Cylindrospermopsin effects on cell viability and redox milieu of Neotropical fish Hoplias malabaricus hepatocytes.

Cylindrospermopsin (CYN) is a cyanotoxin that is cytotoxic to a wide variety of cells, particularly to the hepatocytes. In this study, the toxic effects of purified CYN were investigated in primary cultured hepatocytes of Neotropical fish Hoplias malabaricus. After isolation, attachment, and recovery for 72 h, the cells were exposed for 72 h to 0, 0.1, 1.0, 10, and 100 µg l-1 of CYN. Then, cell viability and a set of oxidative stress biomarker responses were determined. Catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione S-transferase activities were not affected by exposure to CYN. Concentration-dependent decrease of glutathione reductase activity occurred for most CYN-exposed groups, whereas non-protein thiol content increased only for the highest CYN concentration. Lipid peroxidation, protein carbonylation, and DNA damage levels were not altered, but reactive oxygen species levels increased in the cells exposed to the highest concentration of CYN. Cell viability decreased in all the groups exposed to CYN. Thus, CYN may cause a slight change in redox balance, but it is not the main cause of cell death in H. malabaricus hepatocytes.

In vivo genotoxicity of treated water containing the cylindrospermopsin-producer Cylindrospermopsis raciborskii.

Cylindrospermopsin (CYN) is an alkaloid commonly produced by some cyanobacteria that has been implicated in outbreaks of human illness. The aim of this study was to investigate the genotoxicity of Cylindrospermopsis raciborskii cellular content (including CYN) and its byproducts resulting from chlorination during water treatment. DNA damage in blood and liver cells was analysed by the comet assay and micronucleus test (MN). Mice were injected intraperitoneally with the following treatments: (a) physiological saline, (b) treated water, (c) treated water plus C. raciborskii extract (CYN producer strain, CYPO-011 K), (d) C. raciborskii extract (CYN producer strain, CYPO-011 K), (e) C. raciborskii extract (CYN non producer strain), and (f) treated water plus C. raciborskii extract (CYN non producer strain) extract. After 48 h, samples were taken to perform tests (blood and liver cells to the comet assay and bone marrow to MN test). The CYPO-011 K had a genotoxic and mutagenic effects on liver and bone marrow cells. The group that received chlorine-treated water plus CYPO-011 K also exhibited genotoxic effects in the liver, as well as in the blood, and a mutagenic effect in blood marrow cells. The results emphasise the need of improving CYN monitoring in waters bodies in order to reduce the risk of human exposure.

Dualistic evolution of liver damage in mice triggered by a single sublethal exposure to Microcystin-LR.

Microcystins (MCYST) are the most frequently reported cyanotoxins in human poisoning incidents. Despite the well-described mechanism of acute and lethal injury, the sublethal effects of this toxin require further investigation. The aim of this study was to contribute to the knowledge of the variant MCYST-LR effects at sublethal doses by investigating biochemical changes and tissue damage in a murine model. For this purpose, mice were intraperitoneally injected with 45 µg of MCYST-LR/kg body weight. Their organs were collected at 2, 8, 24, 48 or 96 h after injection. Control animals received saline solution. We detected oxidative imbalance in the liver, particularly at 8 h after exposure. Furthermore, biomarkers of liver injury were detected in high concentration in the serum of the exposed animals. Stereological analyses of the liver indicated two different phases in the intoxication process: an initial phase characterized by an increase in steatosis was followed by a second, later phase characterized by increased inflammation and hepatocyte binucleation. Formation of areas of necrosis and increased blood vessel diameter were observed throughout the experimental period. The number of hepatocytes per area unit also decreased. However, these parameters recovered over the period of exposure. MCYST accumulated in liver and was detectable until the end of the monitoring period. These results confirm the necessity for further studies of processes involved in sublethal exposure to MCYST.

Time-dependence of lung injury in mice acutely exposed to cylindrospermopsin.

Cylindrospermopsin is a cyanobacterial toxin of increasing environmental importance, as it can lead to disease if orally or intravenously absorbed. However, its in vivo lung impairment has not been documented. Thus, we aimed at verifying whether cylindrospermopsin can induce lung injury and establish its putative dependence on the time elapsed since exposure. BALB/c mice were intratracheally injected with either saline (NaCl 0.9%, 50 µL, SAL group, n = 12) or a sublethal dose (70 µg/kg) of semi-purified extract of cylindrospermopsin (CYN groups, n = 52). Lung mechanics, histological and biochemical analyses, and cylindrospermopsin presence in lungs and liver were determined in independent groups at 2, 8, 24, 48, and 96 h after cylindrospermopsin instillation. There was a significant increase in static elastance at 24 and 48 h after exposure to cylindrospermopsin, while viscoelastic component of elastance and viscoelastic pressure rose at 48 h. Alveolar collapse augmented in CYN groups at 8 h. A significant increase in polymorphonuclear influx into lung parenchyma, as well as a higher myeloperoxidase activity started off at 24 h. Exposure to cylindrospermopsin increased lipid peroxidation and superoxide dismutase activity and reduced catalase activity in CYN groups. The toxin was detected in lungs and liver of all CYN mice. In conclusion, cylindrospermopsin exposure impaired lung mechanics, which was preceded by lung parenchyma inflammation and oxidative stress.

Grazing impacts of the invasive bivalve Limnoperna fortunei (Dunker, 1857) on single-celled, colonial and filamentous cyanobacteria.

Feeding behavior of the invasive bivalve Limnoperna fortunei in the presence of single-celled, colonial, and filamentous cyanobacteria was tested in laboratory experiments to evaluate the effects of size and shape on mussel feeding. The first hypothesis holds that golden mussel filters more efficiently smaller particles, such as single cells of Microcystis, which could be more easily assimilated by its filtering apparatus. The second hypothesis sustains that L. fortunei filters more efficiently rounded colonies, such as Microcystis, which would be more easily ingested than lengthy filamentous, such as Planktothrix. Filtration rates of golden mussel in the presence of single-celled, colonial and filamentous cyanobacteria were similar. Nevertheless, there was a great difference in the ingestion and pseudofeces production rates. Single cells were widely accepted as food, while filamentous and colonial cyanobacteria were massively expelled as pseudofeces. The results confirmed the first hypothesis that golden mussel prefers to ingest smaller particles. The second hypothesis was rejected since filamentous were preferentially ingested than colonial cyanobacteria. Golden mussel has the potential to remove toxic cells (Microcystis), however this potential would be reduced in cyanobacteria blooms, where colonial forms which are preferentially rejected by L. fortunei, are predominant. In this case, the presence of this invasive bivalve could also enhance the occurrence of blooms by rejecting colonial and filamentous cyanobacteria in pseudofeces.

Feeding behavior of the invasive bivalve Limnoperna fortunei (Dunker, 1857) under exposure to toxic cyanobacteria Microcystis aeruginosa.

The aim of this study was to test the effects of cyanobacteria toxicity on feeding behavior of the golden mussel Limnoperna fortunei. First, it was tested the hypothesis that L. fortunei preferentially graze on non-toxic phytoplankton and reject toxic cyanobacteria. Second, it was tested the hypothesis that toxic cyanobacteria negatively affect feeding and survival of L. fortunei. The present study is the first to evaluate the effects of toxic cyanobacteria on L. fortunei feeding and survival. In the short-term grazing, golden mussel filtration rates were evaluated in the presence of toxic and non-toxic strains of cyanobacteria Microcystis aeruginosa, and non-toxic phytoplankton Nitzschia palea. Highest filtration rates were registered when mussels fed on Nitzschia. Despite that, golden mussel expelled Nitzschia cells in large quantities and preferentially ingested Microcystis cells, both toxic and non-toxic strains. In the long-term grazing, mussels were exposed to toxic and non-toxic strains of Microcystis during 5 days. Filtration rates were not significantly different for toxic and non-toxic Microcystis throughout exposure period. The results have demonstrated cyanobacteria toxicity is not the main factor influencing L. fortunei feeding behavior. Survival of L. fortunei feeding on toxic cyanobacteria shows the potential of this invasive bivalve as a vector to the transference of cyanotoxins to higher trophic levels.

Single sublethal dose of microcystin-LR is responsible for different alterations in biochemical, histological and physiological renal parameters.

Microcystins (MCYSTs) are very stable cyclic peptidic toxins produced by cyanobacteria. Their effects on hepatic tissue have been studied extensively, and they are considered to be a potent hepatotoxin. However, several effects of MCYST on other organs have also been described, but generally in studies using higher doses of MCYST. In the present work, we investigated the effect of a single sublethal dose of MCYST-LR (55 µg/kg) in Wistar rats and analyzed different aspects that influenced renal physiology, including toxin accumulation, excretion, histological morphology, biochemical responses and oxidative damage in the kidney. After 24 h of exposure to MCYST-LR, it was possible to observe an increased glomerular filtration rate (6.28 ± 1.56 vs 2.16 ± 0.48 µl/min per cm(2)) compared with the control group. Increase of interstitial space and collagen deposition corresponded to a fibrotic response to the increased production of reactive oxygen species. The observed decrease of Na(+) reabsorption was due to inhibition of the activity of both Na(+) pumps in proximal tubules cells. We suggested that this modulation is mediated by the effect of MCYST as a phosphatase protein inhibitor that maintains the sustained kinase-mediated regulatory phosphorylation of the ATPases. The observed alteration of Na(+) active transporters lead to damage of renal function, since are involved in regulation of water and solute reabsorption in proximal tubules. The results of this report reinforce the importance of understanding the molecular effects of a single sublethal dose of MCYST-LR, which, in this study, was responsible for macro-alterations found in the renal parenchyma and renal physiology in rats.

Serologic evaluation of human microcystin exposure.

Microcystins are among the most commonly detected toxins associated with cyanobacteria blooms worldwide. Two episodes of intravenous microcystin exposures occurred among kidney dialysis patients during 1996 and 2001. Analysis of serum samples collected during these episodes suggests that microcystins are detectable as free and bound forms in human serum. Our goal was to characterize the biochemical evidence for human exposure to microcystins, to identify uncertainties associated with interpretation of these observed results, and to identify research needs. We analyzed serum samples using enzyme-linked immunosorbent assay (ELISA) methods to detect free microcystins, and gas chromatography/mass spectrometry (GC/MS) to detect 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB). MMPB is derived from both free and protein-bound microcystins by chemical oxidation, and it appears to represent total microcystins present in serum. We found evidence of free microcystins in patient serum for more than 50 days after the last documented exposure. Serum concentrations of free microcystins were consistently lower than MMPB quantification of total microcystins: free microcystins as measured by ELISA were only 8-51% of total microcystin concentrations as detected by the GC/MS method. After intravenous exposure episodes, we found evidence of microcystins in human serum in free and protein-bound forms, though the nature of the protein-bound forms is uncertain. Free microcystins appear to be a small but variable subset of total microcystins present in human serum. Research is needed to elucidate the human toxicokinetics of microcystins, in part to determine how observed serum concentrations can be used to estimate previous microcystin exposure.

Occurrence of toxin-producing cyanobacteria blooms in a Brazilian semiarid reservoir.

We report the occurrence of cyanobacterial blooms and the presence of cyanotoxins in water samples from the Armando Ribeiro Gonçalves reservoir (06 degrees 08 S and 37 degrees 07 W), located in the state of Rio Grande do Norte, in the semiarid region of northeastern Brazil. The cyanobacterial species were identified and quantified during the rainy and dry seasons in the year 2000. Cyanotoxins such as microcystins, saxitoxins and cylindrospermopsins were analyzed and quantified using HPLC and ELISA methods. The mixed toxic blooms of Cylindrospermopsis raciborskii, Microcystis spp (M. panniformis, M. protocystis, M. novacekii) and Aphanizomenon spp (Aphanizomenon gracile, A. cf. manguinii, A. cf. issastschenkoi) were persistent and represented 90-100% of the total phytoplankton species. Toxic cyanobacterial blooms from the Armando Ribeiro Gonçalves reservoir were analyzed and found to have three phases in relation to the annual cycle. During the rainy season, an intense toxic bloom of Cylindrospermopsis raciborskii was recorded along with saxitoxins (3.14 microg.L(-1)). During the transition period, between the rainy and dry seasons, different species of Microscytis occurred and microcystin as high as 8.8 microg.L(-1) was recorded. In the dry season, co-dominance of Cylindrospermopsis raciborskii, Microcystis spp and Aphanizomenon spp occurred and the concentrations of saxitoxin remained very low. Our results indicate the presence of microcystins (8.8 microg.L(-1)) and saxitoxins (3.14 microg.L(-1)) into the crude water, with increasing concentrations from the second fortnight of April to late May 2000. The occurrence of toxic blooms in this reservoir points to a permanent risk of cyanotoxins in supply waters, indicating the need for the implementation of bloom control measures to improve the water quality. Exposure of the local population to cyanotoxins through their potential accumulation in fish muscle must also be considered.

Occurrence of toxin-producing cyanobacteria blooms in a Brazilian semiarid reservoir

Nós relatamos a ocorrência de florescimentos de cianobactérias e a presença de cianotoxinas em amostras de água do reservatório Armando Ribeiro Gonçalves (06º 08Æ S; 37º 07Æ W) situado no Estado do Rio Grande do Norte, na região semi-árida do Brasil. Cianobactérias foram identificadas e quantificadas nos períodos seco e chuvoso do ano 2000. Cianotoxinas tais como, microcistinas, saxitoxinas e cilindrospermopsinas foram quantificadas por HPLC e ELISA. Florescimentos tóxicos mistos de Cylindrospermopsis raciborskii, Microcystis spp (M. panniformis, M. protocystis, M. novacekii) e Aphanizomenon ssp (Aphanizomenon gracile, A. cf. manguinii, A. cf. issastschenkoi) foram persistentes e representaram 90-100% da comunidade fitoplanctônica ao longo do período estudado. No período de chuvas, florescimentos tóxicos de Cylindrospermopsis raciborskii coincidiram com maiores valores de saxitoxinas (3,14 µg.L-1). Entre o período de chuva e estiagem, ocorreram florescimentos tóxicos de Microcytis spp, excedendo o valor mínimo aceitável para consumo humano (8,8 µg.L-1). Na estiagem, baixas concentrações de saxitoxinas foram detectadas em florescimentos menos intensos com co-dominância de Cylindrospermopsis raciborskii, Microcystis spp e Aphanizomenon spp. Nossos resultados revelaram a presença de microcistinas (8,8 µg.L-1) e saxitoxinas (3,14 µg.L-1) na água bruta, a partir da segunda quinzena de abril até o final de maio de 2000. A ocorrência de blooms tóxicos de cianobactérias no reservatório em estudo aponta um risco permanente de cianotoxinas em águas de abastecimento e indica a necessidade da implementação de medidas de controle das florações, visando à melhoria da qualidade da água. A exposição das populações locais às cianotoxinas, pela sua potencial acumulação em musculatura de peixes, também deve ser considerada.

Toxicity of a cyanobacterial extract containing microcystins to mouse lungs

Toxic cyanobacteria in drinking water supplies can cause serious public health problems. In the present study we analyzed the time course of changes in lung histology in young and adult male Swiss mice injected intraperitoneally (ip) with a cyanobacterial extract containing the hepatotoxic microcystins. Microcystins are cyclical heptapeptides quantified by ELISA method. Ninety mice were divided into two groups. Group C received an injection of saline (300 µl, ip) and group Ci received a sublethal dose of microcystins (48.2 µg/kg, ip). Mice of the Ci group were further divided into young (4 weeks old) and adult (12 weeks old) animals. At 2 and 8 h and at 1, 2, 3, and 4 days after the injection of the toxic cyanobacterial extract, the mice were anesthetized and the trachea was occluded at end-expiration. The lungs were removed en bloc, fixed, sectioned, and stained with hematoxylin-eosin. The percentage of the area of alveolar collapse and the number of polymorphonuclear (PMN) and mononuclear cell infiltrations were determined by point counting. Alveolar collapse increased from C to all Ci groups (123 to 262 percent) independently of time, reaching a maximum value earlier in young than in adult animals. The amount of PMN cells increased with time of the lesion (52 to 161 percent). The inflammatory response also reached the highest level earlier in young than in adult mice. After 2 days, PMN levels remained unchanged in adult mice, while in young mice the maximum number was observed at day 1 and was similar at days 2, 3, and 4. We conclude that the toxins and/or other cyanobacterial compounds probably exert these effects by reaching the lung through the blood stream after ip injection.

Toxicity of a cyanobacterial extract containing microcystins to mouse lungs.

Toxic cyanobacteria in drinking water supplies can cause serious public health problems. In the present study we analyzed the time course of changes in lung histology in young and adult male Swiss mice injected intraperitoneally (ip) with a cyanobacterial extract containing the hepatotoxic microcystins. Microcystins are cyclical heptapeptides quantified by ELISA method. Ninety mice were divided into two groups. Group C received an injection of saline (300 microl, ip) and group Ci received a sublethal dose of microcystins (48.2 microg/kg, ip). Mice of the Ci group were further divided into young (4 weeks old) and adult (12 weeks old) animals. At 2 and 8 h and at 1, 2, 3, and 4 days after the injection of the toxic cyanobacterial extract, the mice were anesthetized and the trachea was occluded at end-expiration. The lungs were removed en bloc, fixed, sectioned, and stained with hematoxylin-eosin. The percentage of the area of alveolar collapse and the number of polymorphonuclear (PMN) and mononuclear cell infiltrations were determined by point counting. Alveolar collapse increased from C to all Ci groups (123 to 262%) independently of time, reaching a maximum value earlier in young than in adult animals. The amount of PMN cells increased with time of the lesion (52 to 161%). The inflammatory response also reached the highest level earlier in young than in adult mice. After 2 days, PMN levels remained unchanged in adult mice, while in young mice the maximum number was observed at day 1 and was similar at days 2, 3, and 4. We conclude that the toxins and/or other cyanobacterial compounds probably exert these effects by reaching the lung through the blood stream after ip injection.

Microcystins (cyanobacteria hepatotoxins) bioaccumulation in fish and crustaceans from Sepetiba Bay (Brasil, RJ).

Blooms of cyanobacteria in water bodies cause serious environmental problems and the occurrence of toxic strains are also related with the human health. Aquatic animals could bioaccumulate microcystins (cyanobacteria hepatotoxins) and so, beyond water, the ingestion of contaminated food represents a human health risk. Recently, WHO recommended a maximum concentration of microcystins (MCYSTs) in drinking water and established the tolerable daily intake (TDI) for consumption of cyanobacteria products contends MCYSTs (0.04 microg(-1) kg(-1) day(-1)). Sepetiba Bay is located in the municipal districts of Rio de Janeiro, Mangaratiba and Itaguai; being an important place of fishing activity. Due to the industrial development in the area, this bay is submitted to different environmental impacts, increasing the organic and industrial pollution. A strain of the nanoplanktonic cyanobacteria Synechocystis aquatilis f. aquatilis that produce MCYSTs was already isolated. In this study, we verified MCYSTs presence in muscle tissue of fish and crustaceans, which were harvested monthly in Sepetiba Bay during 11 months, in order to evaluate the potential risk of their ingestion. MCYSTs were analyzed by immunoassay techniques using the ELISA Microcystin Plate Kit (ENVIROLOGIX INC) and the concentration were expressed as microcystin-LR equivalent. The analyses of seston samples, water, muscle tissues showed the presence of this cyanotoxin in all samples and it was verified that 19% of the animals' samples were above the limit recommended by WHO for human consumption. The maximum value found was of 103.3 microg kg(-1) (TDI 0.52 microg kg(-1) day(-1)) and the minimum, was 0.25 microg kg(-1) in crabs muscle tissue (TDI of 0.001 microg kg(-1) day(-1)). Such data demonstrate that, although in low concentrations, there is already a contamination of fish and crustaceans from Sepetiba Bay. We highlight that the recommended limit refers to healthy adult.