Of microcystins

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... 

Figure 5.3 Analysis of 100 ml of (a) surface water and (b) drinking water sample spiked with 0.1 pig/ml of microcystins, using column-switching HPLC 1, microcystin-RR 2, microcystin-YR 3, microcystin-LR. Reprinted from Journal of Chromatography A, 848, H. S. Lee et al, On-line trace enrichment for the simultaneous determination of microcystins in aqueous samples using high performance liquid chromatography with diode-array detection , pp 179-184, copyright 1999, with permission from Elsevier Science.

H. S. Eee, C. K. Jeong, H. M. Eee, S. J. Choi, K. S. Do, K. Kim and Y. H. Kim, On-line trace emicliment foi the simultaneous determination of microcystins in aqueous samples using high peifoi mance liquid cliromatography with diode-aixay detection , /. Chromatogr. 848 179-184 (1999). 

R. W. Moollan, B. Rae and A. Verbeek, Some comments on the detennination of microcystin toxins in water by liigh perfonnance liquid cliromatography. Analyst 121 233-238(1996). 

N. Bouaicha, I. Maatouk, G. Vincent and Y. Levi, A colorimetric and fluoro-metric microplate assay for the detection of microcystin-LR in drinking water without preconcentration, Food Chem. Toxicol., 40 (2002) 1677-1683. 

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...

Rantala A, Fewer DP, Hisbergues M, Rouhiainen L, Vaitomaa J, Bomer T, Sivonen K (2004) Phylogenetic evidence for the early evolution of microcystin synthesis. Proc Natl Acad Sci... 

Zurawell RW, Chen H, Burke JM, Prepas EE (2005) Hepatotoxic cyanobacteria a review of the biological importance of microcystins in freshwater environments. J Toxicol Env Heal B 8 1-37... 

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. 

Cyanobacteria toxins are toxins produced by certain species of blue-green algae that have become a major environmental and public health concern. The behavior of cyanotoxins during chlorination treatment has been recently reviewed by Merel et al. [129]. Chlorination DBFs have been reported only for the hepatotoxins microcystin-LR and cylindrospermopsin. Other cyanotoxins, such as nodularins, saxitoxins, and anatoxins, have yet to be investigated. Different isomers of six chlorination products of microcystin-LR have been characterized dihydroxy-microcystin, monochloro-microcystin, monochloro-hydroxy-microcystin, monochloro-dihydroxy-microcystin, dichloro-dihydroxy-microcystin, and trichloro-hydroxy-microcystin. Only two chlorination DBFs have been reported so far for cylindrospermopsin 5-chloro-cylindros-permopsin and cylindrospermopsic acid [129]. Chlorination of microcystin, cylindrospermopsin, and nodularins seems to reduce the mixture toxicity however, this aspect has not been extensively studied [129]. 

FFepatotoxins are the most commonly encountered toxins involving cyanobacteria, that include the cyclic peptides microcystin and nodularin (Fig. 4). Microcystins are cyclic seven amino acid peptides, and nodularins are cyclic five amino acid peptides. Over 50 different variants of microcystin have been isolated. 

An international intercomparison exercise in the determination of microcystin, carried out by using the most common methods (LC/DAD, ELISA and LC/MS) indicated that LC/DAD is affected by lower precision [234], while the coupling of the LC technique with ELISA permit the achievement of high sensitivity and specificity in the determination of microcystins and nodularin [235] without the need of pre-concentration the method meets the World Health Organization guidelines (1 pg L ). The combination of ELISA characterization and LC analysis with fluorescence, UV, and tandem MS detections, allowed the first identification of cylindrospermopsin, an algal toxin that caused the poisoning of up to 148 persons in Australia [236],... 

Furthermore, the structure of microcystin includes an electrophilic carbon atom, (Fig. 7.26), which is part of the Mdha amino acid. If microcystin is ingested from contaminated water, for example, it is taken up into the liver by an organic anion transporter (OAT) system and therefore is concentrated in the liver. The structure of the microcystins means they are able to associate with the enzymes protein phosphatases, such as PP-1, PP-2A, and PP-2B via hydrophobic and ionic interactions. 

Describe the cellular target and the specific mechanism of toxicity of microcystin LR. 

Fig. 16.1. Chemical structure of microcystin-LR. This image is licensed under the http //www.gnu.org/copyleft/fdl.html GNU Free Documentation License. It uses material from the http //en.wikipedia.org/wiki/Cyanotoxin Wikipedia article.

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. 

J.S. An and W.W. Carmichael, Use of a colorimetric protein phosphatase inhibition assay and enzyme-linked immunosorbent assay for the study of microcystins and nodularins, Toxicon, 32 (1994) 1495-1507. 

C. Rivasseau, P. Racaud, A. Deguin and M.-C. Henion, Development of a bioanalytical phosphatase inhibition test for the monitoring of microcystins in environmental samples, Anal. Chim. Acta, 394 (1999) 243-257. 

L.A. Lawton, C. Edwards and G.A. Codd, Extraction and high-performance liquid chromatography method for the determination of microcystins in raw and treated waters, Analyst, 119 (1994) 1525-1530. 

K. Tsuji, S. Naito, F. Kondo, M.F. Watanabe, S. Suzuki, H. Nakazawa, M. Suzuki, T. Shimada and K.-I. Harada, A clean-up method for analysis of trace amounts of microcystins in lake waters, Toxicon, 32 (1994) 1251-1259. 

C. Edwards, L.A. Lawton, K.A. Beattie, G.A. Codd, S. Pleasance and G.J. Dear, Analysis of microcystins from cyanobacteria by liquid... 

G. Vasas, D. Szydlowska, A. Gaspar, M. Welker, M. Trojanowicz and G. Borbely, Determination of microcystins in environmental samples using capillary electrophoresis, J. Biochem. Biophys. Methods, 66 (2006) 87-97. 

K. Tsuji, H. Masui, H. Uemura, Y. Mori and K.-I. Harada, Analysis of microcystins in sediments using MMPB method, Toxicon, 39 (2001) 687-692. 

M. Campas, D. Szydlowska, M. Trojanowicz and J.-L. Marty, Enzyme inhibition-based biosensor for the electrochemical detection of microcystins in natural blooms of cyanobacteria (2006), submitted for publication. 

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