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.

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.

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.

[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.

[Over-compensatory growth of Microcystis aeruginosa after high temperature stress].

Two groups of Microcystis aeruginosa FACHB 905 cultures, 40 degrees C and 25 degrees c cultures were set in present study. Both of them were cultured for 5 and 10 days before transferred to fresh medium in same cell densities and then cultured under the same conditions at 25 degrees C. The algae which were cultured under 25 degrees C for all the time were set as the control. The growth, chlorophyll a concentration, Fv/Fm, net photosynthetic rate and respiration rate were determined after re-inoculation. The result showed that the high temperature treated groups have lower specific growth rate and lower Fv/Fm than those of control groups (p < 0.01). However, in the middle-later stage of recovery growth, the specific growth rate of 40 degrees C for 5 days group was 0.362, and it was significantly higher than that of control groups of 0.301 (p < 0.05), while the specific growth rate of 40 degrees c for 10 days group was 0.358, and there was no significant difference with control. 40 degrees C for 5 days group showed over-compensatory growth while 40 degrees C for 10 days group showed exact-compensatory growth. It implies that the over-compensatory growth characteristics of Microcysis aeruginosa is an endogenous biological factor that contributing the outbreak of blooms.

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.

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.