Microcystins, phosphatase inhibition

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... 

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. 

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. 

C.J. Ward, K.A. Beattie, E.Y.C. Lee and G.A. Codd, Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria comparisons with high-performance liquid chromatography analysis for microcystins, FEMS Microbiol. Lett., 153 (1997) 465-473. 

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

A combination of PPIA and microcystin immunoassay was proposed by Carmichael et al. (1999) to indicate the potential toxicity of a bloom sample and the concentration of the microcystins. A combined assay, consistent with this principle, was developed by Metcalf et al. (2001) this includes preexposure of the sample to microcystin antibodies, to make microcystins/nodularins that are present biounavailable to the subsequent addition of protein phosphatase enzyme, before assaying for protein phosphatase inhibitoiy activity. The resulting assay, termed the colorimetric immunoprotein phosphatase inhibition ass (CIPPIA), was found to be specific for microcystins and nodularins since the microcystin antibodies protect the protein phosphatase from inhibition by the toxins. Complete protection from inhibition of protein phospliatase by the antibodies indicates that the inhibition of the protein phosphatase in the sample was due to the cyanobacterial toxins. These colorimetric assays showed a good correlation with the HPLC analysis of extracts cyanobacteria. Immunoassays can also be combined with physicochemical methods such as HPLC (Zeck 2001b). In this case, the HPLC method separates the microcystins according to their hydrophobicity and the resulting fractions are analyzed by immunoassay. 

Carmichael, W.W, and An, J. 1999. Using an enzyme linked immunosorbent assay (ELISA) and a protein phosphatase inhibition assay for the detection of microcystins and nodularins. Nat Toxins 7 377-385. 

Metcalf, J.S., Bell, S.G., and Codd, G.A. 2001. Colorimetric immuno-protein phosphatase inhibition assay for specific detection of microcystins and nodularins of cyanobacteria. Appl Environ Microbiol 67 904—909. 

Rapala, I, Erkomaa, K., Kukkonen, I, Sivonen, K., and Lahti K. 2002. Detection of microcystins with protein phosphatase inhibition assay, high performance liquid chromatography-UV detection and enzymelinked immunosorbent assay. Comparison of methods. Analytica ChimicaActa (466) 213- 231. 

Protein phosphatase inhibition has been correlated with the onset of acute microcystin-induced hepatotoxicosis in mice (70) and with microfilament reorganization and cell deformation in isolated hepatocytes (65). Inhibition of these enzymes causes hyperphosphorylation of numerous cytosolic and cytoskeletal proteins in isolated hepatocytes exposed to microcystin (63,65,71). It has recently been shown that at higher concentrations similar morphological effects are produced also in non-hepatocytes (72). 

Jones, G.J. and Orr, P.T., Release and degradation of microcystin following algicide treatment of a Microcystis aeruginosa bloom in a recreational lake, as determined by HPLC and protein phosphatase inhibition assay. Water Res., 28, 871, 1994. 

Another variant of PP2A assay is the one reported by Isobe et al. [166] where a firefly bioluminescence system is used for the detection of protein phosphatase 2A inhibitors, in which luciferin phosphate is hydrolyzed to luciferin and inorganic phosphate by protein phosphatase 2A. The recent commercial availability of the phosphatase enzymes, which obviates the need to isolate them from animal tissues, also makes this approach very attractive. However, not all microcystins variants react with protein phosphatase enzymes to a similar extent [161,163] and the assay is sensitive to protein phosphatase inhibitors other than microcystins, such as okadaic acid, tautomycin, and calyculin A. In addition, the cyanobacterial sample itself may contain phosphatase activity that masks the presence of toxins [160]. As a consequence, the lack of specificity of the protein phosphatase inhibition assays requires that additional confirmatory analytical methods be employed for specific analysis of cyanobacterial toxins. 

The ELISA is currently the most promising method for rapid sample screening for MCs because of its sensitivity, specificity, and ease of operation. These assays are based on the use of monoclonal or polyclonal antibodies. These assays show greater specificity than protein phosphatase inhibition assays but do not indicate the relative toxicides of microcystin and nodularin variants instead, ELISAs rely on the structure of toxins for detection. Therefore cross-reactivities of the different toxins may vary and sensitivity depends on the structure rather than toxicity. 

C. Mackintosh, Determination of Microcystins in Waters and extracts of cyanobacteria by protein phosphatase inhibition assay, Report to the Department of the Environment (1995). 

Microcystins potently inhibit serine/threonine protein phosphatases that dephosphorylate other proteins, an action akin to an on/off switch. The novel hydrophobic amino acid ADDA is essential to its bioactivity. Its action also promotes the formation of tumors. The liver is particularly susceptible to these toxins because unlike many other cell types, these peptides readily penetrate liver cells and are specifically taken up through the bile acid transport pathway. These toxins damage the liver by affecting the maintenance by these phosphatases of the cyto-skeleton, a network of protein filaments. Protein phosphatases degrade the colorless p-nitrophenyl phosphate into a yellow product and this has been... 

After ingestion, microcystins are released from cyano-bacterial cells and are absorbed into the portal circulation from the small intestine via bile acid transporters in the intestinal wall. Microcystins are then accumulated in hepatocytes via similar bile acid transporters on hepato-cyte membranes (Hooser et al., 1991). Microcystins irreversibly inhibit serine/threonine protein phosphatases 1 and 2A (Yoshizawa et al., 1990). Microcystin-LR may also bind to AP synthase, leading to hepatocyte apoptosis (Mikhailov et al., 2003). 

Protein phosphatases are ubiquitous. They are foxmd in all tissues and across species as diverse as mammals, plants, and bacteria, and they play a critical role in the regulation of multiple cellular metabolic pathways. Protein phosphatases reverse tiie active state of kinases through the hydrolytic removal of tiie phosphoryl group from kinases. The protein phosphatases inhibited by microcystins have broad substrate specificity and play roles in the regulation of a wide range of cellular fxmc-tions. Protein phosphatase 2A is highly conserved and is a major downregulator of active protein kinases in eukaryotic cells. Toxic effects in hepatocytes and other... 

From the different cases related to inhibition, the most in-depth studied case is that of pesticides, mainly as inhibitors of cholinesterase enzymes other cases involve inhibition of sarcosine oxidase that allows determining carboxylic acids or inhibition of protein phosphatases, attempted for the resolution of microcystin types. Inhibition of aminooxidases also opens a way for determination of amount and effects of modem antidepressants. 

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. 

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). 

The potent toxin microcystin [blue-green algae inhibit one of the phosphatases (PP2A)] increases mitogenic activity and so is a tumor promoter at low-level exposures. The liver toxicity at high levels of acute exposure is probably also due to disturbances in protein phosphorylation. 

As outlined above, protein phosphorylation is a key process involved in many signal transduction pathways and reversal of this process is catalyzed by a multiplicity of phosphoprotein phosphatases (PPs). Major PPs catalyzing dephosphorylation of phosphoserine or phosphothreonine residues on proteins include PP1 (inhibited by phosphorylated inhibitor protein I-1 and by okadaic acid and microsystins), PP2 (also inhibited by okadaic acid and microcystins), PP2B or calcineurin (CaM-activated and having a CaM-like regulatory subunit) and PP2C (Mg2+-dependent) [18]. These PPs have been found in all eukaryotes so far examined [18, 19]. In addition, a variety of protein phosphotyrosine phosphatases can reverse the consequences of RTK or JAK/STAT receptor activation [20]. 

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. 

On the one hand, protein phosphatase and acetylcholinesterase inhibition assays for microcystin and anatoxin-a(s) detection, respectively, are excellent methods for toxin analysis because of the low limits of detection that can be achieved. On the other hand, electrochemical techniques are characterised by the inherent high sensitivities. Moreover, the cost effectiveness and portability of the electrochemical devices make attractive their use in in situ analysis. The combination of enzyme inhibition and electrochemistry results in amperometric biosensors, promising as biotools for routine analysis. 

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