William R. Evans

Hepatotoxic microcystin diversity in cyanobacterial blooms collected in portuguese freshwaters

Abstract Twelve toxic cyanobacterial bloom samples collected in natural lakes, reservoirs and rivers of Portugal were analysed. Toxicity was evaluated by mouse LD 50 bioassay of the lyophilised samples. The main bloom species present in the samples were Microcystis aeruginosa, Microcystis wesenbergii, Anabaena flos-aquae and Nostoc sp. Toxins were extracted, isolated by reverse phase HPLC and characterised by HPLC amino acid analysis and fast atom bombardment mass spectrometry. Two to seven microcystins were purified from each sample, and a total of seven different toxins were isolated and their structure assigned. MCYST-LR was the most common and its proportion in each sample ranged from 45.5% to 99.8% of the total microcystin contents. MCYST-RR, MCYST-YR and [D-Asp 3 ]MCYST-LR were also identified in the samples. Three less common microcystins, MCYST-HilR, [L-MeSer 7 ]MCYST-LR and [Dha 7 ]MCYST-LR, were found in only one sample. Total MCYST concentration varied from 1.0 to 7.1 μg/mg dry weight of cyanobacteria. Significant relationships between LD 50 -MCYST-LR-total MCYST content were found. The need for monitoring cyanobacteria and their toxins in eutrophic waters that are used for drinking and recreation purposes is discussed.

First report of microcystins from a Brazilian isolate of the cyanobacteriumMicrocystis aeruginosa

This is the first report on microcystins, cyclic heptapeptide hepatotoxins, from Brazilian water supplies. A colony isolate (NPJB-1) of the colonial cyanobacteriumMicrocystis aeruginosa from Lagoa das Garças, São Paulo, was cultured under non-axenic conditions. Exponential phase cells were harvested, concentrated and lyophilized for mouse bioassays and toxin extraction. The LD100 of lyophilized cell suspensions was approximately 31 mg kg−1 (dry cell weight/animal weight). Isolation, purification and characterization of the toxins were carried out by reversed phase HPLC, HPLC amino acid analysis and fast atom bombardment mass spectrometry. Strain NPJB-1 produces two different hepatotoxic heptapeptide microcystins. The main one was microcystin-LR, the most commonly reported microcystin from cyanobacteria. The other was microcystin-LF, the phenylalanine variant of microcystin-LR. This is the first published report for microcystin-LF.

Structure determination and toxicity of a new microcystin from Microcystis aeruginosa strain 205

A new hepatotoxic microcystin was isolated from the cyanobacterium Microcystis aeruginosa strain 205. Its structure was found to be [Dha7]microcystin-RR as determined by amino acid analysis, mass spectrometry and 1H NMR spectroscopy. LD50 value (i.p. mouse) of this toxin was 180 micrograms/kg. The 48 hr lethal concentration (48-hr-LC50) of the toxin for larvae of the yellow fever mosquito, Aedes aegypti, was 14.9 micrograms/ml.

Factors influencing growth and toxin production by cultures of the freshwater cyanobacterium Lyngbya wollei Farlow ex Gomont

Collections of Lyngbya wollei were taken from Guntersville Reservoir, Alabama, over a period of three years. Healthy filaments were isolated and transferred to agar plates of Z-8 and LM6E media. Unialgal isolates were cultured for the study of growth and paralytic shellfish poison (PSP) production. Filaments were extracted and the toxins were detected using high performance liquid chromatography (HPLC) with post column oxidation followed by fluorescence detection. HPLC profiles show that laboratory cultures of L. wollei produced decarbamoyl gonyautoxin 2 and 3, plus several other PSP like toxins whose structures are under investigation. At 26 °C and a light intensity of 11 or 22 µmol m-2 s-1 optimum production of both biomass and toxins occurred. A decrease or increase in temperature or light flux caused a reduction in dry weight or toxicity. Compared to control levels, lower PO4-P and NO3-N and higher calcium levels gave rise to higher biomass and toxicity. Lower calcium, calcium- or PO4-P deficient medium and high NO3-N or PO4-P caused a large decrease in dry weight and toxicity.

Two methyl ester derivatives of microcystins, cyclic heptapeptide hepatotoxins, isolated from Anabaena flos-aquae strain CYA 83/1

Cultured cells of Anabaena flos-aquae strain CYA 83/1, isolated from Lake Edlandsvatn, Norway, produced two microcystin mono-methyl ester derivatives (1 and 2) at the D-Glu unit in addition to microcystin-LR (3), [D-Asp3]microcystin-LR (4), microcystin-RR (5), and [D-Asp3]microcystin-RR (6). Structures of these compounds were assigned based on their amino acid analysis with a Waters Pico Tag HPLC system plus fast atom bombardment mass spectrometry (FABMS), including tandem FABMS, analysis on the two new microcystins, [D-Glu(OCH3)6]microcystin-LR (1) and [D-Asp3, D-Glu(OCH3)6]microcystin-LR (2). Toxicity data were not obtained for 1 and 2 because of the small amounts isolated from the cells.

Isolation of linear peptides related to the hepatotoxins nodularin and microcystins

Abstract Linear peptide 2 , Adda-D-Glu(γ)-Mdhb-D-MeAsp(β)-L-Arg-OH, was isolated from cultured Nodularia spumigena and was analyzed in the cells after one weeks ( 1:2 : 30:1) to eight weeks (>100:1) cultivation. Three linear peptides, Adda-D-Glu(γ)-Mdha-D-Ala-L-Leu-D-MeAsp(β)-L-Arg-OH ( 3 ), L-Leu-D-MeAsp(β)-L-Arg-Adda-D-Glu(γ)-Mdha-D-Ala-OH ( 4 ), and L-Phe-D-MeAsp(β)-L-Arg-Adda-D-Glu(γ)-Mdha-D-Ala-OH ( 5 ) were obtained from a water bloom of Microcystis spp. collected from Homer Lake (Illinois). Some of these linear peptides ( 2 , 3 ) are thought to be biogenetic precursors of nodularin and microcystins. Linear peptides 2 , 3 , and 4 did not show apparent toxicity at 1.0, 1.1, and 2.25 mg/kg, respectively, in a mouse bioassay (ip). Feeding experiments using 13 C-labeled precursors established that the 2-, 6- and 8-methyl and 9-methoxy carbons of the unusual (2 S ,3 S ,8 S ,9 S )-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (Adda) unit of 1 were clearly derived from L-methionine.

Isolation and structures of five microcystins from a Russian Microcystis aeruginosa strain CALU 972

Five microcystins were obtained from Microcystis aeruginosa strain CALU 972 isolated from a hepatotoxic water bloom collected in Lake Kroshnosero (Russia). The structure of a new toxin (1) was determined as [Dha7]microcystin-YR by amino acid analyses and fast atom bombardment mass spectrometry, and the toxins 2, 3, 4, and 5 were assigned the structures as [Dha7]microcystin-LR, [D-Asp3,Dha7]microcystin-LR, [Dha7]microcystin-RR, and [D-Asp3,Dha7]microcystin-RR, respectively, by direct comparison with authentic samples.

Isolation and structures of microcystins from a cyanobacterial water bloom (Finland)

A hepatotoxic cyanobacterial (blue-green algal) water bloom was collected from a constructed water reservoir in Finland. The water bloom contained two cyanobacterial species, Microcystis aeruginosa and Aphanizomenon flos-aquae. Two hepatotoxins, 1 and 2, were isolated from extracts of lyophilized cells. The structures of 1 and 2 were assigned based upon their amino acid analyses on a Waters Pico Tag HPLC system and a chiral GC capillary column (Chirasil Val III), fast atom bombardment mass spectrometry (FABMS), high resolution FABMS, and tandem FABMS data. Toxin 1 was identical to a previously reported compound, [D-Asp3]microcystin-RR. Toxin 2 was new and was assigned the structure [D-Asp3]microcystin-YR.

Two new l-serine variants of microcystins-LR and -RR from Anabaena sp. strains 202 A1 and 202 A2

Two new microcystins, [L-Ser7]microcystin-LR (1) and [L-Ser7]microcystin-RR (2), were isolated from a filamentous fresh water cyanobacterium (blue-green alga), Anabaena sp. strain 202 A1, along with the two major toxins, [Dha7]microcystin-LR (3) and [Dha7]microcystin-RR (4) and their minor components the D-Asp variants [D-Asp3,Dha7]microcystin-LR (5) and [D-Asp3,Dha7]microcystin-RR (6). Anabaena sp. strain 202 A1 also produced another new toxin, whose structure is tentatively proposed as [D-Asp3,L-Ser7]microcystin-XR (7), where X is a leucine homologue. Anabaena sp. strain 202 A2 produced one new microcystin, 1, and three known microcystins, 3, 4, and 5. The structures of the toxins were assigned based on their amino acid analyses, and fast atom bombardment mass spectrometry data.

Isolation of microcystin‐LR from a microcystis (cyanobacteria) waterbloom collected in the drinking water reservoir for porto, Portugal

Abstract Plankton tows collected over the water intake towers on Crestuma reservoir, which is the drinking water supply for about 2,000,000 persons in the Porto, Vila Nova de Gaia, and Gondomar regions near the city of Porto, were found to be hepatotoxic using the mouse intraperi‐toneal bioassay. Phytoplankton in these samples was dominated by the cyanobacteria Microcystis aeruginosa (95%). The lethal dose for 50% of the animals tested of lyophilized bloom material was approximately 30 mg/kg (dry cell weight/animal weight). Using HPLC separation, a single fraction was obtained. Isolation and purification of this fraction resulted in a toxin that was shown to be microcystin‐LR by amino acid and MS analyses. Since microcystin‐LR is a potent hepatotoxin and liver tumor promoter, and since high cell densities of Microcystis aeruginosa producing microcystin‐LR were found near the water intake lines, it shows that a regular monitoring of microcystin(s) should be developed in water used for human consumption to ...