Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line. BMC Pharmacol 8:8

European Commission - Joint Research Centre, Institute for Environment and Sustainability, Rural, Water, and Ecosystem Resources Unit, Via E, Fermi 2749, 21027 Ispra (VA), Italy.
BMC Pharmacology (Impact Factor: 1.84). 02/2008; 8(1):8. DOI: 10.1186/1471-2210-8-8
Source: PubMed


A broad spectrum of cytotoxicity assays is currently used in the fields of (eco)toxicology and pharmacology. To choose an appropriate assay, different parameters like test compounds, detection mechanism, specificity, and sensitivity have to be considered. Furthermore, tissue or cell line can influence test performance. For zebrafish (Danio rerio), as emerging model organism, cell lines are now increasingly used, but few studies examined cytotoxicity in these cell systems. Therefore, we compared four cytotoxicity assays in the zebrafish liver cell line, ZFL, to test four differently acting model compounds. The tests comprised two colorimetric assays (MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, and the LDH assay detecting lactate dehydrogenase activity) and two fluorometric assays (alamarBlue(R) using resazurin, and CFDA-AM based on 5-carboxyfluorescein diacetate acetoxymethyl ester). Model compounds were the pharmaceutical Tamoxifen, its metabolite 4-Hydroxy-Tamoxifen, the fungicide Flusilazole and the polycyclic aromatic hydrocarbon Benzo[a]pyrene.
All four assays performed well in the ZFL cells and led to reproducible dose-response curves for all test compounds. Effective concentrations causing 10% or 50% loss of cell viability (EC10 and EC50 values) varied by a maximum factor of 7.0 for the EC10 values and a maximum factor of 1.8 for the EC50 values. The EC values were not statistically different between the four assays, which is due to the assessed unspecific effects of the compounds. However, most often, the MTT assay and LDH assay showed the highest and lowest EC values, respectively. Nevertheless, the LDH assay showed the highest intra- and inter-assay variabilities and the lowest signal-to-noise ratios. In contrast to MTT, the other three assays have the advantage of being non-destructive, easy to handle, and less time consuming. Furthermore, AB and CFDA-AM can be combined on the same set of cells without damaging the cells, allowing later on their use for the investigation of other endpoints.
We recommend the alamarBlue and CFDA-AM assays for cytotoxicity assessment in ZFL cells, which can be applied either singly or combined.

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    • "The Danio rerio permanent hepatocyte cell line, ZFL, is a model of fishderived cells considered relevant for the identification of toxicological hazard to aquatic organisms (Gajski et al., 2015). Several studies demonstrate the sensitivity of this cell line when exposed to various contaminants , including metals (Chan et al., 2006;Costa et al., 2012;Sandrini et al., 2009;Seok et al., 2007), pesticides (Goulart et al., 2015), pharmaceuticals (Bopp and Lettieri, 2008;Pomati et al., 2007;Gajski et al., 2015), effluents (Christianson-Heiska and Isomaa, 2008) and biodiesel (Cavalcante et al., 2014).⁎ Corresponding author at: Departamento de Ciências Fisiológicas, Universidade Estadual de Londrina, PB: 10011, Londrina, Paraná 86057-970, Brazil. "
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    • "MTT assay: MTT assay was conducted according to the process given in the literary works (Bopp and Lettieri 2008) using control adriamycin. 96-Well plate was used to seed the cells followed by incubation for 24 hours at 37°C. "
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    • "Over the past 50 years, the AB assay has been widely used in studies of cell viability and cytotoxicity for biological and environmental applications (Rampersad, 2012; Vega-Avila and Pugsley, 2011; White et al., 1996). The bioassay can also be used to establish the relative cytotoxicity of agents within various chemical classes (Bopp and Lettieri, 2008; Borra et al., 2009; Mikus and Steverding, 2000; Miret et al., 2006). Using the REDOX indicator resazurin (oxidised form), it is possible to spectrophotometrically measure the cellular proliferation. "
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