Effects of pure microcystin-LR on the transcription of immune related genes and heat shock proteins in larval stage of zebrafish (Danio rerio)

Microcystins (MCs) are cyanobacterial toxins in water blooms that have received increasing attention as a public biohazard for human and animal health. Previous studies were mainly focused on the toxic effects on adult fish, rather than juvenile or larvae, and the response of fish immune system were usually neglected. This paper presents the first data of the effects of microcystin-LR (MC-LR) on transcription of several genes essential for early lymphoid development (Rag1, Rag2, Ikaros, GATA1, Lck and TCR alpha) and heat shock proteins (HSP90, HSP70, HSP60, HSP27) in zebrafish larvae. Relative changes of mRNA transcription were analyzed by real time PCR. The transcription of Rag1, Rag2, Ikaros, GATA1, Lck and TCR alpha were up-regulated when following exposure to 800 mu g/L MC-LR, which may indicate that specific lymphocytes differentiation and TCR/lg arrangement are induced to counteract the toxic effects of MC-LR. It was also interesting to note the dramatically increased transcription of HSP90. HSP70, HSP60 and HSP27, which may indicate their important roles as molecular chaperones under oxidative stress. (C) 2009 Elsevier B.V. All rights reserved.

Oxidative stress response after prolonged exposure of domestic rabbit to a lower dosage of extracted microcystins

Oxidative stress response after prolonged exposure to a low dose of microcystins (MCs) was studied in liver, kidney and brain of domestic rabbits. Rabbits were treated with extracted MCs (mainly MC-LR and MC-RR) at a dose of 2 MC-LReq. mu g/kg body weight or saline solution every 24 h for 7 or 14 days. During the exposure of MCs, increase of lipid peroxidation (LPO) levels were detected in all the organs studied, while antioxidant enzymes responded differently among different organs. The enzyme activities Of Superoxide dismutase (SOD). catalase (CAT) and glutathione reductase (GR) in liver decreased in the MCs treated animals. In brain, there were obvious changes in glutathione peroxidase (GPx) and GR, while only CAT was obviously influenced in kidney. Therefore, daily exposure at a lower dosage of MCs, which mimicked a natural route of MCs. could also induce obvious oxidative stress in diverse organs of domestic rabbits. The oxidative stress induced by MCs in brain was as serious as in liver and kidney, suggesting that brain may also be a target of MCs in mammals. And it seems that animals may have more time to metabolize the toxins or to form an adaptive response to reduce the adverse effects when exposed to the low dose of MCs. (C) 2008 Elsevier B.V. All rights reserved.

Reduction in microcystin concentrations in large and shallow lakes: Water and sediment-interface contributions

Blooms of cyanobacteria, or blue-greens, are known to produce chemicals, such as microcystins, which can be toxic to aquatic and terrestrial organisms. Although previous studies have examined the fate of microcystins in freshwater lakes, primary elimination pathways and factors affecting degradation and loss have not been fully explained. The goal of the present study was to explore sources of algal toxins and investigate the distribution and biodegradation of microcystins in water and sediment through laboratory and field analyses. Water and sediment samples were collected monthly from several locations in Lake Taihu from February 2005 to January 2006. Samples were analyzed for the presence of microcystin. Water and sediment were also used in laboratory studies to determine microcystin degradation rates by spiking environmental samples with known concentrations of the chemical and observing concentration changes over time. Some water samples were found to efficiently degrade microcystins. Microcystin concentrations dropped faster in water collected immediately above lake sediment (overlying water). Degradation in sediments was higher than in water. Based on spatial distribution analyses of microcystin in Lake Taihu, higher concentrations (relative to water concentrations) of the chemical were found in lake sediments. These data suggest that sediments play a critical role in microcystin degradation in aquatic systems. The relatively low levels of microcystins found in the environment are most likely due to bacterial biodegradation. Sediments play a crucial role as a source (to the water column) of bio-degrading bacteria and as a carbon-rich environment for bacteria to proliferate and metabolize microcystin and other biogenic toxins produced by cyanobacteria. These, and other, data provide important information that may be applied to management strategies for improvement of water quality in lakes, reservoirs and other water bodies. (C) 2007 Elsevier Ltd. All rights reserved.

Allelopathic interactions between Potamogelon maackianus and Microcystis aeruginosa

The allelopathic interactions between Potamogeton maackianus and toxic cyanobacteria (Microcystis aeruginosa) were studied. P maackianus inhibited the growth of M. aeruginosa, both in a coexistence culture system and in exudates experimental culture system. M. aeruginosa also showed effects on the secondary metabolic biosynthesis and secreting behavior of P maackianus. The main lipophilic components of the hexane extracts and the exudates from the macrophyte were analyzed through GC-MS determination. The lipophilic components of the hexane extracts and the exudates from P. maackianus were influenced by M. aeruginosa or their released chemicals. Comparing the lipophilic constituents of the hexane extracts with those in the exudates, the results showed that weak polar compounds contained in the macrophytes can be secreted into the surrounding water. Many compounds, such as N-phenyl-2-naphthalenamine and isopropyl myristate, were detected both in the hexane extracts and the exudates. The changes of lipophilic components in the hexane extracts would be a response to the stress of toxic cyanobacteria or their released toxins. Those changes of exudates, especially the increased content of N-phenyl-2-naphthalenamine, might also be an induced defensive behavior mediated by the released toxins from M aeruginosa. The study results about reciprocal allelopathic responses between macrophytes and cyanobacteria can help in the management of eutrophic waters, and is also important information concerning strategies for recovering eutrophic waters.

Distribution and bioaccumulation of microcystins in water columns: A systematic investigation into the environmental fate and the risks associated with microcystins in Meiliang Bay, Lake Taihu

For the purpose of understanding the environmental fate of microcystins (MCs) and the potential health risks caused by toxic cyanobacterial blooms in Lake Taihu, a systematic investigation was carried out from February 2005 to January 2006. The distribution of MCs in the water column, and toxin bioaccumulations in aquatic organisms were surveyed. The results suggested that Lake Taihu is heavily polluted during summer months by toxic cyanobacterial blooms (with a maximum biovolume of 6.7 x 10(8) cells/L) and MCs. The maximum concentration of cell-bound toxins was 1.81 mg/g (DW) and the dissolved MCs reached a maximum level of 6.69 mu g/L. Dissolved MCs were always found in the entire water column at all sampling sites throughout the year. Our results emphasized the need for tracking MCs not only in the entire water column but also at the interface between water and sediment. Seasonal changes of MC concentrations in four species of hydrophytes (Eichhornic crassipes, Potamogeton maackianus, Alternanthera philoxeroides and Myriophyllum spicatum) ranged from 129 to 1317, 147 to 1534, 169 to 3945 and 124 to 956 ng/g (DW), respectively. Toxin accumulations in four aquatic species (Carassius auratus auratu, Macrobrachium nipponensis, Bellamya aeruginosa and Cristaria plicata) were also analyzed. Maximum toxin concentrations in the edible organs and non-edible visceral organs ranged from 378 to 730 and 754 to 3629 ng/g (DW), respectively. Based on field studies in Lake Taihu, risk assessments were carried out, taking into account the WHO guidelines and the tolerable daily intake (TDI) for MCs. Our findings suggest that the third largest lake in China poses serious health threats when serving as a source of drinking water and for recreational use. In addition, it is likely to be unsafe to consume aquatic species harvested in Lake Taihu due to the high-concentrations of accumulated MCs. (C) 2007 Elsevier Ltd. All rights reserved.

Bioaccumulation of the hepatotoxic microcystins in various organs of a freshwater snail from a subtropical Chinese lake, Taihu lake, with dense toxic microcystis blooms

In this paper, we describe the seasonal dynamics of three common microcystins (MCs MC-RR, MC-YR, and MC-LR) in the whole body, hepatopancreas, intestine, gonad, foot, remaining tissue, and offspring of a freshwater snail, Bellamya aeruginosa, from Gonghu Bay of Lake Taihu, China, where dense toxic Microcystis blooms occur in the warm seasons. Microcystins were determined by liquid chromatography electrospray ionization mass spectrum. Microcystin (MC-RR + MC-YR + MC-LR) content of the offspring and gonad showed high positive correlation, indicating that microcystins could transfer from adult females to their young with physiological connection. This study is the first to report the presence of microcystins in the offspring of the adult snail. The majority of the toxins were present in the intestine (53.6%) and hepatopancreas (29.9%), whereas other tissues contained only 16.5%. If intestines are excluded, up to 64.3% of the toxin burden was allocated in the hepatopancreas. The microcystin content in the intestine, hepatopancreas, and gonad were correlated with the biomass of Microcystis and intracellular and extracellular toxins. Of the analyzed foot samples, 18.2% were above the tolerable daily microcystin intake recommended by the World Health Organization (WHO) for human consumption. This result indicates that public health warnings regarding human ingestion of snails from Taihu Lake are warranted. In addition, further studies are needed to evaluate the occurrence by Microcystis in relation to spatial and temporal changes in water quality.

Sorption, degradation and mobility of microcystins in Chinese agriculture soils: Risk assessment for groundwater protection

In the present paper, sorption, persistence, and leaching behavior of three microcystin variants in Chinese agriculture soils were examined. Based on this study, the values of capacity factor and slope for three MCs variants in three soils ranged from 0.69 to 6.00, and 1.01 to 1.54, respectively. The adsorption of MCs in the soils decreased in the following order: RR > Dha(7) LR > LR. Furthermore, for each MC variant in the three soils, the adsorption rate in the soils decreased in the following order: soil A > soil C > soil B. The calculated half-time ranged between 7.9 and 17.8 days for MC-RR, 6.0-17.1 days for MC-LR, and 7.1-10.2 days for MC-Dha(7) LR. Results from leaching experiments demonstrated that recoveries of toxins in leachates ranged from 0-16.7% for RR, 73.2-88.9% for LR, and 8.9-73.1% for Dha 7 LR. The GUS value ranged from 1.48 to 2.06 for RR, 1.82-2.88 for LR, and 1.76-2.09 for Dha(7) LR. Results demonstrated the use of cyanobacterial collections as plant fertilizer is likely to be unsafe in soils. (c) 2006 Elsevier Ltd. All rights reserved.

First report of aphantoxins in China - waterblooms of toxigenic Aphanizomenon flos-aquae in Lake Dianchi

The oligohaline cyanobacterium Aphanizomenon flos-aquae (L.) Ralfs (A. flos-aquae) has been reported in several countries to produce paralytic shellfish poisons (PSPs) or protracted toxic effects. In the past years, A. flos-aquae blooms have occurred annually in the eutrophic Lake Dianchi (300 km(2) in area, located in southwestern China). Material from natural blooms dominated by A. flosaquae was collected and lyophilized. Acute toxicity testing was performed by mouse bioassay using extracts from the lyophilized material. Clear symptoms of PSPs, intoxications were observed. To confirm the production of PSPs, a strain of A. flos-aquae (DC-1) was isolated and maintained in culture. Histopathological effects were studied by examining the organ damages using transmission electron microscopy (TEM). Slight hepatocytic damage with swollen mitochondria was found. The ultrastructural pulmonary lesions were characterized by distortied nuclei and indenting of karyotheca, together with degeneration and tumefaction of mitochondria and endoplasmic reticulum. Control animals injected with acetic acid did not exhibit histopathological damage in any organ. Toxic effects of cultured algal cells on enzymatic systems in the mouse were studied using sublethal doses of extracts. Significant glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) increases, together with decrease of the glutathione (GSH) level, were measured. These results indicated a potential role of PSPs intoxicating and metabolizing in the test animals. HPLC-FLD and LC/MS analysis of extracts from cultured material demonstrated the PSP toxins produced by A. flos-aquae bloom. To the best of our knowledge, this is the first study reporting chemically and toxicologically confirmed PSP toxins related to A. flosaquae in China. (c) 2005 Elsevier Inc. All rights reserved.

Nitrate and phosphate supplementation to increase toxin production by the marine dinoflagellate Alexandrium tamarense

Alexandrium tamarense toxins have great value in biotechnology research as well as important in connection with shellfish poisoning. The influence of nitrate or nitrate and phosphate supplementation on cell biomass and toxin content were investigated in batch cultures. When cultures at low nitrate (88.2 mu M NaNO3) Were supplemented with 793.8 mu M NaNO3 at day 10 the cell density and cellular toxin contents were increased by 6-29% and 20-76%, respectively, compared with controls, and maximal values were 43,600 cells/ml (day 38) and 0.91 pg/cell (day 31). Supplementation with nitrate at day 14 or with nitrate and phosphate at day 10/14 to the cultures did not increase the cell density compared with the non-supplemented middle nitrate or high phosphate (108 mu M NaH2PO4) cultures, respectively, but increased the cellular toxin contents by an average of 52%. The results showed that supplementation with nitrate or with nitrate and phosphate at different growth phases of the cultures increased toxin yield by an average of 46%. Supplementation with nitrate at selected times to maintain continuous low level of nitrate might contribute to the effective increase of toxin yield of A. tamarense. (c) 2005 Elsevier Ltd. All rights reserved.

Optimization of an effective extraction procedure for the analysis of microcystins in soils and lake sediments

Microcystin analysis in sediments and soils is considered very difficult due to low recovery for extraction. This is the primary limiting factor for understanding the fate of toxins in the interface between water and sediment in both the aquatic ecosystem as well as in soils. In the present study, a wide range of extraction solvents were evaluated over a wide range of pH, extraction approaches and equilibration time to optimize an effective extraction procedure for the analysis of microcystins in soils and lake sediments. The number of extractions required and acids in extraction solutions were also studied. In this procedure, EDTA-sodium pyrophosphate solution was selected as an extraction solvent based on the adsorption mechanism study. The optimized procedure proved to be highly efficient and achieved over 90% recovery. Finally, the developed procedure was applied to field soil and sediment sample collected from Chinese lakes during bloom seasons and microcystins were determined in six of ten samples. (c) 2005 Elsevier Ltd. All rights reserved.

Effect of inclusion of blue-green algae meal on growth and accumulation of microcystins in gibel carp (Carassius auratus gibelio)

Six isonitrogenous (crude protein content: 38%) and isoenergetic (gross energy content: 17 kJ g(-1)) diets were formulated to investigate the effects of inclusion of blue-green algae meal on gibel carp (Carassius auratus gibelio). In each diet, 15% of the protein was supplied by fishmeal; the remainder was supplied by soybean meal and blue-green algae meal. Diet 1 was used as control with no blue-green algae meal whereas the content in diets 2-6 was 15.15, 29.79, 44.69, 59.58 and 74.48%, respectively. Each diet was fed to five groups of gibel carp for 12 weeks in a flow-through system. Final body weight and specific growth rate (SGR) of fish fed diet 5 were significantly lower than the control diet (P < 0.05). Mortality of gibel carp increased with increase in algae meal inclusion (P < 0.05), but there was no significant difference between fish fed diets 3-6 (P > 0.05). Feed conversion efficiency (FCE) decreased with the increase in algae meal inclusion (P < 0.05). Fish-fed diet 6 showed the highest feeding rate (P < 0.05), while there were no significant differences among the other groups (P > 0.05). Apparent digestibility coefficient of dry matter, protein, and energy decreased with increasing algae meal inclusion in the diets (P < 0.05). Aspartate aminotransferase (GOT) activity in the liver was not significantly different among groups (P > 0.05). Liver alanine aminotransferase (GPT) activity of fish-fed diets 4, 5 and 6 was significantly lower than the control diet (diet 1; P < 0.05). Microcystins in the muscle, liver, gallbladder, and spleen increased with increasing algae inclusion (P < 0.05).

Seasonal dynamics of the hepatotoxic microcystins in various organs of four freshwater bivalves from the large eutrophic Lake Taihu of subtropical China and the risk to human consumption

So far, little is known on the distribution of hepatotoxic microcystin (MC) in various organs of bivalves, and there is no study on MC accumulation in bivalves from Chinese waters. Distribution pattern and seasonal dynamics of MC-LR, -YR and -RR in various organs (hepatopancreas, intestine, visceral mass, gill, foot, and rest) of four edible freshwater mussels (Anodonta woodiana, Hyriopsis cumingii, Cristaria plicata, and Lamprotula leai) were studied monthly during Oct. 2003-Sep. 2004 in Lake Taihu with toxic cyanobacterial blooms in the summer. Qualitative and quantitative determinations of MCs in the organs were done by LC-MS and HPLC. The major toxins were present in the hepatopancreas (45.5-55.4%), followed by visceral mass with substantial amount of gonad (27.6-35.5%), whereas gill and foot were the least (1.8-5.1%). The maximum MC contents in the hepatopancreas, intestine, visceral mass, gill, foot, and rest were 38.48, 20.65, 1.70, 0.64, 0.58, and 0.61 mu g/g DW, respectively. There were rather good positive correlation in MC contents between intestines and hepatopancreas of the four bivalves (r = 0.75-0.97, p < 0.05). There appeared to be positive correlations between the maximum MC content in the hepatopancreas and the delta(13)C (r = 0.919) or delta(15)N (r = 0.878) of the foot, indicating that the different MC content in the hepatopancreas might be due to different food ingestion. A glutathione (GSH) conjugate of MC-LR was also detected in the foot sample of C. plicata. Among the foot samples analyzed, 54% were above the provisional WHO tolerable daily intake (TDI) level, and the mean daily intakes from the four bivalves were 8-23.5 times the TDI value when the bivalves are eaten as a whole, suggesting the high risk of consuming bivalves in Lake Taihu. (C) 2005 Wiley Periodicals, Inc.

Growth and antioxidant system of the cyanobacterium Synechococcus elongatus in response to microcystin-RR

Microcystins, one type of the cyanobacterial toxins, show a broad range of hazardous effects on other organisms. Most of the researches on the toxic effects of microcystins have involved in animals and higher plants. Little work, however, has been done on evaluating the mechanisms of microcystin toxicity on algae. In this study, the toxicological effects of microcystin-RR (MC-RR) on the cyanobacterium Synechococcus elongatus were investigated. For this purpose, six physio-biochemical parameters (cell optical density, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST)) were tested in algal cells when exposed to 100 mug(-1) microcystin-RR. The results showed that the growth of Synechococcus elongatus ( expressed as optical density) was significantly inhibited compared with the control. At the same time, the treated algae exhibited a pronounced increase in production of ROS and MDA after 6 days exposure to microcystin-RR. Signi. cant changes in GSH levels and GSH-Px, GSH activities were also detected in algal cells, with higher values being observed in the toxin treated algae after 6 days exposure. GST activities in the treated algae exhibited a decline after exposure and rapid augmentation on day 3, thereafter, they kept at a high level when compared to the control group. GSH contents and GSH-Px activities were also significantly raised in the toxin-treated algae cells from day 3, but they showed a sharp decrease on day 4, which was the onward of cell proliferation. These results suggested that oxidative stress manifested by elevated ROS levels and MDA contents might be responsible for the toxicity of microcystin to Synechococcus elongatus and the algal cells could improve their antioxidant ability through the enhancement of enzymatic and non-enzymatic preventive substances.

Tissue distributions and seasonal dynamics of the hepatotoxic microcystins-LR and -RR in a freshwater snail (Bellamya aeruginosa) from a large shallow, eutrophic lake of the subtropical China

Tissue distributions and seasonal dynamics of the hepatotoxic microcystins-LR and -RR in a freshwater snail (Bellamya aeruginosa) were studied monthly in a large shallow, eutrophic lake of the subtropical China during June-November, 2003. Microcystins (MCs) were quantitatively determined by High-Performance Liquid Chromatography (HPLC) with a qualitative analysis by a Finnigan LC-MS system. On the average of the study period, hepatopancreas was the highest in MC contents (mean 4.14 and range 1.06-7.42 mug g(-1) DW), followed by digestive tracts (mean 1.69 and range 0.8-4.54 mug g(-1) DW) and gonad (mean 0.715 and range 0-2.62 mug g(-1) DW), whereas foot was the least (mean 0.01 and range 0-0.06 mug g(-1) DW). There was a positive correlation in MC contents between digestive tracts and hepatopancreas. A constantly higher MC content in hepatopancreas than in digestive tracts indicates a substantial bioaccumulation of MCs in the hepatopancreas of the snail. The average ratio of MC-LR/MC-RR showed a steady increase from digestive tracts (0.44) to hepatopancreas (0.63) and to gonad (0.96), suggesting that MC-LR was more resistant to degradation in the snail. Since most MCs were present in the hepatopancreas, digestive tracts and gonad with only a very small amount in the edible foot, the risk to human health may not be significant if these toxic parts are removed prior to snail consumption. However, the possible transference of toxins along food chains should not be a negligible concern. (C) 2004 Elsevier Ltd. All rights reserved.

Effects of nitrogen forms on the production of cyanobacterial toxin microcystin-LR by an isolated Microcystis aeruginosa

A cyanobacterial strain, which produced high content of microcystin-LR (MC-LR) but no rnicrocystin-RR (MC-RR), was isolated from the hypertrophic Dianchi Lake in China and identified as Microcystis aeruginosa DC-1. Effects of nitrogen containing chemicals and trace elements on the growth and the production of MC-LR by this strain were Studied. In the presence of bicine, compared with urea and ammonium, nitrate greatly promoted the growth and the production of MC-LR. However, leucine and arginine, which were the constitutional components in the molecular structure of MC-LR or RR, inhibited the production of MC-LR. Iron and silicon up to 10mg/L had little effects on the growth of M. aeruginosa DC-1, but the production of MC-LR was apparently enhanced. Under all conditions studied here, only MC-LR but no RR was detected within the cells of M. aeruginosa DC-1. Thus, chemical forms of nitrogen, rather than the usually concerned the total nitrogen, Lind trace elements played important roles in the production of MC toxins during cyanobacterial blooms.