Chromium Induced Toxicity Research
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In the present study, we hypothesize that cytotoxicity, genotoxicity and oxidative stress play a key role in chromium induced toxicity in SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines when exposed for 24 h. Acute toxicity tests were conducted on three fish species namely L. calcarifer, E. suratensis and C. catla by exposing them to different concentration (0, 10, 20, 30, 40 and 50 mg/L) of chromium for 96 h under static conditions and the LC50 was calculated. The percentage cell survival was assessed by multiple endpoints such as MTT, NR, AB and CB assays were performed in seven fish cell lines exposed to different concentrations of chromium and EC50 values of all the four endpoints was calculated. Linear correlations between each in vitro cytotoxicity assay and the in vivo mortality data were highly significant. Microscopic examination of cell morphology indicated cell shrinkage, cell detachment, vacuolations and cell swelling at highest concentration of chromium (50mg/L). The DNA damage and nuclear fragmentation were assessed by comet assay and Hoechst staining, in seven fish lines exposed to different concentrations of chromium. The result of antioxidant parameter obtained show significantly decreased catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GSH) and Glutathione peroxidase (GPx), and increased level of lipid peroxidation (LPO) in all the cell lines after exposure to increasing chromium in a concentration-dependent manner. This results proves that fish cell lines could be used as an alternative to whole fish using cytotoxicity, genotoxicity and oxidative stress assessment after exposure to chromium.
Keywords: Fish cell lines, Chromium, Cytotoxicity, Genotoxicity, Oxidative stress
Heavy metal pollution of water is a serious environmental problem facing the modern world. At global level heavy metals pollution is increasing in the environment due to increase in number of industries (Chidambaram et al. 2009). Industrial effluents are discharged into the sewage canals, rivers and irrigation water, causing major pollution and health hazards (Baddesha and Rao 1986). Many industrial wastewaters contain heavy metals like cadmium, lead, zinc, cobalt and chromium. The toxic heavy metals are mostly absorbed and get accumulated in various plant parts as free metals which may adversely affect the plant growth and metabolism (Barman and Lal 1994). Human beings and cattle are badly affected when these metals are incorporated into food chain as it causes bronchitis and cancer (Khasim et al. 1989; McGrath and Smith 1990; Nath et al. 2005). Among heavy metals, chromium plays a major role in polluting our aquatic environment system. In nature chromium occurs predominately in two valances Cr (III) and Cr (VI). Hexavalent chromium [Cr (VI)] predominates over the Cr (III) form in natural waters. Hexavalent chromium [Cr (VI)] particulates enter the aquatic medium through effluents discharged from leather tanning, textiles, chrome electroplating, metal finishing, dyeing and printing industries and several other industries.
The Cr (VI) penetrates biological membranes easily and causes cellular damage by oxidative stress (Irwin et al. 1997; Begum et al. 2006), its unselective exposure may pose serious effect on aquatic communities including fish. Toxic effects of Cr(VI) on enzymological/biochemical (Al-Akel and Shamsi 1996; Vutukuru et al. 2007; Oner et al. 2008), hematological (Gautam and Gupta 1989; Al-Akel and Shamsi 1996), immunological (Prabakaran et al. 2007) parameters, endocrine toxicity (Mishra and Mohanty 2009) and genotoxicity (Chen et al. 2011) have been reported in many teleosts fishes.
In environmental risk assessment, much of the toxicity test on fish has involved the use of lethality as the endpoint. On the other hand, in vivo bioassay is expensive and requires huge quantity of toxicant. The exposure time is only 24 h as opposed 96 h in bioassay, which could reduce the cost of labor, lab facilities and test time but more importantly allow decisions to be made more rapidly. Nevertheless, toxicity testing with fish is an essential part of environmental risk assessment procedures (Castano et al. 2003). For all these considerations, the development and use of in vitro assays that could measure early stages of toxicity in vertebrates represent an approach that could be very useful to monitoring environmental risk assessment (Walker 1999). Over the last four decades, cell and tissue culture methods have been refined and have now become an essential tool in environmental research. There are a lot of ethical, scientific and economical reasons that support the development of in vitro methods for use in ecotoxicology (Castano and Gomez-Lechon 2005; Bols et al. 2005; Schirmer, 2006; Fent 2007; Taju et al. 2012, 2013, 2014). The use of fish cell lines in environmental toxicology has been reviewed and positively assessed mainly with regards to cytotoxicity (Babich and Borenfreund 1991; Castano et al. 2003; Fent 2001). Cytotoxicity assessments can be readily employed to examine multiple endpoints, including measurements of cell death (apoptosis), cell viability, cellular morphology, cell metabolism, cell attachment/detachment, cell membrane permeability, proliferation, growth kinetics, genotoxicity and oxidative stress (Maracine and Segner 1998; Li and Zhang 2002; Shuilleabhain et al. 2004; Taju et al. 2014).
In the present study, three fish species from three different aquatic environments, Lates calcarifer (Marine), Etroplus suratensis (Brackishwater) and Catla catla (freshwater) were selected as representatives of their respective environments to study their suitability for acute toxicity test to evaluate the potential risk of chromium (Cr). They are excellent food fishes with a good market demand in India, Malaysia, Bangladesh and Pakistan. Some attempts were made to study in vivo acute toxicity in Sea bass, Etroplus and Catla using various toxicants (Chezhian et al. 2010; Azmat and Javed 2011, 2012; Bhat et al. 2012; Taju et al. 2012, 2013). The seven fish cell lines namely SISK and SISS cell lines derived from L. calcarifer (Sahul Hameed et al. 2006; Parameshwaran et al. 2006b), SICH and ICG cell lines derived from C. catla (Ishaq Ahmed et al. 2009b; Taju et al. 2014), and IEE, IEK and IEG cell lines derived from E. suratensis (Sarath Babu et al. 2012) were used as in vitro assays to evaluate the cytotoxicity, genotoxicity and oxidative stress exposed to chromium. The results of in vitro cytotoxicity were compared with the results of in vivo acute toxicity test using fish. The use of these cell lines for toxicity assessment of chromium instead of living fish is recommended.
2. Material and methods
2.1. Chemicals and reagents
Tissue culture media and chemicals were obtained from GIBCO (Invitrogen Corporation, USA). Potassium dichromate (K2Cr2O7), EDTA, Trichloroacetic acid, DTNB [5,5-dithio-bis-(2-nitrobenzoic acid)], Thiobarbituric acid, Hydrogen peroxide, Nitro blue tetrazolium (NBT), Riboflavin, Hydroxylamine-HCl, Triton X-100, Ethidium bromide, Methanol, Acetic acid, Sodium chloride, Sodium hydroxide and Coomassie Blue was purchased from SRL chemicals, India.
2.2. Collection of experimental animals
Lates calcarifer and Etroplus suratensis were collected from Central Institute of Brackishwater Aquaculture (CIBA), Chennai. Catla catla was collected from a local pond in Walajapet, Vellore - District, Tamil Nadu, India. The experimental fishes were 2 - 3 g in body weight. Specimens were transported live in oxygen bags or buckets to the laboratory, acclimatized and maintained for 20-30 days in a salinity range of 5-10 ppt for E. suratensis, 20-25 ppt for L. calcarifer and in freshwater in the case of C. catla (23-28oC) under an ambient photoperiod in the laboratory for 10 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.
2.3. In vivo fish acute toxicity test
Fish acute toxicity tests were conducted by exposing E. suratensis, L. calcarifer and C. catla (N = 10 per aquarium) for 96 h to chromium under static conditions (OECD 203, 1992). Five different concentrations chromium i.e., 0, 10, 20, 30, 40 and 50 mg/L diluted with seawater (5 ppt) and freshwater while control with sea water and freshwater alone were tested to determine the LC50 (concentration at which 50% of the fish population dies). The aquaria had a working volume of 30 lit based on the body weight of fishes (1 g fish/L). Dead fishes 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).
2.4. Fish Cell lines
A total of seven cell lines established from different organs of L. calcarifer (SISS-seabass spleen, SISK-kidney), E. suratensis (IEE - Etroplus eye, IEG - gill, IEK - kidney) and C. catla (SICH - Catla heart, ICG - gill) were tested for their sensitivities to chromium. These fish cell lines were propagated at 28oC in Leibovitz's L-15 medium (pH 7.0 -7.4) with 2mM L-glutamine, 10% foetal bovine serum (FBS), penicillin 100 IU/ml and streptomycin 100 Âµg/ml. The osmolarity ranged from 300 to 360 mOsm kg-1. These cells were sub-cultured every 2-3 days using standard procedure. Cells at exponential growth phase were harvested and used for in vitro cytotoxicity tests.
2.5. In vitro cytotoxicity assay using fish-derived cell lines
SISS, SISK, IEE, IEK, IEG, SICH and ICG cells at exponential growth phase were collected 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 x 104/well and incubated overnight at 28oC. After incubation, the medium was removed and the cells were re-fed with medium containing 0 (control), 10, 20, 30, 40 and 50 mg/L of chromium for 24 h EC50 analysis. Then four endpoints for cytotoxicity, i.e., MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay, Neutral red (NR) uptake assay, Alamar blue assay (AB) and protein concentration for Coomassie blue (CB) assay were determined after 24 h exposure as described by Borenfreund et al. (1988), Borenfreund and Puerner (1985), Taju et al. (2012) and Shopsis and Eng (1985), respectively.
2.5.1. Cell morphology
SISS, SISSK, IEE, IEK, IEG, SICH and ICG cells were plated into a 24 well tissue culture plate at a density of 2Ã-105 cells (in 1 mL growth medium). After overnight growth, supernatants from the culture plates were removed and fresh aliquots of growth medium containing various concentrations of the chromium (0, 10, 20, 30, 40 and 50 mg/L) were exposed for 24 h. Upon incubation, cells were washed with phosphate-buffered saline (PBS, pH 7.4) and the morphological changes were observed under an inverted phase-contrast microscope (Carl Zeiss, Germany) at 100Ã- magnification.
2.6. Assessment of in vitro genotoxicity using fish-derived cell lines
2.6.1. Comet assay
The Single Cell Gel Electrophoresis (comet assay) was performed on SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines according to the method of Singh et al. (1988) with slight modifications in accordance with the protocols of Taju et al. (2014). 5 x 104 cells on 500 Î¼L of complete culture medium were seeded per well in a 24-well-plate. After 24 h incubation, cells were exposed to chromium using the following concentrations: 0 (control), 10, 20, 30, 40, 50 and 60 mg/L. At the end of the exposure period, cells were collected through trypsinization, followed by centrifugation at 1000 rpm for two minutes to obtain the pellet and avoid cell loss. After the centrifugations, the supernatant was discarded and the pellet resuspended in 100 Î¼L of 0.9% agarose in milliQ water (low-melting point agarose, Sigma Aldrich chemicals, USA). The suspensions of cells in agarose were then applied dropwise to microscope slides containing an agarose layer (agarose electrophoresis grade, prepared with a 1% concentration in milliQ water), and kept in a freezer for 10 min. The cells were lysed in freshly made lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, 10% DMSO, 1% Triton X-100, pH 10), for 1 h at 4 Â°C. After rinsing with redistilled water, the slides were placed on the horizontal gel box, covered with the cold alkaline buffer (0.3 M NaOH, 1 mM EDTA, pH >13) and left for 20 min. Electrophoresis was run in the same buffer at 25 V (0.83 V/cm) at 300 mA for 20 min at 4 Â°C. After electrophoresis the slides were neutralized in a cold neutralization buffer (0.4 M Tris-HCl, pH 7.5), for 2 to 5 min, fixed in methanol:acetic acid (3:1) for 5 min and stored in the dark at room temperature. Prior to examination, the slides were rehydrated and stained with 10 Âµg/mL ethidium bromide and examined using a Zeiss Axioplan epifluorescence microscope (Carl Zeiss, Germany). A positive control (5 ÂµM H2O2) was also included in every batch of samples. This strategy was chosen to compare the variation in the distance of migration. The positive control was not included in evaluation. Slides were examined at 100x magnifications using a fluorescence microscope. For each experimental condition 100 randomly chosen cells from two duplicate slides were examined (50 from each slide). In all 100 comets were scored visually according to the relative intensity of the fluorescence in the tail length. The extent of DNA migration was determined as a percentage of DNA in the tail (% tDNA) using an image analysis system comet 5, Kinetic Imaging Ltd.
2.6.2. Assessment Nuclear fragmentation by Hoechst 33258
Nuclear fragmentation of SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines was analyzed with Hoechst 33258. The cells were seeded in 12-well cell culture plates and incubated overnight. Then the cells were treated with different concentrations of chromium (0, 10, 20, 30, 40 and 50 mg/L). Cells were fixed in 4% paraformaldehyde in PBS for 30 min, washed with PBS, and stained with 1 Î¼g/mL Hoechst 33258 in PBS for 30 min. Stained cells were washed twice with PBS. The changes in nuclei were observed with a fluorescent microscope through a UV filter.
2.7. Preparation of cell extract and Biochemical estimations
The SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines were exposed to different concentrations of chromium (0, 10, 20, 30, 40, 50 and 60 mg/L) on 25 cm2 flasks for 24 h. After 24 h they were trypsinized and pelleted by centrifugation at 500Ã-g for 5 min. The cell pellet was washed with PBS (0.1M, pH7.4), resuspended in 500 Âµl chilled homogenizing buffer (250mM sucrose, 12mM Tris-HCl, 0.1mM DTT, pH 7.4) and lysed using Dounce homogenizer. The lysate was centrifuged (8000Ã-g, 10 min, 4 Â°C) and the supernatant (cell extract) was used for various biochemical assays. Protein concentration in the cell extract was estimated by the method of Lowry et al. (1951).
The enzymatic antioxidant superoxide dismutase (SOD) activities were determined by following the procedures described by Kono (1978). Catalase (CAT) activity was determined by following the method described by Aebi (1974). The level of non-enzymatic antioxidant reduced glutathione (GSH) was estimated following the procedures described Saldak and Lindsay (1968). The activity of glutathione peroxidase (GPx) was assayed by the method of Flohe and Gunzler, (1984). The level of lipid peroxidation (LPO) was measured according to the method described Beuge and Aust (1978) based on the reaction with thiobarbituric acid. The results were recorded as Âµmol of TBA reactive substances/mg protein. The enzymatic and non-enzymatic parameters was expressed as Âµmol/mg protein.
2.8. Data analysis
Experiments were performed in triplicate with eight replicates for each exposure concentration. Absolute values of each assay were transformed to control percentages. The results of LC50 and EC50 values were expressed as dilution in (mg/L) of the sample calculated using computerized (EPA, 2000) software. 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. Analysis of variance was used to determine whether groups of variables differed from each other (SPSS, Version 16).
The cumulative percentage mortality in L. calcarifer, E. suratensis and C. catla exposed to different concentrations of chromium was determined at 96 h and the results are presented in Fig 1. The toxic effect of chromium on the survival of fish was found to be concentration and time dependent. The chromium at the concentration of 50 mg/L caused 100%, 96.66% and 90% mortality, respectively, in L. calcarifer, E. suratensis and C. catla, whereas lower concentration of chromium at 10 mg/L caused 26.66%, 16.66% and 20% mortality of L. calcarifer, E. suratensis and C. catla respectively. No mortality was recorded in the control fish even after 96 h exposure. The LC50 values corresponding to 24, 48, 72 and 96 h of exposure of chromium were determined and results are presented in Table 1.
Five different concentrations which ranged from 10 to 50 mg/L of chromium were used to carry out the in vitro toxicity assay in SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines using four cytotoxicity end points (MTT, NR, AB and CB assays) and the results are shown in Fig.2 A-D. The cytotoxicity of chromium to SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines was found to be similar in all the toxic endpoints employed. The lowest concentration of chromium tested (10 mg/L) was found to toxic in all the cell lines particularly SICH and IEK cell lines. The progressive increase in the concentration of chromium led to increase in toxicity when compared to control cells. The MTT, NR, AB and CB cytotoxicity endpoint assays revealed that a 24-h exposure of all the cell lines to different concentrations of chromium produced a dose-dependent reduction in the fraction of viability. The EC50 values and 95% confidence limit values obtained for chromium are summarized in Table 2. Correlations among the endpoints employed in the SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines to study cytotoxicity of chromium have been determined. A general tendency in the sensitivity among the four endpoints could be observed and statistical analysis revealed good correlation with R2 = 0.889-0.927 for all combinations between endpoints (Data not shown).
The in vivo values of L. calcarifer vs. in vitro data of its two cell lines exposed to chromium were highly significant p<0.0l with R2=0.956 (L. calcarifer vs. SISS) and 0.962 (L. calcarifer vs. SISK); R2=0.973 and 0.993; R2=0.980 and 0.975; R2=0.992 and 0.977 for MTT (Fig 3A), NR (Fig 3B), AB(Fig 3C) and CB (Fig 3D), respectively. The in vivo values of E. suratensis were compared with in vitro values of its three cell lines (IEE, IEG and IEK) exposed to chromium and were found to be highly significant p<0.0l with R2=0.985 (E. suratensis vs. IEE), 0.987 (E. suratensis vs. IEK) and 0.968 (E. suratensis vs. IEG); R2=0.980, 0.936 and 0.956; R2=0.961, 0.955 and 0.904 and R2=0.955, 0.939 and 0.974 for MTT (Fig 3E), NR (Fig 3F), AB(Fig 3G) and CB (Fig 3H), respectively. Linear correlations between each in vitro vs. in vivo (C. catla)values of chromium were highly significant p<0.0l with R2=0.991 (C. catla vs, SICH) and 0.993 (C. catla vs, ICG); R2=0.982 and 0.983; 0.974 and 0.990 and 0.987 and 0.984 for MTT (Fig 3I), NR (Fig 3J), AB(Fig 3K) and CB (Fig 3L), respectively.
The prominent morphological changes of the cells exposed to high concentrations of chromium were observed. The changes observed include cell shrinkage, cell detachment, vacuolations and cell swelling in SISS (Fig 4H), SISK (Fig 4I), IEE (Fig 4J), IEK (Fig 4L), IEG (Fig 4L), SICH (Fig 4M) and ICG (Fig 4N) cell lines. In controls, no morphological alterations were observed in the SISS (Fig 4A), SISK (Fig 4B), IEE (Fig 4C), IEK (Fig 4D), IEG (Fig 4E), SICH (Fig 4F) and ICG (Fig 4G) cell lines.
The percentage of DNA damage and the cumulative tail length from 100 cells per sample were measured in SISS, SISK, IEE, IEK, IEG, SICH and ICG cells exposed to different concentrations of chromium (0, 10, 20, 30, 40 and 50 mg/L) and the results are shown in Fig. 5. The length of tail DNA in SISS, SISK, IEE, IEK, IEG, SICH and ICG cells exposed to 10 mg/L of chromium was estimated to be about 1.7%, 2.0%, 1.3%, 1.5%, 2.1%, 1.4% and 1.5%, respectively at a 24-h exposure, and chromium at the concentration of 50 mg/L caused 8.9%, 11.0%, 9.4%, 8.8%, 11.1%, 6.4% and 7.2% of tail DNA migration in SISS, SISK, IEE, IEK, IEG, SICH and ICG cells, respectively (Fig. 5). Comet results of chromium exposed SISS, SISK, IEE, IEK, IEG, SICH and ICG cells showed a dose dependent increase in tail DNA (%) compared to the control cells, which gave the extent of DNA damage.
The SISS, SISK, IEE, IEK, IEG, SICH and ICG cells were exposed to chromium for 24 h at different concentrations (0, 10, 20, 30, 40 and 50 mg/L) and the results are shown in Fig. 6A-N. Apoptotic cells were identified by Hoechst staining of condensation and fragmentation of the nuclei as shown in SISS cells (Fig. 6H), SISK cells (Fig. 6I), IEE cells (Fig. 6J), IEK cells (Fig. 6K), IEG cells (Fig. 6L), SICH cells (Fig. 6M) and ICG cells (Fig. 6N) at higher concentration i.e. 50 mg/L of chromium exposed for 24 h, while no nuclear changes were observed in control cells are shown in SISS cells (Fig. 6A), SISK cells (Fig. 6B), IEE cells (Fig. 6C), IEK cells (Fig. 6D), IEG cells (Fig. 6E), SICH cells (Fig. 6F)and ICG cells (Fig. 6G).
The level of antioxidant parameters such as SOD, CAT, GPx, GSH and LPO was measured in SISS, SISK, IEE, IEK, IEG, SICH and ICG cells exposed to different concentrations of chromium and the results were presented in Fig 7A-E. Regarding oxidative stress biomarkers, no significant change was observed in SOD, CAT, GSH and LPO levels in the SISS, SISK, IEE, IEK, IEG, SICH and ICG cells exposed to lower concentrations i.e. 10 mg/L of chromium when compared to the control cells. However, when these cell lines were exposed to 50 mg/L of chromium, the activity of SOD (~2.1, ~2.3, ~1.5, ~1.3, ~2.3, ~1.2 and ~2.2 fold in SISS, SISK, IEE, IEK, IEG, SICH and ICG cells respectively in Fig 7A), CAT (~5.2, ~6.8, ~5.3, ~7.4, ~6.4, ~5.2 and ~4.6 fold in SISS, SISK, IEE, IEK, IEG, SICH and ICG cells respectively Fig 7B) and level GSH (~1.6, ~1.5, ~1.3, ~1.6, ~1.5, ~1.8 and ~1.3 fold in SISS, SISK, IEE, IEK, IEG, SICH and ICG cells respectively Fig 7C) and GPx (~1.2, ~1.1, ~1.0, ~1.2, ~1.1, ~0.9 and ~1.3 fold in SISS, SISK, IEE, IEK, IEG, SICH and ICG cells respectively Fig 7D) decreased was found to be significantly (*P<0.05) in all the cell lines when compared to the control cell lines. Whereas the level of LPO (~1.3, ~1.5, ~1.0, ~1.4, ~1.2, ~1.4 and ~0.7 fold in SISS, SISK, IEE, IEK, IEG, SICH and ICG cells respectively Fig 7E) was increased and found to be significant (*P<0.05) in all the cell lines when compared to the control cell lines. Chromium resulted in the dose-dependent decrease of SOD, CAT, GPx, GSH levels and LPO increased, which indicated the oxidative stress in the SISS, SISK, IEE, IEK, IEG, SICH and ICG cells.
Heavy metals constitute a main group of aquatic pollutants due to their bioacuumulative and non-biodegradable properties (Velma and Tchounwou 2010). Their excessive contamination of aquatic ecosystems has evoked major environmental and health concerns worldwide (Vutukuru et al. 2007). Chromium is the sixth most abundant heavy metal in the earth crust (U.S. EPA 1984). Fish and Fish cell lines constitute an excellent model to understand the mechanistic aspects of metal toxicity (Taju et al. 2014). In this study, we have examined the in vivo toxicity in three fish species in different environment i.e. L. calcarifer (Marine water), E. suratensis (brackish water) and C. catla (Fresh water), and in vitro cytotoxicity, oxidative stress and genotoxicity of the three same fish cell lines, SISS, SISK (Seabass spleen and kidney cell lines), IEE, IEK, IEG (Etroplus eye, kidney and gill cell lines), SICH and ICG (Catla heart and gill cell lines) an exposure to chromium. The results of this study clearly show that the fish cell lines experienced oxidative stress by modulating the antioxidant enzyme, exhibited DNA damage, nuclear fragmentation and microscopic morphological changes in the SISS, SISK, IEE, IEK, IEG, SICH and ICG cells. The LC50 values of chromium were determined as 30.22, 33.83 and 30.64 mg/L respectively in L. calcarifer, E. suratensis and C. catla, respectively at 96 h of exposure in this study. Recently, Mishra and Mohanty (2009) reported the LC50 values of chromium on Channa punctatus at 96 h of exposure as 41.75 mg/L. The LC50 values observed by Mishra and Mohanty (2009) were found to be higher when compared to L. calcarifer, E. suratensis and C. catla and this indicates that the L. calcarifer, E. suratensis and C. catla were found to more sensitive to chromium.
Seven fish cell lines derived from L. calcarifer (SISS & SISK), E. suratensis (IEE, IEK and IEG) and C. catla (SICH and ICG) were applied to evaluate the cytotoxicity of chromium using MTT, AB, NR and cell protein (CB) assays. The results of in vitro assays were compared with the results of in vivo test to determine the suitability of these fish cell lines for toxicological studies to replace the use of whole fish. The evaluation of cytotoxicity of chemical substances using animal cells has been carried out by many workers (Ekwall 1980a, 1983; Metcalfe 1971; Muir 1983a, 1983b; Paganuzzi et al. 1981; Benoit et al. 1987). Four commonly used endpoint assays (MTT, NR AB and cell protein assay CB) were employed in the present study using SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines of E. suratensi, C. catla and L. calcarifer to evaluate the in vitro cytotoxicity of chromium. The main observation was that the cytotoxicity was closely associated in all the seven cell lines independent of the toxic endpoints employed. This not only supports the observations of Ekwall (1995) and Li and Zhang (2002) that most cell lines have a similar results to toxicants when toxicity is measured by different endpoints, corresponding to inhibition or destruction of basal functions and structures, and also suggests that endpoints employed in the present study can also be used to predict acute cytotoxicity. Tan et al. (2008) have used six fish cell lines to study the toxicity of four heavy metals: cadmium, chromium, zinc, and copper by using two cytotoxicity endpoints MTT and CB assays. The results revealed that carp epithelioma cells are least tolerant to chromium. The NR uptake assay is a useful method for comparing the relative acute cytotoxicity of metals in vitro with metal and chemicals toxicity studies in whole fish in vivo (Brandao et al. 1992; Ryan and Hightower 1994; Taju et al. 2013). In the present study, we employed that SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines for cytotoxicity assessment of chromium by using four endpoints. Our results show that there is no significant difference between all the four endpoints.
Segner (1994) reported that the relationship of the in vitro cytotoxicity values to in vivo fish toxicity data is less satisfying and that this might be due to the inconsistency of the in vivo values. As observed in the present study, a positive relationship of acute lethal potency in fish with in vitro cytotoxicity has been found by Fry et al. (1990). Castano et al. (1996) found good correlations between in vivo and in vitro for each endpoint and 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 correlation of in vitro cytotoxicity of metals with in vivo toxicity data was evaluated by comparing the 24 h NR50 results of R1 cells to 96 h LC50 data of different fish species. The rvalues (R1 cell line) were 0.64 for the relation between LC50, data of golden ide and bluegill sunfish, 0.58 for golden ide and rainbow trout in soft water, and 0.68 for golden ide and rainbow trout in hard water (Segner et al. 1994). In the present study, in vitro cytotoxicity of chromium with in vivo results was evaluated by comparing the 24 h MTT, NR, AB and CB data of seven Indian fish cell lines to 96 h data of three fish species (L. calcarifer, E. suratensis and C. catla). A good correlation was found between in vitro of seven fish cell lines compared with in vivo values of whole fish exposed to chromium for 24 h and 96 h respectively, with r=0.902 to 0.99. The results revealed that the four endpointsvalues were closely correlated to whole fish in vivo values and that the linear correlation between each in vitro parameter and the in vivo data were found to be highly significant. The results of in vitro assays using SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines of E. suratensis, C. catla and L. calcarifer were correlated with those obtained from in vivo assay using the same species of fish (L. calcarifer, E. suratensis and C. catla). Based on the results of the present study we recommend the use of these seven cell lines instead of living fish for toxicity assessment of metal salts and environmental contaminants.
The present study showed that chromium induced genotoxicity in SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines by comet assay. DNA damage was observed in SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines exposed to chromium in a concentration dependent manner. The DNA damage at higher test concentrations in SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines could be due to the elevated levels of tail DNA in all cell lines compared to their controls cells. Induction of ROS under metallic stress could attack the DNA and damage its integrity. Our present results are similar to the previous reports (Iqbal Ahmad et al. 2006; Velma and Tchounwou 2010; 2013) DNA damage in gill and kidney of Anguilla anguilla L. exposed to chromium with or without pre-exposure to Î²-naphthoflavone. In another study, medaka fin cell lines exposed to Cr (VI) to examine the genotoxic potentials, have observed DNA double strand breaks and chromosome damage in a concentration dependent manner (Goodale et al. 2008). Abbas et al (2007) studied the genotoxic potential of Cr (VI) in Orechromis spp. The fish were exposed to sub lethal concentrations (43.7 mg/L) of Cr (VI) for 24 and 96h. The results showed polymorphic bands in long time exposed fish whereas no polymorphic bands in short time exposed fish were seen (Velma and Tchounwou, 2010).
The Hoechst 33342 is a widely used in vitro genotoxicity testing in fundamental research for assessing nuclear fragmentation and condensation nuclei for different industrial chemicals, environmental biomonitoring and human population monitoring (Speit and Rothfuss 2012, Taju et al. 2014). In the present study, the Hoechst stain showed how chromium can induce genotoxic damage in SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines. Our nuclear fragmentation results compare favourably with recent studies on mammalian cells (CHO-K1) which have been used to investigate the genotoxic potential of chromium (VI) at different concentrations (Thompson et al. 2012). Thompson et al. (2012) conducted the nuclear fragmentation assay assays to confirm the genotoxicity of chromium in CHO-K1 cells, and reported a dose-dependent nuclear damage in the Hoechst assay. In the current work, nuclear fragmentation and condensation of nuclei was observed dose-dependent manner in all the seven fish cell lines exposed to chromium by Hoechst.
In the present study antioxidant parameters such as CAT, SOD, GSH and GPx activity decreased significantly in SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines exposed to different concentration of chromium when compared to the control cells. The decrease was found to be concentration dependent manner. The current study confirms the role of oxidant-antioxidant imbalance as a result of chromium exposure in SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines. All the seven fish cell lines had elevated level of LPO, decreased expression of non-enzymatic antioxidant (GSH) and as well as enzymatic antioxidants (SOD, CAT and GPx) compared to control cell lines. Our result is in conformity with the report of Velma and Tchounwou, (2010) who reported in vivo chromium induced oxidative stress in liver and kidney tissues of Goldfish, Carassius auratus as evidenced by the decreased level of CAT, SOD and GPx levels and increased level of LPO. Recently Kumari et al. (2014) reported a significant decrease oxidative biomarkers CAT and SOD activities, and increased level of GSSG-R in liver, gill, muscle and brain tissues of Labeo rohita exposed to chromium at different time intervals. According to another report, oxidative biomarkers such as CAT, SOD, GSH levels was significantly decreased and LPO level increased in Labeo rohita exposed to different heavy metals at different time interval (Khalid et al. 2015).
In future, fish cell lines will be utilized as a biological model for evaluating the cytotoxicity, genotoxic effect and oxidative stress of chemical pollutants in environmental samples and will also become a standard practice in toxicological study. The advantages of using in vitro assay include time saving, low cost, ease of operation and reduction in the number of fish required.
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