Routine culture of zebrafish liver cells (ZFL)
The zebrafish (Danio rerio) liver cell line ZFL  was obtained from ATCC (Promochem). Cells were cultured in medium composed of 50% Leibovitz L-15, 35% DMEM, and 15% Ham F-12, supplemented with 15 mM HEPES, 0.15 g/L NaHCO3, 0.01 mg/mL insulin, 50 ng/mL epidermal growth factor (EGF), and 5% fetal bovine serum (FBS) as proposed by ATCC. Cells were cultured at 28°C in 75 cm2 cell culture flasks (Stratagene). Cells were subcultured every 5–7 days. For this purpose, they were first rinsed with 1 mL of 0.25% trypsin EDTA (Gibco), and then detached with 1 mL of trypsin EDTA. Trypsination was stopped by addition of 5 mL of 10% FBS containing medium. Cells were centrifuged for 5 min at 300 g. Cell pellets were resuspended in 5% FBS containing medium and either split to new flasks or plated in 96 well flat-bottom culture plates for cytotoxicity assays.
Exposure of ZFL cells for cytotoxicity assessment
For comparison of the four cytotoxicity assays, cells were exposed to four differently acting model compounds: the polycyclic aromatic hydrocarbon Benzo[a]pyrene (BaP; purity ≥ 97%, Fluka), the fungicide Flusilazole (Flus; purity 99.8%, Riedel-de Haën), the pharmaceutical Tamoxifen (Tam) as Tamoxifen citrate (purity ≥ 99%, Sigma) and its metabolite trans-4-Hydroxy-Tamoxifen (4-OHT; purity ≥ 98%, Sigma). Each compound was dissolved in dimethyl sulfoxide (DMSO) and diluted in a way that all stock solutions were 200 times concentrated compared to the final test concentrations. Concentration ranges were 30 pM-1.97 μM for BaP, 5 nM-5 μM for Flus, 5 nM-11 μM for Tam, and 0.2–43 μM for 4-OHT, which is in the range of in vivo studies with waterborne exposure of zebrafish for BaP, Tam, and 4-OHT [20–22, 29]. For Flusilazole, no studies on fish are available.
For finding the optimal biological test conditions, in the beginning different cell densities were applied using two-fold dilution series in the range of 780–100,000 cells/well and cell viability tests were performed after 24 h. 25,000 cells/well turned out to be in the optimum range for all of the tests. Thus, cells were plated in 96 well flat-bottom plates at an initial cell density of 25,000 cells/well in 200 μL of medium and allowed to settle for 24 h. In parallel, wells containing only medium without cells were prepared and used later on for background correction. After the pre-incubation period, for each test compound and each concentration, 1 μL of the DMSO stock solutions was added so that the final DMSO content was 0.5%. All tests were performed at least three times and each time in triplicate.
The MTT assay was performed using the cell proliferation kit I MTT (Roche) according to the manufacturer's protocol. In brief, after 24 h of incubation with the test compounds, 100 μL of medium were removed from each well. Then, 10 μL of the MTT solution were added and plates incubated for 4 h at 28°C. Since the manufacturer recommends incubation at 37°C and ZFL cells have to be incubated at 28°C, we tested different incubation times with MTT (2, 4, and 6 h) to establish optimized conditions. No relevant differences were observed for the different time points, so that the 4 h incubation was used. After the incubation with MTT, the solubilization solution was added and plates incubated over night. The next day, absorption of the produced formazan was measured at 570 nm with 680 nm as reference wavelength using a microplate reader (Infinite 200, Tecan). For data evaluation, background and reference wavelength corrected absorption values were averaged for the triplicates and expressed as "% cytotoxicity" referring to the untreated control containing only the solvent DMSO.
A second set of cells was exposed to the test compounds for the LDH assay, which was performed using the Cytotoxicity Detection Kit LDH (Roche). For this assay, a positive control, leading to 100% cytotoxicity by lysing the cells completely, was included in the assay. The positive control was 2% Triton X-100 solution in the assay medium, as proposed by the manufacturer. After pre-incubation of the cells, before addition of the test compounds, the growth medium was exchanged from medium containing 5% FBS to medium containing only 1% FBS. Then, test compounds in DMSO were dosed and plates incubated as for the other assays for 24 h. For testing the released LDH activity, 100 μL of culture medium were transferred to a new 96 well plate. 100 μL of the reaction solution from the kit, containing the detection dye and the catalyst were then added and absorption was measured after 30 min at 490 nm with 655 nm as reference wavelength in an ELISA reader (Model 680, Biorad). As for the other assays, background values from wells without cells were subtracted and average values for the triplicates calculated. Cytotoxicity was then calculated according to the following equation: Cytotoxicity (%) = (experimental value - DMSO control)/(positive control - DMSO control) × 100.
The above described final test conditions were the result of preceding tests to optimize the assay described in the following: it was reported that FBS can lead to high background values in the LDH assay . Therefore, its influence was first assessed comparing the normally used culture medium with 5% FBS to a reduced FBS concentration of 1%. This was done in presence of 2% Triton X-100. The positive control substance Triton X-100 has to be added, since the LDH assay does not detect the viable but only damaged or completely lysed cells.
Combined alamarBlue® and CFDA-AM assay
The fluorogenic indicator dyes alamarBlue® (AB) (BioSource, Invitrogen) and CFDA-AM (5-carboxyfluorescein diacetate acetoxymethyl ester; Molecular Probes, Invitrogen) were used in combination on a third set of cells, since it was shown before that their fluorescent products can be detected at different non-interfering wavelengths . AB was obtained as ready-to-use stock solution, which had to be diluted 200 times to obtain the working solution. CFDA-AM powder was dissolved in DMSO to a stock solution of 4 mM, which was diluted 1000 times to reach the 4 μM working solution. Working solutions were prepared in 1× Earle's-G medium , which was investigated in preceding experiments to support ZFL cell viability during the assay incubation period. For this purpose, cells were exposed to the dyes dissolved in culture medium without FBS or in Earle's-G medium. Since Earle's-G medium showed the same good correlations between cell number and fluorescence and lower background values it was chosen for standard test conditions. The AB/CFDA-AM assay was then performed according to . In brief, after 24 h of incubation with the test compounds, the medium was aspirated off completely and 100 μL/well of the alamarBlue/CFDA-AM working solution were added. After 30 min of incubation, fluorescence was measured at 530 nm excitation/590 nm emission for AB and 485 nm/535 nm for CFDA-AM, respectively, using a fluorescence plate reader (Infinite 200, Tecan). For data evaluation, average values of triplicates were calculated after background correction and all average values for the treatments referred to the DMSO control, in order to express data as "% cytotoxicity".
Data evaluation and statistical analysis
For each single assay, data were evaluated separately for three independent experiments. Dose-response curves were fitted using a nonlinear-regression sigmoidal dose-response curve model provided in the GraphPad Prism software (GraphPad software, Inc., San Diego, USA). EC10 and EC50 values were derived from these fitted curves for the single experiments. Final EC-values (Table 1) were calculated as average of three independent experiments with the standard deviation of the mean indicating the variation. Data from the different assays were statistically compared using One way ANOVA analysis with Tukey's test as post-ANOVA analysis (p < 0.05).
Intra-assay variabilities were calculated as coefficient of variance (CV), based on the triplicate values within each individual experiment. In a second step, CV values for the three individual experiments for each tested compound were determined calculating the median CV in a low (0–20%) and high (80–100%) cytotoxicity range. The inter-assay variability was assessed based on the EC10 and EC50 values. Median CV values were calculated using the EC values from each three individual experiments for each tested compound. Signal-to-noise ratios were calculated based on Tam exposure experiments for each compound concentration tested. Average absorption or fluorescence signals from wells with differently treated cells were divided by the average signal from wells containing only the test medium without cells, representing the background values. Then, median values for the low (0–20%) and high (80–100%) cytotoxicity range were calculated.