Article

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

ABSTRACT

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.

Download full-text

Full-text

Available from: PubMed Central · License: CC BY
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: This work aimed to evaluate the effects of different dilutions of gasoline water-soluble fraction (GSF) on Danio rerio hepatocyte cell line (ZFL). Two tests were used to assess cell viability, MTT reduction assay (MTT) and the trypan blue (TB) exclusion test. Oxidative stress was evaluated through the quantification of reactive oxygen species (ROS) and the assessment of the total antioxidant capacity against peroxyl radicals (ACAP) and the comet assay was employed to assess DNA damage. ZFL cells were exposed to 5, 10, 25 and 50% GSF or only to saline for 1, 3 and 6h. The GSF exhibited concentration-dependent cytotoxicity, and longer exposure times resulted in lower cell viability as indicated by both MTT and TB assays. The establishment of oxidative stress in cells exposed to GSF was not observed at any exposure period and the lower ROS levels could be related to the increased antioxidant capacity after 6-h exposure. DNA damage was significantly increased after exposure to GSF at the three experimental times. Taking together these results show that GSF has a genotoxic potential at the lower concentrations and becomes cytotoxic at higher concentrations and that ZFL can be considered a good biological model for in vitro toxicological studies.
    Full-text · Article · Nov 2015 · Toxicology in Vitro
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: This study was aimed to evaluate anticancer potential of newer synthesize 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines and its derivatives. All newly furnished scaffolds were subjected to screening for their in vitro anticancer potential against DU-145 and PC-3 prostate cancer cell lines using SRB and MMT bioassays. The structures of final compounds were confirmed with the aid of FT-IR, 1H NMR, 13C NMR spectroscopy and CHN analysis. Bioassay studies suggested that all thiadiazines were promising cytotoxic agents with % cytotoxicity ranging from 44.39-71.24%, whereas potent GI50 level in the range 11.96-32.51 µg/mL and results were comparable to the potencies of control drugs adriamycin and doxorubicin. Variation of heterocyclic pharmacophores along with the C-5 position of 1,2,4-triazole in terms of quinoline, quinazoline, coumarin and pyridine lead to the different SAR predictions in which quinoline and benzimidazole moieties found most promising.
    Preview · Article · Apr 2015 · Bangladesh Journal of Pharmacology
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Comparisons of 2D and 3D cell culture models in literature have indicated differences in cellular morphology and metabolism, commonly attributed the better representation of in vivo conditions of the latter cell culture environment. Thus, interest in the use of 3D collagen gels for in vitro analysis has been growing. Although comparative studies to date have indicated an enhanced resistance of cells on collagen matrices against different toxicants, in the present study it is demonstrated that non-adapted protocols can lead to misinterpretation of results obtained from classical colorometric dye-based cytotoxic assays. Using the well established Alamar Blue assay, the study demonstrates how the transfer from 2D substrates to 3D collagen matrices can affect the uptake of the resazurin itself, affecting the outcome of the assay. Using flow cytometry, it is demonstrated that the cell viability is unaffected when cells are grown on collagen matrices, thus the difference seen in the fluorescence is a result of a dilution of the resazurin dye in the collagen matrix, and an increased uptake rate due to the larger cell surface exposed to the surrounding environment, facilitating more effective diffusion through the cellular membrane. The results are supported by a rate equation based simulation, verifying that differing uptake kinetics can result in apparently different cell viability. Finally, this work highlights the feasibility to apply classical dye-based assays on collagen based 3D cell culture models. However, the diffusion and bioavailability of test substances in 3D matrices used in in vitro toxicological assays must be considered and adaption of the protocols is necessary for direct comparison with the traditional 2D models. Moreover, the observations made based on the resazurin dye can be applied to drugs or nanoparticles which freely diffuse through the collagen matrices, thus affecting the effective concentration exposed to the cells.
    No preview · Article · Oct 2014 · Toxicology in Vitro
Show more