Hepatic Histopathological Characteristics and Antioxidant Response of Phytoplanktivorous Silver Carp Intraperitoneally Injected with Extracted Microcystins

Objective To investigate the hispathological characteristics and antioxidant responses in liver of silver carp after intraperitoneal administration of microcystins （MCs） for further understanding hepatic intoxication and antioxidation mechanism in fish. Methods Phytoplanktivorous silver carp was injected intraperitoneally （i.p.） with extracted hepatotoxic microcystins （mainly MC-RR and -LR） at a dose of 1000 μg MC-LReq./kg body weight, and liver histopathological changes and antioxidant responses were studied at 1, 3, 12, 24, and 48 h, respectively, after injection. Results The damage to liver structure and the activities of hepatic antioxidant enzymes including catalase （CAT）, superoxide dismutase （SOD）, and glutathione peroxide （GPX） were increased in a time-dependent manner. Conclusion In terms of clinical and histological signs of intoxication and LD50 （i.p.） dose of MC-LR, silver carp appears rather resistant to MCs exposure than other fishes. Also the significantly increased SOD activity in the liver of silver carp suggests a higher degree of response to MCs exposure than CAT and GPX.

Accumulation of microcystins in water and economic fish in Phayao Lake, and fish ponds along the Ing River tributary in Chiang Rai, Thailand

This study determined the levels of microcystins in water and fish from Phayao Lake, Phayao Province and selected fish ponds along the Ing River tributary in Chiang Rai Province. Samples were collected monthly for 8 months (January to August 2011 for Phayao Lake, and November 2008 to June 2009 for fish ponds) and were analyzed by HPLC. The highest total microcystin-LR levels in water and fish in Phayao Lake were recorded in April 2010 at 2.60 ± 2.48 μg·L-1 and 0.20 ± 0.03 μg·kg-1 dry weight, respectively. Microcystis aeruginosa Kütz were the dominant species (271.6 ± 72.4 mm3/m3) in the lake. Colony number of Microcystis spp showed a positive correlation with soluble orthophosphate (r2 = 0.77). Similarly, Nile tilapia ponds surveyed along the tributary in Chiang Rai were contaminated with microcystins as well. The highest concentration detected in water was in March 2009 (0.58 ± 0.24 μg·L-1), whilst the maximum concentration in fish was recorded in April 2009 (2.68 ± 0.51 μg·kg-1 dry weight). Microcystis spp. dominated the pond waters and was positively correlated with chlorophyll a (r2=0.80) and soluble nitrate (r2=0.71). The highest concentration of the cyanobacteria was recorded in February 2009 at 4272.5 ± 62.3 mm3/m3. Results showed that total microcystin-LR concentration in fish in Chiang Rai ponds were higher than in Phayao Lake. This study suggested the possible health risks associated with the bioaccumulation of microcystins in fish (Nile tilapia) cultivated in fish ponds along the tributary in Chiang Rai and in Phayao Lake.

Gastrointestinal toxicity induced by microcystins

Microcystins(MCs) are produced by certain bloomforming cyanobacteria that can induce toxicity in various organs, including renal toxicity, reproductive toxicity, cardiotoxicity, and immunosuppressive effects. It has been a significant global environmental issue due to its harm to the aquatic environment and human health. Numerous investigators have demonstrated that MC exposure can induce a widespread epidemic of enterogastritis with symptoms similar to food poisoning in areas close to lakes. Both in vivo and in vitro studies have provided evidence of positive associations between MC exposure and gastrointestinal toxicity. The toxicity of MCs on the gastrointestinal tract is multidimensional. MCs can affect gastrointestinal barrier function and shift the structure of gut microbiota in different gut regions. Furthermore, MCs can inhibit the secretion of gastrointestinal digestive enzymes and the release of inflammatory cytokines, which affects the expression of immune-related genes in the intestine. The damage of the intestine is closely correlated to MC exposure because the intestine is the main site for the digestion and absorption of nutrients. The damage to the gastrointestinal tract due to MCs was summarized from different aspects, which can be used as a foundation for further exploration of molecular damage mechanisms.

Isolation of Microcystins from the Cyanobacterium Planktothrix rubescens Strain No80

Three minor microcystins have been isolated from a Planktothrix rubescens strain.Their structures have been elucidated by one-and two-dimensional NMR spectroscopy and high-resolution tandem mass spectrometry as the compounds[Asp^(3),(E)-Dhb^(7)]MC-LY(1),[Asp^(3),(E)-Dhb^(7)]MC-HtyW(2),and[Asp^(3),(E)-Dhb^(7)]MC-LW(3).The amino acids found at the variable positions 2 and 4 of the microcystin core structure are in accordance with the predicted amino acid substrate activation selectivities of the non-ribosomal peptide synthetases McyA and McyB described earlier for this strain.All structural microcystin variants produced by this strain were shown to inhibit protein phosphatase 1 in the nanomolar range.

Rapid Determination of Three Kinds of Microcystins in Environmental Water Samples by Disk SPE-Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

A method of rapidly detecting three kinds of microcystins( MCs) in environmental water samples by using disk SPE- ultra high performance liquid chromatography- tandem mass spectrometry( UPLC- MS / MS) was established. Firstly,environmental water samples were extracted by disk SPE column( C_(18)),and three kinds of MCs were separated by Waters BEH C_(18) chromatographic column with acetonitrile- 0. 2% formic acid solution as the mobile phase. After the gradient elution separation,the external standard method was used for quantitative and qualitative analysis under MRM of UPLC- MS / MS. The results showed that the three kinds of MCs in the range of 0. 05- 10 μg / L showed good linear relation,and the correlation coefficients were higher than 0. 999 4,while the method detection limit was 0. 04 ng / L. Under 0. 1,1,and 5 μg / L standard addition for the same environmental sample,the average recovery was 82. 8%- 108. 8%,and the relative standard deviation of determination results was2. 1%- 10. 1%( n = 6). This method is rapid,sensitive and accurate,so it can be effectively applied in the monitoring of MCs in environmental water samples.

Influences of Cyanobacterial Toxins Microcystins on the Seedling of Plants

Cyanobacterial blooms associated by their toxins have been increasing in frequency in fresh water bodies throughout the world. Among the cyanobacterial toxins, microcystins (MC) are the most common and cause severe adverse impacts on plants, aquatic organisms and human beings. In this study, the effects of MC (at the concentrations of 20 and 200 μg·L-1) from field water and crude extract of cyanobacterial scum (mainly Microcystis spp.) from the Dau Tieng Reservoir, Vietnam, on the seedlings of three plants, Brassica rapa-chinensis, B. narinosa and Nasturtium officinale, were investigated for over a period of 7 days. The results showed that MC reduced the fresh weight, root and shoot length of the exposed seedlings. In addition, abnormalities of leaf shape and color of B. rapa-chinensis under exposure to MC were observed. The results implied that MC were taken up and might be accumulated in the seedlings possessing potential risk to consumers as seedlings of these plants are a common food source for Vietnamese. To the best of our knowledge, this is the first report on the effects of MC on B. rapa-chinensis, B. narinosa and N. officinale.

New Records of Microcystins in Some Bulgarian Water Bodies of Health and Conservational Importance

Microcystins cause acute hepatotoxicity and chronic liver tumor promotion. This study presents the results of HPLC DAD analyses and their LC-MS confirmation of samples from five Bulgarian water bodies (reservoirs Stoudena, Pchelina, Bistritsa and lakes Dourankoulak, Vaya). The total concentration of microcystins in water samples ranged from 0.1 to 26.5 μg/l. The amount of microcystins in the biomasses ranged from 11.4 to 49.6 μg/g (d.w.). The high percent of positive samples in which the most toxic microcystin-LR is recorded, can serve as a strong alarm for the necessity of a serious study and relevant discussion of the problem with responsible authorities at national level.

Seven microcystins from Microcystis waterbloom in Lake Dalai,China

Seven types of microcystins,isolated from Microcystis waterbloom in Lake Dalai, were characterized.The major toxins:MCYST-LR,MCYST-RR,[D-Asp^3]MCYST-LR and [Dha^7]MCYST-LR were identified by high performance liquid chromatography(HPLC),as com- pared with the authentic microcystins.The minor toxins:MCYST FR,[L-Mser^7]MCYST-LR and an unknown MCYST which was most likely to be MCYST-(H_4)YR were identified with frit- fast atom bombardment liquid chromatography/mass spectrometry(Frit-FAB LC/MS)and amino acid analysis.The toxigenic diversity in blue-green algae(cyanobacteria)was discussed.

Identification and Determination of Microcystins in Source Water and Waterbloom Sample From Meiliang Bay,Taihu Lake,China

Objective To identify and determine the congener and level of microcystins in the source water of Taihu Lake. Methods Improved method of SPE combined with HPLC was employed to detect the concentration and varieties of microcystins in source water and bloom samples collected from Meiliang Bay, Taihu Lake. Results The contents of two predominant microcystin components, MC-RR, and MC-LR, were relatively high in samples during warm months and correlated with the phase of algae growth. The maximum concentrations of MC-RR and MC-LR in water sample reached 3.09±0.53μg/L and 2.39±0.41μg/L during the period of water bloom in September 2004, respectively. Even without waterbloom, the concentration of MC-LR in source water sample was still higher than the guideline value. Conclusion The status of microcystin pollution in this region is serious and measures to monitor and control the growth of cyanobacteria are urgently needed.

Characterization of chlorine dioxide as disinfectant for the removal of low concentration microcystins

Microcystins, which represents one kind of cancerogenic organic compounds, is abundant in eutro- phication water. The effects of reaction factors on chlorine dioxide （C102） for removal of low-concentration Micro- cystin-LR, Microcystin-RR, and Microcystin-YR in water as well as the reaction mechanisms was investigated by using enzyme-linked immunosorbent assay （ELISA） kit and gas chromatography-mass spectrometry （GC-MS）. The results showed that MC-LR, MC-RR, and MC-YR could be efficiently decomposed by C102. The degradation efficiency was shown positively correlated to the concen- tration of C102 and reaction time; while the effect of reaction temperature and pH is slight. The kinetic constants and activation energies of the reaction ofMC-LR, MC-RR, and MC-YR with C102 are determined as 459.89, 583.15, 488.43 L.（mol.min）I and 64.78, 53.01, 59.15kJ.mok1, respectively. As indicated by high performance liquid chromatography mass spectrometer （HPLC-MS） analysis, degradation should be accomplished via destruction of Adda group by oxidation, with the formation of dihydroxy substituendums as end products. This study has provided a fundamental demonstration of C102 serving as oxidizing disinfectant to eliminate microcystins from raw water source.

Detoxification strategy of Microcystis aeruginosa to the toxicity of Cd(Ⅱ):role of EPS in alleviating toxicity

Although many studies have found that cadmium(Cd)can be toxic to microalgae,only a few reports focused on the role of extracellular polymeric substances(EPS)in Cd(Ⅱ)detoxification.The biochemical and physiological endpoints of Microcystis aeruginosa,including the composition and functional groups of soluble EPS(SL-EPS),loosely bound EPS(LB-EPS),and tightly bound EPS(TB-EPS),were detected to elucidate the toxicity and detoxification mechanisms of Cd(Ⅱ)for cyanobacteria.Toxicological and physiological assays on M.aeruginosa showed that the 0.25-mg/L Cd(Ⅱ)resulted in a larger inhibition on growth and F_(v)/F_(m).Nevertheless,Cd(Ⅱ)significantly induced much higher contents of superoxide dismutase(SOD),intracellular microcystin LR(MC-LR),extracellular MC-LR,and EPS.Scanning electron microscopy with energy dispersive X-ray spectroscopy confirmed that Cd(Ⅱ)was absorbed into the EPS layer.Fourier transform infrared spectrum analysis revealed that the functional groups bound with Cd(Ⅱ)of algae biomass,SL-EPS,LB-EPS,and TB-EPS were somewhat different.The C=O/C=N groups ofδ-lactam or protein were their prominent functional groups,suggesting that amide or proteins in the EPS played a key role in the adsorption in Cd(Ⅱ).The concentration of 0.25 mg/L of Cd(Ⅱ)may change the chemical structure of EPS by altering the production of protein-like substances containing tryptophan.This study indicated that M.aeruginosa could detoxify Cd(Ⅱ)stress via induction of antioxidant capacity(higher SOD activity and MC synthesis),EPS production,and modification in chemical structure of EPS.

An Automatic Solid Phase Extraction and Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry for Determination of Seven Microcystins at Ultra-Trace Levels in Surface Water

A method was developed for the detection of seven microcystins(microcystin-LR, RR, YR, LA, LY, LW and LF) in surface water using automatic solid-phase extraction(A-SPE) coupled with ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS). The automated solid-phase extraction system was used to extract microcystins(MCs) from water samples. UPLC-MS/MS was used to determine MCs concentrations in just 5 min. Method detection limits were from 0.3 to 0.9 ng/L, microcystin recoveries ranged from 83.8% to 114%, and the relative standard deviation(RSD) varied from 5.6% to 12.5%. This analytical approach was found to be simple, highly sensitive, accurate, which required little manual operation. Additionally, to validate this analytical method, A-SPE+UPLC-MS/MS was applied to characterize the concentration of MCs in Taihu Lake, Wuxi, China.

Bifurcation analysis of modeling biodegradation of Microcystins

In this paper, a model with time delay describing biodegradation of Microcystins (MCs) is investigated. Firstly, the stability of the positive equilibrium and the existence of Hopf bifurcations are obtained. Furthermore, an explicit algorithm for determining the direction and the stability of the bifurcating periodic solutions is derived by using the normal form theory and center manifold argument. Finally, some numerical simulations are carried out to illustrate the applications of the results.

Spatio-temporal dynamics of cyanobacterial abundance and toxicity in a Mediterranean hypereutrophic lake

The Mediterranean basin is considered one of the most vulnerable areas in the world under the impacts of global warming and changes in precipitation patterns.Oubeira,a shallow and polymictic freshwater lake located in northeastern Algeria(36°50′N,08°23′E),has recently experienced a significant proliferation of harmful cyanobacteria resulting in the generation of toxins.We carried out this study in order to understand the succession patterns of dominant cyanobacteria and cyanotoxin production and the factors driving this in Oubeira Lake.A total of 26 cyanobacterial genera were identified,and among them Microcystis and Planktothrix accounted for more than 60%of the overall cell abundance.The summer/fall period was dominated by Microcystis,Planktothrix,and in lesser extend by Cylindrospermum,Cylindrospermopsis.During the fall/winter transition,Dolichospermum,Pseudanabaena,and Aphanizomenon were the dominant genera.Statistically,the bloom-forming cyanobacteria showed significant differences between months but not between stations.Intracellular microcystins(MC-LR)was detected in all collected samples(0.62-and 19.14-μg MC-LR equivalent/L),but appeared in high concentrations throughout the period of dominance of Microcystis and Planktothrix.Microcystis was more sensitive to nutrients than to temperature.Planktothrix was more dependent on temperature than on nutrients,which explains their coexistence during summer-fall period.However,both genera are positively correlated with MC-LR and would probably be the main producers of microcystins.Pseudanabaena,Dolichospermum,and Aphanizomenon co-occurred at the end of Planktothrix dominance period(December).Aphanizomenon and Pseudanabaena were correlated negatively with temperature and positively with water conductivity.Dolichospermum showed a strong positive correlation with MC-LR.Oubeira Lake,could serve as a model of how cyanobacteria blooms may develop in lakes within Mediterranean climates.

微囊藻毒素的神经毒性及其与神经退行性疾病的关系

微囊藻毒素(Microcystins,MCs)是蓝藻产生的一类天然毒素,对肝、神经等多个器官具有毒性。中毒人群或动物表现出一系列的神经毒性症状,引起了越来越多研究学者的关注。同时MCs可以通过一种有机阴离子转运多肽,经血脑屏障转运至脑组织中进行分布和蓄积,造成神经系统发育异常,损害神经系统功能,并可能引发相关的神经退行性疾病如阿尔茨海默病(Alzheimer’s disease,AD)、帕金森病(Parkinson’s disease,PD)的发生。一系列的蛋白质组学研究发现,MCs可能是通过影响脑组织中细胞骨架、氧化应激及能量代谢等方面对神经系统发育与功能产生一定的损伤。对MCs的神经毒性研究进展进行了概述,对其神经毒性作用机制进行了初步探讨,并对其进一步的研究提出了展望。

A method to extract algae toxin of microcystin-LR

A simple and low-cost method to obtain cyanobacterial toxin microcystin-LR(MC-LR) was developed. A new strain of Microcystis aeruginosa, named DC-1, producing microcystin-LR but not microcystin-RR, was separated from the field blooming algae samples of Dianchi Lake, in southwest of China. Following three times' freeze and thaw treatment, the cultivated DC-1 cells were extracted with 40% methanol in water. The extract was centrifuged and the supernatant applied to a Hydrophilic-Lipophilic Balance(HLB) SPE cartridge. Eluted impurities with a certain gradient from 30% to 50% methanol in water, MC-LR was finally eluted from the HLB cartridge with 60% methanol in water, and samples containing 3.85% to 14.8% of MC-LR were obtained. These MC-LR samples may be used in adsorption and biodegradation experiments instead of using expensive standard reagents.

Ecological damage of submerged macrophyte Myriophyllum spicatum by cell extracts from microcystin(MC)-and non-MC-producing cyanobacteria,Microcystis

To explore how decomposed Microcystis-dominant cyanobacterial blooms affect submerged macrophytes,the submerged plant Myriophyllum spicatum was exposed to cell extracts from microcystin(MC)-and non-MC-producing Microcystis strains in a laboratory experiment.Results showed that both Mcracystis cell extracts exerted obvious damages to plant biomass,photosynthesis,primary and secondary metabolism measures,and resistance of plant antioxidant systems,with MC-producing Microcystis having stronger effects due to the presence of MCs.Cyanotoxins other than MCs responsible for the negative effects from both strains needs further identification.The Shannon diversity and Chao1 indices of epiphytic and planktonic bacteria were decreased by the cell extracts from both Microcystis strains.However,epiphytic and planktonic bacterial communities responded differently to cell extracts at the genus level.The dominant genera of planktonic bacteria including Enterobacter,Pseudomonas,and Novosphingobium from phylum Proteobacteria,Chryseobacterium from phylum Bacteroidetes,and Microbacterium from Actinobacteriota in the treatments with cell extracts were previously reported to have strains with algicidal and MC-degrading capabilities.B acterial genes associated with energy production and conversion,amino acid transport and metabolism,and inorganic ion transport and metabolism,were more abundant in both treatments than the control for planktonic bacteria,but less abundant for epiphytic bacteria.We speculate that planktonic bacterial communities have the potential to use and degrade substances derived from Microcystis cell extracts,which may be beneficial for M.spicatum to alleviate damages from Microcystis.Further research is needed to verify the structure and function dynamics of epiphytic and planktonic bacteria in the interaction between cyanobacteria and submerged macrophytes.

BLUE-GREEN ALGAE TOXINS AND LIVER CANCER

Microcystins (MCYSTs) isolated from blue-green algae,are hepatotoxic polypeptides.It will induce severe intrahepatic hemorrhage and liver necrosis at low concentrations in rats and mice.MCYST- LR is one of MCYSTs which consists of 2 variable L- amino acids(leucine and arginine),3 D-amino acids and 2 unusualamino acids(including Adda).MCYSTs bind to protein phosphatase 1 and 2A,and strongly inhibit their activities.The resultant increase of phosphoprotein was referred to be involved in tumor-promoting activity in liver.According to the above results and animal study,MCYST-LR is a potent liver tumor promoter.There were 9 positive from 30 samples of pond-ditch water in high endemic county-Haimen by high-peformance liquid chromatograph and 3 already confirmed by liquid chromograph/mass spectrometer.The quantities of MCYSTs were different between drinking water of liver cancer cases and controls groups.122±0.057and 0.072±0.044μg/200ml respectively) by ELISA. It is not easy to remove by conventional water treatment procedures.The relationship between MCYSTs and oncogenes and anti-oncogenes are under studying.

Evaluation of Size Structure in Freshwater Cyanobacterial Populations: Methods to Quantify Risk Associated with Changes in Biomass and Microcystin Concentrations

Cyanobacterial populations in surface waters, including drinking water supplies and recreational waters, represent an ever present challenge for resource managers. As communities continuously respond to external and internal processes, dynamic profiles of composition, dominance, growth and toxigenicity emerge. In this study measures of size structure and biomass, quantified using light microscopy and fluorometry, were used to estimate microcystin concentrations through linear regression analysis. Toxigenic profiles using cyanobacterial biomass were developed for lakes dominated by Microcystis spp. and Dolichospermum spp., influenced by both genus-specific pigment concentrations as well as microcystin concentrations. Community composition (Log %MIC) and biomass were used to describe microcystin concentrations in mixed assemblages, where composition was the first input variable. The accessory photopigment phycocyanin was used to describe the linear relationship between the daily growth and net microcystin production rates in the bloom-forming Microcystis spp. samples, suggesting that this size-fractionated sample may provide indications of potential toxigenicity in the whole lake water sample. Future investigations using fluorometric evaluation of cyanobacterial populations could provide additional applications and metrics for use by resource managers to quantify risk association with elevated cyanotoxin concentrations.

Toxic Cyanobacteria in Four Brazilian Water Supply Reservoirs

Cyanobacterial blooms have become a worldwide concern due to the production of toxins harmful to humans and animals. In Brazil and worldwide, microcystins are the most frequently found cyanotoxin in water bodies. Four important reservoirs in Brazil’s Southeast and Northeast regions were sampled to identify the cyanobacteria community and the occurrence of potential toxin-producing species in the country’s public supply reservoirs. A total of 14 taxa were identi-fied, 11 of which are known as potential toxin producers. Potentially toxic cyanobacteria were recorded at concentra-tions above 20,000 cells.ml-1 in all samples from all four reservoirs, thus requiring microcystin monitoring in drinking water according to Brazilian legislation. Although the sample from Mundaú reservoir showed the highest concentration of microcystins in water, it had one of the lowest values of cells.ml-1, which demonstrates the non-correspondence between these two parameters. This calls into question the existence of a minimum level of potentially toxic cyanobacteria cells to merit the monitoring of microcystins in the treated water from these sources.