, Volume 68, Issue 1, pp 143–155 | Cite as

Pyocyanin induced in vitro oxidative damage and its toxicity level in human, fish and insect cell lines for its selective biological applications

  • P. Priyaja
  • P. Jayesh
  • Rosamma Philip
  • I. S. Bright Singh
Original Research


Pyocyanin is a redox active phenazine pigment produced by Pseudomonas aeruginosa, with broad antibiotic activity having pharmacological, aquaculture, agriculture and industrial applications. In the present work cytotoxicity induced by pyocyanin is demonstrated in a human embryonic lung epithelial cell line (L-132), a rainbow trout gonad cell line (RTG-2) and a Spodoptera frugiperda pupal ovarian cell line (Sf9). For toxicity evaluation, cellular morphology, mitochondrial function (XTT), membrane leakage of lactate dehydrogenase, neutral red uptake, affinity of electrostatic binding of protein with sulforhodamine B dyes, glucose metabolism, and reactive oxygen species, were assessed. Results showed that higher pyocyanin concentration is required for eliciting cytotoxicity in L-132, RTG-2 and Sf9. The microscopic studies demonstrated that the cell lines exposed to pyocyanin at higher concentrations alone showed morphological changes such as clumping and necrosis. Among the three cell lines L-132 showed the highest response to pyocyanin than the others. In short, pyocyanin application at concentrations ranging from 5 to 10 mg l−1 were not having any pathological effect in eukaryotic systems and can be used as drug of choice in aquaculture against vibrios in lieu of conventional antibiotics and as biocontrol agent against fungal and bacterial pathogens in agriculture. This is besides its industrial and pharmacological applications.


Pyocyanin Toxicity IC50 Vibriosis Antibacterial Antifungal 



This work was funded by Department of Biotechnology, Government of India under the project, Program Support in Marine Biotechnology (Order No.: BT/PR4012/AAQ/03/204/2003). The first author thanks DBT for Fellowship.


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefGoogle Scholar
  2. Anjaiah V, Cornelis P, Koedam N (2003) Effect of genotype and root colonization in biological control of fusarium wilts in pigeon pea and chickpea by Pseudomonas aeruginosa PNA1. Can J Microbiol 49:85–91CrossRefGoogle Scholar
  3. Arunkumar G, Rao SG, Shivananda PG (1997) Anti-Staphylococcal activity of Pseudomonas aeruginosa. Curr Sci 72:580–582Google Scholar
  4. Bano N, Musarrat J (2003) Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent. Curr Microbiol 46:324–328CrossRefGoogle Scholar
  5. Britigan BE, Rasmussen GT, Cox CD (1997) Augmentation of oxidant injury to human pulmonary epithelial cells by the Pseudomonas aeruginosa siderophore pyochelin. Infect Immun 65:1071–1076Google Scholar
  6. Chaerun SK, Tazaki K, Asada R, Kogure K (2004) Bioremediation of coastal areas 5 years after the Nakhodka oil spill in the Sea of Japan: isolation and characterization of hydrocarbon-degrading bacteria. Environ Int 30:911–922CrossRefGoogle Scholar
  7. Chythanya R, Karunasagar I, Karunasagar I (2002) Inhibition of shrimp pathogenic vibrios by a marine Pseudomonas I-2 strain. Aquaculture 208:1–10CrossRefGoogle Scholar
  8. Costa AL, Cusmano V (1975) Anti-mycotic activity of pyocyanin in vitro and in vivo on a pathogenic strain of Candida albicans. Gen Bacteriol Virol Immunol 66:297–308Google Scholar
  9. De Meyer G, Audenaert K, Hofte M (1999) Pseudomonas aeruginosa 7NSK2-induced systemic resistance in tobacco depends on in planta salicylic acid accumulation but is not associated with PR1a expression. Eur J Plant Pathol 105:513–517CrossRefGoogle Scholar
  10. Decker T, Lohman-Matthes ML (1988) A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis (TNF) activity. J Immunol Methods 15:61–69CrossRefGoogle Scholar
  11. Dive D (1973) Action of exocellular pigments secreted by Pseudomonas aeruginosa on the growth and division of Colpidium campylum. Protistologica 9:315–318Google Scholar
  12. Fernández RO, Pizarro RA (1997) High-performance liquid chromatographic analysis of Pseudomonas aeruginosa phenazines. J Chromatogr 771:99–104CrossRefGoogle Scholar
  13. Gloyne LS, Grant GD, Perkins AV, Powell KL, McDermott CM, Johnson PV, Anderson GJ, Kiefel M, Dukie SA (2011) Pyocyanin-induced toxicity in A549 respiratory cells is causally linked to oxidative stress. Toxicol In Vitro 25:1353–1358CrossRefGoogle Scholar
  14. Hasanuzzaman M, Umadhay-Briones KM, Zsiros SM, Morita N, Nodasaka Y, Yumoto I, Okuyama H (2004) Isolation, identification and characterization of a novel, oil-degrading bacterium, Pseudomonas aeruginosa T1. Curr Microbiol 49:108–114CrossRefGoogle Scholar
  15. Hassan HM, Fridovich I (1980) Mechanism of the antibiotic action of pyocyanine. J Bacteriol 141:156–163Google Scholar
  16. Imlay JA (2008) Cellular defenses against superoxide and hydrogen peroxide. Annu Rev Biochem 77:755–776CrossRefGoogle Scholar
  17. Jose D, Jayesh P, Gopinath P, Mohandas A, Singh ISB (2014) Potential application of β1,3 glucanase from an environmental isolate of Pseudomonas aeruginosa MCCB 123 in fungal extraction. Indian J Exp Biol 52:89–96Google Scholar
  18. Kerr JR, Taylor GW, Rutman A, Hoiby N, Cole PJ, Wilson R (1999) Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth. J Clin Pathol 52:385–387CrossRefGoogle Scholar
  19. Korzeniewski C, Callewaert DM (1983) An enzyme-release assay for natural cytotoxicity. J Immunol Methods 64:313–320CrossRefGoogle Scholar
  20. Kumar RS, Ayyadurai N, Pandiaraja P, Reddy AV, Venkateswarlu Y, Prakash O, Sakthivel N (2005) Characterization of antifungal metabolite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad-spectrum antifungal activity and biofertilizing traits. J Appl Microbiol 98:145–154CrossRefGoogle Scholar
  21. Lee YK, Kim HW, Liu CL, Lee HK (2003) A simple method for DNA extraction from marine bacteria that produce extracellular materials. J Microbiol Methods 52:245–250CrossRefGoogle Scholar
  22. Morrison MM, Seo ET, Howie JK, Sawyer DT (1978) Flavin model systems. 1. The electrochemistry of 1-hydroxyphenazine and pyocyanine in aprotic solvents. J Am Chem Soc 100:207–211CrossRefGoogle Scholar
  23. Muller M (2006) Premature cellular senescence induced by pyocyanin, a redox-active Pseudomonas aeruginosa toxin. Free Radic Biol Med 41:1670–1677CrossRefGoogle Scholar
  24. Muller M, Li Z, Maitz PK (2009) Pseudomonas pyocyanin inhibits wound repair by inducing premature cellular senescence: role for p38 mitogen-activated protein kinase. Burns 35:500–508CrossRefGoogle Scholar
  25. Ohfuji K, Sato N, Hamada-Sato N, Kobayashi T (2004) Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonas aeruginosa. Biosens Bioelectron 19:1237–1244CrossRefGoogle Scholar
  26. Pai SS, Anas AA, Jayaprakash NS, Priyaja P, Sreelakshmi B, Philip R, Mohandas A, Bright Singh IS (2010) Penaeus monodon larvae can be protected from Vibrio harveyi infection by preemptive treatment of rearing system with antagonistic or non-antagonistic bacterial probiotics. Aquac Res 41:847–860CrossRefGoogle Scholar
  27. Pham TH, Boon N, Maeyer K, Hofte M, Rabaey K, Verstraete W (2008) Use of Pseudomonas species producing phenazine-based metabolites in the anodes of microbial fuel cells to improve electricity generation. Appl Microbiol Biotechnol 80:985–993CrossRefGoogle Scholar
  28. Preetha R, Jose S, Prathapan S, Vijayan KK, Jayaprakash NS, Philip R, Bright Singh IS (2010) An inhibitory compound produced by Pseudomonas with effectiveness on Vibrio harveyi. Aquac Res 41:1452–1461Google Scholar
  29. Priyaja P (2012) Pyocyanin (5-methyl-1-hydroxyphenazine) produced by Pseudomonas aeruginosa as antagonist to vibrios in aquaculture: over expression, downstream process and toxicity. Ph.D. Thesis, Cochin University of Science and Technology, IndiaGoogle Scholar
  30. Priyaja P, Jayesh P, Correya NS, Sreelakshmi B, Sudheer NS, Philip R, Bright Singh IS (2014) Antagonistic effect of Pseudomonas aeruginosa isolates from various ecological niches on Vibrio species pathogenic to crustaceans. J Coast Life Med 2:76–84Google Scholar
  31. Rabaey K, Verstraete W (2005) Microbial fuel cells: novel biotechnology for energy generation. Trends Biotechnol 23:291–298CrossRefGoogle Scholar
  32. Rangarajan S, Saleena LM, Vasudevan P, Nair S (2003) Biological suppression of rice diseases by Pseudomonas spp. under saline salt condition. Plant Soil 251:73–82CrossRefGoogle Scholar
  33. Rattanachuay P, Duangporn K, Manee T, Teruhiko N, Hiroshi K (2011) Anti-vibrio compounds produced by Pseudomonas sp. W3: characterisation and assessment of their safety to shrimps. World J Microbiol Biotechnol 27:869–880CrossRefGoogle Scholar
  34. Reddy GSN, Aggarwal RK, Matsumoto GI, Shivaji S (2000) Arthrobacter flavus sp.nov., a psychrophilic bacterium isolated from a pond in McMurdo Dry Valley, Antartica. Int J Syst Evol Microbiol 50:1553–1561CrossRefGoogle Scholar
  35. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  36. Tang X, Zhu Y, Meng Q (2007) Enhanced crude oil biodegradability of Pseudomonas aeruginosa ZJU after preservation in crude-oil containing media. World J Microbiol Biotechnol 23:7–14CrossRefGoogle Scholar
  37. Vichai V, Kirtikara K (2006) Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 1:1112–1116CrossRefGoogle Scholar
  38. Vijayan KK, Singh ISB, Jayaprakash NS, Alavandi SV, Pai SS, Preetha R, Rajan JJS, Santiago TC (2006) A brackish water isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaied and non-penaied rearing systems. Aquaculture 251:192–200CrossRefGoogle Scholar
  39. Vukomanovic DV, Zoutman DE, Stone JA, Marks GS, Brien JF, Nakatsu K (1997) Electrospray mass-spectrometric, spectrophotometric and electrochemical methods do not provide evidence for the binding of nitric oxide by pyocyanine at pH 7. Biochem J 322:25–29CrossRefGoogle Scholar
  40. Warren JB, Loi R, Rendell NB, Taylor GW (1990) Nitric oxide is inactivated by the bacterial pigment pyocyanin. Biochem J 266:921–923Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • P. Priyaja
    • 1
  • P. Jayesh
    • 1
  • Rosamma Philip
    • 2
  • I. S. Bright Singh
    • 1
  1. 1.National Centre for Aquatic Animal HealthCochin University of Science and TechnologyKochiIndia
  2. 2.Department of Marine Biology, Microbiology and Biochemistry, School of Marine SciencesCochin University of Science and TechnologyKochiIndia

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