Isolation and structural characterization of microcystin-LR and three minor oligopeptides simultaneously produced by Radiocystis feernandoi (Chroococcales, Cyanobacteriae): a Brazilian toxic cyanobacterium.

Several blooms of cyanobacteria naturally occurring in freshwater reservoirs have been associated to numerous fatalities and cases of livestock and human poisoning. Microcystins (Mcs) are the most frequently found cyclic heptapeptide toxins in the cyanobacterial extracts. In previous work, Radiocystis fernandoi (strain SPC 714) lyophilized extracts were found to be hepatotoxic to mice with LD100 of about 60 mg kg(-1) and Mc LR was suggested as responsible for that toxicity. Here, we describe the isolation of four oligopeptides from R. fernandoi methanol extract by reversed-phase high performance liquid chromatography (RP-HPLC). The major component, which eluted with 65% acetonitrile from acetonitrile/water gradient, was identified as Mc-LR and its structure was confirmed by the presence of molecular related ion species [M+H]+ at m/z 996.3, ([M+H-Adda])+ at m/z 861.5, [Arg-Adda-Glu+H]+ at m/z 599.8, and [PhCH2CH(OMe)]+ at m/z 135.1 in the ESI spectra. Two components corresponding to small signals eluted from C18 column, respectively, with 44 and 45% acetonitrile had their structures proposed as isomers of aeruginosin derivatives showing molecular ions at m/z 651.7 and a [CHOI]+ immonium at m/z 140.1. Finally, the structure of the third minor and most hydrophobic component (68% acetonitrile elution) isolated from R. fernandoi extract seemed to correspond to a cyclic cyanopeptolin like micropeptin K139, a trypsin inhibitor firstly isolated from Microcystis aeruginosa, showing similar ions fragmentation pattern and [M+H]+ at m/z 987.6 in its ESI spectra.

Toxic cyanobacteria and microcystin concentrations in a public water supply reservoir in the Brazilian Amazonia region.

Toxic cyanobacteria in public water supply reservoirs represent a serious health risk as they can release potent cyanotoxins into the water. In the present study we analyzed surface water collected from the Utinga Reservoir, the main source of drinking water for the city of Belem-PA, in order to characterize the reservoir's cyanobacterial biota and to determine the toxicity of these organisms and the concentration of microcystins in raw and treated water. The cyanobacterial biota included potentially toxic genera such as Aphanizomenon, Microcystis, Nostoc, Oscillatoria, Planktothrix and Radiocystis. Mouse bioassays revealed a 43.6% frequency of hepatotoxic strains, including 30.8% Microcystis viridis strains and 12.8% Radiocystis fernandoi strains, with the lowest LD(100) (ip) of 45 and 75 mg kg(-1) body weight, respectively. Subacute hepatotoxicity was observed for Aphanizomenon cf. gracile and Coelomoron pusillum strains. HPLC analysis confirmed the production of microcystins at maximum concentrations of 4.22 microg mg(-1) dry weight for M. viridis and 2.47 microg mg(-1) for R. fernandoi. Microcystins at concentrations of up to 1.25 microg L(-1) detected in raw water by ELISA, together with a cyanobacterial density of 20,000 cells mL(-1), represents the first report of a bloom of cyanobacteria for an Amazonian water body.

Microcystin production by Radiocystis fernandoi (Chroococcales, Cyanobacteria) isolated from a drinking water reservoir in the city of Belém, PA, Brazilian Amazonia region.

During the monitoring of toxic cyanobacteria in the Utinga Reservoir, which is the main drinking water supply for the city of Belém, PA, Brazil, a Radiocystis fernandoi strain (SPC714) was isolated. This non-axenic strain was submitted to a toxicity bioassay with mice and microcystin production analyzed by HPLC-DAD. The species was identified based on cultured and natural preserved material. Morphometric, developmental and reproductive characteristics were analyzed. The strain was cultured in liquid ASM-1 medium, at 25+/-1 degrees C, at an incident irradiance of 20 micromol photon m(-2)s(-1) and constant aeration. At the end of the exponential growth phase, cells were lyophilized and submitted to toxicity tests. The strain showed high toxicity to mice, by intraperitoneal route, with an approximate LD100 of 60 mg kg(-1) of body weight, producing characteristic symptoms of hepatotoxicity. Analyses performed by HPLC-DAD confirmed the production of microcystins, in a concentration of 3.83 microg mg(-1) of lyophilized cells. This is the first reference related to the toxicity of the genus Radiocystis.