Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line

Background: 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® 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. Results: 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. Conclusions: We recommend the alamarBlue and CFDA-AM assays for cytotoxicity assessment in ZFL cells, which can be applied either singly or combined.JRC.H.5-Rural, water and ecosystem resource

Heat, air and moisture transport modelling in ventilated cavity walls

Cavity walls are a widely used external wall type in north-western Europe with a good moisture tolerance in cool humid climates. In this work, a cavity wall configuration with a brick veneer outside leaf and a wood fibre board inside leaf is analysed with a newly developed coupled computational fluid dynamics-heat, air and moisture model. Drying of the outside or inside cavity leaf, both for summer and winter conditions was analysed. The new model was compared with a widely used simulation tool for building envelope analysis (WUFI (R)) that uses a simplified modelling approach for the convection in the cavity. The study showed that the simplified model overestimated the drying and moistening rates of the cavity wall compared to the detailed model. For both models the drying of the outer leaf was mainly determined by the outside conditions, and the outside leaf dried out mainly to the outside and not to the cavity. For the inside leaf, however the cavity ventilation was of major importance in drying. The study revealed that the simplified model could not be used to evaluate the drying potential of a ventilated cavity because it overestimated the ventilation effect systematically. The simplified model would in such case indicate lower moisture contents than in reality and consequently lower risk for mould growth, wood rot or other structural damage. Only detailed modelling of the convection in the cavity, as in the new model, leads to a correct evaluation of ventilated cavity walls