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Toxic Effects Produced by Microcystins from a Natural Cyanobacterial Bloom and a Microcystis aeruginosa Isolated Strain on the Fish Cell Lines RTG-2 and PLHC-1

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Abstract

Toxic cyanobacterial blooms are a worldwide problem, causing serious water pollution and public health hazard to humans and livestock. The intact cells as well as the toxins released after cellular lysis can be responsible for toxic effects in both animals and humans and are actually associated with fish kills. Two fish cell lines—PLHC-1 derived from a hepatocellular carcinoma of the topminnow Poeciliopsis lucida and RTG-2 fibroblast-like cells derived from the gonads of rainbow trout Oncorhynchus mykiss were exposed to several concentrations of extracts from a natural cyanobacterial bloom and a Microcystis aeruginosa–isolated strain. After 24 hours, morphologic and biochemical changes (total protein content, lactate dehydrogenase leakage, neutral red uptake, methathiazole tetrazolium salt metabolization, lysosomal function, and succinate dehydrogenase [SDH] activity) were investigated. The most sensitive end point for both cyanobacterial extracts in PLHC-1 cells was SDH activity, with similar EC50 values (6 μM for the cyanobacterial bloom and 7 μM for the isolated strain). RTG-2 cells were less susceptible according to SDH activity, with their most sensitive end point lysosomal function with an EC50 of 4 μM for the M. aeruginosa–isolated strain and 72 μM for the cyanobacterial bloom. The lysosomal function was stimulated at low concentrations, although SDH activity increased at high doses, indicating lysosomal and energetic alterations. Increased secretion vesicles, rounding effects, decreased cell numbers and size, hydropic degeneration, esteatosis, and apoptosis were observed in the morphologic study. Similar sensitivity to the M. aeruginosa–isolated strain was observed in both cell lines, whereas the cyanobacterial bloom was more toxic to the PLHC-1 cell line.

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References

  • Aune T, Berg K (1986) Use of freshly prepared rat hepatocytes to study toxicity of blooms of the blue-green algae Microcystis aeruginosa and Oscillatoria agardhii. J Toxicol Environ Health 19:325–336

    CAS  Google Scholar 

  • Baltrop JA, Owen TC, Cory AH, Cory JG (1991) 5-((carboxyphenyl) –3- (4,5- dimethylthiazol)- 3- (4-sulfophenyl) tetrazolium, inner salt (MTS) and related analogs of 2- (4,5- dimethyl thiazolyl) –2,5- diphenyltetrazolium bromide (MTT) decreasing to purple water soluble formazans as cell viability indicators. Bioorg Med Chem Lett 1:611

    Google Scholar 

  • Batista T, de Sousa G, Suput JS, Rahmani R, Suput D (2003) Microcystin-LR causes the collapse of actin filaments in primary human hepatocytes. Aquat Toxicol 65:85–91

    Article  CAS  Google Scholar 

  • Botha N, Gehringer MM, Downing T, van de Venter M, Shepard EG (2004) The role of microcystin-LR in the induction of apoptosis and oxidative stress in CaCo2 cells. Toxicon 43:85–92

    CAS  Google Scholar 

  • Borenfreund E, Puerner JA (1984) A simple quantitative procedure using monolayer cultures for cytotoxicity assays. J Tissue Cult Methods 9:7–9

    Google Scholar 

  • Bouaïcha N, Maatouk I (2004) Microcystin-LR nodularin induce intracellular glutathione alteration, reactive oxygen species production and lipid peroxidation in primary cultured rat hepatocytes. Toxicol Lett 148:53–63

    Article  Google Scholar 

  • Cameán AM, Molina R, Moreno IM, Pichardo S, Jos A, Moyano R (2005) Biochemical and pathological characterization of Microcystin-induced liver and kidney damage in Tilapia (Oreochromis spp). J Physiol Biochem 61:201–202

    Google Scholar 

  • Carbis CR, Rawlin GT, Grant P, Mitchell GF, Anderson JW, McCauley I (1997) A study of feral carp, Cyprinus carpio L., exposed to Microcystis aeruginosa at lake Mokoan, Australia, and possible implications for fish health. J Fish Dis 20:81–91

    Article  Google Scholar 

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

    CAS  Google Scholar 

  • Castaño A, Tarazona JV (1995) The use of cultured cells in environmental toxicology: In vitro toxicity tests. In: Cajaraville MP (ed) Servicio editorial de la Universidad del País Vasco. Bilbao, Spain, pp 279–288

    Google Scholar 

  • Chong MWK, Gu KD, Lam PKS, Yang M, Fong WF (2000) Study on the cytotoxicity of microcystin-LR on cultured cells. Chemosphere 41:143–147

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Duffy PA, Flint OP (1987) In vitro dermal irritancy tests. In: Atterwill CK, Steele CE (eds) In vitro methods in toxicology. Cambridge University Press, Cambridge, UK, pp 279–297

    Google Scholar 

  • Eriksson JE, Toivola DM, Reinikainen M, Rabergh CMI, Meriluoto JAO (1994) Testing of toxicity in cyanobacteria by cellular assays. In: Detection methods for cyanobacterial toxins. Codd GA, Jefferies TM, Keevil CW, Potter E (eds). The Royal Society of Chemistry, Cambridge, UK, pp 75–84

    Google Scholar 

  • 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

    Google Scholar 

  • Fisher WJ, Dietrich DR (2000) Pathological and biochemical characterization of microcystin-induced hepatopancreas and kidney damage in carp (Cyprinus carpio). Toxicol Appl Pharmacol 164:73–81

    Google Scholar 

  • Fladmark KE, Serres MH, Larsen NL, Yasumoto T, Aune T, Doskeland SO (1998) Sensitive detection of apoptogenic toxins in suspension cultures of rat and salmon hepatocytes. Toxicon 36:1101–1114

    Article  CAS  Google Scholar 

  • Fladmark KE, Brustugun OT, Mellgren G, Krakstad C, Boe R, Vintermyr OK, Schulman H, Doskeland SO (2002) Ca2+/calmodulin-dependent protein kinase II is required for microcystin-induced apoptosis. J Biol Chem 277:2804–2811

    Article  CAS  Google Scholar 

  • Ghosh S, Khan SA, Wickstrom M, Beasley V (1995) Effects of microcystin-LR on actin and the actin-associated proteins a-actinin and talin in hepatocytes. Nat Toxins 3:405–414

    CAS  Google Scholar 

  • Heinze R, Fastner J, Neumann U, Chorus I (2001) Testing cyanobacterial toxicity with primary rat hepatocyte and cell-line assays. In: Chorus I (ed) Cyanotoxins, occurrence, causes, consequences. Springer, Berlin, Germany, pp 317–324

    Google Scholar 

  • Jos A, Repetto G, Ríos JC, Hazen MJ, Molero MI, del Peso A, et al. (2003) Ecotoxicological evaluation of carbamazepine using an in vitro test battery. Toxicol In Vitro 17:525–532

    Article  CAS  Google Scholar 

  • Magalhães VF, Marinho MM, Domingos P, Oliveira AC, Costa SM, Azevedo LO et al. (2003) Microcystins (cyanobacteria hepatotoxins) bioaccumulation in fish and crustaceans from Sepetiba Bay (Brasil, RJ). Toxicon 42:289–295

    Article  Google Scholar 

  • Mohamed ZA, Carmichael ZA, Hussein AA (2003) Estimation of microcystins in the freshwater fish Oreochromis niloticus in an Egyptian fish farm containing a Microcystis bloom. Environ Toxicol 18:137–141

    Article  CAS  Google Scholar 

  • Montagnolli W, Zamboni A, Luvizotto-Santos R, Yunes JS (2004) Acute effects of Microcystis aeruginosa from the Patos Lagoon Estuary, Southern Brazil, on the microcrustacean Kalliapseudes schubartii (Crustacea: Tanaidacea). Arch Environ Contam Toxicol 46:463–469

    Article  CAS  Google Scholar 

  • Moreno I, Cameán A, Tavares MJ, Pereira p, Franca S (2003a) Toxicity of cyanobacteria isolated from the Guadiana River. Aquat Ecosyst Health Manage 6:409–415

    CAS  Google Scholar 

  • Moreno IM, Mate A, Repetto G, Vázquez CM, Cameán AM (2003b) Influence of microcystin-LR on the activity of membrane enzymes in rat intestinal mucosa. J Physiol Biochem 59:293–299

    CAS  Google Scholar 

  • Moreno I, Pereira p, Franca S, Cameán A (2004) Toxic cyanobacteria strains isolated from blooms in the Guadiana River (Southwest of Spain). Biol Res 37:405–417

    CAS  Google Scholar 

  • Nasri AB, Bouaïcha N, Fastner J (2004) First report of a microcystin-containing bloom of the cyanobacteria Microcystis spp. in Lake Oubeira, Eastern Algeria. Arch Environ Contam Toxicol 46:197–202

    CAS  Google Scholar 

  • Oberemm A, Becker J, Codd GA, Steinberg C (1999) Effects of cyanobacterial toxins and aqueous crude extracts of cyanobacteria on the development of fish and amphibians. Environ Toxicol 14:77–88

    Article  CAS  Google Scholar 

  • Pichardo S, Jos A, Zurita JL, Salguero M, Camean AM, Repetto G (2005, in press) The use of the fish cell lines RTG-2 and PLHC-1 to compare the toxic effects produced by microcystins LR and RR. Toxicol In Vitro 19: 865–873

    Article  CAS  Google Scholar 

  • Repetto G, Sanz p (1993) Neutral red uptake, cellular growth and lysosomal function: In vitro effects of 24 metals. Altern Lab Anim 21:501–507

    Google Scholar 

  • Repetto G, Jos A, Hazen MJ, Molero ML, del Peso A, Salguero M, et al. (2001) A test battery for the ecotoxicological evaluation of pentachlorophenol. Toxicol In Vitro 15:503–509

    CAS  Google Scholar 

  • Repetto G, del Peso A, Jos A (2003) Ecotoxicological characterization of complex mixtures. In: Mothersill C, Austin B (eds). In vitro methods in aquatic toxicology. Springer Praxis, Chichester, UK, pp 295–326

    Google Scholar 

  • Teneva I, Asparuhova D, Dzhambazov B, Mladenov R, Schirmer K (2002) The freshwater cyanobacterium Lyngbya aerugineo-coerulea produces compounds toxic to mice and to mammalian and fish cells. Environ Toxicol 18:9–20

    Google Scholar 

  • Zegura B, Sedmak B, Filipic M (2003) Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2. Toxicon 41:41–49

    Article  CAS  Google Scholar 

  • Zimba PV, Khoo L, Gaunt p, Carmichael WW, Brittain S (2001) Confirmation of catfish mortality from Microcystis toxins. J Fish Dis 24:741–747

    Article  Google Scholar 

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Acknowledgments

The investigators thank the Spanish CICYT (Projects No. AGL 2002-02622 and PPQ 2002-03717) for the financial support for this study.

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Authors and Affiliations

  1. Area of Toxicology, Faculty of Pharmacy, University of Seville, Seville, Spain

    S. Pichardo, A. Jos, A. M. Camean & G. Repetto

  2. National Institute of Toxicology and Forensic Sciences, Avda Dr Fedriani s/n, Seville, 41009, Spain

    J. Zurita, M. Salguero & G. Repetto

Authors
  1. S. Pichardo
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  2. A. Jos
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  3. J. Zurita
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  4. M. Salguero
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  5. A. M. Camean
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  6. G. Repetto
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Correspondence to G. Repetto.

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Pichardo, S., Jos, A., Zurita, J. et al. Toxic Effects Produced by Microcystins from a Natural Cyanobacterial Bloom and a Microcystis aeruginosa Isolated Strain on the Fish Cell Lines RTG-2 and PLHC-1. Arch Environ Contam Toxicol 51, 86–96 (2006). https://doi.org/10.1007/s00244-005-0122-8

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  • DOI: https://doi.org/10.1007/s00244-005-0122-8

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