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Ecotoxicology

, Volume 23, Issue 7, pp 1326–1335 | Cite as

Optimization of NRU assay in primary cultures of Eisenia fetida for metal toxicity assessment

  • Amaia Irizar
  • Daniel Duarte
  • Lucia Guilhermino
  • Ionan Marigómez
  • Manu Soto
Article

Abstract

Coelomocytes, immunocompetent cells of lumbricids, have received special attention for ecotoxicological studies due to their sensibility to pollutants. Their in vitro responses are commonly quantified after in vivo exposure to real or spiked soils. Alternatively, quantifications of in vitro responses after in vitro exposure are being studied. Within this framework, the present study aimed at optimizing the neutral red uptake (NRU) assay in primary culture of Eisenia fetida coelomocytes for its application in soil toxicity testing. Optimized assay conditions were: earthworm depuration for 24 h before retrieving coelomocytes by electric extrusion; 2 × 105 seeded cells/well (200 µl) for the NRU assay and incubation for 1 h with neutral red dye. Supplementation of the culture medium with serum was not compatible with the NRU assay, but coelomocytes could be maintained with high viability for 3 days in a serum-free medium without replenishment. Thus, primary cultures were used for 24 h in vitro toxicity testing after exposure to different concentrations of Cd, Cu, Ni and Pb (ranging from 0.1 to 100 μg/ml). Primary cultures were sensitive to metals, the viability declining in a dose-dependent manner. The toxicity rank was, from high to low, Pb > Ni > Cd > Cu. Therefore, it can be concluded that the NRU assay in coelomocytes in primary cultures provides a sensitive and prompt response after in vitro exposure to metals.

Keywords

Eisenia fetida Coelomocyte Primary cultures In vitro exposure Metal 

Notes

Acknowledgments

This research was supported by the Basque Government (ETORTEK IE10-273; SAIOTEK S-PC13UN028; and Grant to Consolidated Research Group, GIC07/26-IT-393-07; IT-810-13) and the University of the Basque Country (Research & Formation Unit in Ecosystem Health Protection, UFI 11/37). A.I has been recipient of a predoctoral fellowship from the Basque Government.

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Abdul Rida AMM, Bouché MB (1997) Earthworm toxicology: from acute to chronic tests. Soil Biol Biochem 29:699–703CrossRefGoogle Scholar
  2. Adamowicz A (2005) Morphology and ultrastructure of the earthworm Dendrobaena veneta (Lumbricidae) coelomocytes. Tissue Cell 37:125–133CrossRefGoogle Scholar
  3. Asensio V, Kille P, Morgan AJ, Soto M, Marigómez I (2007) Metallothionein expression and neutral red uptake as biomarkers of metal exposure and effect in Eisenia fetida and Lumbricus terrestris exposed to Cd. Eur J Soil Biol 43:233–238CrossRefGoogle Scholar
  4. Asensio V, Rodríguez-Ruiz A, Garmendia L, Andre J, Kille P, Morgan AJ, Soto M, Marigómez I (2013) Towards an integrative soil health assessment strategy: A three tier (integrative biomarker response) approach with Eisenia fetida applied to soils subjected to chronic metal pollution. Sci Total Environ 442:344–365Google Scholar
  5. Balls M, Fentem JH (1999) The validation and acceptance of alternatives to animal testing. Toxicol In Vitro 13:837–846CrossRefGoogle Scholar
  6. Bierkens J, Klein G, Corbisier P, van den Heuvel R, Verschaeve R, Weltens R, Schoeters G (1998) Comparative sensitivity of 20 bioassays for soil quality. Chemosphere 37:2935–2947CrossRefGoogle Scholar
  7. Bilej M, Prochazkova P, Silverowa M, Jaskova R (2010) Earthworm immunity. Adv Exp Med Biol 708:66–79Google Scholar
  8. Borenfreund E, Puerner JA (1985) A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J Tissue Cult Methods 9:7–9CrossRefGoogle Scholar
  9. Brousseau P, Fugère N, Bernier J, Coderre D, Nadeau D, Poirier G, Fournier M (1997) Evaluation of earthworm exposure to contaminated soil by cytometric assay of coelomocytes phagocytosis in Lumbricus terrestris (Oligochaeta). Soil Biol Biochem 29:681–684CrossRefGoogle Scholar
  10. Cholewa J, Feeney GP, O’Reilly M, Sturzenbaum SR, Morgan AJ, Plytycz B (2006) Autofluorescence in eleocytes of some earthworm species. Folia Histochem Cytobiol 44:65–71Google Scholar
  11. Cooper EL (1974) Phylogeny of leukocytes: earthworm coelomocytes in vitro and in vivo. In: Lindhal-Kiessling K, Osoba D (eds) Lymphocyte recognition and effector mechanisms. Proceedings of the eighth leukocyte culture conference. Academic Press, New York, pp 155–162Google Scholar
  12. Di Marzio WD, Saenz ME, Lemiere S, Vasseur P (2005) Improved single-cell gel electrophoresis assay for detecting DNA damage in Eisenia foetida. Environ Mol Mutagen 46:246–252CrossRefGoogle Scholar
  13. Diogène J, Dufour M, Poirier GG, Nadeau D (1997) Extrusion of earthworm coelomocytes: comparison of the cell populations recovered from the species Lumbricus terrestris, Eisenia fetida and Octolasion tyrtaeum. Lab Anim 31:326–336CrossRefGoogle Scholar
  14. Eyambe G, Goven AJ, Fitzpatrick LC, Venables B, Cooper EL (1991) Extrusion protocol for use in chronic immunotoxicity studies with earthworms (Lumbricus terrestris) coelomic leukocytes. Lab Anim 25:61–67CrossRefGoogle Scholar
  15. Falkner E, Appl H, Eder C, Losert UM, Schoffl H, Pfaller W (2006) Serum free cell culture: the free access online database. Toxicol In Vitro 20:395–400CrossRefGoogle Scholar
  16. Fischer AB (1985) Factors influencing cadmium uptake and cyto-toxicity in cultured-cells. Xenobiotica 15:751–757CrossRefGoogle Scholar
  17. Fugère N, Brousseau P, Krystyniak K, Coderre D, Fournier M (1996) Heavy-metal specific inhibition of phagocytosis and different in vitro sensitivity of heterogeneous coelomocytes from Lumbicus terrestris (Oligochaeta). Toxicology 109:157–166CrossRefGoogle Scholar
  18. Hamed SS, Kauschke E, Cooper EL (2002) Cytochemical properties of earthworm coelomocytes enriched by Percoll. In: Beschin A, Bilej M, Cooper EL (eds) A new model for analyzing antimicrobial peptides with biomedical applications. IOS Press, Ohmsha, pp 29–37Google Scholar
  19. Hartung T, Ball M, Bardouille C, Blanck O, Coecke S, Gstraunthaler G, Levvis D (2002) Good cell culture practice—ECVAM Good Cell Culture Practice Task Force report 1. Atla Altern Lab Anim 30:407–414Google Scholar
  20. Hayashi Y, Engelmann P, Foldbjerg R, Szabo M, Somogyi I, Pollak E, Molnar L, Autrup H, Sutherland DS, Scott-Fordsmand J, Heckmann LH (2012) Earthworms and humans in vitro: characterizing evolutionarily conserved stress and immune responses to silver nanoparticles. Environ Sci Technol 46:4166–4173Google Scholar
  21. Hendawi M, Sauvé S, Ashour M, Brousseau P, Fournier M (2004) A new ultrasound protocol for extrusion of coelomocyte cells from the earthworm Eisenia fetida. Ecotoxicol Environ Saf 59:17–22CrossRefGoogle Scholar
  22. Homa J, Niklinska M, Plytycz B (2003) Effect of heavy metals on coelomocytes of the earthworm Allolobophora chlorotica. Pedobiologia 47:640–645Google Scholar
  23. Koziol B, Markowicz M, Kruk J, Plytycz B (2006) Riboflavin as a source of autofluorescence in Eisenia fetida coelomocytes. Photoch Photobiol 82:570–573Google Scholar
  24. Kurek A, Homa J, Plytycz B (2002) Earthworm coelomocytes: convenient model for basic and applied sciences. In: Beschin A, Bilej M, Cooper EL (eds) A new model for analyzing antimicrobial peptides with biomedical applications. IOS press, Ohmsha, pp 38–46Google Scholar
  25. Kurek A, Homa J, Kauschke E, Plytycz B (2007) Characteristics of coelomocytes of the stubby earthworm, Allolobophora chlorotica (Sav.). Eur J Soil Biol 43:S121–S126Google Scholar
  26. Liu B, Guo Y, Wang J, Xu R, Wang X, Wang D, Zhang LQ, Xu YN (2010) Spectroscopic studies on the interaction and sonodynamic damage of neutral red (NR) to bovine serum albumin (BSA). J Lumin 130:1036–1043CrossRefGoogle Scholar
  27. Maboeta MS, Reinecke SA, Reinecke AJ (2003) Linking lysosomal biomarker and population responses in a field population of Aporrectodea caliginosa (Oligochaeta) exposed to the fungicide copper oxychloride. Ecotoxicol Environ Saf 56:411–418CrossRefGoogle Scholar
  28. Maleri RA, Fourie F, Reinecke AJ, Reinecke SA (2008) Photometric application of the MTT- and NRU-assays as biomarkers for the evaluation of cytotoxicity ex vivo in Eisenia andrei. Soil Biol Biochem 40:1040–1048CrossRefGoogle Scholar
  29. OECD (1984) Guideline for the testing of chemicalsGoogle Scholar
  30. Plytycz B, Rozanowska R, Seljelid R (1992) Quantification of neutral red pinocytosis by adherent cells: comparative studies. Folia Biol (Krakow) 40:3–9Google Scholar
  31. Plytycz J, Homa B, Koziol M, Rozanowska M, Morgan AJ (2006) Riboflavin content in autofluorescent earthworm coelomocytes is species-specific. Folia Histochem Cytobiol 44:275–280Google Scholar
  32. Plytycz B, Klimek M, Homa J, Tylko G, Kolaczkowska E (2007) Flow cytometric measurement of neutral red accumulation in earthworm coelomocytes: novel assay for studies on heavy metal exposure. Eur J Soil Biol 43:116–120CrossRefGoogle Scholar
  33. Plytycz B, Kielbasa E, Grebosz A, Duchnowski M, Morgan AJ (2010) Riboflavin mobilization from eleocyte stores in the earthworm Dendrodrilus rubidus inhabiting aerially-contaminated Ni smelter soil. Chemosphere 81:199–205CrossRefGoogle Scholar
  34. Repetto G, del Peso A, Zurita JL (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3:1125–1131CrossRefGoogle Scholar
  35. Ruby MV, Schoof R, Brattin W, Goldade M, Post G, Harnois M, Mosby DE, Casteel SW, Berti W, Carpenter M, Edwards D, Cragin D, Chappell W (1999) Advances in evaluating the oral bioavailability of inorganics in soil for use in human health risk assessment. Environ Sci Technol 33:3697–3705CrossRefGoogle Scholar
  36. Sanchez-Hernandez JC (2006) Earthworm biomarkers in ecological risk assessment. Rev Environ Contam Toxicol 188:85–126Google Scholar
  37. Sauvé S, Hendawi M, Brousseau P, Fournier M (2002) Phagocytic response of terrestrial and aquatic invertebrates following in vitro exposure to trace elements. Ecotoxicol Environ Saf 52:21–29CrossRefGoogle Scholar
  38. Scaps P, Grelle C, Decamps M (1997) Cadmium and lead accumulation in the earthworm Eisenia fetida (Savigny) and its impact on cholinesterase and metabolic pathway enzyme activity. Comp Biochem Physiol 7(116):3Google Scholar
  39. Scott-Fordsmand JJ, Weeks JM, Hopkin SP (1998) Uptake and toxicity of spiked nickel to earthworm Eisenia fetida in a range of chinese soils. Environ Toxicol 30:2586–2593Google Scholar
  40. Seibert H, Morchel S, Gulden M (2002) Factors influencing nominal effective concentrations of chemical compounds in vitro: medium protein concentration. Toxicol In Vitro 16:289–297CrossRefGoogle Scholar
  41. Spurgeon DJ, Hopkin SP, Jones DT (1994) Effects of Cadmioum, coppe, lead and zinc on growth, reproduction and survival of the earthworm Eisenia fetida (Savigny): assessing the environmental impacts of poit-source metal contamination in terrestrial ecosystems. Environ Pollut 84:1213–1300CrossRefGoogle Scholar
  42. Spurgeon DJ, Svendsen C, Rimmer VR, Hopkin SP, Weeks JM (2000) Relative sensitivity of life-cycle and biomarker responses in four earthworm species exposed to zinc. Environ Toxicol Chem 19:1800–1808CrossRefGoogle Scholar
  43. Spurgeon DJ, Ricketts H, Svendsen C, Morgan AJ, Kille P (2005) Hierarchical responses of soil invertebrates (earthworms) to toxic metal stress. Environ Sci Technol 39:5327–5334CrossRefGoogle Scholar
  44. Svendsen C, Spurgeon DJ, Hankard PK, Weeks JM (2004) A review of lysosomal membrane stability measured by neutral red retention: is it a workable earthworm biomarker? Ecotoxicol Environ Saf 57:20–29CrossRefGoogle Scholar
  45. Toupin J, Marks DH, Cooper EL, Lamoureux G (1977) Earthworm coelomocytes in vitro. In vitro Cell Dev Biol 13:218–222Google Scholar
  46. Valembois P, Rouch P, Du Pasquier L (1973) Dégradation in vitro de protéines étrangéres par les macrophages du lombricien Eisenia foetida (Sav.) C.R.Séances Acad Sci III (Série D) 277:57–65Google Scholar
  47. van der Ploeg MJ, van den Berg JH, Bhattacharjee S, de Haan LH, Ershov DS, Fokkink RG, Zuilhof H, Rietjens IMCM, van den Brink NW (2012) In vitro nanoparticle toxicity to rat alveolar cells and coelomocytes from the earthworm Lumbricus rubellus. Nanotoxicology 8(1):28–37CrossRefGoogle Scholar
  48. van der Valk J, Brunner D, De Smet K, Fex Svenningsen A, Honegger P, Knudsen LE, Lindl T, Noraberg J, Price A, Scarino ML, Gstraunthaler G (2010) Optimization of chemically defined cell culture media—replacing fetal bovine serum in mammalian in vitro methods. Toxicol In Vitro 24:1053–1063CrossRefGoogle Scholar
  49. van Eeckhout HS, De Schamphelaere KAC, Heijerick DG, Van Sprang PA, Janssen CR (2005) Bioavailability and aging of nickel in soils: invertebrate toxicity testing. Draft Final Report. Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University, Ghent, BelgiumGoogle Scholar
  50. van Gestel CAM, Koolhaas JE, Hamers T, van Hoppe M, van Roovert M, Korsman C, Reinecke SA (2009) Effects of metal pollution on earthworm communities in a contaminated floodplain area: linking biomarker, community and functional responses. Environ Pollut 157:895–903CrossRefGoogle Scholar
  51. Weeks JM, Svendsen C (1996) Neutral red retention by lysosomes from earthworm (Lumbricus rubellus) coelomocytes: a simple biomarker of exposure to soil copper. Environ Toxicol Chem 15:1801–1805CrossRefGoogle Scholar
  52. Weyermann J, Lochmann D, Zimmer A (2005) A practical note on the use of cytotoxicity assays. Int J Pharm 288:369–376Google Scholar
  53. Xiao NW, Song Y, Ge F, Liu XH, Ou-Yang ZY (2006) Biomarkers responses of the earthworm Eisenia fetida to acetochlor exposure in OECD soil. Chemosphere 65:907–912CrossRefGoogle Scholar
  54. Yan ZG, Wang BX, Xie DL, Zhou Y, Guo G, Xu M, Bai L, Hou H, Li F (2011) Uptake and toxicity of spiked nickel to earthworm Eisenia fetida in a range of Chinese soils. Environ Toxicol Chem 30:2586–2593CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Amaia Irizar
    • 1
  • Daniel Duarte
    • 2
  • Lucia Guilhermino
    • 2
  • Ionan Marigómez
    • 1
  • Manu Soto
    • 1
  1. 1.Cell Biology & Environmental Toxicology Research Group, Research Centre for Experimental Marine Biology & Biotechnology (PIE) & Zoology & Animal Cell Biology Department (Faculty of Science & Technology)University of the Basque CountryBilbaoSpain
  2. 2.Laboratory of Ecotoxicology, Institute of Biomedical Sciences of Abel Salazar (ICBAS)University of PortoPortoPortugal

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