Automated Image-Based Fluorescence Screening of Mitochondrial Membrane Potential in Daphnia magna: An Advanced Ecotoxicological Testing Tool

Abstract

This study demonstrated the strengths of in vivo molecular staining coupled with automated imaging analysis in Daphnia magna. A multiwell plate protocol was developed to assess mitochondrial membrane potential using the JC-1 dye. The suitability of five common anesthetics was initially tested, and 5% ethanol performed best in terms of anesthetic effects and healthy recovery. The staining conditions were optimized to 30 min staining with 2 μM JC-1 for best J-aggregate formation. The protocol was validated with the model compound carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and used to measure the effect of four environmental contaminants, 2,4-dinitrophenol, triclosan, n-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), and ibuprofen, on mitochondrial health. Test organisms were imaged using an automated confocal microscope, and fluorescence intensities were automatically quantified. The effect concentrations for CCCP were lower by a factor of 30 compared with the traditional OECD 202 acute toxicity test. Mitochondrial effects were also detected at lower concentrations for all tested environmental contaminants compared to the OCED 202 test. For 2,4-dinitrophenol, mitochondria effects were detectable after 2 h exposure to environmentally relevant concentrations and predicted organism death was observed after 24 h. The high sensitivity and time efficiency of this novel automated imaging method make it a valuable tool for advancing ecotoxicological testing.

Keywords: 2,4-dinitrophenol; 6PPD; NAMs; carbonyl cyanide 3-chlorophenylhydrazone; high-content imaging; high-content screening, JC-1; ibuprofen; pharmaceuticals, ecotoxicology; triclosan.

Figure 1

Image analysis: creation of a segmentation mask was based on the difference in brightness in the transmitted light (TL) image of D. magna. This mask was applied to the corresponding fluorescence images (Cy3, FITC) to quantify fluorescence intensities of JC-1 staining.

Figure 2

Images show an overlay of nuclei staining with DAPI (blue) and the JC-1 signal in the Cy3 channel (red) after staining D. magna with 0.5, 1.0, 2.0, or 5.0 μM for 30 min. Column I (A1, B1, C1, and D1) was acquired with a 4× air objective and shows the 2D projection of 50–100 z-stacks. Images in column II (A2, B2, C2, and D2) were acquired with a 20× water immersion objective focusing on D. magna head. A representative z-layer was selected. Column III (A3, B3, C3, and D3) is a digital zoom of the images in column II.

Figure 3

Graphs show the immobilization of D. magna (blue) and corresponding red/green ratio of the JC-1 signal (red) from the automated image-based screening of the mitochondrial membrane potential after 2 and 24 h of exposure to carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The data are derived from three independent experiments, with each individual data point representing the average of five replicates. The corresponding concentration–response model and 95% confidence intervals are shown.

Figure 4

Effect on ATP levels after 2 h of exposure to 250 and 2500 μg/L CCCP. Values represent mean ± SD from four independent experiments with ten individuals for each concentration. Statistically significant differences from control are indicated as follows: *p < 0.05 (ANOVA followed by Dunnet’s multiple comparison test).

Figure 5

Graphs show the immobilization of D. magna (blue) and corresponding red/green ratio of the JC-1 signal (red) from the automated image-based screening of the mitochondrial membrane potential after 2 and 24 h of exposure to 2,4-dinitrophenol, triclosan, N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), and ibuprofen. The data are derived from three independent experiments, with each individual data point representing the average of five replicates. The corresponding concentration–response model and 95% confidence intervals are shown.