Analytical and Bioanalytical Chemistry

, Volume 400, Issue 4, pp 1015–1029 | Cite as

Free Ca2+ as an early intracellular biomarker of exposure of cyanobacteria to environmental pollution

  • Ana Lilia Barrán-Berdón
  • Ismael Rodea-Palomares
  • Francisco Leganés
  • Francisca Fernández-PiñasEmail author
Paper in Forefront


Calcium functions as a versatile messenger in a wide variety of eukaryotic and prokaryotic cells. Cyanobacteria are photoautotrophs which have a great ecological impact as primary producers. Our research group has presented solid evidence of a role of calcium in the perception of environmental changes by cyanobacteria and their acclimation to these changes. We constructed a recombinant strain of the freshwater cyanobacterium Anabaena sp. PCC 7120 that constitutively expresses the calcium-binding photoprotein apoaequorin, enabling in-vivo monitoring of any fluctuation in the intracellular free calcium concentration of the cyanobacterium in response to any environmental stimulus. The “Ca2+ signature” is the combination of changes in all Ca2+ signal properties (magnitude, duration, frequency, source of the signal) produced by a specific stimulus. We recorded and analyzed the Ca2+ signatures generated by exposure of the cyanobacterium to different groups of environmental pollutants, for example cations, anions, organic solvents, naphthalene, and pharmaceuticals. We found that, in general, each group of tested chemicals triggered a specific calcium signature in a reproducible and dose-dependent manner. We hypothesize that these Ca2+ signals may be related to the cellular mechanisms of pollutant perception and ultimately to their toxic mode of action. We recorded Ca2+ signals triggered by binary mixtures of pollutants and a signal induced by a real wastewater sample which could be mimicked by mixing its main constituents. Because Ca2+ signatures were induced before toxicity was evident, we propose that intracellular free Ca2+ may serve as an early biomarker of exposure to environmental pollution.


Aequorin Biomarker Ca2+ signature Cyanobacterium Environmental pollution Pollutant interaction 



This research was funded by the Spanish Ministry of Science and Innovation (grant CGL2010-15675, sub-programme BOS) and by the Comunidad de Madrid grant S-2009/AMB/1511 (Microambiente-CM) .

Supplementary material

216_2010_4209_MOESM1_ESM.pdf (721 kb)
ESM1 (PDF 721 kb)


  1. 1.
    Potts M (1994) Desiccation tolerance of prokaryotes. Microbiol Rev 58(4):755–805Google Scholar
  2. 2.
    Bachmann T (2003) Transforming cyanobacteria into bioreporters of biological relevance. Trends Biotechnol 21(6):247–249CrossRefGoogle Scholar
  3. 3.
    Rawson DM, Willmer AJ, Turner AP (1989) Whole-cell biosensors for environmental monitoring. Biosensors 4(5):299–311CrossRefGoogle Scholar
  4. 4.
    Clapham DE (1995) Calcium signaling. Cell 80(2):259–268CrossRefGoogle Scholar
  5. 5.
    Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1(1):11–21CrossRefGoogle Scholar
  6. 6.
    Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signalling: Dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4(7):517–529CrossRefGoogle Scholar
  7. 7.
    Dominguez DC (2004) Calcium signalling in bacteria. Mol Microbiol 54(2):291–297CrossRefGoogle Scholar
  8. 8.
    McAinsh MR, Brownlee C, Hetherington AM (1997) Calcium ions as second messengers in guard cell signal transduction. Physiol Plant 100(1):16–29CrossRefGoogle Scholar
  9. 9.
    Rudd JJ, Franklin-Tong VE (2001) Unravelling response-specificity in ca2+ signalling pathways in plant cells. New Phytol 151(1):7–33CrossRefGoogle Scholar
  10. 10.
    McAinsh MR, Pittman JK (2009) Shaping the calcium signature. New Phytol 181(2):275–294CrossRefGoogle Scholar
  11. 11.
    Kudla J, Batistic O, Hashimoto K (2010) Calcium signals: The lead currency of plant information processing. Plant Cell 22(3):541–563. doi: 10.1105/tpc.109.072686 CrossRefGoogle Scholar
  12. 12.
    Gangola P, Rosen B (1987) Maintenance of intracellular calcium in Escherichia coli. J Biol Chem 15 (262(26):):2570-2574Google Scholar
  13. 13.
    Bush DS, Jones RL (1990) Measuring intracellular ca levels in plant cells using the fluorescent probes, indo-1 and fura-2: Progress and prospects. Plant Physiol 93(3):841–845CrossRefGoogle Scholar
  14. 14.
    Hinkle PM, Shanshala ED 2nd, Nelson EJ (1992) Measurement of intracellular cadmium with fluorescent dyes. Further evidence for the role of calcium channels in cadmium uptake. J Biol Chem 267(35):25553–25559Google Scholar
  15. 15.
    Knight MR, Campbell AK, Smith SM, Trewavas AJ (1991) Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature 352(6335):524–526CrossRefGoogle Scholar
  16. 16.
    Knight MR, Smith SM, Trewavas AJ (1992) Wind-induced plant motion immediately increases cytosolic calcium. Proc Natl Acad Sci USA 89(11):4967–4971CrossRefGoogle Scholar
  17. 17.
    Brini M, Marsault R, Bastianutto C, Alvarez J, Pozzan T, Rizzuto R (1995) Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation. J Biol Chem 270(17):9896–9903CrossRefGoogle Scholar
  18. 18.
    Torrecilla I, Leganes F, Bonilla I, Fernandez-Pinas F (2000) Use of recombinant aequorin to study calcium homeostasis and monitor calcium transients in response to heat and cold shock in cyanobacteria. Plant Physiol 123(1):161–176. doi: 10.1104/pp.123.1.161 CrossRefGoogle Scholar
  19. 19.
    Torrecilla I, Leganes F, Bonilla I, Fernandez-Pinas F (2004) A calcium signal is involved in heterocyst differentiation in the cyanobacterium Anabaena sp. PCC7120. Microbiology 150(Pt 11):3731–3739CrossRefGoogle Scholar
  20. 20.
    Torrecilla I, Leganés F, Bonilla I, Fernández-Piñas F (2001) Calcium transients in response to salinity and osmotic stress in the nitrogen-fixing cyanobacterium Anabaena sp. PCC7120, expressing cytosolic apoaequorin. Plant Cell Environ 24(6):641–648CrossRefGoogle Scholar
  21. 21.
    Torrecilla I, Leganés F, Bonilla I, Fernández-Piñas F (2004) Light-to-dark transitions trigger a transient increase in intracellular Ca2+ modulated by the redox state of the photosynthetic electron transport chain in the cyanobacterium Anabaena sp. PCC7120. Plant Cell Environ 27(7):810–819CrossRefGoogle Scholar
  22. 22.
    Leganes F, Forchhammer K, Fernandez-Pinas F (2009) Role of calcium in acclimation of the cyanobacterium Synechococcus elongatus PCC 7942 to nitrogen starvation. Microbiology 155(Pt 1):25–34CrossRefGoogle Scholar
  23. 23.
    Schafer S, Bickmeyer U, Koehler A (2009) Measuring Ca2+-signalling at fertilization in the sea urchin Psammechinus miliaris: Alterations of this Ca2+-signal by copper and 2, 4, 6-tribromophenol. Comp Biochem Physiol C Toxicol Pharmacol 150(2):261–269CrossRefGoogle Scholar
  24. 24.
    Biagioli M, Pinton P, Scudiero R, Ragghianti M, Bucci S, Rizzuto R (2005) Aequorin chimeras as valuable tool in the measurement of Ca2+ concentration during cadmium injury. Toxicology 208(3):389–398CrossRefGoogle Scholar
  25. 25.
    Wang SS, Chen L, Xia SK (2007) Cadmium is acutely toxic for murine hepatocytes: Effects on intracellular free Ca2+ homeostasis. Physiol Res 56(2):193–201Google Scholar
  26. 26.
    Ogunbayo OA, Lai PF, Connolly TJ, Michelangeli F (2008) Tetrabromobisphenol a (TBBPA), induces cell death in tm4 sertoli cells by modulating Ca2+ transport proteins and causing dysregulation of Ca2+ homeostasis. Toxicol In Vitro 22(4):943–952CrossRefGoogle Scholar
  27. 27.
    Kozlova O, Zwinderman M, Christofi N (2005) A new short-term toxicity assay using Aspergillus awamori with recombinant aequorin gene. BMC Microbiol 5:40CrossRefGoogle Scholar
  28. 28.
    Dondero F, Dagnino A, Jonsson H, Capri F, Gastaldi L, Viarengo A (2006) Assessing the occurrence of a stress syndrome in mussels (Mytilus edulis) using a combined biomarker/gene expression approach. Aquat Toxicol 78(Suppl 1):S13–S24CrossRefGoogle Scholar
  29. 29.
    Ohta M, Suzuki T (2007) Participation of the inositol phospholipid signaling pathway in the increase in cytosolic calcium induced by tributyltin chloride intoxication of chlorophyllous protozoa Euglena gracilis z and its achlorophyllous mutant sm-zk. Comp Biochem Physiol C Toxicol Pharmacol 146(4):525–530CrossRefGoogle Scholar
  30. 30.
    Dondero F, Jonsson H, Rebelo M, Pesce G, Berti E, Pons G, Viarengo A (2006) Cellular responses to environmental contaminants in amoebic cells of the slime mould Dictyostelium discoideum. Comp Biochem Physiol C Toxicol Pharmacol 143(2):150–157CrossRefGoogle Scholar
  31. 31.
    Steinkellner H, Mun-Sik K, Helma C, Ecker S, Ma TH, Horak O, Kundi M, Knasmuller S (1998) Genotoxic effects of heavy metals: Comparative investigation with plant bioassays. Environ Mol Mutagen 31(2):183–191CrossRefGoogle Scholar
  32. 32.
    Barbosa JS, Cabral TM, Ferreira DN, Agnez-Lima LF, de Medeiros SR Genotoxicity assessment in aquatic environment impacted by the presence of heavy metals. Ecotoxicol Environ Saf 73 (3):320-325Google Scholar
  33. 33.
    Cherry N, Shaik K, McDonald C, Chowdhury Z Manganese, arsenic, and infant mortality in Bangladesh: An ecological analysis. Arch Environ Occup Health 65 (3):148-153Google Scholar
  34. 34.
    Jones H, Visoottiviseth P, Bux MK, Fodenyi R, Kovats N, Borbely G, Galbacs Z (2008) Case reports: Arsenic pollution in Thailand, Bangladesh, and Hungary. Rev Environ Contam Toxicol 197:163–187CrossRefGoogle Scholar
  35. 35.
    Ohe T, Watanabe T, Wakabayashi K (2004) Mutagens in surface waters: A review. Mutat Res 567(2–3):109–149Google Scholar
  36. 36.
    Li X, Zhang T, Min X, Liu P (2010) Toxicity of aromatic compounds to Tetrahymena estimated by microcalorimetry and qsar. Aquat Toxicol 98(4):322–327CrossRefGoogle Scholar
  37. 37.
    Rosal R, Rodriguez A, Perdigon-Melon JA, Petre A, Garcia-Calvo E, Gomez MJ, Aguera A, Fernandez-Alba AR (2010) Occurrence of emerging pollutants in urban wastewater and their removal through biological treatment followed by ozonation. Water Res 44(2):578–588CrossRefGoogle Scholar
  38. 38.
    Andreozzi R, Raffaele M, Nicklas P (2003) Pharmaceuticals in stp effluents and their solar photodegradation in aquatic environment. Chemosphere 50(10):1319–1330CrossRefGoogle Scholar
  39. 39.
    Rodea-Palomares I, Fernández-Piñas F, González-García C, Leganés F (2009) Use of lux-marked cyanobacterial bioreporters for assessment of individual and combined toxicities of metals in aqueous samples. In: Handbook on cyanobacteria: Biochemistry, biotechnology and applications. Nova Science Publishers, Inc, USAGoogle Scholar
  40. 40.
    Rodea-Palomares I, Petre AL, Boltes K, Leganes F, Perdigon-Melon JA, Rosal R, Fernandez-Pinas F (2010) Application of the combination index (ci)-isobologram equation to study the toxicological interactions of lipid regulators in two aquatic bioluminescent organisms. Water Res 44(2):427–438CrossRefGoogle Scholar
  41. 41.
    Rosal R, Rodea-Palomares I, Boltes K, Fernandez-Pinas F, Leganes F, Gonzalo S, Petre A (2010) Ecotoxicity assessment of lipid regulators in water and biologically treated wastewater using three aquatic organisms. Environ Sci Pollut Res Int 17(1):135–144CrossRefGoogle Scholar
  42. 42.
    Rosal R, Rodriguez A, Perdigon-Melon JA, Mezcua M, Hernando MD, Leton P, Garcia-Calvo E, Aguera A, Fernandez-Alba AR (2008) Removal of pharmaceuticals and kinetics of mineralization by O3/H2O2 in a biotreated municipal wastewater. Water Res 42(14):3719–3728CrossRefGoogle Scholar
  43. 43.
    USEPA (2002) Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organism (fifth edition). U.S. Environmental Protection Agency, Washington, DCGoogle Scholar
  44. 44.
    Rodea-Palomares I, Gonzalez-Garcia C, Leganes F, Fernandez-Pinas F (2009) Effect of pH, EDTA, and anions on heavy metal toxicity toward a bioluminescent cyanobacterial bioreporter. Arch Environ Contam Toxicol 57(3):477–487CrossRefGoogle Scholar
  45. 45.
    Pitta TP, Sherwood EE, Kobel AM, Berg HC (1997) Calcium is required for swimming by the nonflagellated cyanobacterium Synechococcus strain WH8113. J Bacteriol 179(8):2524–2528Google Scholar
  46. 46.
    Nazarenko LV, Andreev IM, Lyukevich AA, Pisareva TV, Los DA (2003) Calcium release from Synechocystis cells induced by depolarization of the plasma membrane: Mscl as an outward ca2+ channel. Microbiology 149(Pt 5):1147–1153CrossRefGoogle Scholar
  47. 47.
    Waditee R, Hossain GS, Tanaka Y, Nakamura T, Shikata M, Takano J, Takabe T, Takabe T (2004) Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria. J Biol Chem 279(6):4330–4338CrossRefGoogle Scholar
  48. 48.
    Kang MS, Jeong JY, Seo JH, Jeon HJ, Jung KM, Chin MR, Moon CK, Bonventre JV, Jung SY, Kim DK (2006) Methylmercury-induced toxicity is mediated by enhanced intracellular calcium through activation of phosphatidylcholine-specific phospholipase c. Toxicol Appl Pharmacol 216(2):206–215CrossRefGoogle Scholar
  49. 49.
    Hassenklöver T, Predehl S, Pilli J, Ledwolorz J, Assmann M, Bickmeyer U (2006) Bromophenols, both present in marine organisms and in industrial flame retardants, disturb cellular Ca2+ signaling in neuroendocrine cells (pc12). Aquat Toxicol 76(1):37–45CrossRefGoogle Scholar
  50. 50.
    Michelangeli F, Ogunbayo OA, Wootton LL, Lai PF, Al-Mousa F, Harris RM, Waring RH, Kirk CJ (2008) Endocrine disrupting alkylphenols: Structural requirements for their adverse effects on Ca2+ pumps, Ca2+ homeostasis & sertoli tm4 cell viability. Chem Biol Interact 176(2–3):220–226CrossRefGoogle Scholar
  51. 51.
    Dingemans MM, van den Berg M, Bergman A, Westerink RH (2010) Calcium-related processes involved in the inhibition of depolarization-evoked calcium increase by hydroxylated pbdes in pc12 cells. Toxicol Sci 114(2):302–309CrossRefGoogle Scholar
  52. 52.
    Kawano T, Kadono T, Furuichi T, Muto S, Lapeyrie F (2003) Aluminum-induced distortion in calcium signaling involving oxidative bursts and channel regulation in tobacco by-2 cells. Biochem Biophys Res Commun 308(1):35–42CrossRefGoogle Scholar
  53. 53.
    Lane R, Ghazi SO, Whalen MM (2009) Increases in cytosolic calcium ion levels in human natural killer cells in response to butyltin exposure. Arch Environ Contam Toxicol 57(4):816–825CrossRefGoogle Scholar
  54. 54.
    Fonnum F, Mariussen E (2009) Mechanisms involved in the neurotoxic effects of environmental toxicants such as polychlorinated biphenyls and brominated flame retardants. J Neurochem 111(6):1327–1347CrossRefGoogle Scholar
  55. 55.
    Errakhi R, Dauphin A, Meimoun P, Lehner A, Reboutier D, Vatsa P, Briand J, Madiona K, Rona JP, Barakate M, Wendehenne D, Beaulieu C, Bouteau F (2008) An early Ca2+ influx is a prerequisite to thaxtomin a-induced cell death in arabidopsis thaliana cells. J Exp Bot 59(15):4259–4270CrossRefGoogle Scholar
  56. 56.
    Domingues I, Agra AR, Monaghan K, Soares AMVM, Nogueira AJA (2010) Cholinesterase and glutathione-s-transferase activities in freshwater invertebrates as biomarkers to assess pesticide contamination. Environ Toxicol Chem 29(1):5–18CrossRefGoogle Scholar
  57. 57.
    Chou TC (2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58(3):621–681CrossRefGoogle Scholar
  58. 58.
    Llabjani V, Trevisan J, Jones KC, Shore RF, Martin FL (2010) Binary mixture effects by PBDE congeners (47, 153, 183, or 209) and PCB congeners (126 or 153) in mcf-7 cells: Biochemical alterations assessed by ir spectroscopy and multivariate analysis. Environ Sci Technol 44(10):3992–3998CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Ana Lilia Barrán-Berdón
    • 1
  • Ismael Rodea-Palomares
    • 1
  • Francisco Leganés
    • 1
  • Francisca Fernández-Piñas
    • 1
    Email author
  1. 1.Departamento de Biología, Facultad de CienciasUniversidad Autónoma de MadridMadridSpain

Personalised recommendations