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Oxidative stress induced by microcystins was evaluated in an estuarine worm, Laeonereis acuta (Nereididae). Ten organisms were exposed to lyophilized cells of a toxic Microcystisaeruginosa strain RST9501 ( approximately 2 microg/mL microcystins, MC); 10 were exposed to lyophilized cells of a nontoxic Aphanotece sp. strain RSMan92 and 10 were maintained without cyanobacterial cells. Exposure time was 48 h. The enzymatic antioxidant defenses, as well as the oxidative damage, were analyzed. Toxic and nontoxic cyanobacteria lowered catalase activity with no changes in glutathione reductase and glutathione-S-transferase activities. This may have led to toxin intracellular accumulation, which should favor oxidative stress generation, observed by the high lipid peroxide and DNA-protein crosslink levels in the group exposed to MC.

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Microcystins produced by cyanobacteria are potent inhibitors of some protein phosphatases, but recent evidence also indicates its potential to generate oxidative stress. In the present study, the effects of microcystin raw extracts (Mic; 0.01 and 20microg/L) and purified okadaic acid (OA; 0.01 and 10microg/L) on short- and long-term memory alteration and antioxidant and oxidative damage were investigated in hippocampus of rats. The results showed an amnesic effect with 0.01 and 20microg/L Mic on retrieval and only with 0.01microg/L Mic on spatial learning. Parallel to these effects oxidative damage was observed as evidenced by augmented levels of lipid peroxides and DNA damage and the absence of antioxidant responses in terms of total oxyradical scavenging capacity. Phase II reactions catalyzed by glutathione-S-transferase were not modified after microcystins exposure. Overall this study showed physiological events (retrieval and spatial learning) that can be related to the classical toxic effects of microcystins (i.e., phosphatase inhibition). In addition, evidence of alternative toxicity mechanisms via oxidative stress generation was also obtained. The fact that organic anion transporter polypeptides (OATP) involved in microcystins uptake are expressed not only in liver but also in brain points to the environmental relevance of the observed effects.

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Microcystins are usually the predominant cyanotoxins present in both drinking and recreational waters after cyanobacterial blooms. Their classic toxic effect is hepatotoxicity through inhibition of serine/threonine phosphatases. However, recent studies also reported oxidative stress generation and disruption of ion regulation in aquatic organisms after microcystins exposure. In the present study, aqueous extracts of Microcystis aeruginosa were administered to the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura) by gavage in variable doses (from 34 to 860 microg kg(-1)) and exposure times (6, 12, and 72 h). A control group was exposed to saline solution. Analyzed variables included oxygen consumption, lipid peroxidation (LPO), enzyme activities (glutathione S-transferases or GST; alanine aminotransferase or ALT; aspartate aminotransferase or AST; and lactate dehydrogenase or LDH), glycogen, and microcystins content. Oxygen consumption increased in organisms exposed for 12h to 860 microg kg(-1) of microcystins and a similar result was observed after 72 h at doses equal to or higher than 34 microg kg(-1). LPO levels increased in doses equal to or higher than 34 microg kg(-1) after 72 h. GST and LDH activities increased after 12 h (at a dose of 860 microg kg(-1)), but ALT and AST activities remained unaltered in all experimental conditions. Glycogen content decreased after 72 h exposure at doses equal to or higher than 172 microg kg(-1). After 12h of exposure to 860 microg kg(-1) of microcystins, the concentration found in the hepatopancreas of C. granulatus was 13.17+/-0.56 microg kg(-1). In crabs exposed to doses higher than 172 microg kg(-1) during 72 h this value raised to 32.14+/-4.12 microg kg(-1). The obtained results indicated that microcystins exposure led the tissue to an oxidative stress condition (high LPO levels), at least in part favored by the augment of oxygen consumption, altering the glycogen metabolism. GST responses were only observed in the short-term experiment (12 h) and no effect on classical markers of vertebrate liver damage (ALT and AST) was observed. Although the hepatopancreas from C. granulatus accumulated a relatively low concentration of toxins, it was enough to induce physiological and biochemical disturbances.

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Antioxidant responses and oxidative stress were evaluated in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura) after oral microcystin administration. Responses were evaluated through antioxidant enzyme activities (catalase-(CAT), superoxide dismutase, glutathione-S-transferase- (GST)). Nonproteic sulfhydril (NP-SH) groups, oxygen consumption, lipid peroxides (LPO), and oxidized proteins were also measured. Microcystin administration increased the oxygen consumption. GST activity and NP-SH concentration showed transient increases and CAT activity showed a peak and then a reduction. Oxidative damage was evidenced with regard to LPO content and suggested by the inhibition of CAT activity at the end of the experiment, indicating that the antioxidant response induced by the toxin was insufficient. A lowering in the number of hepatopancreatic B cells should be related to microcystin elimination.

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Microcystins are hepatotoxins suspected to generate oxidative stress. This mechanism was evaluated in gills of the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura). Adult male crabs were fed ground beef with or without vitamin E (600 mg/kg). Microcystin (1.21 microg/kg) was daily administered through forced ingestion, for 7 days. After exposure, catalase activity was reduced in posterior gills of crabs supplemented with vitamin E. A lower increment in glutathione S-transferase activity (GST) was observed in organisms pretreated with vitamin E and then exposed to microcystin with respect to those exposed to the toxin but not pretreated with the vitamin. Pretreatment with vitamin E also increased nonproteic sulfhyrdil groups and this effect was not observed after microcystin exposure. The fact that supplementation with antioxidants such as vitamin E modulates GST activity indicates the direct or indirect involvement of microcystin in oxidative stress generation.

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Toxic blooms of the cyanobacterium Microcystis aeruginosa, a microcystin producer, have been observed in the past two decades in the Patos Lagoon estuary (southern Brazil). This cyanobacterium reaches the estuary from northern waters and accumulates as toxic blooms in the shallow margins of the environment. Microcystins are phosphatase (PP1 and PP2A) inhibitors and cause animal death via alteration of the liver cell cytoskeletons and intrahepatic hemorrhage. The massive accumulation of toxic material affects the survival of several benthonic estuarine local organisms. The tanaidacea Kalliapseudes schubartii is a benthonic estuarine species which occurs at high densities throughout the year in mixohaline areas of the Patos Lagoon. This microcrustacean is of high ecological relevance and plays an important role in the estuarine food web, as it is consumed on a large scale by estuarine fish. This work verifies the acute toxicity of aqueous extracts of M. aeruginosa RST9501 and of sediments spiked with lyophilized material of the same strain on K. schubartii; it also evaluates the sublethal effects on tanaidacean oxygen consumption rates and glycogen levels under acute exposure to M. aeruginosa aqueous extracts. The strain M. aeruginosa RST9501 was cultured in BGN/2 medium. The aqueous extracts were prepared using the lyophilized material from the strain cultures. Acute tests were performed over 96 h at a salinity of 15, at six toxic concentrations, and resulted in an average 96-h LC50 of 1.44 mg ml(-1). The spiked sediment tests were performed with a 10-day duration, using the lyophilized material in three proportions of powder/sediment and showed an average LC50 of 1.79 mg ml(-1). Oxygen consumption was determined after 24 and 48 h of incubation in adult organisms exposed to sublethal aqueous extract concentrations and showed a significant increase at the highest concentrations. This suggests alterations in the organism's metabolism by exposure to the cyanobacterium extract. The glycogen levels were determined with a commercial kit (Glicox 500; DOLES Ltd.); after 24 and 48 h the dosages were administered in the same organisms utilized in the oxygen consumption test and did not demonstrate significant differences. The results demonstrate the possible risks of intoxication to which the natural populations of K. schubartii were exposed in the environment and emphasize the importance of studies involving sublethal concentrations of M. aeruginosa to other organisms of the trophic web in this aquatic system.

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Microcystins are toxins produced by cyanobacteria, being toxic to aquatic fauna. It was evaluated alternative mechanisms of microcystins toxicity, including oxidative stress and histopathology in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda, Grapsidae). Microcystins was administered to crabs (MIC group) over 1 week, whereas the control (CTR group) received the saline from cyanobacteria culture medium. At day 7, catalase activity was higher in the MIC than in the CTR group, although a decrease of activity was verified in both groups with respect to time 0. Glutathione-S-transferase activity augmented in MIC with respect to CTR, suggesting a higher conjugation rate of the toxins with glutathione. No differences were detected in the superoxide dismutase activity. Lipid peroxidation remained stable in both groups. Histopathological analyses showed that the number of B cells decreased significantly in the CTR as a possible effect of starvation, while no significant change was observed in the MIC group. The hepatopancreas from the MIC group exhibited some necrotic tubules and melanin-like deposits. Overall, results showed that some enzymes of the antioxidant defense system were activated after microcystins exposure, this response being able to maintain lipid peroxidation levels, but insufficient to completely prevent histological damage.

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The objective of this work was to evaluate mechanisms of microcystin toxicity on crustacean species. Adult male crabs of Chasmagnathus granulatus (13.97+/-0.35 g) acclimated to low salinity (2 per thousand ) were injected with saline (control) or Microcystis aeruginosa aqueous extract (39.2 microg/l) at 24 h intervals for 48 h. After the exposure period, the anterior and posterior gills were dissected, measuring Na(+),K(+)-ATPase and glutathione-S-transferase (GST) activity. Total oxyradical scavenging capacity (TOSC) and lipid peroxides (LPO) content were also determined. Na(+),K(+)-ATPase activity in anterior gills was significantly lower in crabs injected with toxin than in control crabs, while no significant difference in the enzyme activity was detected in posterior gills. Both sodium and chloride concentration in the hemolymph were not affected by toxin exposure. Significant changes in GST activity were detected in posterior gills, with higher values being observed in the toxin-injected crabs. Crabs exposed to microcystin also showed a significant increase in the TOSC value against peroxyl radicals, for both anterior and posterior gills. Lipid peroxides level did not change in both gill types after exposure to the toxin. The increased levels of TOSC suggest the occurrence of a crab response against oxidative stress induced by toxin injection, which prevents lipid peroxidation.

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Recent discoveries indicate that microcystins affect enzymes, such as Na(+),K(+)-ATPase, involved in ion regulation of aquatic animals, through K(+)-dependent phosphatase inhibition. In vitro studies showed the inhibitory effect of Microcystis aeruginosa extracts on Na(+),K(+)-ATPase and K(+)-dependent phosphatase activities in gills of Chasmagnathus granulata (Decapoda, Grapsidae). Extracts of M. aeruginosa were prepared from lyophilized or cultures cells of the cyanobacterium. For lyophilized cells, IC(50) values were estimated as 0.46 microg/L (95% confidence interval [CI]=0.40-0.52 microg/L) and 1.31 microg/L (95% CI=1.14-1.51 microg/L) for Na(+),K(+)-ATPase and K(+)-dependent phosphatase, respectively. However, extracts prepared from cultured cells presented a much lower inhibitory potency against both enzymes. Gas chromatography revealed long-chain fatty acids in the lyophilized cell extracts, indicating that they are in part responsible for the enzyme inhibition. In vivo studies showed that the toxin inhibited Na(+),K(+)-ATPase activity in anterior gills, whereas an increased augmented activity of glutathione-S-transferase was observed in both kind of gills, indicating that the crab has increased its ability to conjugate the toxin. No significant differences in hemolymph sodium or chloride concentration were detected. This result is in agreement with the lack of effects of microcystin on Na(+),K(+)-ATPase activity of posterior (osmoregulating) gills.

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The effects of aqueous extracts from a cyanobacteria species, Anabaena spiroides, on fish (Odontesthes argentinensis), crab (Callinectes sapidus), and purified eel acetylcholinesterase (AChE) activity were studied. In vitro concentrations of A. spiroides aqueous extract that inhibited 50% of enzyme activity (IC50) were 23.0, 17.2, and 45.0 mg/L of lyophilized cyanobacteria for eel, fish, and crab AChE, respectively. Eel AChE inhibition follows pseudo-first-order kinetics, the same expected for organophosphorus pesticides. Inhibition of purified eel AChE using mixtures of bioxidized malathion and aqueous extract of A. spiroides showed a competitive feature (p < 0.05), suggesting that the toxin(s) could be structurally similar to an organophosphorus pesticide and that toxins present in the aqueous extract inhibit the active site of the enzyme. The inhibition recovery assays using 2-PAM (0.3 mM) showed that (1) bioxidized malathion inhibited 27.0 +/- 1.1% of crab and 36.5 +/- 0.1% of eel AChE activities; (2) with bioxidized malathion + 2-PAM the registered inhibition was 13.2 +/- 2.1% and 3.7 +/- 0.5% in crab and eel AChE, respectively; (3) the aqueous extract from A. spiroides inhibited 17.4 +/- 2.2% and 59.9 +/- 0.5% of crab and eel AChE activity, respectively; and (4) aqueous extract + 2-PAM inhibited 22.3 +/- 2.6 and 61.5 +/- 0.2% of crab and eel AChEs. The absence of enzyme activity recovery after 2-PAM exposure could imply that the enzyme aging process was extremely quick.

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[D-Leu1]Microcystin-LR was identified as the most abundant microcystin from a laboratory strain of the cyanobacterium Microcystis sp. isolated from a hepatotoxic Microcystis bloom from brackish waters in the Patos Lagoon estuary, southern Brazil. Toxicity of [D-Leu1]microcystin-LR, according to bioassay and protein phosphatase inhibition assay, was similar to that of the commonly-occurring microcystin-LR, which was not detectable in the Patos Lagoon laboratory isolate. This is the first report of a microcystin containing [D-Leu1] in the cyclic heptapeptide structure of these potent cyanobacterial toxins.

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Diversas florações da cianobactéria Microcystis aeruginosa têm sido observadas nas águas do estuário da Lagoa dos Patos nos últimos 15 anos, sem o devido estudo de sua distribuição e ocorrência ou do risco de contaminação do ecossistema aquático. Neste estudo, foram realizadas a identificação e a quantificação das cianobactérias da região estuarina da Lagoa dos Patos, particularmente M. aeruginosa, incluindo um levantamento quali-quantitativo dos principais grupos microfitoplanctônicos em relação à sua distribuição geográfica no estuário. Evidenciou-se que, ao longo de 12 meses, Microcystis aeruginosa esteve presente na região estuarina, com valor máximo de 1,3.106cél.Lû1 em dezembro de 1994 e mínimo de 1,5.105 cél.Lû1 em agosto de 1995, evidenciando a região norte do estuário da Lagoa dos Patos como sua principal fonte de entrada. Os períodos de maior abundância celular e colonial dessa cianobactéria foram consistentes com os resultados obtidos para os níveis de clorofila-a nas águas de superfície. Foram realizados testes de toxicidade (DL50 - 24 h) em camundongos com os extratos das florações de M. aeruginosa e foram determinadas, através de HPLCûDAD, as concentrações e algumas das variantes da hepatotoxina microcistina. As florações de M. aeruginosa observadas foram consideradas altamente tóxicas, com concentrações celulares de toxinas atingindo valores maiores que 1 mg.mgû1 p.s. e DL50 - 24 h menores que 100 mg.Kgû1 p.c. Encontraram-se diversas variantes da toxina microcistina, sendo microcistinaûLR a principal.

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Several blooms of Microcystis aeruginosa have been observed in the Patos Lagoon estuary during the last fifteen years without a proper investigation of their ecological importance or possible toxicity. The present study has identified and quantified the presence of cyanobacteria in the Patos Lagoon estuary, particularly of M. aeruginosa. During this survey, identification and quantification of the main phytoplankton groups were done in relation to geographical distribution in the estuary. The presence of M. aeruginosa colonies in the estuarine region confirmed their superficial distribution throughout the estuarine waters during twelve months with a maximum of 1, 3.10(6) cells. L-1 in December, 1994 and a minimum of 1, 5.10(5) cells. L-1 in August, 1995 and also confirmed that M. aeruginosa originated from waters in the north of the estuary. The period of the highest cell and colonies densities was coincident with high chlorophyll-a levels in surface waters. Toxicity of M. aeruginosa bloom material was determined by bioassay and concentrations of hepatotoxins microcystins were identified by HPLC-DAD. M. aeruginosa blooms were considered highly toxic, presenting a 24 h-LD50 lower than 100 mg.Kg-1 b.w. and a toxin content higher than 1 microgram.mg-1 d.w. Several microcystin variants were found in the extracts with microcystin-LR predominating.

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