SpringerLink
Log in

Establishment and characterization of permanent cell line from gill tissue of Labeo rohita (Hamilton) and its application in gene expression and toxicology

  • Original Research
  • Published:
Cell Biology and Toxicology Aims and scope Submit manuscript

Abstract

Rohu gill cell line (LRG) was established from gill tissue of Indian major carp (Labeo rohita), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 10 % foetal bovine serum (FBS). This cell line has been sub-cultured more than 85 passages over a period of 2 years. The LRG cell line consists of both epithelial and fibroblastic-like cells. The cells were able to grow at a wide range of temperatures from 22 to 32 °C, the optimum temperature being 28 °C. The growth rate of gill cells increased as the FBS proportion increased from 2 to 20 % at 28 °C. The plating efficiency was also high (34.37 %). The viability of the LRG cell line was 70–80 % after 6 months of storage in liquid nitrogen. The karyotype analysis revealed a diploid count of 50 chromosomes. The gill cells of rohu were successfully transfected with pEGFP-N1. Amplification of mitochondrial Cox1 gene using primers specific to L. rohita confirmed the origin of this cell line from L. rohita. The cytotoxicity of malathion was assessed in LRG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Acute toxicity assay on fish was conducted by exposing L. rohita for 96 h to malathion under static conditions. Statistical analysis revealed good correlation with r 2 = 0.946–0.990 for all combinations between endpoints employed. Linear correlations between each in vitro effective concentration 50 and the in vivo lethal concentration 50 data were highly significant.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Germany)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

AB:

Alamar Blue

CB:

Coomassie Blue

DMSO:

Dimethyl sulphoxide

EC50 :

Effective concentration 50

FBS:

Foetal bovine serum

HBSS:

Hank's balanced salt solution

L-15:

Leibovitz's L-15

LC50 :

Lethal concentration 50

LRG:

Labeo rohita Gill cell line

MTT:

3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NR:

Neutral Red

OPs:

Organophosphorus

PCR:

Polymerase chain reaction

References

  • Anonymous. United States Environmental Protection Agency technical report. 2005.

  • Avella M, Berhaut J, Payan P. Primary culture of gill epithelial cells from the sea bass Dicentrarchus labrux. In Vitro Cell Dev Biol. 1994;30:41–9.

    Article  Google Scholar 

  • Babich H, Rosenberg DW, Borenfreund E. In vitro cytotoxicity studies with the fish hepatoma cell line, lhc-1 (Poeciliopsis lucida). Ecotoxicol Environ Saf. 1991;21:327–36.

    Article  PubMed  CAS  Google Scholar 

  • Bahich H, Borenfreund E. Cytotoxicity and genotoxicity assays with cultured fish cells: a review. Toxicol In Vitro. 1991;5:91–100.

    Article  Google Scholar 

  • Bejar J, Borrego JJ, Alvarez MC. A continuous cell line from the cultured marine fish gilt-head seabream (Sparus aurata L.). Aquaculture. 1997;150:143–53.

    Article  Google Scholar 

  • Borenfreund E, Babich H, Martin-Alguacil N. Comparison of two in vitro cytotoxicity assays: the neutral red and tetrazolium (MTT) tests. Toxicol in Vitro. 1988;2:1–6.

    Article  PubMed  CAS  Google Scholar 

  • Borenfreund E, Puerner JA. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett. 1985;24:119–24.

    Article  PubMed  CAS  Google Scholar 

  • Castano A, Bols N, Braunbeck T, Dierickx P, Halder M, Isomaa B, Kawahara K, Lee LEJ, Mothersill C, Part P, Repetto G, Sintes JR, Rufli H, Smith R, Wood C, Segner H. The use of fish cells in ecotoxicology. Report and recommendations of the ECVAM workshop. ATLA. 2003;47(31):317–51.

    Google Scholar 

  • Castano A, Cantarino MJ, Castillo F, Tarasona JV. Correlation between the RTG-2 cytotoxicity test EC50 and in vivo LC50 rainbow trout bioassay. Chemosphere. 1996;32:2141–57.

    Article  CAS  Google Scholar 

  • Chen SL, Sha ZX, Ye HQ. Establishment of a pluripotent embryonic cell line from sea perch blastula embryo. Aquaculture. 2003;218:141–51.

    Article  CAS  Google Scholar 

  • Chen TR. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res. 1977;104:255–62.

    Article  PubMed  CAS  Google Scholar 

  • Chen XY, Shao JZ, **ang L, Liu X. Involvement of apoptosis in malathion-induced cytotoxicity in a grass carp (Ctenopharyngodon idellus) cell line. Comp Biochem Physiol C. 2006;142:36–45.

    Google Scholar 

  • Chi SC, Hu WW, Lo BJ. Establishment and characterization of a continuous cell line (GF-1) derived from grouper, Epinephelus coioides (Hamilton): a cell line susceptible to grouper nervous necrosis virus (GNNV). J Fish Dis. 1999;22:173–82.

    Article  Google Scholar 

  • Dayeh VR, Bols NC, Schirmer K, Lee LEJ. The use of fish-derived cell lines for investigation of environmental contaminants. Current protocols in toxicology. New York: Wiley; 2003. p. 1–17.

  • Fernandez-Puentes C, Novoa B, Figueras A. A new fish cell line derived from turbot (Scophthalmus maximus L.) TV-1. B Eur Assoc Fish Pat. 1993a;13:94–6.

    Google Scholar 

  • Fernandez-Puentes C, Novoa B, Figueras A. Initiation of a cell line from turbot (Scophthalmus maximus L.). In Vitro Cell Dev Biol-An. 1993b;29:899–900.

    Article  Google Scholar 

  • Flano E, Lopez-Fierro P, Alvarez F, Razquin B, Villena A. Splenic cultures from rainbow trout, Oncorhynchus mykiss: establishment and characterization. Fish and Shellfish Immun. 1998;8:589–606.

    Article  Google Scholar 

  • Freiberg EF, Masover GK. Mycoplasma detection in cell culture by concomitant use of bisbenzamide and fluoresceinated antibody. In Vitro Cell Dev Biol. 1990;26:585–8.

    Article  PubMed  CAS  Google Scholar 

  • Freshney RI. Culture of animal cells: a manual of basic technique. New York: Wiley-Liss; 1994.

    Google Scholar 

  • Gulden M, Seibert H. Impact of bioavailability on the correlation between in vitro cytotoxic and in vivo acute fish toxic concentrations of chemicals. Aquat Toxicol. 2005;72:327–37.

    Article  PubMed  Google Scholar 

  • Hessling JJ, Miller S, Levy L. A direct comparison of procedures for the detection of mycoplasma in tissue culture. J Immunol Methods. 1980;38:315–24.

    Article  PubMed  CAS  Google Scholar 

  • Hightower LE, Renfro JL. Recent applications of fish cell culture to biomedical research. J Exp Zool. 1988;248(3):290–302.

    Article  PubMed  CAS  Google Scholar 

  • Hunkapiller MW, Lujan E. Purification of microgram quantities of proteins by polyacrylamide gel electrophoresis. In: Shively J, editor. Methods of protein microcharacterization. Clifton: Humana; 1986. p. 89–101.

    Chapter  Google Scholar 

  • Ishaq Ahmed VP, Chandra V, Sudhakaran R, Rajesh Kumar S, Sarathi M, Sarath Babu V, Ramesh B, Sahul Hameed AS. Development and characterization of cell lines derived from rohu, Labeo rohita (Hamilton), and catla, Catla catla (Hamilton). J Fish Dis. 2009;32:211–8.

    Article  Google Scholar 

  • Isomaa B, Lilius H, Rabergh C. Aquatic toxicology in vitro: a brief review. ATLA. 1994;22:243–53.

    Google Scholar 

  • Kang MS, Oh MJ, Kim YJ, Kawai K, Jung SJ. Establishment and characterization of two cell lines derived from flounder, Paralichthys olivaceus (Temminck & Schlegel). J Fish Dis. 2003;26:657–65.

    Article  PubMed  CAS  Google Scholar 

  • Katiha PK, Jena JK, Pillai NGK, Chakraborty C, Dey MM. Inland aquaculture in India: past trend, present status and future prospects. Aquacul Econ Manag. 2005;9:237–64.

    Article  Google Scholar 

  • Knauer K, Lampert C, Valero-Gonzalez J. Comparison of in vitro and in vivo acute fish toxicity in relation to toxicant made of action. Chemosphere. 2007;68:1435–41.

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–5.

    Article  PubMed  CAS  Google Scholar 

  • Lakra WS, Bhonde RR. Development of primary cell culture from the caudal fin of an Indian major carp, Labeo rohita (Ham.). Asian Fish Sci. 1996;9:149–52.

    Google Scholar 

  • Lakra WS, Raja-Swaminathan T, Joy KP. Development, characterization, conservation and storage of fish cell lines: a review. Fish Physiol Biochem. 2011;37:1–20.

    Article  PubMed  CAS  Google Scholar 

  • Lo CF, Leu JH, Ho CH, Chen CH, Peng SE, Chen YT, Yeh PY, Huang CJ, Wang CH, Kou GH. Detection of baculovirus associated with white spot syndrome (WSSV) in penaeid shrimps using polymerase chain reaction. Dis Aquat Org. 1996;25:133–41.

    Article  CAS  Google Scholar 

  • Manna M, Chakraborty P. Analysis of lactate and malate dehydrogenase enzyme profiles of selected major carps of wetland of Calcutta. J Environ Biol. 2012;33:763–7.

    CAS  Google Scholar 

  • Matei VE. Changes in the cellular ultrastructure of the gill epithelium in Tilapia under the action of cadmium on the fish. Tsitol. 1993;35:34–41.

    CAS  Google Scholar 

  • Mohan CV, Shankar KM. Epidemiological analysis of epizootic ulcerative syndrome of fresh and brackish water fishes of Karnataka. India Curr Sci. 1994;66:656–8.

    Google Scholar 

  • Na N, Guo HR, Zhang SC, Li ZL, Yin LC. In vitro and in vivo acute toxicity of fenpyroximate to flounder Paralichthys olivaceus and its gill cell line FG. Aquat Toxicol. 2009;92(2):76–85.

    Article  PubMed  CAS  Google Scholar 

  • Nicholson BL, Danner DJ, Wu JL. Three new continuous cell lines from marine fishes of Asia. In Vitro Cell Dev Biol. 1987;23:199–204.

    Article  Google Scholar 

  • OECD 203. OECD guideline for testing chemicals. Test no. 203: acute fish test. 1992.

  • Parameswaran V, Ishaq Ahmed VP, Ravi S, Bhonde RR, Sahul Hameed AS. Development and characterization of two new cell lines from milkfish (Chanos chanos) and grouper (Epinephelus coioides) for virus isolation. Mar Biotechnol. 2007;9:281–91.

    Article  PubMed  CAS  Google Scholar 

  • Prakasam A, Sethupathy S, Lalitha S. Plasma and RBCs antioxidant status in occupational male pesticide sprayers. Clin Chim Acta. 2001;310:107–12.

    Article  PubMed  CAS  Google Scholar 

  • Rahim SM, Delaunoy JP, Laurent P. Identification and immunocytochemical localization of two different carbonic anhydrase isoenzymes in teleostean fish erythrocytes and gill epithelia. Histochemie. 1988;89:451–9.

    Article  CAS  Google Scholar 

  • Rao KS, Joseph MA, Shankar KM, Mohan CV. Primary cell culture from explants of heart tissue of Indian major carps. Curr Sci. 1997;73:374–5.

    Google Scholar 

  • Reddy ATV, Ayyanna K, Yellamma K. Cypermethrin induced modulations in lipid metabolism of fresh water teleost. Tilapia mossambica. Biochem Int. 1991;23:963–7.

    PubMed  CAS  Google Scholar 

  • Reddy ATV, Yellamma K. Cypermethrin induced changes in nitrogen metabolism of fish. Tilapia mossambica. Biochem Int. 1991;23:649–54.

    Article  PubMed  CAS  Google Scholar 

  • Rocha A, Ruiz S, Coll JM. Improvement of transfection efficiency of epithelioma papulosum cyprinid carp cells by modification of cell cycle and use of an optimal promoter. Mar Biotechnol. 2004;6:401–10.

    Article  PubMed  CAS  Google Scholar 

  • Sahul Hameed AS, Parameswaran V, Ravi S, Singh B, Ramesh B. Establishment and characterization of India’s first marine fish cell line (SISK) from kidney of sea bass, (Lates calcarifer). Aquaculture. 2006;257:92–103.

    Article  Google Scholar 

  • Sarath Babu V, Chandra V, Nambi KSN, Majeed SA, Taju G, Patole MS, Hameed ASS. Development and characterization of novel cell lines from Etroplus suratensis and their applications in virology, toxicology and gene expression. J Fish Biol. 2012;80:312–34.

    Article  PubMed  CAS  Google Scholar 

  • Sathe PS, Basu A, Mourya DT, Marathe BA, Gogate SS, Banerjee K. A cell line from the gill tissues of Indian cyprinoid Labeo rohita. In Vitro Cell Dev Biol. 1997;33:425–7.

    Article  CAS  Google Scholar 

  • Segner H. Fish cell lines as a tool in aquatic toxicology. In: Braunbeck T, Hinton DE, Streit DE, editors. Fish ecotoxicology. Basel: Birkhauser; 1998.

    Google Scholar 

  • Sender S, Böttcher K, Cetin Y, Gros G. Carbonic anhydrase in the gills of seawater-and freshwater-acclimated flounders Platichthys flesus: purification, characterization, and immunohistochemical localization. J Histochem Cytochem. 1999;47:43–50.

    Article  PubMed  CAS  Google Scholar 

  • Shopsis C, Eng B. Rapid cytotoxicity testing using a semi-automated protein determination on cultured cells. Toxicol Lett. 1985;26(1):1–8.

    Article  PubMed  CAS  Google Scholar 

  • Singh S, Sahai S. Changes in gill structure induced by BHC, lindane and endosulfan in the fresh-water teleost Puntius ticto (Ham.). Folia Morphol (Praha). 1990;38:423–32.

    CAS  Google Scholar 

  • Storm JE, Karl KR, Doull J. Occupational exposure limits for 30 organophosphate pesticides based on inhibition of red blood cell acetylcholinesterase. Toxicol. 2000;150:1–29.

    Article  CAS  Google Scholar 

  • Suresh A, Sivaramakrisha B, Victoriamma PC, Radhakrisshnaiah K. Shifts in protein metabolism in some organs of fresh water fish Cyprinus carpio under mercury stress. Biochem Int. 1991;24:379–89.

    PubMed  CAS  Google Scholar 

  • Taju G, Abdul Majeed S, Nambi KSN, Sarath Babu V, Vimal S, Kamatchiammal S, Sahul Hameed AS. Comparison of in vitro and in vivo acute toxicity assays in Etroplus suratensis (Bloch, 1790) and its three cell lines in relation to tannery effluent. Chemosphere. 2012;87:55–61.

    Article  PubMed  CAS  Google Scholar 

  • Talbot PV, Knobler RL, Buchmeier M. Western and dot immunoblotting analysis of viral antigens and antibodies: application to murine hepatitis virus. J Immunol Methods. 1984;73:177–88.

    Article  PubMed  CAS  Google Scholar 

  • Thenmozhi C, Vignesh V, Thirumurugan R, Arun S. Impacts of malathion on mortality and biochemical changes of freshwater fish Labeo rohita. Iran. J Environ Health Sci Eng. 2011;8(4):325–32.

    CAS  Google Scholar 

  • Tong SL, Lee H, Miao HZ. The establishment and partial characterization of a continuous fish cell line FG-9307 from the gill of flounder Paralichthys olivaceus. Aquaculture. 1997;156:327–33.

    Article  Google Scholar 

  • Tong SL, Miao HZ, Li H. Three new continuous fish cell lines of SPH, SPS and RSBF derived from sea perch (Lateolabrax japonicus) and red sea bream (Pagrosomus major). Aquaculture. 1998;169:143–51.

    Article  Google Scholar 

  • Wise Sr JP, Winn RN, Renfro JL. Generating new marine cell lines and transgenic species. J Exp Zoo. 2002;15:292–5.

    Google Scholar 

  • Wolf K, Quimby MC. Established eurythermic line of fish cells in vitro. Science. 1962;135:1065–6.

    Article  PubMed  CAS  Google Scholar 

  • Zhang SM, Reddy PVGK. On the comparative karyomorphology of three Indian major carps, Catla catla (Ham.), Labeo rohita (Ham.) and Cirrhinus mrigala (Ham.). Aquaculture. 1991;97:7–12.

    Article  Google Scholar 

Download references

Acknowledgments

The first author is a recipient of INSPIRE Fellowship from the Department of Science and Technology, Government of India, New Delhi, India. The authors thank the management of C. Abdul Hakeem College, Melvisharam, India for providing the facilities to carry out this work. This work was funded by the Department of Biotechnology, Government of India, New Delhi, India.

Author information

Authors and Affiliations

  1. OIE Reference Laboratory for WTD, PG & Research Department of Zoology, C. Abdul Hakeem College, 632 509, Melvisharam, Vellore District, Tamil Nadu, India

    S. Abdul Majeed, K. S. N. Nambi, G. Taju, N. Sundar Raj, N. Madan & A. S. Sahul Hameed

Authors
  1. S. Abdul Majeed
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. K. S. N. Nambi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. G. Taju
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. N. Sundar Raj
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. N. Madan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. S. Sahul Hameed
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A. S. Sahul Hameed.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Abdul Majeed, S., Nambi, K.S.N., Taju, G. et al. Establishment and characterization of permanent cell line from gill tissue of Labeo rohita (Hamilton) and its application in gene expression and toxicology. Cell Biol Toxicol 29, 59–73 (2013). https://doi.org/10.1007/s10565-012-9237-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10565-012-9237-7

Keywords

Search

Navigation