Novel metabolites in cyanobacterium Cylindrospermopsis raciborskii with potencies to inhibit gap junctional intercellular communication.

Despite intensive research into toxic bloom-forming cyanobacteria, the majority of their metabolites remain unknown. The present study explored in detail a novel bioactivity identified in cyanobacteria, i.e. inhibition of gap junctional intercellular communication (GJIC), a marker of tumor promotion. The extracellular mixture (exudate) of the cyanobacterial strain Cylindrospermopsis raciborskii (SAG 1.97) was fractionated by semi-preparative reversed phase HPLC, and the fractions assessed for their potencies to inhibit GJIC. Two non-polar fractions that significantly inhibited GJIC were further fractionated, tested and analyzed using multiple mass spectrometric methods. Investigations led to the identification of a putative chemical compound (molecular formula C18H34O3, m/z 299.2581 for the [M+H](+) ion) responsible for observed bioactivities. Specific inhibitors of signaling pathways were used to screen for biochemical mechanisms beyond GJIC inhibition, and the results indicate the involvement of ERK1/2 kinases via a mechanism related to the action of epidermal growth factor EGF but clearly distinct from other anthropogenic tumor promoters like polychlorinated biphenyls or polycyclic aromatic hydrocarbons. The chemical and in vitro toxicological characterizations of the newly described metabolite provide important insights into the still poorly understood health impacts of complex toxic cyanobacterial blooms and indicate that currently applied monitoring practices may underestimate actual risks.

LC-MS analyses of microcystins in fish tissues overestimate toxin levels-critical comparison with LC-MS/MS.

Microcystins are cyclic peptide toxins with hepatotoxic and tumour-promoting properties which are produced in high quantities in freshwater cyanobacterial water blooms, and several studies have reported microcystin accumulation in fish with possible food transfer to humans. In this study, we provide the first comparison of liquid chromatography with single mass-spectrometric and with tandem mass-spectrometric detection for analyses of microcystins in complex fish tissue samples. Use of traditional single mass spectrometry (i.e. monitoring of ions with m/z 519.5 for microcystin-RR and m/z 995.5 for microcystin-LR) was found to provide false-positive responses, thus overestimating the concentrations of microcystins in the tissue samples. More selective tandem mass spectrometry seems to provide more reliable results. The concentrations of microcystins detected by tandem mass spectrometry in fish from controlled-exposure experiments were more than 50% lower in comparison with concentrations obtained by single mass spectrometry. Extensive analyses of edible fish parts-muscles (148 fish specimens from eight different species from five natural reservoirs with dense cyanobacterial water blooms)-showed negligible microcystin concentrations (all analyses below the limit of detection; limit of detection of 1.2-5.4 ng/g fresh weight for microcystin-RR, microcystin-YR and microcystin-LR in multiple reaction monitoring mode). Our findings have practical consequences for critical re-evaluation of the health risks of microcystins accumulated in fish.

Effects of cyanobacterial biomass on the Japanese quail.

Mortality of wild aquatic birds has recently been attributed to cyanobacterial toxins. Despite this, no experimental studies on the effects of defined doses of microcystins administered orally to birds exist. In this experiment, four groups of male Japanese quails daily ingesting 10ml of Microcystis biomass containing 0.045, 0.459, 4.605 or 46.044mug of microcystins, respectively, for 10 and 30 days, showed no mortality. Histopathological hepatic changes in birds after the biomass exposure included cloudy swelling of hepatocytes, vacuolar dystrophy, steatosis and hyperplasia of lymphatic centres. On subcellular level, shrunken nuclei of hepatocytes containing ring-like nucleoli, cristolysis within mitochondria and vacuoles with pseudomyelin structures were present. Vacuolar degeneration of the testicular germinative epithelium was found in two exposed males. Statistically significant differences in biochemical parameters were on day 10 of exposure only. They comprised increased activities of lactate dehydrogenase and a drop in blood glucose in birds receiving the highest dose of the biomass. Principal component analysis revealed a pattern of responses in biochemical parameters on day 10 that clearly separated the two greatest exposure groups from the controls and lower exposures. The results indicate that diagnosis of microcystin intoxication solely based on clinical biochemical and haematological parameters is hardly possible in birds.