Identification and characterization of hydrophobic microcystins in Canadian freshwater cyanobacteria.

Hepatotoxic microcystins produced by cyanobacteria in freshwater lakes represent a significant health hazard to humans and agricultural livestock. Liquid chromatography (LC)-linked protein phosphatase (PPase) bioassay analysis of blooms of Microcystis aeruginosa produced in a Canadian drinking water lake identified several PPase inhibitors with significantly greater hydrophobicity than microcystin-LR, based on their retention time on C18 reverse phase LC columns. Seven PPase inhibitors were purified to homogeneity by bioassay-guided fractionation involving Sephadex LH-20 chromatography and two-step reverse phase at pH 6.5 and 2.0. One of the PPase inhibitors, isolated in a final yield of 1.5 micrograms/g lyophilized cyanobacteria, was identified as microcystin-LL by amino acid analysis and mass spectrometry. A further PPase inhibitor (20 ng/g cyanobacteria) was identified as microcystin-LL but with D-Ala replaced by an unknown amino acid. Four PPase inhibitors (< 20 ng/g cyanobacteria) were characterized by amino acid analysis and identified as microcystin-LV, -LM, -LF and -LZ (where Z represents an unknown hydrophobic amino acid). A further microcystin was also identified (< 10 ng/g cyanobacteria) in which arginine was apparently absent. The biological activity of the seven microcystins as inhibitors of the catalytic subunit of protein phosphatase-1 (PP-1c) was compared with microcystin-LR and motuporin (a hydrophobic analogue of nodularin). All of the compounds inhibited PP-1c with IC50 values of 0.06-0.4 nM, consistent with their identification as microcystins. These findings further demonstrate the applicability of a sensitive PPase bioassay for the identification of variant microcystins in the natural environment.

A unified bioscreen for the detection of diarrhetic shellfish toxins and microcystins in marine and freshwater environments.

Capillary electrophoresis (CE) coupled with liquid chromatography (LC)-linked protein phosphatase (PPase) bioassay was used to detect sensitivity both diarrhetic shellfish toxins and hepatotoxic microcystins in marine and freshwater samples. This procedure provided a quantitative bioscreen for the rapid optical resolution of either of these toxin families in complex mixtures such as cultured marine phytoplankton, contaminated shellfish and cyanobacteria (natural assemblages). Following detection, identified toxins were purified by an enzyme bioassay-guided two-step LC protocol. Using the latter approach, at least four microcystins were rapidly isolated from a cyanobacteria bloom (largely Microcystis aeruginosa) collected from a Canadian drinking-water lake, including a novel microcystin termed microcystin-XR, where X is a previously unidentified hydrophobic amino acid of peptide residue molecular mass 193 Da. The unified CE/LC-linked PPase bioscreen described provides a powerful capability to dissect multiple toxin profiles in marine or freshwater samples contaminated with either okadaic acid or microcystin classes of toxin.

Identification of protein phosphatase inhibitors of the microcystin class in the marine environment.

Toxins produced by marine phytoplankton represent a severe global health hazard to humans that eat seafood and are also responsible for massive natural fish kills in specialized bloom situations. Tumour-promoting hepatotoxins from the freshwater microcystin/nodularin class were identified in Northeastern Pacific Ocean, Eastern Canadian and European mussels for the first time. These hepatotoxins were detected at biologically active levels up to three-fold higher than accepted quarantine levels for the diarrhetic shellfish toxin okadaic acid (OA), based on their activity (in microcystin-LR equivalent units) in a liquid chromatography (LC)-linked protein phosphatase bioassay. The presence of microcystins/nodularins in oceanic shellfish identifies a potentially novel class of intoxication which is also prevalent in other forms of marine aquatic life, namely sponges and fish. The widespread presence of prokaryotic microcystins and nodularins in the marine environment may be indicative of the importance of signal transduction pathways involving potent inhibition of protein phosphatases in early marine eukaryotes.