Cyanobacterial microcystins

Cyanobacteria - the Jekyll and Hyde of marine organisms - are a novel source of potential new pharmaceutical compounds (2618-2620, 2662). On the other hand, toxic cyanobacterial blooms in lakes, rivers, and water storage reservoirs have occurred worldwide (2621, 2663, 2664). For example, 60 patients in a Brazil hemodialysis unit died after drinking water from a lake contaminated with cyanobacterial microcystins (2622), not unlike the toxicity of red tides (2623). Cyanobacteria also produce the highly toxic neurotoxin, p-N-methylamino-L-alanine, which may be produced by all cyanobacteria (2624, 2665). 

C. MacKintosh, K.A. Beattie, S. Klumpp, P. Cohen and G.A. Codd, Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants, FEBS Lett., 264 (1990) 187-192. 

Humble, A.V, Gadd, G.M. and Codd, G.A. (1997) Binding of copper and zinc to three cyanobacterial microcystins quantified by differential pulse polarography. Water Res., 31,1679-1686. 

In the Slimmer of 1989, Rutland Water, the largest man-made lake in Western Europe and which supplies potable water to approximately 500 000 people in the East of England, contained a heavy bloom of Microcystis aeruginosa. By the end of the summer, a number of sheep and dogs had died after drinking from the bloom and concentrated scum. Analysis revealed that the cyanobacterial bloom material was toxic to laboratory mice, and that rumen contents from a poisoned sheep contained fivemicrocystin variants.Microcystins were detected in waters used for recreation in Australia at concentrations greater than 1 mg per... 

Daphnia assay, the brine shrimps are exposed to different concentrations of toxicant, and the toxicity is expressed as the LCjo value. Extracts of cyanobacterial blooms and laboratory cultures, containing microcystins or anatoxin-a, have been found to be toxic towards brine shrimp," and fractionation of such extracts resulted in brine shrimp fatalities only with fractions containing microcystins." " ... 

In vitro cytotoxicity assays using isolated cells have been applied intermittently to cyanobacterial toxicity testing over several years." Cells investigated for suitability in cyanobacterial toxin assays include primary liver cells (hepatocytes) isolated from rodents and fish, established permanent mammalian cell lines, including hepatocytes, fibroblasts and cancerous cells, and erythrocytes. Earlier work suggested that extracts from toxic cyanobacteria disrupted cells of established lines and erythrocytes," but studies with purified microcystins revealed no alterations in structure or ion transport in fibroblasts or erythrocytes,... 

A powerful tool now employed is that of diode array detection (DAD). This function allows peaks detected by UV to be scanned, and provides a spectral profile for each suspected microcystin. Microcystins have characteristic absorption profiles in the wavelength range 200-300 nm, and these can be used as an indication of identity without the concomitant use of purified microcystin standards for all variants. A HPLC-DAD analytical method has also been devised for measurement of intracellular and extracellular microcystins in water samples containing cyanobacteria. This method involves filtration of the cyanobacteria from the water sample. The cyanobacterial cells present on the filter are extracted with methanol and analysed by HPLC. The filtered water is subjected to solid-phase clean-up using C g cartridges, before elution with methanol and then HPLC analysis. 

The ability to identify and quantify cyanobacterial toxins in animal and human clinical material following (suspected) intoxications or illnesses associated with contact with toxic cyanobacteria is an increasing requirement. The recoveries of anatoxin-a from animal stomach material and of microcystins from sheep rumen contents are relatively straightforward. However, the recovery of microcystin from liver and tissue samples cannot be expected to be complete without the application of proteolytic digestion and extraction procedures. This is likely because microcystins bind covalently to a cysteine residue in protein phosphatase. Unless an effective procedure is applied for the extraction of covalently bound microcystins (and nodiilarins), then a negative result in analysis cannot be taken to indicate the absence of toxins in clinical specimens. Furthermore, any positive result may be an underestimate of the true amount of microcystin in the material and would only represent free toxin, not bound to the protein phosphatases. Optimized procedures for the extraction of bound microcystins and nodiilarins from organ and tissue samples are needed. 

It is obvious from the provisional risk assessment values for microcystins, and, being of the same order of magnitude of mammalian toxicity, similar values may be calculated for the cyanobacterial neurotoxins, that sensitive detection methods are required to detect these low concentrations of toxins. Of the biological methods of detection discussed earlier, the mouse and invertebrate bioassays are not sensitive enough without concentration of water samples, in that they are only able to detect mg of microcystins per litre. Only the immunoassays (ng-/rg 1 and the protein phosphatase inhibition assays (ng O... 

R. W. MacKintosh, K. N. Dalby, D. G. Campbell, P. T. W. Cohen, P. Cohen and C. MacKintosh, The cyanobacterial toxin microcystin binds covalently to cysteine-273 on protein phosphatase 1 , FEBS Lett. 371 236-240 (1995). 

Hepatotoxins include microcystins, which are cyclic heptapeptides (Fig. 5.1a) and cylindrospermopsin, a sulfated guanidinium alkaloid (Fig. 5. lb). Microcystins bind to certain protein phosphatases responsible for regulating the distribution of cytoskeletal proteins (Zurawell et al. 2005 Leflaive and Ten-Hage 2007). Hepatocytes exposed to microcystins eventually undergo cellular deformation, resulting in intra-hepatic bleeding and, ultimately, death (Carmichael 2001 Batista et al. 2003). In contrast, cylindrospermopsin appears to have a different mode of activity, possibly involving inhibition of protein or nucleotide synthesis (Codd et al. 1999 Froscio et al. 2003 Reisner et al. 2004). Nevertheless, microcystins are the most common cyanotoxins isolated from cyanobacterial blooms (Sivonen and Jones 1999). 

Fig. 5.1 Common cyanobacterial hepatotoxins. (a) Generalized structure of microcystin, a cyclic heptapeptide. Note that X and Z are L-amino acids. For example, microcystin-LR possesses lysine and arginine residues at X and Z, respectively, (b) Cylindrospermopsin, a hepatotoxic alkaloid from Cylindrospermopsis raceborskii...

Significant concentrations of cyanotoxins have been found to accumulate in the tissues of macroinvertebrates such as mollusks and crustaceans, presenting an indirect route of exposure for invertebrates, fish, and aquatic mammals at higher trophic levels (Negri and Jones 1995). In natural systems, mortality among benthic invertebrate herbivores is probably low because most bloom-forming bacteria are planktonic and only periodically come into contact with the benthos. Nevertheless, Kotak et al. (1996) determined that enhanced mortality of snails at the end of a bloom cycle in Canadian lakes was due to consumption of Microcystis cells that had formed a scum on the surface of macrophytes. Oberemm et al. (1999) found that aqueous microcystins, saxitoxins, and anatoxin-a all resulted in developmental delays in fish and salamander embryos. Interestingly, more severe malformations and enhanced mortality were observed when larvae were exposed to crude cyanobacterial extracts than to pure toxins applied at natural concentrations (Oberemm et al. 1999). 

One role of cyanobacterial allelochemicals may be to alter the motility and distribution of competing photoautotrophs. In a recent study, Kearns and Hunter (2001) examined the effects of toxic metabolites from the filamentous cyanobacterium A. flos-aquae on a unicellular phytoplankton species, Chlamydomonas rein-hardtii. A. flos-aquae synthesizes both microcystins as well as anatoxins, providing the authors with an ecologically relevant opportunity to assess the individual and combinatorial effects of these toxins on an alga. 

McElhiney J, Lawton LA (2005) Detection of the cyanobacterial hepatotoxins microcystins. Toxicol Appl Pharmacol 203 219-230... 

Dahlmann, J., Budakowski, W.R. and Luckas, B., Liquid chromatography-electrospray ionisation-mass spectrometry based method for the simultaneous determination of algal and cyanobacterial toxins in phytoplankton from marine waters and lakes followed by tentative structural elucidation of microcystins, /. Chromatogr., 994, 1-2, 45, 2003. 

Our research group is working on the development of electrochemical biosensors for the detection of microcystin and anatoxin-a(s), based on the inhibition of protein phosphatase and acetylcholinesterase, respectively. These enzyme biosensors represent useful bioanalytical tools, suitable to be used as screening techniques for the preliminary yes/no detection of the toxicity of a sample. Additionally, due to the versatility of the electrochemical approach, the strategy can be applied to the detection of other cyanobacterial toxins. 

The developed biosensor should be, in principle, applicable to any sample suspicious of containing microcystins. This includes water samples, cyanobacterial cells and infected organisms. Whereas water samples could be directly applied to the biosensor, specific extraction protocols are required for the detection of the toxin in cyanobacteria and other organisms. In all the cases, matrix effects should be evaluated. 

Protein phosphatase inhibition-based biosensor for amperometric microcystin detection in cyanobacterial cells... 

Cyanobacterial (Blue-Green Bacteria) Toxins. Cyanobacterial poisonings were first recognized in the late 1800s. Human poisonings are rare however, kills of livestock, other mammals, birds, fish, and aquatic invertebrates are common. It is caused by a variety of biotoxins and cytotoxins, including anatoxin, microcystin, and nodularin produced by several species of cyanobacteria, including Anabaena, Aphanizomenon, Nodularia, Oscillatoria, and Microcystis. The main contamination problems include all eutrophic freshwater rivers, lakes, and streams. 

While protein kinases are responsible for the phosphorylation of their substrates, protein phosphatases perform the opposite duty, removing phosphate groups from their substrates, thus countering the functional impact of the kinases. The two major types of protein phosphatases are the serine/threonine phosphatases and tyrosine phosphatases. Several natural compounds with potent serine/threonine phosphatase inhibitory activity have been identified, including the cyanobacterial metabolite microcystin [105,106]. This compound labels its targets via a Michael addition of a noncatalytic active site cysteine residue with an acceptor in the macrocyclic peptide backbone [107]. A fluorescent probe based on microcystin was synthesized by Shreder et al., and its use in Jurkat lysates identified two previously undescribed phosphatase targets of microcystin, PP-4 and PP-5 [108]. Whereas serine/threonine... 

Cyanobacterial toxins produced and released by cyanobacteria in freshwater around the world are well documented [158,159]. Microcystins are the most common of the cyanobacterial toxins found in water, as well as being the ones most often responsible for poisoning animals and humans who come into contact with toxic blooms and contaminated water [ 160]. Acute exposure results in hepatic injury, which can in extreme cases prove fatal. One such incident occurred that resulted in the death of over 50 dialysis patients due to the use of microcystin-contaminated water in the treatment [161]. Chronic exposure due to the presence of microcystin in drinking water is thought to be a contributing factor in primary fiver cancer (PLC) through the known tumour-promoting activities of these compounds [162],... 

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