Comparative evaluation of the cytotoxicity sensitivity of six fish cell lines to four heavy metals in vitro

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Toxicology in Vitro 22 (2008) 164–170 www.elsevier.com/locate/toxinvit

Brief communication

Comparative evaluation of the cytotoxicity sensitivity of six fish cell lines to four heavy metals in vitro Fengxia Tan a, Min Wang a

a,*

, Weimin Wang a, Yuanan Lu

a,b,*

Animal Cell Culture Laboratory, Fisheries College, Huazhong Agriculture University, Wuhan, Hubei 430070, PR China b Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu, HI 96822, USA Received 26 July 2007; accepted 20 August 2007 Available online 7 September 2007

Abstract To establish the potential use of cell cultures as a simple and sensitive biological tool to detect environmental pollutants, six cell lines established from several fish species including GCF (grass carp fins), CIK (Ctenopharyngodon idellus kidney), EPC (epithelioma papulosum cyprini), CCO (channel catfish ovary), BB (brown bullhead caudal trunk) and FHM (fathead minnow muscle) were tested and compared for their cytotoxic sensitivity to four heavy metals: cadmium (Cd), chromium (Cr), zinc (Zn), and copper (Cu). Following a 24-h exposure to these metal salts at selected concentrations, test cells were characterized by morphology, viability and proliferation. Our results indicate that all these metal salts are cytotoxic to these fish cell lines, but at varied levels. Calculated inhibitory concentration (IC50) values revealed that the cytotoxicity of Cr and Cd was significantly more pronounced than that of the other two metal salts. Comparative analysis of these fish cell lines showed that C. idellus kidney (CIK) cells are the most sensitive cell line to copper, epithelioma papulosum cyprini (EPC) cells are more sensitive than other cells to Cr and Zn, while channel catfish ovary (CCO) cell line is the most sensitive one to Cd. In conclusion, CIK, EPC and CCO could potentially be sensitive bio-indicators for the initial monitoring and assessment of acute cytotoxicity of heavy metals in the aquatic environment.  2007 Published by Elsevier Ltd. Keywords: In vitro cytotoxicity; Fish cell line; Heavy metal; Aquatic environment, 50% inhibition concentration (IC50)

1. Introduction

Abbreviations: BB, a fish cell line derived from brown bullhead muscle; BSA, Bovine Serum Albumin; CB, Coomassie Blue; CCO, a fish cell line derived from channel catfish ovary; Cd, cadmium; CIK, a fish cell line derived from grass carp kidney; Cr, chromium; Cu, copper; DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO, dimethyl sulfoxide; EPC, a fish cell line derived from common carp epithelioma; FHM, a fish cell line derived from fathead minnow muscle; GCF, a fish cell line derived from grass carp fins; IC50, 50% inhibition concentration; M199, Medium 199; MEM, Minimal Essential Medium; MTT, thiazolyl blue tetrazolium bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NR, neutral red; RT, room temperature; NCS, new cattle serum; Zn, zinc. * Corresponding authors. Address: Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu, HI 96822, USA. Fax: +1 808 956 5818 (Y. Lu); +86 27 8728 2465 (M. Wang). E-mail addresses: [email protected] (M. Wang), ylu@pbrc. hawaii.edu (Y. Lu). 0887-2333/$ - see front matter  2007 Published by Elsevier Ltd. doi:10.1016/j.tiv.2007.08.020

A large number of chemical pollutants enter the aquatic environment each year. These pollutants, which include some heavy metals, are known to be potentially cytotoxic to biota and present a health threat to the public. Thus, establishment of a sensitive biological monitoring system for early detection and ecotoxicological evaluation is required. Due to the inherent economic and ethical constraints associated with in vivo tests using live organisms, the utilization of in vitro biological test systems, such as cell cultures derived from aquatic animal species, is preferred. Applying cell cultures to ecotoxicological assessment offers a number of well-described advantages as compared to in vivo animal tests (Fryer and Lannan, 1994). Culturing established fish cells in vitro is relatively rapid, cost-effective, readily reproducible, and can be easily adapted to

F. Tan et al. / Toxicology in Vitro 22 (2008) 164–170

automated high-throughput screening technologies (Castano and Gomez-Lechon, 2005; Castano et al., 2003; Fent, 2001). These characteristics make it feasible to examine a large variety of chemical pollutants in environmental samples. Additionally, in vitro cytotoxicity assessments can be readily employed to examine multiple parameters, including measurements of cell death, viability, morphology, metabolism, cell attachment/detachment, cell membrane permeability, proliferation and growth kinetics (Maracine and Segner, 1998; Schirmer et al., 1998; Li and Zhang, 2001; Nı´ Shu´illeabha´in et al., 2004). Furthermore, in vitro fish cell assays are able to generate comparable results on relative potency ranking and effect of toxicants, allowing employment of cell culture systems to reduce, define and replace in vivo acute lethality tests (Segner, 2004). An initial attempt to use a fish cell line to study aquatic toxicants was conducted by Rachlin and Perlmutter who examined the possibility of using the muscle cells of fathead minnow to assess the cytotoxicity of Zn2+(Rachlin and Perlmutter, 1968). Establishment of more and more cell cultures from marine and mammalian species has promoted the rapid development of cell cultures as sensitive acute bioassays for the assessment of toxicological risks associated with chemical pollutants worldwide (Keddy et al., 1995; Ma´tlova´ et al., 1995; Huuskonen et al., 1998; Segner, 1998; Olabarrieta et al., 2001; Repetto et al., 2001; Choi and Oris, 2003; Nı´ Shu´illeabha´in et al., 2004; Segner, 2004; Wang et al., 2004; G} ulden et al., 2005). In 1995, Ekwall proposed that a lethal dose of chemicals in environmental samples is the dose that actually kills an organism through toxicity at the cellular level (i.e., cytotoxicity). This forms the principal basis for relating in vitro cytotoxicity data to in vivo acute lethality (Ekwall, 1995). Since then, many tests have been conducted to explore the potential use of cell culture based cytotoxicity tests as an essential alternative to in vivo experimental assays (Bru¨schweiler et al., 1995; Castano et al., 1995, 1996; Dierickx and Bredael-Rozen, 1996; Vega et al., 1996; Clemedson et al., 1998; Segner, 1998; Fent, 2001; Repetto et al., 2001; Castano et al., 2003; Segner, 2004). Today, employment of fish cell lines as in vitro biological methods for assessing the ecotoxicological effects of pollutant chemicals in the environment is becoming an established technique for the regular practice of predicting any acute cytotoxicity in vivo (Segner, 1998; Fent, 2001; Castano et al., 2003; Caminada et al., 2006). In the near future, it is anticipated that the utilization of fish cell lines as a biological model for evaluating the cytotoxicity of pollutant chemicals in environmental samples will become a standard practice. To facilitate the use of established in vitro cell lines from aquatic animals as valuable biological tools to monitor and detect environmental pollutant chemicals, it is necessary to test more cell lines comparatively to identify the more sensitive cell cultures as bio-indicators to common toxic chemicals. To address this, six fish cell lines were tested for their sensitivity to four heavy metal salts (cadmium, chromium, zinc and copper) commonly reported in China’s aquatic

165

environment. The tested cell lines included GCF (grass carp fin) and CIK (Ctenopharyngodon idellus kidney), two established cell lines from the grass carp, which is one of the four major fish species found in China. These cell lines were also compared to four other previously established cell lines, which included BB (brown bullhead), CCO (channel catfish ovary), EPC (epithelioma of carp) and FHM (fathead minnow). In this paper, we describe the employment of the MTT (thiazolyl blue tetrazolium bromide) and CB (Coomassie Blue) assays and demonstrate a diversified sensitivity of these cell lines to the four tested metal salts. This new information will be useful for future selection of fish cell lines for cytotoxicity assessment of particular environmental pollutants. 2. Materials and methods 2.1. Chemicals Four metal salts of analytical grade were used in this cytotoxic study, including: CdCl2 Æ 2.5H2O, K2Cr2O7, ZnCl2, CuSO4 Æ 5H2O. These metals were selected for cytotoxic evaluation in fish cell lines because they are commonly found in the aquatic environment and possess ecotoxicological potential. Stock solutions of these metal salts were prepared with sterile ddH2O to a concentration of 0.1 M. For the cytotoxicity assays, ten serial dilutions of each salt stock were made in the selected cell culture medium to final concentrations of 1, 3, 5, 7, 10, 30, 50, 70, 100 and 150 lM for Cd and Cr, and 10, 30, 50, 70, 100, 200, 300, 400, 500, and 600 lM for Cu and Zn, respectively. 2.2. Cell culture 2.2.1. Cell culture medium Three cell culture media, including M199 (Medium 199), MEM (Minimal Essential Medium), and DMEM (Dulbecco’s Modified Eagle’s Medium), were used to culture selected fish cells in this study. These media were supplemented with 10% (v/v) of new cattle serum (NCS) (Gibco, UK), penicillin (100 IU/ml) and streptomycin (100 lg/ml) (Sigma-Aldrich, UK). Coomassie Blue G-250, BSA (Bovine Serum Albumin) and MTT were from Amresco (USA). 2.2.2. Fish cell lines A total of six cell lines established from different fish species were tested for their sensitivities to the four metal salts (Table 1). These fish cells were propagated at 25– 28 C in one of the three cell growth media and subcultured every 3–4 days. Cells at exponential growth phase were harvested for the cytotoxicity tests. 2.3. Cytotoxicity assays Cytotoxicity assays were performed in 96-well tissue culture plates (Greiner, Germany). Two types of assays were

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Table 1 A list of fish cell lines used in this cytotoxicity assays Nameninfo

Species

Tissue origin

Growth medium

Incubation temperature (C)

Reference

BB CCO CIK EPC FHM GCF

Brom bullhead (Ictalurus nebulosus) Channel catfish (Ictalurus punctatus) Grass carp (Ctenopharyngodon idellus) Carp (Ctenopharyngodon cyprini) Fathead minnow (Pimephales promelas) Grass carp (Ctenopharyngodon idella)

Caudal trunk Ovary Kidney Epithelioma Muscle Fins

MEM + 10% NCS DMEM + 10% NCS M199 + 10% NCS MEM + 10% NCS M199 + 10% NCS MEM + 10% NCS

25 28 26 25 26 25

Wolf and Quimby (1969) Bowser and Plumb (1980) Zuo et al. (1986) Fijan et al. (1983) Gravell and Malsberger (1965) Lu et al. (1990)

employed to measure cell viability and cellular protein content. Fish cells at exponential proliferation phase were harvested, seeded in 96-well plates at 2 · 104 cells/well in 100 ll of cell medium, and incubated for 24 h at their optimal growth temperature. Prior to metal exposure, the growth medium was discarded and replaced with the medium containing varied concentrations of heavy metal (eight wells/concentration) and incubated for 24 h. Wells containing no cells but just cell culture medium alone (blank controls) and cells with medium only (negative control) were used as controls on each 96-well plate. The cytotoxicity was determined in at least three independent experimental tests that were performed eight times per experiment for each of the metals. 2.3.1. Cell viability by MTT reduction assay The MTT test is based on the cellular uptake of MTT and its subsequent reduction in the mitochondria of living cells to MTT formazan (a dark, water insoluble, and blue product). Dead cells are almost completely negative for this cleavage activity. Following the 24-h exposure to the toxic chemicals, cells were incubated with 25 ll/well of MTT solution (5 mg/ml) in selected media for 4 h at 26 ± 1 C. The MTT-containing medium was removed and the intracellular formazan crystals were solubilized and extracted with DMSO (dimethyl sulfoxide). After incubation for 15 min at room temperature (RT), the plates were transferred to the microplate reader (BIO-680, BIORAD, USA) to measure the absorbance of the extracted solution at 570 nm (Gonc¸alves et al., 1998). Cell viability was expressed as a fraction of the negative control (cells with medium only). 2.3.2. Coomassie Blue dye protein assay The Coomassie Blue assay is a method to measure the total amount of cellular proteins based on the binding of protein molecules to Coomassie dye under acidic condition (Shopsis and Eng, 1985). In brief, following exposure to the toxic chemicals, the medium was removed from the plates and the cells were washed with PBS and lysed with 50 ll/ well of 0.1 M NaOH. The plates were incubated at 26 ± 1 C for 1 h and then 200 ll of Coomassie Blue solution (10 mg Coomassie Blue G-250, dissolved in 5 ml of 95% ethanol and 10 ml of 85% phosphoric acid, and then diluted to the final volume of 100 ml with distilled water) were added to each well. After an additional 20 min incu-

bation at RT, the absorbance of each well was measured at 570 nm. Serial dilutions of 1–100 mg/ml BSA dissolved in 0.1 M NaOH were used for the protein standard. Cell viability was expressed as a fraction of the negative control (cells with medium only) (Li and Zhang, 2001). 2.4. Data analysis Experiments were performed in triplicate with eight replicates for each exposure concentration. Data was statistically analyzed with Statistic 6.0 (StatSoft Software, USA). The individual data points of the concentration– response cytotoxicity tables are presented as the arithmetic mean percent inhibition relative to the control standard deviation (SD). Cell viability and the logarithm of concentration were fitted with the recursive equation (a linear regression model). The IC50-value was deduced from the normalized data and the recursive equation. 3. Results and discussion The MTT and CB cytotoxicity assays revealed that a 24h exposure of the six cell lines to different concentrations of the four heavy metals produced a dose-dependent reduction in the fraction of viable cells. The dose-response to each heavy metal is given in Tables 2 and 3. We considered the lowest concentration of each heavy metal that had a significant difference from the control (P < 0.05, t-test) to be the lowest cytotoxic dose. The results of the MTT assay showed that the lowest cytotoxic concentrations of cadmium in the six cell lines ranged from 3.0 lM to 7.0 lM. The CCO cells show the most sensitivity at 3.0 lM; the BB and CIK cells are affected at 5.0 lM; and for cell lines EPC, FHM, and GCF, the concentration threshold is 7.0 lM (Table 2). The lowest toxic concentrations of chromium against these six cells ranged from 3.0 lM to 10.0 lM. Under described experimental conditions, BB, EPC, and GCF are more sensitive to chromium than the other three cell lines (Table 2). The lowest toxic concentrations of zinc for these six cell lines varied from 10 lM to 70 lM (Table 3). As indicated in the Table 3, EPC cells appear to be the most sensitive, followed by BB, FHM, and then CIK, while CCO and GCF are the least sensitive ones. The lowest toxic concentrations for copper ranged from 30 lM to 300 lM for these fish cell lines. The cytotoxic effect of this chemical was detected at 30 lM for

F. Tan et al. / Toxicology in Vitro 22 (2008) 164–170

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Table 2 Viability (%) of six cell lines exposed to cadmium (CdCl2 Æ 2.5H2O) and chromium (K2Cr2O7) Conc. (lM)

BB (MTT)

(CB)

(MTT)

(CB)

(MTT)

(CB)

(MTT)

(CB)

(MTT)

(CB)

(MTT)

(CB)

Cd 0 1 3 5 7 10 30 50 70 100 150

100 109 ± 9 89 ± 5 76 ± 3* 67 ± 3* 58 ± 2* 30 ± 7* 17 ± 4* 8 ± 2* 2 ± 2* 0 ± 6*

100 105 ± 11 99 ± 12 83 ± 8 72 ± 6* 61 ± 5* 25 ± 5* 8 ± 10* 0 ± 2* 0 ± 2* 0 ± 6*

100 99 ± 6 76 ± 4* 65 ± 3* 58 ± 2* 50 ± 1* 27 ± 6* 16 ± 3* 9 ± 1* 2 ± 1* 0 ± 4*

100 100 ± 4 79 ± 11 68 ± 8* 61 ± 7* 54 ± 5* 31 ± 11* 20 ± 6* 14 ± 3* 6 ± 3* 0 ± 4*

100 109 ± 2* 96 ± 6 91 ± 3* 82 ± 3* 72 ± 2* 42 ± 1* 28 ± 1* 19 ± 3* 9 ± 3* 0 ± 1*

100 109 ± 7 95 ± 9 84 ± 7 77 ± 5* 69 ± 4* 45 ± 10* 34 ± 5* 27 ± 2* 19 ± 1* 10 ± 5*

100 113 ± 7 101 ± 2 99 ± 1 89 ± 1* 79 ± 9* 46 ± 5* 31 ± 3* 21 ± 1* 10 ± 1* 0 ± 2*

100 118 ± 12 107 ± 7 93 ± 5 84 ± 4* 73 ± 3* 43 ± 8* 28 ± 4* 19 ± 1* 9 ± 5* 0 ± 1*

100 107 ± 13 97 ± 6 94 ± 6 86 ± 5* 78 ± 4* 51 ± 2* 39 ± 1* 31 ± 1* 23 ± 1* 13 ± 1*

100 119 ± 3* 106 ± 7 101 ± 5 91 ± 4 81 ± 3* 48 ± 8* 33 ± 4* 23 ± 1* 12 ± 2* 0 ± 5*

100 108 ± 7 94 ± 8 89 ± 8 79 ± 7* 68 ± 5* 35 ± 1* 19 ± 2* 9 ± 4* 0 ± 4* 0 ± 4*

100 111 ± 12 106 ± 7 89 ± 5 78 ± 4* 67 ± 3* 32 ± 7* 15 ± 3* 4 ± 1* 0 ± 3* 0 ± 6*

Cr 0 1 3 5 7 10 30 50 70 100 150

100 100 ± 3 90 ± 1* 79 ± 10* 72 ± 9* 64 ± 7* 41 ± 3* 30 ± 2* 23 ± 2* 16 ± 1* 7 ± 2*

100 105 ± 7 91 ± 4 80 ± 3* 72 ± 2* 64 ± 2* 40 ± 6* 29 ± 3* 22 ± 2* 14 ± 2* 5 ± 4*

100 114 ± 12 107 ± 6 98 ± 4 90 ± 11 81 ± 2* 69 ± 10* 50 ± 5* 38 ± 8* 25 ± 8* 10 ± 9*

100 116 ± 10 112 ± 7 102 ± 6 99 ± 6 95 ± 5 81 ± 7* 61 ± 12* 48 ± 7* 34 ± 2* 18 ± 4*

100 108 ± 11 103 ± 8 98 ± 7 95 ± 7 85 ± 3* 53 ± 6* 38 ± 2* 29 ± 3* 18 ± 1* 7 ± 6*

100 106 ± 17 105 ± 10 100 ± 2 92 ± 7 82 ± 6* 52 ± 4* 38 ± 10* 28 ± 5* 19 ± 2* 7 ± 5*

100 90 ± 6 58 ± 11* 43 ± 5* 33 ± 2* 23 ± 1* 0 ± 1* 0 ± 2* 0 ± 3* 0 ± 4* 0 ± 6*

100 100 ± 6 66 ± 5* 50 ± 3* 40 ± 4* 28 ± 4* 17 ± 7* 15 ± 2* 8 ± 2* 4 ± 4* 1 ± 2*

100 102 ± 9 95 ± 9 97 ± 7 84 ± 8 71 ± 4* 29 ± 11* 9 ± 9* 0 ± 7* 0 ± 7* 0 ± 8*

100 105 ± 6 100 ± 5 99 ± 4 95 ± 3 81 ± 3* 36 ± 2* 15 ± 8* 2 ± 2* 0 ± 2* 0 ± 7*

100 100 ± 4 85 ± 2* 73 ± 1* 65 ± 1* 56 ± 6* 29 ± 9* 17 ± 5* 9 ± 10* 0 ± 5* 0 ± 2*

100 95 ± 7 79 ± 4* 67 ± 6* 59 ± 5* 50 ± 6* 24 ± 5* 12 ± 4* 4 ± 5* 0 ± 2* 0 ± 4*

*

CCO

CIK

EPC

FHM

GCF

Significant difference from treatment without metal (P < 0.05, t-test).

Table 3 Viability (%) of six cell lines exposed to zinc (ZnCl2) and copper (CuSO4 Æ 5H2O) Conc. (lM)

BB

CCO

CIK

EPC

FHM

GCF

(MTT)

(CB)

(MTT)

(CB)

(MTT)

(CB)

(MTT)

(CB)

(MTT)

(CB)

(MTT)

(CB)

Zn 0 10 30 50 70 100 200 300 400 500 600

100 100 ± 7 78 ± 5* 62 ± 4* 52 ± 3* 42 ± 2* 21 ± 1* 9 ± 1* 0 ± 2* 0 ± 3* 0 ± 5*

100 100 ± 6 79 ± 4* 64 ± 3* 53 ± 3* 43 ± 1* 22 ± 3* 10 ± 3* 1 ± 3* 0 ± 3* 0 ± 2*

100 112 ± 12 95 ± 8 91 ± 10 77 ± 8* 63 ± 5* 35 ± 7* 18 ± 2* 7 ± 4* 0 ± 4* 0 ± 3*

100 112 ± 15 103 ± 7 100 ± 4 86 ± 2* 70 ± 11* 39 ± 3* 21 ± 2* 8 ± 5* 0 ± 7* 0 ± 10*

100 100 ± 2 78 ± 11 65 ± 9* 56 ± 7* 48 ± 2* 30 ± 11* 20 ± 3* 13 ± 5* 7 ± 4* 3 ± 5*

100 100 ± 9 81 ± 6* 69 ± 5* 61 ± 6* 53 ± 3* 37 ± 1* 27 ± 4* 21 ± 1* 15 ± 4* 11 ± 2*

100 78 ± 4* 53 ± 3* 42 ± 2* 34 ± 2* 27 ± 1* 11 ± 1* 2 ± 1* 0 ± 2* 0 ± 4* 0 ± 7*

100 80 ± 7* 56 ± 5* 45 ± 4* 38 ± 3* 30 ± 3* 15 ± 3* 6 ± 3* 0 ± 3* 0 ± 2* 0 ± 2*

100 98 ± 2 71 ± 2* 56 ± 3* 47 ± 4* 37 ± 5* 17 ± 2* 6 ± 3* 0 ± 2* 0 ± 1* 0 ± 4*

100 95 ± 10 73 ± 7* 58 ± 5* 49 ± 6* 38 ± 3* 18 ± 3* 7 ± 4* 0 ± 5* 0 ± 4* 0 ± 2*

100 106 ± 15 98 ± 8 96 ± 8 82 ± 6* 67 ± 1* 38 ± 7* 21 ± 6* 9 ± 4* 0 ± 5* 0 ± 3*

100 107 ± 10 100 ± 7 98 ± 5 83 ± 4* 68 ± 3* 38 ± 4* 21 ± 5* 9 ± 4* 0 ± 2* 0 ± 2*

Cu 0 10 30 50 70 100 200 300 400 500 600

100 113 ± 13 110 ± 7 106 ± 8 98 ± 8 93 ± 8 82 ± 10 62 ± 4* 47 ± 4* 36 ± 7* 27 ± 10*

100 115 ± 8 106 ± 5 105 ± 4 101 ± 3 92 ± 2* 85 ± 12 63 ± 9* 48 ± 8* 36 ± 7* 27 ± 6*

100 104 ± 10 100 ± 8 90 ± 7 81 ± 6* 73 ± 5* 56 ± 3* 46 ± 3* 39 ± 3* 33 ± 2* 29 ± 3*

100 109 ± 15 102 ± 12 90 ± 10 82 ± 9 73 ± 8* 56 ± 4* 47 ± 5* 40 ± 4* 34 ± 3* 30 ± 3*

100 98 ± 3 76 ± 3* 60 ± 2* 50 ± 2* 39 ± 5* 18 ± 3* 5 ± 3* 0 ± 2* 0 ± 2* 0 ± 4*

100 99 ± 7 76 ± 8* 62 ± 5* 52 ± 3* 41 ± 7* 21 ± 8* 10 ± 7* 1 ± 10* 1 ± 7* 1 ± 7*

100 100 ± 7 76 ± 6* 61 ± 5* 51 ± 6* 40 ± 5* 20 ± 4* 8 ± 3* 0 ± 4* 0 ± 2* 0 ± 4*

100 104 ± 9 89 ± 7 73 ± 6* 62 ± 6* 51 ± 5* 29 ± 4* 16 ± 5* 7 ± 3* 0 ± 3* 0 ± 2*

100 100 ± 3 80 ± 3* 67 ± 2* 59 ± 2* 50 ± 2* 33 ± 1* 23 ± 1* 16 ± 1* 11 ± 1* 6 ± 1*

100 96 ± 11 85 ± 9 71 ± 5* 62 ± 3* 52 ± 6* 32 ± 4* 21 ± 5* 13 ± 6* 7 ± 7* 2 ± 8*

100 116 ± 16 114 ± 18 103 ± 15 95 ± 14 78 ± 9 27 ± 11* 0 ± 15* 0 ± 9* 0 ± 5* 0 ± 1*

100 118 ± 7* 103 ± 11 104 ± 5 98 ± 4 90 ± 5 47 ± 10* 16 ± 8* 0 ± 5* 0 ± 2* 0 ± 6*

*

Significant difference from treatment without metal (P < 0.05, t-test).

CIK, EPC, and FHM, 70 lM for CCO and nearly 200 lM or more for BB and GCF (Table 3). Comparative analyses of cytotoxicity in these fish cell lines indicate that copper is the least cytotoxic. Amidst

the lowest toxic concentration data shown in Tables 2 and 3, each heavy metal at lower and non-cytotoxic concentrations, such as 1.0 lM for cadmium and chromium and 10.0 lM for zinc and copper, showed an increase in

F. Tan et al. / Toxicology in Vitro 22 (2008) 164–170

absorbance in the MTT and CB assays for most cells. This was interpreted as a hormesis effect, which has been previously observed in PLHC-1 cells exposed to cadmium (Ryan and Hightower, 1994) and RTG-2 cells exposed to zinc salts (Nı´ Shu´illeabha´in et al., 2004). Hormesis is often associated with an increase in cell proliferation compared to controls (Nı´ Shu´illeabha´in et al., 2004). Table 4 shows the IC50-values for heavy metals in each of the six cell lines. Data ranged from 4.0 ± 0.7 lM (chromium with EPC cells) to 379 ± 51 lM (copper with BB cells). The relatively low IC50-values for cadmium and chromium indicate a higher cytotoxicity of these chemicals to the cells. As shown in Table 4, the IC50-values for zinc and copper, with regards to cell viability ranged from 35 lM to 150 lM and from 70 lM to 379 lM, respectively, suggesting their low cytotoxic nature as compared to cadmium and chromium. The percentage of dead cells reached 50% only when high concentrations of these chemical salts were used. Based on the IC50-values for the four heavy metals, copper has the lowest cytotoxic impact on these six fish cell lines. These results are consistent with the report by Maracine and Segner (1998) who assessed the acute metal cytotoxicity in RTG-2 cells by means of the neutral red (NR) uptake inhibition assay and showed that the most toxic metal, in terms of NR50, was Hg, followed by Cd, Zn, Cu, Pb and Ni. However, a report published by Xiang et al. (2001) ranked the cytotoxicity of five heavy metals in the order of Cd > Hg > Pb > Cu > Cr > As. The cytotoxicity of these six metal ions was tested using ZC-7901 cells by means of the MTT reduction assay. The diverse cytotoxicity responses to these heavy metals may reflect the different experimental settings employed with fish cell lines. We considered the cell line having the lowest IC50-value to be the most sensitive cells to a particular heavy metal. As shown in Table 4, the sensitivities of these six cell lines to different heavy metals varied extensively. Grass carp kidney cells (CIK) appear to be the most sensitive cells to copper (IC50 = 70 ± 1.3 lM) while GCF is more sensitive to

Table 4 IC50 (±SD) of six cell lines to four metals (lM) measured by MTT reduction and CB assay Cell line

Method

Cadmium

Chromium

Zinc

Copper

BB

MTT assay CB assay MTT assay CB assay MTT assay CB assay MTT assay CB assay MTT assay CB assay MTT assay CB assay

14 ± 3.6 14 ± 4.0 10 ± 3.5 12 ± 7.9 22 ± 0.7 24 ± 6.0 26 ± 2.4 28 ± 3.5 32 ± 1.3 28 ± 3.5 18 ± 0.6 17 ± 2.5

20 ± 2.1 19 ± 3.6 50 ± 5.3* 66 ± 8.8* 33 ± 1.9 32 ± 4.9 4 ± 0.7 5 ± 1.9 17 ± 5.5* 21 ± 4.9* 13 ± 2.2* 10 ± 2.5*

76 ± 3.2* 79 ± 2.9* 138 ± 49 156 ± 94 91 ± 10 112 ± 54 35 ± 2.7 40 ± 4.8 63 ± 1.4 67 ± 4.9 150 ± 47 152 ± 34

379 ± 51 386 ± 64 253 ± 49 261 ± 71 70 ± 1.3 74 ± 4.7 72 ± 3.5 103 ± 37 101 ± 17 106 ± 50 146 ± 90 192 ± 29

CCO CIK EPC FHM GCF

IC50 = concentration causing a 50% inhibition in cell viability. * Significant difference between MTT assay and CB assay (P < 0.05, t-test).

chromium and cadmium. On the other hand, carp epithelioma cells (EPC) are least tolerant to chromium (IC50 = 4.0 ± 0.7 lM) and zinc (IC50 = 35 ± 2.7 lM) among these six cell lines and channel catfish ovary cells (CCO) are the most sensitive cells to cadmium (IC50 = 12 ± 7.9 lM). The varying cell sensitivities to these toxic chemicals may largely be attributed to the differences in their aquatic animal species and tissue/organ origins. To validate the cytotoxicity results by the MTT technique, the cytotoxic sensitivities of the six fish cell lines to the heavy metals were also evaluated using the CB assay to measure the total synthesized protein activities of test cells as compared to control cells. Despite some minor variations regarding the lowest cytotoxicity concentration, the IC50values obtained from both assays appeared very similar (Table 4). A significant correlation (R = 0.9927; P < 0.0001) between these two methods was ascertained by a regression equation (y = 1.0132x + 0.0286, R2 = 0.9847). This closely represented the ideal straight-line case of y = x, with y corresponding to the Ln IC50 (MTT assay) and x corresponding to the Ln IC50 (CB assay) (Fig. 1). Similar results from these two assays suggest validated cytotoxicities of the metals in these fish cell lines. These results were not unexpected since it was reported in the literature that the IC50-values from the MTT and CB assays were in the same order of magnitude for assessing the cytotoxicity of compounds using other fish cell lines (Li and Zhang, 2001). Currently, RTG-2 and PHLC-1 cells are widely used in testing the cytotoxicity of heavy metals. However, our results show that CCO or CIK would be better biological systems for monitoring toxic metals in aquatic environment since they are more sensitive than the RTG-2 or PHLC-1. The cytotoxic dose (NR50-value) of cadmium against RTG-2 for a 24-h exposure was 120 lM (Maracine and Segner, 1998). We have shown that the cytotoxicity of cadmium against the CCO cell line is 10 lM, indicating an increased sensitivity to cadmium toxicity by over 10 times. Similarly, the cytotoxicity of copper to PHLC-1 revealed an IC50 value of 700 lM by the CB assay (G} ulden et al., 2005). The same test in our studies show that the cytotoxic dose of copper to CIK is 74 lM, suggesting that the CIK cells are far more sensitive than the PHLC-1 cells to copper.

Ln IC50 for CB assay

168

7 6 5 4 3 2 1 0

y = 1.0132x + 0.0286 2 R = 0.9847

0

1

2

3 4 5 Ln IC50 for MTT assay

6

7

Fig. 1. Linear regression of the Ln IC50-values in the MTT and the cell protein content assays. The line shows a 1:1 correlation where Ln IC50 (MTT assay) = Ln IC50 (CB assay).

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In summary, comparative analyses of cytotoxicity assays show that the general sequence of toxicity of these four heavy metals to these six fish cell lines is Cd and Cr > Zn > Cu. The MTT and CB assays demonstrated that these fish cell lines have differential cell sensitivities depending on the particular chemical tested. Among these six fish cells, CIK is most sensitive to Cu, and EPC is most sensitive to Cr and Zn, while CCO is very sensitive to Cd. Thus, CIK, EPC and CCO can be potentially valuable biological tools for risk assessment of these particular chemicals. Further comparison and evaluation of these cell lines with others, including those derived from marine mammal species, is essential for determining an optimal cell culture system as the most sensitive bio-indicator to certain toxic chemicals in the aquatic environment. In addition, testing a broadened set of other heavy metals would further increase current understanding of possibly using these cell-lines as potential bio-indicator systems to monitor and detect other hazardous pollutants in the aquatic environment. Acknowledgements The authors thank Dr. Ning Wang (Columbia Environmental Research Center, Columbia, Missouri, USA) for comments on an early draft of the manuscript. This study was supported by the ‘‘948’’ Grant from China Ministry of Agriculture (2005Z37) and the Science and Technology Foundation of Hubei Province (2005AA401C41), PR China. References Bowser, P.R., Plumb, J.A., 1980. Channel catfish virus: comparative replication and sensitivity of cell lines from channel catfish ovary and the brown bullhead. Journal of Wildlife Diseases 16, 451–454. Bru¨schweiler, B.J., Wu¨rgler, F.E., Fent, K., 1995. Cytotoxicity in vitro of organotin compounds to fish hepatoma cells PLHC-1 (Poeciliopsis lucida). Aquatic Toxicology 32, 143–160. Caminada, D., Escher, C., Fent, K., 2006. Cytotoxicity of pharmaceuticals found in aquatic systems: comparison of PLHC-1 and RTG-2 fish cell lines. Aquatic Toxicology 79, 114–123. Castano, A., Gomez-Lechon, M.J., 2005. Comparison of basal cytotoxicity data between mammalian and fish cell lines: a literature survey. Toxicology In Vitro 19, 695–705. Castano, A., Vega, M.M., Tarazona, J.V., 1995. Acute toxicity of selected metals and phenols on RTG-2 and CHSE-214 fish cell line. Bulletin of Environmental Contamination and Toxicology 55, 222–229. Castano, A., Cantarino, M.J., Castillo, P., Tarazona, J.V., 1996. Correlations between the RTG-2 cytotoxicity test IC50 and in vivo LC50 rainbow trout bioassay. Chemosphere 32, 2141–2157. Castano, A., Bols, N., Braunbeck, T., Dierickx, P., Halder, M., Isomaa, B., Kawahara, K., Lee, L-E.J., Mothersill, P., Pa¨rt, P., Repetto, G., Sintes, J.R., Wood, C., Segner, H., 2003. The use of fish cell in ecotoxicology. ATLA 31, 317–351. Choi, J., Oris, J.T., 2003. Assessment of the toxicity of anthracene photomodification products using the topminnow (Poeciliopsis lucida) hepatoma cell line (PLHC-1). Aquatic Toxicology 65, 243–251. Clemedson, C., Andersson, M., Aoki, Y., Barile, F.A., Bassi, A.M., Calleja, M.C., Castano, A., Clothier, R.H., Dierickx, P., Ekwall, B., Ferro, M., Fiskesjo, G., Garza-Ocanas, L., Go´mez-Lecho´n, M.J., Gulden, M., Hall, T., 1998. MEIC evaluation of acute systemic toxicity. Part IV. In vitro results from 67 toxicity assays used to test

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