Respiratory toxicity of cyanobacterial aphantoxins from Aphanizomenon flos-aquae DC-1 in the zebrafish gill.

Aphantoxins from Aphanizomenon flos-aquae are frequently identified in eutrophic waterbodies worldwide. These toxins severely endanger environmental safety and human health due to the production of paralytic shellfish poisons (PSPs). Although the molecular mechanisms of aphantoxin neurotoxicity have been studied, many questions remain to be resolved such as in vivo alterations in branchial histology and neurotransmitter inactivation induced by these neurotoxins. Aphantoxins extracted from a naturally isolated strain of A. flos-aquae DC-1 were determined by high performance liquid chromatography. The basic components of the isolated aphantoxins identified were gonyautoxin 1 (GTX1), gonyautoxin 5 (GTX5), and neosaxitoxin (neoSTX), which comprised 34.04, 21.28, and 12.77% of the total, respectively. Zebrafish (Danio rerio) was administrated 5.3 or 7.61mg STX equivalents (eq)/kg (low and high doses, respectively) of the A. flos-aquae DC-1 aphantoxins by intraperitoneal injection. Histological alterations and changes in neurotransmitter inactivation in the gills of zebrafish were investigated for 24h following exposure. Aphantoxin exposure significantly increased the activities of gill alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and resulted in histological alterations in the gills during the first 12h of exposure, indicating the induction of functional and structural damage. Gill acetylcholinesterase (AChE) and monoamine oxidase (MAO) activities were inhibited significantly, suggesting an alteration of neurotransmitter inactivation in zebrafish gills. The observed alterations in gill structure and function followed a time- and dose-dependent pattern. The results demonstrate that aphantoxins or PSPs lead to structural damage and altered function in the gills of zebrafish, including changes in histological structure and increases in the activities of AST and ALT. The inhibition of the activities of AChE and MAO suggest that aphantoxins or PSPs could induce respiratory toxicity in the zebrafish gill. Furthermore, these parameters may be used as bioindicators for investigating aphantoxin exposure and cyanobacterial blooms in nature.

Antioxidative responses in zebrafish liver exposed to sublethal doses Aphanizomenon flos-aquae DC-1 aphantoxins.

Aphanizomenon flos-aquae secretes paralytic shellfish poisons (PSPs), termed aphantoxins, and endangers environmental and human health via eutrophication of water worldwide. Although the molecular mechanism of neuronal PSP toxicity has been well studied, several issues remain unresolved, notably the in vivo hepatic antioxidative responses to this neurotoxin. Aphantoxins extracted from a natural isolate of A. flos-aquae DC-1 were resolved by high performance liquid chromatography. The primary components were gonyautoxins 1 and 5 and neosaxitoxin. Zebrafish (Danio rerio) were treated intraperitoneally with either 5.3 or 7.61 (low and high doses, respectively) µg saxitoxin (STX) equivalents (eq)/kg of A. flos-aquae DC-1 aphantoxins. Antioxidative responses in zebrafish liver were examined at different timepoints 1-24h post-exposure. Aphantoxin administration significantly enhanced hepatic malondialdehyde (MDA) content 1-12h post-exposure, indicative of oxidative stress and lipid peroxidation. By contrast, levels of reduced glutathione (GSH) in zebrafish liver declined significantly after 3-24h exposure, suggesting that GSH participates in MDA metabolism. A significant upregulation of the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) was observed, suggesting that aphantoxins induce lipid peroxidation in zebrafish liver and are likely to be hepatotoxic. Hepatic levels of MDA and GSH, and of the three enzymes (SOD, CAT, and GPx), therefore provide potential biomarkers for studying environmental exposure to aphantoxins/PSPs from cyanobacterial blooms.

Morphological alterations and acetylcholinesterase and monoamine oxidase inhibition in liver of zebrafish exposed to Aphanizomenon flos-aquae DC-1 aphantoxins.

Aphanizomenon flos-aquae is a cyanobacterium that produces neurotoxins or paralytic shellfish poisons (PSPs) called aphantoxins, which present threats to environmental safety and human health via eutrophication of water bodies worldwide. Although the molecular mechanisms of this neurotoxin have been studied, many questions remain unsolved, including those relating to in vivo hepatic neurotransmitter inactivation, physiological detoxification and histological and ultrastructural alterations. Aphantoxins extracted from the natural strain of A. flos-aquae DC-1 were analyzed by high-performance liquid chromatography. The main components were gonyautoxins 1 and 5 (GTX1, GTX5) and neosaxitoxin (neoSTX), which comprised 34.04%, 21.28%, and 12.77% respectively. Zebrafish (Danio rerio) were exposed intraperitoneally to 5.3 or 7.61 µg STX equivalents (eq)/kg (low and high doses, respectively) of A. flos-aquae DC-1 aphantoxins. Morphological alterations and changes in neurotransmitter conduction functions of acetylcholinesterase (AChE) and monoamine oxidase (MAO) in zebrafish liver were detected at different time points 1-24h post-exposure. Aphantoxin significantly enhanced hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and histological and ultrastructural damage in zebrafish liver at 3-12 h post-exposure. Toxin exposure increased the reactive oxygen species content and reduced total antioxidative capacity in zebrafish liver, suggesting oxidative stress. AChE and MAO activities were significantly inhibited, suggesting neurotransmitter inactivation/conduction function abnormalities in zebrafish liver. All alterations were dose- and time-dependent. Overall, the results indicate that aphantoxins/PSPs induce oxidative stress through inhibition of AChE and MAO activities, leading to neurotoxicity in zebrafish liver. The above parameters may be useful as bioindicators for investigating aphantoxins/PSPs and cyanobacterial blooms in nature.

Lipid peroxidation and antioxidant responses in zebrafish brain induced by Aphanizomenon flos-aquae DC-1 aphantoxins.

Aphanizomenon flos-aquae is a cyanobacterium that is frequently encountered in eutrophic waters worldwide. It is source of neurotoxins known as aphantoxins or paralytic shellfish poisons (PSPs), which present a major threat to the environment and human health. The molecular mechanism of PSP action is known, however the in vivo effects of this neurotoxin on oxidative stress, lipid peroxidation and the antioxidant defense responses in zebrafish brain remain to be understood. Aphantoxins purified from a natural isolate of A. flos-aquae DC-1 were analyzed using high performance liquid chromatography. The major components of the toxins were gonyautoxins 1 and 5 (GTX1 and GTX5, 34.04% and 21.28%, respectively) and neosaxitoxin (neoSTX, 12.77%). Zebrafish (Danio rerio) were injected intraperitoneally with 7.73 µg/kg (low dose) and 11.13 µg/kg (high dose) of A. flos-aquae DC-1 aphantoxins. Oxidative stress, lipid peroxidation and antioxidant defense responses in the zebrafish brain were investigated at various timepoints at 1-24h post-exposure. Aphantoxin exposure was associated with significantly increased (>1-2 times) reactive oxygen species (ROS) and malondialdehyde (MDA) in zebrafish brain compared with the controls at 1-12h postexposure, suggestive of oxidative stress and lipid peroxidation. In contrast, reduced glutathione (GSH) levels in the zebrafish brain exposed to high or low doses of aphantoxins decreased by 44.88% and 41.33%, respectively, after 1-12h compared with the controls, suggesting that GSH participated in detoxification to ROS and MDA. Further analysis showed a significant increase in the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) compared with the controls, suggesting elimination of oxidative stress by the antioxidant response in zebrafish brain. All these changes were dose and time dependent. These results suggested that aphantoxins or PSPs increased ROS and MDA and decreased GSH in zebrafish brain, and these changes induced oxidative stress. The increased activity of SOD, CAT and GPx demonstrated that these antioxidant enzymes could play important roles in eliminating excess ROS and MDA. These results also suggest that MDA, ROS, GSH and these three antioxidant enzymes in the brain of zebrafish may act as bioindicators for investigating A. flos-aquae DC-1 aphantoxins or PSPs and algal blooms in nature.

[Effects of nitrate on organic removal and microbial community structure in the sediments].

The strategy promoted pollutant degradation and transformation under the anaerobic circumstance by adding nitrate as an electron acceptor has been widely used in sediment bioremediation. However, few literature reports on organic removal characteristics and microbial community responses in the contaminated river sediment under the nitrate reduction condition. Methods including the polar and non-polar chemical fractionation, relative abundance detection of organic matters by GC-MS were combined and applied to investigate organic removals and PCR-DGGE analysis was used for microbial community structures in sediment incubation systems with or without calcium nitrate addition. The results indicated that the addition of calcium nitrate could significantly enhance removal efficiencies of organic pollutants. The removal efficiency of total organic carbon (TOC) and the total peak area of organic matters in GC-MS were 47.25% and 29.55% which were higher than those of the control. The effect descending order of organic pollutants was: silicon materials > alkanes > polycyclic aromatic hydrocarbons > heterocyclic compounds > olefins > benzene homologues > polar compounds > phthalates > aldehydes and ketones > alkyl esters. And removal rates of silicon materials, the persistent organic pollutants, benzene homologues and heterocyclic compounds were 46.73%, 36.25%, 23.19% and 35.92% which were higher than those of the control. The PCR-DGGE profile of bacterial 16S rDNA V3 fragments showed obviously different microbial community structures between the treatment and the control systems. Blastn analysis revealed that sequences of 10 predominant bands from DGGE profile were closely related to Proteobacteria, Actinobacteria, Clostridia, Chloroflexi, Caldiserica and uncultured bacterium. The research findings provide some helpful scientific information for promoting organic pollutant removal of river sediment by nitrate reduction.

Zebrafish locomotor capacity and brain acetylcholinesterase activity is altered by Aphanizomenon flos-aquae DC-1 aphantoxins.

Aphanizomenon flos-aquae (A. flos-aquae) is a source of neurotoxins known as aphantoxins or paralytic shellfish poisons (PSPs) that present a major threat to the environment and to human health. Generally, altered neurological function is reflected in behavior. Although the molecular mechanism of action of PSPs is well known, its neurobehavioral effects on adult zebrafish and its relationship with altered neurological functions are poorly understood. Aphantoxins purified from a natural isolate of A. flos-aquae DC-1 were analyzed by HPLC. The major analogs found in the toxins were the gonyautoxins 1 and 5 (GTX1 and GTX5; 34.04% and 21.28%, respectively) and the neosaxitoxin (neoSTX, 12.77%). Zebrafish (Danio rerio) were intraperitoneally injected with 5.3 and 7.61 µg STXeq/kg (low and high dose, respectively) of A. flos-aquae DC-1 aphantoxins. The swimming activity was investigated by observation combined with video at 6 timepoints from 1 to 24 h post-exposure. Both aphantoxin doses were associated with delayed touch responses, reduced head-tail locomotory abilities, inflexible turning of head, and a tailward-shifted center of gravity. The normal S-pattern (or undulating) locomotor trajectory was replaced by a mechanical motor pattern of swinging the head after wagging the tail. Finally, these fish principally distributed at the top and/or bottom water of the aquarium, and showed a clear polarized distribution pattern at 12 h post-exposure. Further analysis of neurological function demonstrated that both aphantoxin doses inhibited brain acetylcholinesterase activity. All these changes were dose- and time-dependent. These results demonstrate that aphantoxins can alter locomotor capacity, touch responses and distribution patterns by damaging the cholinergic system of zebrafish, and suggest that zebrafish locomotor behavior and acetylcholinesterase can be used as indicators for investigating aphantoxins and blooms in nature.

[Response of the artificial cyanobacterial crusts to low temperature and light stress and the micro-structure changes under laboratory conditions].

Low temperature and light are noticeable environmental conditions commonly experienced by cyanobacterial crusts growing in desert areas. Here we reported the effects of low temperature and light on the morphology, physiological characteristics and ultrastructural changes of artificial cyanobacterial crust. Firstly artificial cyanobacterial crusts were formed by inoculating Microcoleus vaginatus Gom. and Scytonema javanicum (Kütz.) Born et Flah onto shifting sand in Petri dishes. Then, the artificial cyanobacterial crusts were selected as the experimental materials and subjected to the following treatments: 28 degrees C + 60 microE x (m2 x s)(-1) (control), 10 degrees C + 60 microE x (m2 x s)(-1), 2 degrees C +60 microE x (m2 x s)(-1) and 2 degrees C + dark. On the 0th, 5th and 12th days during the experimental period, biomass (expressed as Chl-a), photosynthetic activities (optimal quantum yield, Fv/Fm), exopolysaccharide (EPS), scytonemin, carotenoid and C-phycocyanin contents of the crusts in different treatments were determined. We also observed the ultrastructural changes of the cyanobacterial crusts in the control and 2 degrees C treatments by means of scan electron microscope (SEM). Moreover, the morphological properties such as crust color, crust thickness and crust dry weight etc. were also examined. The results indicated that the morphology of the treated crusts suffered unfavorable effect under light and low temperature stress, and Chl-a, Fv/Fm, EPS, scytonemin and carotenoid contents as well as C-phycocyanin content of the treated crusts were all significantly lower than those of the crusts under control conditions (P < 0.05). When the cyanobacterial crusts were treated for 12 days under 2 degrees C + 60 microE (m2 x s)(-1), Chl-a, Fv/Fm, EPS, scytonemin and carotenoid contents as well as C-phycocyanin content within the crusts decreased by 61.48%, 94.89%, 66.37%, 31.01%, 59.38%, and 65.91%, respectively. Obvious destruction in ultrastructure was observed in the cyanobacterial crust under cold stress, such as the presence of numerous honeycombs within the crusts and the sparse and loose appearance of the algal filaments, etc. The research verified that the acquired treatments had negative effects on the morphology, growth and microstructures of the cyanobacterial crusts, and the cooperation of low temperature and dark could provide effective protection for the morphological, physiological and microstructural features of the crust subjected to cold and light stress. The aim of this study was to primarily discuss the responses of cyanobacterial crusts to low temperature and light stress, and to offer a basic understanding of cyanobacterial crusts against extreme environments in fields, which may have certain academic significance for researches interested in cyanobactrial crusts.

UV-B-induced oxidative damage and protective role of exopolysaccharides in desert cyanobacterium Microcoleus vaginatus.

UV-B-induced oxidative damage and the protective effect of exopolysaccharides (EPS) in Microcoleus vaginatus, a cyanobacterium isolated from desert crust, were investigated. After being irradiated with UV-B radiation, photosynthetic activity (Fv/Fm), cellular total carbohydrates, EPS and sucrose production of irradiated cells decreased, while reducing sugars, reactive oxygen species (ROS) generation, malondialdehyde (MDA) production and DNA strand breaks increased significantly. However, when pretreated with 100 mg/L exogenous EPS, EPS production in the culture medium of UV-B stressed cells decreased significantly; Fv/Fm, cellular total carbohydrates, reducing sugars and sucrose synthase (SS) activity of irradiated cells increased significantly, while ROS generation, MDA production and DNA strand breaks of irradiated cells decreased significantly. The results suggested that EPS exhibited a significant protective effect on DNA strand breaks and lipid peroxidation by effectively eliminating ROS induced by UV-B radiation in M. vaginatus.

The role of glutathione metabolism in tolerance of tobacco BY-2 suspension cells to microcystin-RR.

This study was undertaken to investigate the role of the glutathione-involved detoxifying mechanism in defending the tobacco BY-2 suspension cells against microcystin-RR (MC-RR). Analysis showed that exposure of the cells to different concentrations of MC-RR (0.1, 1 and 10 microg/mL) for 0-6 days resulted in a time and concentration-dependent decrease in cell viability and increase in reactive oxygen species (ROS) content. Reduced glutathione (GSH) and total glutathione (tGSH) content as well as glutathione reductase (GR), glutathione peroxidase (GPX) and glutathione-S-transferase (GST) activities significantly increased after 3-4 days exposure in the highest two concentration treated groups, while decreased until reaching the control values except for GPX at day 6. Oxidized glutathione (GSSG) content markedly increased compared with control in high concentration MC-RR treated group after 6 days exposure. The GSH/GSSG ratio was much higher than control in 10 microg/mL MC-RR treated group at day 4, but after 6 days exposure, the ratios in all treated groups were lower than that of the control group.

Effects of iron on growth, pigment content, photosystem II efficiency, and siderophores production of Microcystis aeruginosa and Microcystis wesenbergii.

Changes in growth, photosynthetic pigments, and photosystem II (PS II) photochemical efficiency as well as production of siderophores of Microcystis aeruginosa and Microcystis wesenbergii were determined in this experiment. Results showed growths of M. aeruginosa and M. wesenbergii, measured by means of optical density at 665 nm, were severely inhibited under an iron-limited condition, whereas they thrived under an iron-replete condition. The contents of chlorophyll-a, carotenoid, phycocyanin, and allophycocyanin under an iron-limited condition were lower than those under an iron-replete condition, and they all reached maximal contents on day 4 under the iron-limited condition. PS II photochemical efficiencies (maximal PS II quantum yield), saturating light levels (I(k)) and maximal electron transport rates (ETR(max)) of M. aeruginosa and M. wesenbergii declined sharply under the iron-limited condition. The PS II photochemical efficiency and ETR(max) of M. aeruginosa rose , whereas in the strain of M. wesenbergii, they declined gradually under the iron-replete condition. In addition, I ( k ) of M. aeruginosa and M. wesenbergii under the iron-replete condition did not change obviously. Siderophore production of M. aeruginosa was higher than that of M. wesenbergii under the iron-limited condition. It was concluded that M. aeruginosa requires higher iron concentration for physiological and biochemical processes compared with M. wesenbergii, but its tolerance against too high a concentration of iron is weaker than M. wesenbergii.

Toxicity of microcystins in the isolated hepatocytes of common carp (Cyprinus carpio L.).

The toxicity of hepatotoxic microcystins produced mainly by Microcystis aeruginosa in mammals and fishes was well studied in recent years. However, there were scarcely reports in toxic effects of microcystins on isolated hepatocytes of fishes, especially investigation of microcystin-induced apoptosis and/or necrosis in carp hepatocytes. In the present study, the isolated hepatocytes of common carp were exposed to various concentrations of microcystins (0.01, 0.1, 1, 10, 100, 1000 microg L(-1)) for 2, 4, 8, 16 and 24h, respectively, and cytotoxicity of microcystins in the toxin-treated cells was determined. Results of this study showed that cytotoxicity of microcystins on carp hepatocytes was time and dose-dependent, and the approximate LC(50) of microcystins in carp hepatocytes was 169.2 microg L(-1). The morphological changes typical of apoptosis, such as blebbing of cell membrane, condensation and fragmentation of cell nucleus were observed in the hepatocytes exposed to microcystins (1, 10 and 100 microg L(-1)) using fluorescence and differential interference contrast microscopy. Agarose gel electrophoresis of DNA demonstrated a typical apoptotic "ladder pattern" in microcystin-treated hepatocytes after 16 h of exposure. Results of the present study indicated that the form of cell death in microcystin-treated hepatocytes depend on the exposure dose of toxin. When lower concentration of microcystins (10 and 100 microg L(-1)) was used for exposure, carp hepatocytes died in apoptosis while, when higher one used (1000 microg L(-1)), they died in the form of necrosis.

Selective control of toxic Microcystis water blooms using lysine and malonic acid: an enclosure experiment.

Three enclosures (10 x 10 x 1.5-1.3 m in depth) were set beside Dianch Lake, Kunming, People's Republic of China, for the period from July 28 to August 26, 2002. The enclosures were filled with cyanobacterial (Microcystis aeruginosa) water bloom-containing lake water. Lake sediment that contained macrophytes and water chestnut seeds was spread over the entire bottom of each enclosure. Initially, 10 g/m(2) of lysine was sprayed in Enclosure B, and 10 g/m(2) each of lysine and malonic acid were sprayed together in Enclosure C. Enclosure A remained untreated and was used as a control. The concentrations of lysine, malonic acid, chlorophyll a, and microcystin as well as the cell numbers of phytoplankton such as cyanobacteria, diatom, and euglena were monitored. On day 1 of the treatment, formation of cyanobacterial blooms almost ceased in Enclosures B and C, although Microcystis cells in the control still formed blooms. On day 7 Microcystis cells in Enclosure B that had been treated with lysine started growing again, whereas growth was not observed in Microcystis cells in Enclosure C, which had been treated with lysine and malonic acid. On day 28 the surface of Enclosure B was covered with water chestnut (Trapa spp.) and the Microcystis blooms again increased. In contrast, growth of macrophytes (Myriophllum spicatum and Potamogeton crispus) was observed in Enclosure C; however, no cyanobacterial blooms were observed. Lysine and malonic acid had completely decomposed. The microcystin concentration on day 28 decreased to 25% of the initial value, and the pH shifted from the initial value of 9.2 to 7.8. We concluded that combined treatment with lysine and malonic acid selectively controlled toxic Microcystis water blooms and induced the growth of macrophytes.

Physiological and biochemical analyses of microcystin-RR toxicity to the cyanobacterium Synechococcus elongatus.

Freshwater Microcystis may form dense blooms in eutrophic lakes. It is known to produce a family of related cyclic hepatopeptides (microcystins, MC) that constitute a threat to aquatic ecosystems. Most toxicological studies of microcystins have focused on aquatic animals and plants, with few examining the possible effects of microcystins on phytoplankton. In this study we chose the unicellular Synechococcus elongatus (one of the most studied and geographically most widely distributed cyanobacteria in the picoplankton) as the test material and investigated the biological parameters: growth, pigment (chlorophyll-a, phycocyanin), photosynthetic activity, nitrate reductase activity, and protein and carbohydrate content. The results revealed that microcystin-RR concentrations above 100 microg x L(-1) significantly inhibited the growth of Synechococcus elongatus. In addition, a change in color of the toxin-treated algae (chlorosis) was observed in the experiments. Furthermore, MC-RR markedly inhibited the synthesis of the pigments chlorophyll-a and phycocyanin. A drastic reduction in photochemical efficiency of PSII (F(v)/F(m)) was found after a 96-h incubation. Changes in protein and carbohydrate concentrations and in nitrate reductase activity also were observed during the exposure period. This study aimed to evaluate the mechanisms of microcystin toxicity on a cyanobacterium, according to the physiological and biochemical responses of Synechococcus elongatus to different doses of microcystin-RR. The ecological role of microcystins as an allelopathic substance also is discussed in the article.

Reactive oxygen species and antioxidant enzymes activity of Anabaena sp. PCC 7120 (Cyanobacterium) under simulated microgravity.

It was found that reactive oxygen species in Anabaena cells increased under simulated microgravity provided by clinostat. Activities of intracellular antioxidant enzymes, such as superoxide dismutase, catalase were higher than those in the controlled samples during the 7 days' experiment. However, the contents of glutathione [correction of gluathione], an intracellular antioxidant, decreased in comparison with the controlled samples. The results suggested that microgravity provided by clinostat might break the oxidative/antioxidative balance. It indicated a protective mechanism in algal cells, that the total antioxidant system activity increased, which might play an important role for algal cells to adapt the environmental stress of microgravity.

pkn22 (alr2502) encoding a putative Ser/Thr kinase in the cyanobacterium Anabaena sp. PCC 7120 is induced by both iron starvation and oxidative stress and regulates the expression of isiA.

In cyanobacteria, the isiA gene is required for cell adaptation to oxidative damage caused by the absence of iron. We show here that a putative Ser/Thr kinase gene, pkn22 (alr2052), is activated by iron deficiency and oxidative damage in Anabaena sp. PCC 7120. A pkn22 insertion mutant is unable to grow when iron is limiting. pkn22 regulates the expression of isiA (encoding CP43'), but not of isiB (encoding flavodoxin) and psbC (CP43). Fluorescence measurement at 77 K reveals the absence of the typical signature of CP43' associated with photosystem I in the mutant under iron-limiting conditions. We propose that Pkn22 is required for the function of isiA/CP43' and constitutes a regulatory element necessary for stress response.

[Lipid peroxidation in microalgae cells under simulated microgravity].

Objective. To provide direct evidences for effects of microgravity on structure and function of plasma membrane. Method. Malondialdehyde (MDA) content was examined on the basis of quantitative reaction of both MDA and thiobarbituric acid (TBA), and electrolyte leaking was determined with conductometer model DDS-11A. Result. Experiments showed that under simulated microgravity, lipid peroxidation and the content of MDA increased. Meanwhile, the membrane permeability increased in cells of two microalgae: Anabaena sp PCC7120 and Synechococcus 7942. Conclusion. Our results suggest that there is some commonness between microgravity stress and certain other environmental stresses. And cellular membrane might be the site of perception of gravity in unicells without special gravity sensitive structure, such as alga cells.