyses of the liver (Figure 7) and kidneys (Figure 8) showed pathological changes in these organs. In the liver of mice exposed to the cyanobacterial extract was observed infiltration of leukocytes and increased number of necrotic cells (Figure 7B).

Figure 7.

Histopathology of the liver from a mouse treated in  vivo with: (A) PBS (control group); (B) Heteroleiblenia extract; (C) Heteroleiblenia growth medium. (1) infiltration of leukocytes, (2) necrotic cells with pyknosis, (3) haemorrhage. The original magnification was 400x.

Control mice (n=3)

Heteroleiblenia extract treated mice (n=3)

Heteroleiblenia growth medium treated mice (n=3)

Body weight before treatment (g)

23.8 ± 0.6

23.8 ± 0.8

22.7 ± 0.3

Body weight after treatment (g)

24.3 ± 0.3

23.1 ± 0.3

22.4 ± 0.4

No

No

No

Parameters

Symptoms of neurotoxity Death of mice after 24 h

No

No

No

Liver weight (g)

0.95 ± 0.03

0.97 ± 0.04

0.97 ± 0.03

Spleen weight (g)

0.058 ± 0.04

0.058 ± 0.02

0.057 ± 0.03

Kidney weight (g)

0.15 ± 0.001

0.15 ± 0.002

0.15 ± 0.001

Table 1.

Effects of H. kuetzingii on BALB/c mice after i.p. injection of cyanobacterial extract or growth medium.

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The histopathological analysis of kidneys of mice treated with Heteroleiblenia extract showed breaking of the epithelium integrity of the glomerular capsule and lack of differentiation of Bowman’s space, which collects the glomerular filtrate (Figure 8B). Changes in the proximal and distal convoluted tubules as well as leukocyte infiltration and congestion of the blood vessels were also observed (Figure 8B). Kidney samples from mice treated with growth medium showed enlarged Bowman’s capsules and enlarged areas of the macula densa, which is known to act as a sensor for the concentration of sodium cations and/or chloride anions (Figure 8C).

Histological changes of the liver were more pronounced in samples of mice exposed to the growth medium, where massive hemorrhages, leukocyte infiltration and necrosis were present (Figure 7C).

3.4 ELISA and HPLC analysis for presence of microcystins/nodularins and saxitoxins

Figure 8.

Histopathology of the kidney from a mouse treated in vivo with: (A) PBS (control group); (B) Heteroleiblenia extract; (C) Heteroleiblenia growth medium. (1) breaking of the epithelium integrity of the glomerular capsule, (2) lack of differentiatied Bowman’s space, (3) changes in the proximal and distal convoluted tubules, (4) leukocyte infiltration, (5) enlarged Bowman’s capsule, (6) enlarged areas of the macula densa. The original magnification was 400x.

Cyanotoxins

Heteroleiblenia extract

Heteroleiblenia growth medium

Microcystins/nodularins (ppb)

0.05

ND

Saxitoxins (ppt)

60

10

Table 2. 1224

Data from the performed ELISA analyses showed presence of 0.05 ppb microcystins/nodularins in the extract, but not in the growth medium (Table 2). The detected concentration was at the lower detection limit of the used ELISA kit for microcystins. Saxitoxins of 60 ppt and 10 ppt were detected by ELISA in the extract and the growth medium, respectively (Table 2). The lower detection limit for this assay is 10 ppt. Figure 9 shows representative HPLC chromatograms of: a standard mixture solution containing anatoxin-a, saxitoxins and microcystin-LR (Figure 9A); Heteroleiblenia extract (Figure 9B); and growth medium in which H. kuetzingii was grown (Figure 9C). The extract of H. kuetzingii exhibited several peaks (Figure 9B). The retention time for one of them correspnded to anatoxin-α (7.54 min) and other two are potential variants of the microcystins (44.05 min and 47.02 min). It is well known that there are more than 80 variants of microcystins. An additional peak at 10.98 min retention time was also strikingly observed. This peak could be a member of the paralytic shellfish toxins (PSTs) different from the saxitoxin (STX) – probably dacarbamoylsaxitoxin (dcSTX) or gonyautoxin (GTX). Unfortunately, the accurate identification of this peak is not achievable by the method used, especially without the appropriate standard. No peaks were observed in the growth medium (Figure 9C). Obtained HPLC results are in agreement with the ELISA analyses.

Content of microcystins/nodularins and saxitoxins in Heteroleiblenia extract and Heteroleiblenia growth medium determined by ELISA. ND – not detected.

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Figure 9.

Analysis of cyanotoxins by HPLC. (A) a mixture of standard cyanotoxins; (B) Heteroleiblenia extract; (C); Heteroleiblenia growth medium. A volume of 200 μL of each sample was used for HPLC analysis under the conditions described in Experimental Procedures.

4. Discussion To examine the toxic potential of the H. kuetzingii extract and the growth medium in which H. kuetzingii was grown, we have used two primary cell cultures – mouse kidney cells and mouse fibroblasts. It has been discussed that freshly isolated cells or their primary cultures more closely resemble the in vivo environment of the respective cell type compared to the cell lines, which lack expression of key functions (e.g. organic anion transport) of their in vivo correspondents as

a result of prolonged cultivation [29]. Studies on the mechanism of action of the microcystins, indicated the need of such organic anion transporters in order for the toxic substance to be able to enter the cell [30,31]. It was found that OATP (organic anion transporting polypeptides) are expressed in many cell types including enterocytes, hepatocytes and renal epithelial cells [32]. The expression of OATP may be determines the high sensitivity of the used in our study primary kidney cells, where after 48 h of treatment the toxicity reached 60% with a clear dose-dependent effect. 1225

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Primary skin fibroblasts were chosen as a test system since the skin is one of the main routes for local exposure to xenobiotics from the environment. On the other hand, Lyngbya species are main producers of the dermatotoxins lyngbyatoxin and aplysiatoxin [33,34]. During the first 24 h of exposure to the test solutions, no significant changes in the cell survival were observed. After 48 h, the toxicity reached 50-56%. In addition, we have performed assessment of the cytotoxicity by using two cell lines (A549 and 3T3). The cell line A549 was chosen for this experiment, since it has been found that these cells express the organic aniontransporting polypeptides OATP1B1 and OATP1B3 [35], which mediate the cellular uptake of microcystins. On the other hand, these cells also express the major target protein phosphatases (PP1 and PP2A) inhibited by the microcystins [35]. As an alternative cell line to the primary mouse fibroblasts we have selected the cell line 3T3. Results showed a similar trend observed for the primary cell cultures. A dose- and time-dependent loss of cell viability was observed in the assays performed with both cell lines. The decrease of cell viability and proliferation of A549 cells was more pronounced compared to the 3T3 cells. These differences might be attributed to the expression of the OATP1B1 and OATP1B3 on the A549 cells. The fish cell line RTgill-W1 is another test system used in our study. This epithelial cell line was derived from gill explants of trout (Oncorhynchus mykiss) [25]. According to Lee et al. [36] this cell line allows the detailed study of both the cytotoxicity and biotransformation of chemicals that cause cytotoxicity, with a greater efficiency than an in vivo study of the gills. RTgill-W1 cells were used to evaluate the toxicity of industrial waste water, polycyclic aromatic hydrocarbons and heavy metals [37-42]. The lower sensitivity of the fish cells toward microcystins (compared to the mammalian cells) in our study could be explained with the absence of detectable mRNA levels of organic anion transporter polypeptides [43]. Boaru et al. [43] showed also that microcystin-LR causes damage of the subcellular structures. All indicators of cytotoxicity (MTT, alamarBlue™, CFDA-AM and Neutral red) detect changes in the cell functions. The fact that different cytotoxicity tests give similar results suggests that there is a common mechanism for direct toxic mode of action. We assume that it is linked to disruption of the normal functions of the cell membranes since all four indirect methods evaluate the membrane integrity. The great advantage of the biological methods for identification of natural toxicants is the possibility for detection of combined toxicity caused by a mix of known and unknown substances with a common or 1226

similar pattern of toxicity [44]. Our choice of methods is based on the understanding that in the environment most cyanotoxins are produced from different algae and during the state of “blooms” they are presented as a mix of toxins, whose interactions unfortunately are not yet studied [45]. The assessment of the toxicity in this study was made without preliminary data for the existing toxic substances in the samples, yet the results from the in vitro tests showed a correlation with the in vivo data for histopathological changes in the liver and kidney of the experimental animals. Several previous studies on the mechanism of action of the microcystin and its forms, determine the liver as a primary site of accumulation of this group cyanotoxins [46,47]. It is known that the microcystins inhibit protein phosphatase activity (PP1 and PP2A), leading to hyperphosphorylation of many cellular proteins, including those of the hepatocellular cytoskeleton [48-52]. The fatal loss of control of regulatory phosphorylation causes loss of cell-cell contacts and massive bleeding in the liver. We have observed a slight increase of the liver size of the treated mice, but this was much less pronounced compared to other reported data of hepatotoxic effects. The histopathological analysis of the liver of the experimental animals treated with H. kuetzingii extract showed lymphocyte infiltration and increased number of the necrotic cells. Massive infiltration of leukocytes and erythrocytes, hemorrhages and necrosis were observed in the liver samples of the mice treated with growth medium in which H. kuetzingii was grown. However, the structural changes in the tissue in both experimental groups were much less pronounced than expected during intoxication with cyanotoxins and particularly with hepatotoxins. It should be taken in account that the effect of these toxins depends on their concentration in the sample. ELISA for microcystins and nodularins showed presence of these toxins in very low concentrations. The HPLC analysis of the extract of H. kuetzingii also indicated the presence of microcystins. On the other hand, fat deposits, which are typical during intoxication with cylindrospermopsin, were not observed in the liver of both groups of treated mice suggesting absence of this toxin. The structure and function of the renal system makes the kidney particularly vulnerable to the toxic action of xenobiotics [29]. It was found that renal epithelial cells of the proximal nephron are a target for nephrotoxic compounds due to a large number of transport systems and the presence of xenobiotic metabolizing enzymes such as cytochrome P-450, glucuronyl transferase, sulfotransferases, glutathione S-transferases and others [53]. The histopathological analysis of kidneys from mice

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treated with extract of H. kuetzingii showed alteration of the integrity of the proximal and distal convoluted tubules, infiltration of leukocytes and congestion of the blood vessels. In addition, loss of the integrity of Bowman’s capsule, lack of differentiated Bowman’s space and collapsed glomeruli filled with leukocytes was also observed. Similar histopathological changes were observed in mouse kidney after treatment with MC-YR [54,55]. Taken together with the HPLC results we could conclude that H. kuetzingii produce intracellular microcystins. Although the behavior of the experimental animals treated with Heteroleiblenia extract showed no symptoms of neurotoxicity, the ELISA tests for saxitoxins detected presence of this group neurotoxins in a concentration of 60 ppt. An additional unknown peak at 10.98 min was also detected by HPLC analisys. Taking in account the fact that clearing of the neurotoxins is performed

by simple glomerular filtration, we could assume that presence of neurotoxins caused disruption of the integrity of Bowman’s capsule and Bowman’s space observed by the kidney histology. The macula densa is known to act as a sensor for the concentration of sodium cations and/or chloride anions, and histological analysis of kidney from mice treated with growth medium showed enlargement of both Bowman’s space and macula densa, suggesting the presence of other unknown toxic metabolites as well. In conclusion, this is the first report for toxigenicity of the cyanobacterial genus Heteroleiblenia. In vitro and in vivo analyses of H. kuetzingii revealed the presence of cytotoxic effects. The performed ELISA and HPLC analyses detected saxitoxins and microcystins at low concentrations. Our results indicate a need for monitoring of the distribution and abundance of the Heteroleiblenia species in the freshwater basins.

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