Microcystin-LR

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.

Figure 8. Left The cyclic heptapeptide hepatotoxin microcystin-LA (cyanoginosin-LA) produced by the colonial cyanobacterium Microcystis aeruginosa strain WR-70 (UV-010). MW = 909. Right The cyclic heptapeptide hepatotoxin microcystin-LR (cyanoginosin-LR) produced by a waterbloom of the colonial cyanobacterium Microcystis aeruginosa collected in Lake Akersvatn, Norway, 1984-85 MW=994, 69J1).

Useful serine/threonine protein phosphatase inhibitors include microcystin-LR (which inhibits protein phosphatases 1, 2A, and 2C, and related enzymes) and /1-glycerophosphate. Sodium fluoride may also be employed. Sodium orthovanadate inhibits protein tyrosine phosphatases. 

In this laboratory, we also include the metal ion chelators EDTA (ethylene diamine tetraacetic acid binds, e.g., Mg2 1 -ions) and EGTA (ethylene glycol-bis(2-aminoethyl)-Al,iV,iV/,iV/,-tetraacetic acid binds, e.g., Ca2+-ions) in our lysis buffers. These agents help prevent phosphatase action (by the metal ion-dependent phosphatase PP2C, which is not inhibited by microcystin-LR), metal (Ca2+) dependent proteinases, and protein kinases, which require divalent cations such as Mg2 1 (and, in some cases, also Ca2+). We also use a mix of proteinase inhibitors that inhibit a broad range of proteolytic enzymes, including serine and cysteine proteinases. 

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...

Cells of C. reinhardtii were exposed to cell-free filtrates from A. flos-aquae, pure microcystin-LR or anatoxin-a, or combinations of the toxins. Both the position of the cells and the chlorophyll-a concentration of the cultures were observed for 12 days. Exposure to crude extracts as well as to combinations of the toxins significantly decreased chlorophyll levels in the cultures. Furthermore, these compounds were all capable of paralyzing the algae and thus promoted the settlement of C. reinhardtii cells. One intriguing aspect of this dynamic interaction is the separate finding that C. reinhardtii may actually induce toxin synthesis in A. flos-aquae (Kearns and Hunter 2000), essentially signaling its own demise. 

Komarek J, Kling H (2003) Filamentous cyanobacteria. In Wehr JD, Sheath RG (eds) Freshwater algae of North America ecology and classification. Academic, San Diego, CA, pp 117-196 Kotak BG, Zurawell RW, Prepas EE, Holmes CFB (1996) Microcystin-LR concentration in aquatic food web compartments from lakes of varying trophic status. Can J Fish Aquatic Sci 53 1974-1985... 

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]. 

These natural toxins are heptapeptides produced by cyanobacteria, which are associated with algal blooms. These substances are a hazard to wild and farm animals and sometimes humans who come in contact with contaminated water. There are a number of these toxins, some of which such as microcystin LR are hepatotoxic, causing damage to both hepatocytes and endothelial cells. The toxins have some unusual structural features, incorporating three D-amino acids and two very unusual ones, namely, methyldehydro alanine (Mdha) and amino-methoxy-trimethyl-phenyl-decadi-enoic acid (Adda) (Fig. 7.26). 

Figure 7.26 The structure of the hepatotoxic cyclic heptapeptide microcystin LR. L-Arginine and L-leucine are variable amino acids. The reactive unsaturated group is indicated by the star. Abbreviations Adda, amino-methoxy-trimethyl-phenyl-decadienoic acid Mdha, methyldehydro-alanine Masp, methyl D-/so-aspartate D-Glu, D-/soglutamate D-Ala, D-alanine.

These natural toxins are heptapeptides produced by cyanobacteria and have unusual structural features, incorporating three D-amino acids. Microcystin LR is hepatotoxic, as a result of inactivation of protein phosphatases, which leads to breakdown of the cytoskeleton and cell death. 

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

McCarthy and coworkers have used this strategy en route to ADDA conjugates [93, 94], residues that are found in the cyclic peptides microcystin LA 73, microcystin LR 74, and nodularin R 75, Fig. 5. 

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.

In order to demonstrate the viability of the approach, protein phosphatase inhibition was first performed with the enzyme in solution and detected by colorimetric methods. Two microcystin variants, microcystin-LR and microcystin-RR, were used. Both enzymes were inhibited by these toxins, although to a different extent. The 50% inhibition coefficients (IC50) towards microcystin-LR were 0.50 and 1.40 pgL 1 (concentrations in the microtitre well) for the Upstate and the GTP enzymes, respectively. Hence, the Upstate enzyme was more sensitive. The IC50 towards microcystin-RR were 0.95 and 2.15 pgL-1 for the Upstate and the GTP enzymes, respectively. As expected, microcystin-LR was demonstrated to be a more potent inhibitor. 

Fig. 16.5. Colorimetric standard curves for the inhibition of PP2A from GTP Technology immobilised on different supports by (a) microcystin-LR (MC-LR) and (b) microcystin-RR (MC-RR). Inhibition is expressed as percentage of the control (no microcystin). Concentrations refer to those in the well. Reprinted from Campas et at. [86], with permission from Elsevier.

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. 

K.I. Harada, K. Matsuura, M. Suzuki, M.F. Watanabe, S. Oishi, A.M. Dahlem, V.R. Beasley and W.W. Carmichael, Isolation and characterization of the minor components associated with microcystins LR and RR in the cyanobacterium (blue-green algae), Toxicon, 28 (1990) 55-64. 

Microcystin-LR (MC-LR) (Sigma, France) standard solutions first prepared in 50 50 methanokwater and subsequently diluted in 30 mM tris-HCl, 2mM dithiothreitol (DTT), 2mM ethylene diamine tetraacetic acid (EDTA), 0.2mgmL 1 bovine serum albumin (BSA), 20 mM MgCl2 buffer (pH 8.4) (prepared in Milli-Q water). 

Campbell, D.L., Lawton, L.A., Beattie, K.A. and Codd, G.A. (1994) Comparative assessment of the specificity of the brine shrimp and Microtox assays to hepatotoxic (microcystin-LR-containing) cyanobacteria, Environmental Toxicology and Water Quality 9 (1), 71-77. 

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