Skip to main content
SpringerLink
Log in

Inhibition equivalency factors for microcystin variants in recombinant and wild-type protein phosphatase 1 and 2A assays

  • Research Article
  • Published:
Environmental Science and Pollution Research Aims and scope Submit manuscript

Abstract

In this work, protein phosphatase inhibition assays (PPIAs) have been used to evaluate the performance of recombinant PP1 and recombinant and wild-type PP2As. The enzymes have been compared using microcystins-LR (MC-LR) as a model cyanotoxin. Whereas PP2ARec provides a limit of detection (LOD) of 3.1 μg/L, PP1Rec and PP2AWild provide LODs of 0.6 and 0.5 μg/L, respectively, lower than the guideline value proposed by the World Health Organization (1 μg/L). The inhibitory potencies of seven MC variants (-LR, -RR, -dmLR, -YR, -LY, -LW and -LF) have been evaluated, resulting on 50 % inhibition coefficient (IC50) values ranging from 1.4 to 359.3 μg/L depending on the MC variant and the PP. The PPIAs have been applied to the determination of MC equivalent contents in a natural cyanobacterial bloom and an artificially contaminated sample, with multi-MC profiles. The inhibition equivalency factors (IEFs) have been applied to the individual MC quantifications determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the estimated MC-LR equivalent content has been compared to PPIA results. PPIAs have demonstrated to be applicable as MC screening tools for environmental applications and to protect human and animal health.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • An J, Carmichael WW (1994) Use of a colorimetric protein phosphatase inhibition assay and enzyme linked immunosorbent assay for the study of microcystins and nodularins. Toxicon 32:1495–1508

    Article  CAS  Google Scholar 

  • Barco M, Lawton LA, Rivera J, Caixach J (2005) Optimization of intracellular microcystin extraction for their subsequent analysis by high-performance liquid chromatography. J Chromatogr A 1074:23–30

    Article  CAS  Google Scholar 

  • Bouaïcha N, Maatouk I, Levi VY (2002) A colorimetric and fluorometric microplate assay for the detection of microcystin-LR in drinking water without preconcentration. Food Chem Toxicol 40:1677–1683

    Article  Google Scholar 

  • Blom JF, Jüttner F (2005) High crustacean toxicity of microcystin congeners does not correlate with protein phosphatase inhibitory activity. Toxicon 46:465–470

    Article  CAS  Google Scholar 

  • Brooks WP, Codd GA (1988) Immunoassay of hepatotoxic cultures and water blooms of cyanobacteria using Microcystis areuginosa peptide toxin polyclonal antibodies. Environ Technol Lett 9:1343–1348

    Article  CAS  Google Scholar 

  • Burch MD (2008) Effective doses, guidelines and regulations. In: Hudnell HK (ed) Cyanobacterial harmful algal blooms. Springer, New York, pp 831–854

    Chapter  Google Scholar 

  • Campàs M, Marty J-L (2007) Highly sensitive amperometric immunosensors for microcystin detection in algae. Biosens Bioelectron 22:1034–1040

    Article  Google Scholar 

  • Campàs M, Szydlowska D, Trojanowicz M, Marty J-L (2005) Towards the protein phosphatase-based biosensor for microcystin detection. Biosens Bioelectron 20:1520–1530

    Article  Google Scholar 

  • Campbell DL, Lawton LA, Beattie KA, Codd GA (1994) Comparative assessment of the specificity of the brine shrimp and Microtox assays to hepatotoxic (microcystin-LR containing) cyanobacteria. Environ Toxicol Water Qual 9:71–77

    Article  CAS  Google Scholar 

  • Carmichael WW (1994) The toxins of cyanobacteria. Sci Am 270:78–86

    Article  CAS  Google Scholar 

  • Chu FS, Huang X, Wei RD (1990) Enzyme-linked immunosorbent assay for microcystins in blue-green algal blooms. J AOAC Int 73:451–456

    CAS  Google Scholar 

  • Dawson RM (1998) The toxicology of microcystins. Toxicon 36:953–962

    Article  CAS  Google Scholar 

  • De Figueiredo DR, Azeiteiro UM, Esteves SM, Gonçalves FJM, Pereira MJ (2004) Microcystin-producing blooms-a serious global public health issue. Ecotox Environ Safe 59:151–163

    Article  Google Scholar 

  • Edwards C, Lawton LA, Beattie KA, Codd GA, Pleasance S, Dear GJ (1993) Analysis of microcystins from cyanobacteria by liquid chromatography with mass spectroscopy using atmospheric-pressing ionisation. Rap Commun Mass Spectrom 7:714–721

    Article  CAS  Google Scholar 

  • Falconer IR (1993) Measurement of toxins from blue-green algae in water and foodstuffs. In: Falconer IR (ed) Algal Toxins in Seafood and Drinking Water. Academic Press, London, pp 165–175

    Chapter  Google Scholar 

  • Fischer A, Hoeger SJ, Stemmer K, Feurstein DJ, Knobeloch D, Nussler A, Dietrich DR (2010) The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro: a comparison of primary human hepatocytes and OATP-transfected HEK293 cells. Toxicol Appl Pharmacol 245:9–20

    Article  CAS  Google Scholar 

  • Heresztyn T, Nicholson BC (2001) Determination of cyanobacterial hepatotoxins directly in water using a protein phosphatase inhibition assay. Water Res 35:3049–3056

    Article  CAS  Google Scholar 

  • Hoeger SJ, Schmid D, Blom JF, Ernst B, Dietrich DR (2007) Analytical and functional characterization of microcystins [Asp(3)]MC-RR and [Asp(3), Dhb(7)]MC-RR: consequences for risk assessment? Environ Sci Technol 41:2609–2616

    Article  CAS  Google Scholar 

  • Ikehara T, Imamura S, Oshiro N, Ikehara S, Shinjo F, Yasumoto T (2008) A protein phosphatase 2A (PP2A) inhibition assay using a recombinant enzyme for rapid detection of microcystins. Toxicon 51:1368–1373

    Article  CAS  Google Scholar 

  • Ikehara T, Imamura S, Sano T, Nakashima J, Kuniyoshi K, Oshiro N, Yoshimoto M, Yasumoto T (2009) The effect of structural variation in 21 microcystins on their inhibition of PP2A and the effect of replacing Cys269 with glycine. Toxicon 54:533–544

    Article  Google Scholar 

  • Jochimsen EM, Carmichael WW, An J, Cardo DM, Cookson ST, Holmes CEM, Antunes MBC, Filho DAM, Lyra TM, Barreto VST, Azevedo SMFO, Jarvis WR (1998) Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. N Engl J Med 338:873–878

    Article  CAS  Google Scholar 

  • Kuiper-Goodman T, Falconer I, Fitzgerald J (1999) Cyanobacterial toxins. In: Chorus I, Batram J (eds) Toxic Cyanobacteria in Water-A Guide to their Public Health Consequences, Monitoring and Management, WHO, E. & F.P. Spon, London, pp 113–153

  • Lawton LA, Edwards C, Codd GA (1994) Extraction and high-performance liquid chromatography method for the determination of microcystins in raw and treated waters. Analyst 119:1525–1530

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Monks NR, Liu S, Xu Y, Yu H, Bendelow AS, Moscow JA (2007) Potent cytotoxicity of the phosphatase inhibitor microcystin LR and microcystin analogues in OATP1B1- and OATP1B3-expressing HeLa cells. Mol Cancer Ther 6:587–598

    Article  CAS  Google Scholar 

  • Nishiwaki-Matsushima R, Ohta T, Nishiwaki S, Suganuma M, Kohyama K, Ishikawa T, Carmichael WW, Fujiki H (1992) Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J Cancer Res Clin Oncol 118:420–424

    Article  CAS  Google Scholar 

  • Ortelli D, Edder P, Cognard E, Jan P (2008) Fast screening and quantitation of microcystins in microalgae dietary supplement products and water by liquid chromatography coupled to time of flight mass spectrometry. Anal Chim Acta 617:230–237

    Article  CAS  Google Scholar 

  • Perron MC, Qui B, Boucher N, Bellemare F, Juneau P (2012) Use of chlorophyll a fluorescence to detect the effect of microcystins on photosynthesis and photosystem II energy fluxes of green algae. Toxicon 59:567–577

    Article  CAS  Google Scholar 

  • Pouria S, de Andrade A, Barbosa J, Cavalcanti RL, Barreto VT, Ward CJ, Preiser W, Poon GK, Neild GH, Codd GA (1998) Fatal microcystin intoxication in haemodialysis unit in Caruaru, Brazil. Lancet 352:21–26

    Article  CAS  Google Scholar 

  • Pyo D, Lee J, Choi E (2004) Enzyme-linked immunosorbent assay detection of microcystins using new monoclonal antibodies. J Immunoassay Immunochem 25:227–239

    Article  CAS  Google Scholar 

  • Rinehart KL, Harada KI, Namikoshi M, Chen C, Harvis CA, Munro MHG, Blunt JW, Mulligan PE, Beasley VR, Dahlem AM, Carmichael WW (1988) Nodularin, microcystin, and the configuration of Adda. J Am Chem Soc 110:8557–8558

    Article  CAS  Google Scholar 

  • Sheng JW, He M, Shi HC (2007) A highly specific immunoassay for microcystin-LR detection based on a monoclonal antibody. Anal Chim Acta 603:111–118

    Article  CAS  Google Scholar 

  • Spoof L, Karlsson K, Meriluoto J (2001) High-performance liquid chromatographic separation of microcystins and nodularin, cyanobacterial peptide toxins, on C18 and amide C16 sorbents. J Chromatogr A 909:225–236

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Ueno Y, Nagata S, Tsutsumi T, Hasegawa A, Yoshida F, Suttajit M, Mebs D, Pütsch M, Vasconcelos V (1996) Survey of microcystins in environmental water by a high sensitive immunoassay based on monoclonal antibody. Nat Toxins 4:271–276

    Article  CAS  Google Scholar 

  • Ufelmann H, Krueger T, Lucas B, Schrenk D (2012) Human and rat hepatocyte toxicity and protein phosphatase 1 and 2A inhibitory activity of naturally occurring desmethyl-microcystins and nodularins. Toxicology 293:59–67

    Article  CAS  Google Scholar 

  • Vesterkvist PSM, Misiorek JO, Spoof LEM, Toivola DM, Meriluoto JAO (2012) Comparative cellular toxicity of hydrophilic and hydrophobic microcystins on Caco-2 cells. Toxins 4:1008–1023

    Article  CAS  Google Scholar 

  • Welker M, von Döhren H (2006) Cyanobacterial peptides—nature’s own combinatorial biosynthesis. FEMS Microbiol Rev 30:530–556

    Article  CAS  Google Scholar 

  • WHO (1998) Guidelines for drinking-water quality, Addendum to vol. 1, 2nd ed. World Health Organization, Geneva

  • Yang B, Xu J-Z, Ding T, Wu B, Jing S, Ding S-J, Chen H-L, Sheng C-Y, Jiang Y (2009) A novel method to detect seven microcystins in hard clam and corbicula fluminea by liquid chromatography–tandem mass spectrometry. J Chromatogr A 877:3522–3528

    CAS  Google Scholar 

  • Yoshizawa S, Matsushima R, Watanabe MF, Hard K, Ichihara A, Carmichael WW, Fujiki H (1990) Inhibition of protein phosphatases by microcystin and nodularin associated with hepatotoxicity. J Cancer Res Clin Oncol 116:609–614

    Article  CAS  Google Scholar 

  • Zeck A, Eikenberg A, Weller MG, Niessner R (2001) Highly sensitive immunoassay based on a monoclonal antibody specific for [4-arginine]microcystins. Anal Chim Acta 441:1–13

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work has been funded by INIA (2008-00084-00-00) and MINECO (BIO2011-26311). Dr. Campàs acknowledges financing support from MINECO and the European Social Fund through the Ramón y Cajal Programme. Diana Garibo acknowledges a scholarship from the Comissionat per a Universitats i Recerca of the Departament d’Innovació, Universitats i Empresa of the Generalitat de Catalunya. Dr. del Valle acknowledges support from the Catalonia program ICREA Academia. The authors acknowledge Dr. Quesada from the Universidad Autónoma de Madrid (Spain) for kindly providing the natural bloom sample from the Alcántara reservoir, and Dr. Mimendia for advice and fruitful conversations.

Author information

Authors and Affiliations

  1. IRTA, Carretera de Poble Nou km 5.5, 43540, Sant Carles de la Ràpita, Spain

    Diana Garibo, Jorge Diogène & Mònica Campàs

  2. Mass Spectrometry Laboratory/Organic Pollutants, IDAEA-CSIC, C/Jordi Girona 18, 08034, Barcelona, Spain

    Cintia Flores & Josep Caixach

  3. Sensors and Biosensors Group, Universitat Autònoma de Barcelona, Edifici Cn, Campus de Bellaterra, 08034, Barcelona, Spain

    Xavier Cetó, Beatriz Prieto-Simón & Manel del Valle

Authors
  1. Diana Garibo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cintia Flores
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xavier Cetó
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Beatriz Prieto-Simón
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Manel del Valle
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Josep Caixach
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jorge Diogène
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mònica Campàs
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mònica Campàs.

Additional information

Responsible editor: Philippe Garrigues

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 172 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Garibo, D., Flores, C., Cetó, X. et al. Inhibition equivalency factors for microcystin variants in recombinant and wild-type protein phosphatase 1 and 2A assays. Environ Sci Pollut Res 21, 10652–10660 (2014). https://doi.org/10.1007/s11356-014-3065-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11356-014-3065-7

Keywords

Search

Navigation