Cell Biology and Toxicology

, Volume 29, Issue 1, pp 59–73 | Cite as

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

  • S. Abdul Majeed
  • K. S. N. Nambi
  • G. Taju
  • N. Sundar Raj
  • N. Madan
  • A. S. Sahul Hameed
Original Research

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 r2 = 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.

Keywords

Labeo rohita Fish cell line In vitro Cytotoxicity Malathion 

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

Introduction

The Indian carps, namely rohu, Labeo rohita (Hamilton); mrigal, Cirrhinus cirriosus (Bloch); and catla, Catla catla (Hamilton) are the most edible fishes in India and the best suitable carps for their cultivable qualities (Katiha et al. 2005; Manna and Chakraborty 2012). The rohu is the most important freshwater fish cultured in India, Bangladesh and other adjacent countries in the region. The L. rohita is a natural inhabitant of the riverine system of northern and central India and the rivers of Pakistan, Bangladesh and Myanmar. The adoption of intensive farming practices, unregulated use of inputs and inbreeding in hatcheries has led to increased disease occurrence in tropical carps. There have been several incidences of mass mortality of carps in culture systems suspected to be caused by microbial diseases, particularly of viral aetiology (Mohan and Shankar 1994).

Tissue culture and the development of cell lines from fish are of priority interest for pathogen detection, toxicology, carcinogenesis, cellular physiology and genetic regulation and expression. The first fish cell line was developed in 1962 from gonad of rainbow trout (Oncorhynchus mykiss) and designated as RTG-2, and even now this cell line has tremendous applications in virological and toxicological studies (Wolf and Quimby 1962). Since then the work on developing fish cell lines is progressing and the number of fish cell lines increased tremendously to 283 (Lakra et al. 2011).

Species-specific cell lines are important tools for studying toxicology, carcinogenesis and gene regulation and expression in fish (Hightower and Renfro 1988; Bahich and Borenfreund 1991; Wise et al. 2002) and are also essential for isolating and identifying fish viruses. In India, primary cell cultures from gill tissue of C. cirriosus and L. rohita have been developed (Sathe et al. 1997), and attempts have been made to develop primary cultures from a variety of other tissues such as heart and caudal fin of Indian major carp (Lakra and Bhonde 1996; Rao et al. 1997; Ishaq Ahmed et al. 2009).

Fish cell cultures are finding application in toxicology for evaluating the effects of various chemicals, pesticides and industrial wastes and in the study of carcinogenesis as in vitro models for investigating cell transformation by fish viruses, chemical agents and the interaction of viruses and chemical carcinogens. Cell lines of Etroplus suratensis were evaluated for their potential use as screening tools for the ecotoxicological assessment of tannery effluent to replace the use of whole fish (Taju et al. 2012).

The in vitro tests would also satisfy a societal desire to avoid the use of whole animals in toxicological tests. Efforts have been made over the past three decades to find out whether in vitro cytotoxicity assays can be used for aquatic hazard assessment as a substitute to in vivo tests (Isomaa et al. 1994; Segner 1998; Castano et al. 2003; Gulden and Seibert 2005; Knauer et al. 2007; Na et al. 2009).

For centuries, pesticides were utilized in agriculture to enhance food production by controlling disease vectors and eradicating harmful pests (Prakasam et al. 2001). Organophosphorus compounds are more commonly used as insecticides (Storm et al. 2000). Malathion is a non-systemic and wide spectrum organophosphate insecticide. It was one of the earliest organophosphate insecticides developed (introduced in 1950s). It was used for agricultural and non-agricultural purposes. Once malathion is introduced into the environment, it may cause serious trouble to aquatic organisms and is notorious for causing severe metabolic disturbances in non-target species, like fish and freshwater mussels (Anonymous 2005). The present study described the development and characterization of gill cell line of rohu and its application in gene expression and to test the toxicity of malathion.

Materials and methods

Primary cell culture and routine maintenance

Live healthy fingerlings of L. rohita of 5–10 g were collected from local fish farms and transported live to the laboratory and were maintained in the laboratory in sterile aerated fresh water containing 1,000 IU/ml penicillin and 1,000 μg/ml streptomycin for 24 h at room temperature (28 ± 2 °C). The fish were anaesthetized in ice-cold water, immersed in 5 % chlorex for 5 min, wiped with 70 % ethanol and dissected. Gill tissues were taken aseptically, minced into small pieces (approximately 1 mm3 in size) and washed three times in Leibovitz's L-15 (GIBCO) medium containing antibiotics (500 IU/ml penicillin, 500 μg/ml streptomycin and 2.5 μg/ml Fungizone). The tissue fragments were inoculated into 25 cm2 cell culture flasks containing 5 ml of complete growth medium (L-15 supplemented with 15 % foetal bovine serum), antibiotics (penicillin 100 IU/ml, streptomycin 100 μg/ml) and Fungizone (2.5 μg/ml). The flasks were incubated at 28 °C in a normal atmosphere incubator, and half of the medium was changed every 3 or 4 days. Upon reaching 95 % confluence, the cells were sub-cultured at a ratio of 1:2–1:3 following the standard trypsinization method using trypsin–EDTA solution (trypsin 0.25 %, EDTA 0.02 %) in phosphate-buffered saline (PBS).

Growth studies

The gill cells of L. rohita were exposed to different temperatures and foetal bovine serum (FBS) concentrations to determine the optimum temperature and FBS concentration for their optimum growth. The cells (LRG cell line at 37th passage level) were seeded at a concentration of 106 cells ml−1 in 25 cm2 tissue culture flasks and incubated at 28 °C for 2 h for attachment. Then, the batches of culture flasks were incubated at temperatures of 22, 28, 32, 37 and 40 °C for growth studies. For five consecutive days, cell densities were measured using a haemocytometer and expressed as cells per square millimetre following the protocol of Tong et al. (1997). The experiments were conducted in triplicate. Cell growth in different concentrations of FBS (2, 5, 10, 15 and 20 %) was carried out at 28 °C using the same procedure as described above.

Cell-plating efficiency

Plating efficiency was determined at its 32nd passage by seeding different concentrations of cells (100, 500 or 1,000 cells 25 cm−2 in a tissue culture flask) in duplicates. Then, batches of culture flasks were incubated at 28 °C in L-15 medium containing 10 % FBS. The medium was discarded after 8 days of incubation. The cells were then fixed with 10 % formalin for 15 min, stained with 5 ml of crystal violet for 10 min, rinsed with tap water and air-dried. The stained colonies were then counted under an inverted microscope, and plating efficiency was calculated as described by Freshney (1994). The population-doubling time of the LRG cell line was calculated using the methodology described by (Freshney 1994) by cell counts in a hemocytometer.

Cryopreservation

The established cell line was cryopreserved using standard protocol and viability of cells after cryopreservation in liquid nitrogen was evaluated by the method described by Chen et al. (2003a). For cryopreservation, 48 or 72 h old cultures (LRG) at different passage levels were used. The cells were cryopreserved in L-15 medium with 10 % FBS and 10 % dimethyl sulphoxide at a density of 106 cells ml−1 and stored in liquid nitrogen (−196 °C).

Chromosomal analysis

Chromosomal analysis was carried out on LRG cell line at different passage levels, using the protocol described by Freshney (1994) and Sahul Hameed et al. (2006). Chromosome counts were performed in >100 metaphase plates of LRG cell line.

Transfection

The gene expression of the LRG cells was assessed following the protocol described by Rocha et al. (2004). Cells at the 34th passage were seeded at a density of 5 × 105 cells per well in a six-well plate. Sub-confluent monolayers were transfected with pEGFP-N1 plasmid using lipofectamine 2000 (Invitrogen Corporation, Grand Island, NY, USA). After 24 h, the green fluorescence signal was observed under a fluorescence microscope (Carl Zeiss, Germany).

Mitochondrial Cox1 gene analysis by polymerase chain reaction

The mitochondrial Cox1 gene of L. rohita was analysed by PCR using the DNA obtained from tissues and cell line developed from the L. rohita for authentication of origin of cell line from the same species. Template DNA was extracted from tissues of fish and cell line following the method described by Lo et al. (1996). Briefly, the samples were homogenized separately in NTE buffer (0.2 m NaCl, 0.02 m Tris–HCl, 0.02 m EDTA, pH 7.4) and centrifuged at 3,000×g at 4 °C, after which the supernatant fluids were placed in fresh centrifuge tubes together with an appropriate amount of digestion buffer (100 mm NaCl, 10 mm Tris–HCl, pH 8.0, 50 mm EDTA, pH 8.0, 0.5 % sodium dodecyl sulphate and 0.1 mg ml−1 proteinase K). After incubation at 65 °C for 2 h, the digests were deproteinized by successive phenol/chloroform/isoamyl alcohol extraction, and DNA was recovered by ethanol precipitation, drying and resuspension in TE buffer. The forward (5′-GCT CTT GGG TTC ATC TTC-3′) and reverse (5′-GCA TAG GCA TCT GGG TAG-3′) (GenBank accession no. NC_017608) primers were used for amplifying mitochondrial Cox1 gene of L. rohita. A fragment of 324 bp was amplified. PCR was carried out in an Eppendorf thermal cycler (Eppendorf AG, Hamburg, Germany). Each PCR reaction was in a 25-μl volume containing both forward and reverse primers (10 μM, 0.5 μl each), MgCl2 (25 mM, 1.5 μl), dNTPs (2 mM, 2.0 μl), PCR buffer (10 × 2.5 μl), Taq-DNA (1 U, 0.5 μl) (Bangalore Genei, India), template DNA (0.3–0.4 μl) and nucleic acid-free water. PCR cycling conditions included an initial denaturation at 95 °C for 5 min, followed by 30 cycles of 40 s at 95 °C, 50 s at 55 °C and 40 s at 72 °C with a final extension of 10 min at 72 °C. Amplified products were analysed in 1 % agarose gels stained with ethidium bromide and visualized with ultraviolet transilluminator.

Mycoplasma detection by direct fluorescent assay

The cells were seeded in cover slips and incubated for 24–72 h at 28 °C to give a confluence of 80–90 %. Without decanting the medium, 2 ml of carnoy's fixative (3:1,100 % methanol–glacial acetic acid) was added gently to the dish containing the cover slip and incubated at room temperature for 2 min. The medium was aspirated and fresh fixative was added. The fixative was then aspirated, and the cover slip was allowed to air dry. A working stock solution of Hoechest 33258 stain (Sigma-Aldrich, USA) was made at a concentration of 0.05 μg/ml in Hank's balanced salt solution, without phenol red, at pH 7.0. Approximately 1 ml of 0.05 μg/ml of stain was incubated on a dried cover slip for 10 min. The cover slips were then washed three times with distilled water and mounted in 0.1 M acetate buffer (pH 5.5) on a microscope slide. When almost dry, the slide was sealed and examined under a fluorescent microscope (Carl Zeiss, Germany) ×500 magnification.

SDS–PAGE analysis and electroelution

The proteins of gill tissue of rohu were analysed by 12 % sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (Laemmli 1970). The tissue sample was mixed with Laemmli sample buffer, boiled for 5 min and electrophoresed at a constant current of 30 mA. After electrophoresis, the gel was stained with Coomassie Brilliant Blue. Molecular weight standards were co-electrophoresed. The prominent protein of 30 kDa molecular weight observed in the gill tissue sample was electroeluted from the gel using the method of Hunkapiller and Lujan (1986). A preparative SDS–PAGE was run with gill tissue sample. After the run, the gel was soaked in pre-chilled KCl (0.4 M). The prominent protein band was excised, and gel slice was minced into small pieces (less than 1 mm) with a sterile razor blade. The gel pieces were put into a dialysis bag with TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH 8), and the bag was kept in horizontal electrophoretic tank filled with TE buffer. Constant power supply (50 mA) was set and run for 6 h. After elution, the sample was dialysed and concentrated by a Speed vac evaporator. The purified gill protein was estimated and confirmed on SDS–PAGE.

Production of polyclonal antiserum

Antiserum was raised against 30 kDa protein of gill epithelium in 12-week-old Swiss white mice. The mice were injected subcutaneously with electroeluted protein with Freund's complete adjuvant followed by a booster with incomplete adjuvant 7 days later. The blood was collected 7 days after the booster injection, and the serum was separated.

Western blot analysis

Western blot analysis to detect 30-kDa protein in gill tissue and gill cell line of rohu was carried out by the method described by Talbot et al. (1984).

In vivo fish acute toxicity test

L. rohita with body weight of 2–3 g were obtained from a local fish farm and acclimatized in 100 l fibreglass tank at 23–28 °C under an ambient photoperiod in the laboratory for 7 days prior to experiments. The fish were fed with commercial pellet feed twice a day and starved for 24 h before and during the experiments. Acute toxicity tests were conducted by exposing L. rohita (N = 10 fish per tank) for 96 h to malathion (50 % emulsified concentration, EC) under static conditions (OECD 203 1992). Six concentrations of malathion (5, 10, 15, 20, 25 and 30 mg/l) diluted with freshwater and control with freshwater alone were tested to determine the lethal concentration 50 (LC50) (concentration at which 50 % of the fish population dies). The tank had a working volume of 30 L (three tanks per each concentration) based on the body weight of fish (1 g/l). Dead fish were counted and removed immediately every day. All the experiments were conducted in triplicates. Mortalities were recorded following the guideline for fish acute toxicity (OECD 203 1992).

In vitro cytotoxicity assay using rohu gill cell line

Gill cell line of L. rohita at exponential growth phase was harvested and diluted to a concentration of 105 cells/ml in Leibovitz’s L-15 medium with 10 % FBS. After agitation, the cells were added to each well of 96-well tissue culture plates at the concentration of 2 × 104/well and incubated overnight at 28 °C. After incubation, the medium was removed, and the cells were re-fed with medium containing 0 (control), 5, 10, 15, 20, 25 and 30 μg/ml of malathion for 24 h EC50 analysis. Then four endpoints for toxicity, i.e. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Neutral Red (NR) uptake assay, Alamar Blue (AB) assay and protein concentration assay were determined after 24 h exposure.

Cell morphology analysis

Cells were plated into a 35-mm tissue culture plate at a density of 2 × 105 cells (in 2 ml growth medium). After overnight growth, supernatants from the culture plates were removed, and fresh aliquots of growth medium containing six different concentrations of malathion were added. Upon incubation, cells were washed with PBS (pH 7.4), and the morphological changes were observed under an inverted phase-contrast microscope (Carl Zeiss, Germany) at ×200 magnification.

MTT assay

MTT assay described by Borenfreund et al. (1988) is based on inhibition by chemical injury of the reduction of soluble yellow MTT tetrazolium salt to a blue insoluble MTT formazan product by mitochondrial succinic dehydrogenase. After a 24-h exposure period, the test medium was replaced by 20 μl of 5 mg/ml MTT (Sigma, St. Louis, MO, USA) in PBS. After incubation for 4 h at 20 °C, the solution was removed carefully, and the cells were rinsed twice with PBS rapidly. Then dimethyl sulphoxide was added at the rate of 150 μl/well to solubilize the purple formazan crystals produced. Absorbance of each well was measured at 490 nm and the EC50 value calculated.

Neutral red uptake assay

NR uptake assay was carried out based on the procedure described by Borenfreund and Puerner (1985). This assay measures the inhibition of cell growth, which is based on the absorbance of the vital dye NR by living, but not by dead, cells. After 24 h exposure, the test medium in each well was replaced by 200 μl L-15 medium containing 50 μg/ml of NR and incubated in situ for 3 h at 28 °C. The cells were then rinsed with warm PBS to remove the NR dye and then de-stained with 200 μl solution containing glacial acetic acid, ethanol and water in the ratio of 1:50:49, respectively. After rapid agitation for 10 min at room temperature, the absorbance of the solution in each well was measured at 550 nm with a microplate reader (Multiscan EX Thermo Electron Corporation), and the EC50 value (concentration of test agent which causes 50 % inhibition in NR uptake) was determined.

Alamar Blue reduction assay

Alamar Blue assay was carried out to assess the metabolic activity using the water soluble reagent, AB as described by Dayeh et al. (2003) and by Trek Diagnostic Systems, Inc., Westlake, OH, USA (reagent source). Alamar Blue was diluted with L-15 medium to 10 % (v/v), filter-sterilized and stored in the dark at 4 °C. After removing the test medium, 150 μl of this solution was added into each well, and 2 h later, the wells were read with a fluorometric multiwell plate reader (Multiscan EX Thermo Electron Corporation) at respective excitation and emission wavelengths of 530 (± 30) and 595 (± 35) nm.

Coomassie Blue dye protein assay

The cell protein assay using Coomassie Blue (CB) dye as described by Shopsis and Eng (1985) was carried out to investigate the cell growth by the alteration of the total cellular protein. After 24 h exposure, the test medium was removed, and the cells were washed with PBS and then lysed in 50 μl of 0.1 mM NaOH per well for 1 h at 28 °C. Then 200 μl of Coomassie Blue solution (10 mg Coomassie Blue G-250, dissolved in 5 ml of 95 % ethanol, 10 ml of 85 % phosphoric acid and then diluted to the final volume of 100 ml with distilled water) was added to each well and incubated for 20 min at RT. The absorbance at 570 nm was then measured. Serial dilutions of 1–100 mg/ml BSA dissolved in 0.1 mM NaOH were used for the protein standard. The cytotoxicity was expressed as a fraction of the negative control (cells with media alone).
$$ {{{\%\;\mathrm{Cytotoxicity}=\mathrm{OD}\;\mathrm{of}\;\mathrm{test}\;\mathrm{sample}}} \left/ {{\mathrm{OD}\;\mathrm{of}\;\mathrm{Control}\times 100}} \right.} $$

Data analysis

Experiments were performed in triplicate and eight replicates for in vivo and in vitro, respectively, for each exposure concentration. Absolute values of each assay were transformed to control percentages. The results of LC50 and EC50 values were calculated using computerized software (Trimmed Spearman-Karber, Program Version 1.5). The individual data points of the concentration response cytotoxicity graph were presented as the arithmetic mean percent inhibition relative to the control standard error (SE). Cell viability and the concentration were fitted scatter plots with the regressive equation (a linear regression model). The strength of the r2 value was used to determine whether a linear or quadratic relationship was assumed.

Results

Primary culture and subculture of rohu gill cells

Cell cultures were initiated from gill tissues of rohu by explant method. The emergence of cells began from the edges of seeded gill tissue explants, and it was observed during the fourth day following tissue explanting (Fig. 1a). A confluent monolayer around the explants of gill tissue was observed after the 15th day of implantation. However, the LRG cells grew continuously and were sub-cultured at intervals of 5–7 days. The cells were split at the ratio of 1:2–1:3. The initial subcultures of this cell line consisted of both epithelial and fibroblast like cells. After 57 subcultures, LRG cells were bio-transformed. Morphologically, LRG cell line is composed of both epithelial and fibroblast-like cells with diameter of 25–30 μm (Fig. 1b, c). The LRG cell line of rohu has been sub-cultured more than 89 times since its initiation in June 2010.
Fig. 1

Phase-contrast photomicrographs of the rohu gill cell line. a Primary culture on fourth day following tissue explanting, b subcultured at passage 33 at low magnification and c subcultured at passage 33 at high magnification. Scale bar 50 μm

Effect of temperature and FBS concentration

The LRG cell line exhibited different growths at different temperatures (Fig. 2a). The optimum temperature for maximum growth was found to be 28 °C; however, significant growth was also observed at a wide range of temperatures from 22 to 32 °C. No significant growth was observed at 37 and 40 °C. The growth rate of rohu gill cells increased as the FBS proportion increased from 2 to 20 % at 28 °C. The LRG cells exhibited poor growth at 2 and 5 % concentrations of FBS, relatively good growth at 10 %, but maximum growth occurred at the concentrations of 15 and 20 % FBS (Fig. 2b).
Fig. 2

Growth responses of the LRG cell line at the 37th passage to selected a temperature, b foetal bovine serum concentrations and c photograph of colony formation at low cell density (×100 magnification)

Plating efficiency

Plating efficiency of the LRG cell line was determined at seeding concentrations of 100, 500 and 1,000 cells. Moderately high plating efficiency was observed with LRG cells [17.3 (±1.3), 25.56 (± 1.03) and 34.37 (± 2.39) %], respectively (Fig. 2c). The doubling time of rohu gill cell line was approximately 43 h.

Cryopreservation and revival

Evaluation of the viability of LRG cells stored in liquid nitrogen (−196 °C) established the capability of cells to survive following 6 months of storage. More than 70–80 % of cells from each vial remained viable even after 6 months and retained the ability to attach and grow at 28 °C. Following storage, no obvious alterations in morphology or growth pattern were observed for LRG cells.

Chromosomal number analysis

The results of chromosomal analysis of 100 metaphase plates prepared from the LRG cell line of L. rohita at different passage levels revealed that the chromosome numbers varied from 35 to 55. The modal number (2n) was 50 (Fig. 3a, b). The chromosome morphologies of LRG cell line were mostly telocentric.
Fig. 3

Chromosome number distribution at a passages 42 and b metaphase. In total, 100 metaphases were counted

Transfection

Transfection of the LRG cell line was successfully analysed using pEGFP-N1 with lipofectamine 2000. Clear and strong green fluorescent expression of EGFP in LRG cells could be detected as early as 24 h after transfection (Fig. 4a).
Fig. 4

a Expression of GFP gene in LRG cell line at passage 53rd transfected with pEGFP-N1 and b agarose gel electrophoresis of PCR products from LRG cells and rohu muscle. Lane M 100 bp marker; lane N negative control; lane 1 muscle tissue of rohu; lane 2 LRG cell line

Mitochondrial Cox1 gene analysis

DNA was isolated from the LRG cell line and gill tissue of L. rohita. Amplification of the extracted DNA using the primers designed from the genomic sequence of mitochondrial Cox1 gene of L. rohita revealed the expected PCR product of 324 bp (Fig. 4b). These results indicate conclusively that the cell line originated from L. rohita.

Mycoplasma detection

The LRG cell line showed brightly stained nuclei. No pinpoints or filamentous pattern of fluorescence on the cell surface were observed (Fig. 5a). Thus, the LRG cell line was confirmed to be free from mycoplasma contamination.
Fig. 5

a LRG cell line stained with Hoechest 33258 and image capture under fluorescent microscope (×500 magnification) and b western blot analysis of 30 kDa protein in newly established cell line and rohu fish tissue. Lane M marker; lane 1 LRG cell line; lane 2 gill tissue; lane 3 muscle; lane 4 heart; lane 5 liver; lane 6 kidney

Western blot analysis

The western blot analysis revealed a distinct band of 30 kDa protein in LRG cell line and gill tissue, whereas no background coloration was observed in the other organs of rohu fish (Fig. 5b). Thus, this confirmed that LRG cell line originated from gill tissue of rohu fish.

In vivo acute toxicity

The fish were exposed to six different concentrations of malathion, and mortality of fish was recorded. The results are presented in Table 1. Malathion at the concentration of 30 mg/l caused 100 % mortality in L. rohita, whereas lower concentration of malathion at 5 mg/l caused 6.66 % mortality. The LC50 value was determined as 14.09 mg/l at 96 h exposure to malathion. No mortality was observed in the control group of fish.
Table 1

Cumulative percent mortality of L. rohita exposed to six different concentrations of malathion for 96 h (mean ± SE)

Concentrations of malathion (%, w/v) (mg/l)

No. of fish exposed

No. of dead fish

Cumulative mortality (%)

LC50 value at 96 h (95 % upper–lower confidence limit)

0

30

0

0

 

5

30

2

6.66 ± 0.86

10

30

7

23.00 ± 5.77

14.09

15

30

14

46.60 ± 3.33

(12.29–16.15)

20

30

22

73.30 ± 3.33

 

25

30

28

93.30 ± 5.67

30

30

30

100.0 ± 0.00

In vitro cytotoxicity

In vitro cytotoxicity test was carried out to test the toxicity of malathion in LRG cells of L. rohita using four widely used endpoint assays, namely MTT, NR, AB and cell protein assays (CB). A total of six concentrations which ranged from 5 to 30 μg/ml of malathion were used to carry out the in vitro toxicity assay in LRG cells using four basal cytotoxicity end points, and the results are shown in Fig. 6a. The lowest concentration of malathion (5 μg/ml) was found to be toxic to LRG cells. The progressive increase in the concentration of malathion led to increase in toxicity when compared to control. EC50 values of the malathion resulting in 50 % inhibition of cytotoxicity parameters after 24 h exposure to malathion were calculated, and the results show that EC50 value at 24 h (95 % upper–lower confidence limit) was found to be 12.88 μg/ml (11.42–14.53), 14.07 μg/ml (12.36–16.02), 13.47 μg/ml (12.16–14.93) and 12.47 μg/ml (11.68–14.12) for MTT50, NR50, AB50 and CB50, respectively. Correlations among the endpoints employed in the LRG cells of L. rohita to study cytotoxicity of malathion have been determined. A general tendency in the sensitivity among the four endpoints could be observed. Statistical analysis revealed good correlation with r2 = 0.903–0.969 for all combinations between endpoints.
Fig. 6

a In vitro cytotoxicity of malathion to gill cell line of L. rohita after 24 h exposure by MTT, NR uptake, AB assay and cell protein assay. The individual data points are expressed as the arithmetic mean percentage of control (mean ± SE) (n = 8 replicate). Linear regressions between each in vitro EC50 and in vivo LC50; b MTT EC50 vs. LC50, c NR EC50 vs. LC50, d AB EC50 vs. LC50 and e CB EC50 vs. LC50

Linear correlations between each in vitro parameter (EC50) and the in vivo LC50 data of malathion were determined, and the results are shown in Fig. 6b–e. Linear correlations between each in vitro EC50 and the in vivo LC50 data of malathion were highly significant p < 0.001 with r2 = 0.946–0.990. Prominent morphological changes of the LRG cells due to toxicity of malathion include cell shrinkage, cell detachment, vacuolations and cell swelling. No morphological changes were observed in control cells (Fig. 7).
Fig. 7

Morphological alterations (cell detachment, cell shrinkage, cell damage and vacuolations) were observed in rohu gill cell line exposed to different concentrations of malathion, while no morphological changes were observed in control cells and 100 % destruction in 30 μg/ml of malathion. a Control cell line, b 5 μg/ml, c 10 μg/ml, d 15 μg/ml, e 20 μg/ml and f 25 μg/ml

Discussion

In the present study, a new cell line from the gill of Indian major carp, L. rohita, was successfully established and characterized. The LRG cell line has been passaged more than 85 times over a period of 2 years. The LRG cell line was established by means of the explant technique. The explant technique has many advantages over the trypsinization method in terms of rapidity, ease and maintenance of cell interactions and the avoidance of enzymatic digestion which can damage the cell surface (Avella et al. 1994). Moreover, the explants in vitro are supposed to be structurally closer to the organ in vivo than cultures obtained using cell suspensions (Flano et al. 1998; Sahul Hameed et al. 2006).

The temperature range for growth of LRG cell line was 22–32 °C with optimum growth at 28 °C, identical with reported fish cell lines of rohu and catla (RE & CB) cell lines (Ishaq Ahmed et al. 2009); PK, GK, PH cell lines (Nicholson et al. 1987); FG-9307 cell line (Tong et al. 1997); SPH, SPS, RSBF cell lines (Tong et al. 1998); FFN FSP cell lines (Kang et al. 2003) and IEE, IEG, IEB, IEK cell lines (Sarath Babu et al. 2012). The LRG cell line is well adapted to grow in Leibovitz's L-15 supplemented with 10 % FBS. Fernandez-Puentes et al. (1993a, b) have reported that Leibovitz's L-15 cell culture medium is the best medium for the growth of fish cells when compared to other culture media. The growth rate of the cells improved as the FBS concentration increased from 2 to 20 %. However, a 10 % concentration of FBS also provided relatively good growth and is therefore cost-effective for maintenance of this cell line.

The plating efficiency was determined for LRG cell line, and the results revealed a high plating efficiency (34 %) when the cells were seeded at the rate of 1,000 cells flask−1. Chi et al. (1999) recorded a plating efficiency of 21 % in GF-1 cell line at 50th passage. Cryopreservation of cell lines is necessary for long-term storage, and LRG cell line was well adapted for cryopreservation. Seventy to 80 % of stored cells were recovered and grew to confluency in 5 days at the optimum temperature of 28 °C. The recovery rate of some other fish cell lines was 50 % for SAF-1 (Sparus aurata) (Bejar et al. 1997), 73 % for GF-1 (Epinephelus coioides) (Chi et al. 1999) and 90 % for SIGE and SIMH (E. coioides and Chanos chanos) (Parameswaran et al. 2007). The modal chromosome number (2n) in LRG cell line was found to be 50, which was identical to the modal chromosome number reported earlier by (Zhang and Reddy 1991).

The results of transfection experiments revealed that LRG cell line of L. rohita was efficient in gene expression and could thus be used to evaluate promoter efficiency in various recombinant constructs and expression studies. The expression of pEGFP-N1 in LRG cells could be detected as early as 24 h of transfection. Mitochondrial DNA, particularly mitochondrial Cox1 gene, has frequently been used in species identification, classification and in establishing phylogenetic relationships among aquatic and mammalian species. To further confirm the origin of the newly established LRG cell line, gill tissue DNA was extracted from fish, and cellular DNA was extracted from the cells at early passage and amplified by PCR using specific primer pairs targeting the conserved regions of mitochondrial Cox1 gene of L. rohita. The PCR of mitochondrial Cox1 confirm that the LRG cell line originated from L. rohita.

The identification of mycoplasma in cell culture by Hoechst has been employed by several investigators (Chen 1977; Hessling et al. 1980; Freiberg and Masover 1990) as in the present study. It is clear from the results obtained that no extranuclear fluorescence was observed in the LRG cell line. In the present study, polyclonal antiserum was raised against 30 kDa protein of fish gill epithelium. The western blot analysis revealed a band corresponding to protein of 30 kDa from LRG cell line and homogenate of rohu gills. Similar studies have been carried out using western blot to detect 30 kDa protein in fish gill tissue (Rahim et al. 1988; Sender et al. 1999).

Acute toxicity of malathion to Indian major carp rohu was found to be 14.09 mg/l. A similar report has been previously noted in L. rohita exposed to malathion (LC50 15 mg/l) at 96 h (Thenmozhi et al. 2011). In the present study, the cell line developed from gill tissue of rohu fish was used as an in vitro assay to assess the cytotoxicity of malathion, and the results were compared with the results of in vivo assay to make use of LRG cell line for toxicological study as a substitute for whole fish. Other fish cell lines such as PLHC-1 cell line of Poeciliopsis lucida and ZC-7901 cell line of Ctenopharyngodon idellus have been used as in vitro assay as in the present study to test the cytotoxicity of malathion (Babich et al. 1991; Chen et al. 2006).

Castano et al. (1996) found good correlations between in vivo LC50s and in vitro EC50s for each endpoint and also for the cytotoxicity index and suggested the applicability of the RTG-2 cell line as an alternative protocol to estimate the acute toxicity of chemicals on fish without using live animals. The results of the present study revealed that the four EC50 values were closely correlated to whole fish LC50 values and that the linear correlation between each in vitro parameter and the LC50 data were found to be highly significant. The results of in vitro assays using a gill cell line of L. rohita were correlated with those obtained from in vivo assay using the same species of fish (L. rohita). The results of the present study indicate the possibility of using the gill cell line instead of living fish for toxicity assessment of pesticides and environmental contaminants. To confirm this, more studies need to be carried out using different chemical substances with toxic properties. In future, fish cell lines will be utilized as a biological model for evaluating the cytotoxicity of chemical pollutants in environmental samples and will also become a standard practice in toxicological studies. The advantages of using in vitro assay include time saving, low cost, ease of operation and reduction in the number of fish required.

The gill of fish is an organ responsible for osmoregulation, acid–base balance, nitrogen excretion and metabolism of circulating hormones. On account of its streamlined anatomical position and constant contact with water, it is badly affected by toxic compounds and infective agents (Sathe et al. 1997). Many aquatic pollutants are assayed by the damage they cause to the ultrastructure of the gill epithelium (Matei 1993), histopathological changes (Singh and Sahai 1990), perturbation in carbohydrate metabolism (Reddy and Yellamma 1991b), nitrogen metabolism (Reddy et al. 1991) or protein metabolism (Suresh et al. 1991). However, these studies had been carried out on intact gills in vivo. In vitro approaches have not been fully exploited due to lack of a cell culture system and standard assay protocol.

In conclusion, the LRG cell line was developed and characterized from freshwater fish L. rohita. This cell line has the potential to serve as a useful tool for freshwater fish conservation genetics, toxicology and gene expression. Further studies are required to study the suitability of LRG cell line for the isolation of viruses of freshwater fish and toxicological studies. Based on the results of the present study, we recommend the use of this cell line as a substitute for living fish for toxicity assessment of pesticides and environmental pollutants.

Notes

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.

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Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • S. Abdul Majeed
    • 1
  • K. S. N. Nambi
    • 1
  • G. Taju
    • 1
  • N. Sundar Raj
    • 1
  • N. Madan
    • 1
  • A. S. Sahul Hameed
    • 1
  1. 1.OIE Reference Laboratory for WTD, PG & Research Department of ZoologyC. Abdul Hakeem CollegeMelvisharam, Vellore DistrictIndia

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