Large-scale assessment of the zebrafish embryo as a possible predictive model in toxicity testing

Abstract

Background: In the drug discovery pipeline, safety pharmacology is a major issue. The zebrafish has been proposed as a model that can bridge the gap in this field between cell assays (which are cost-effective, but low in data content) and rodent assays (which are high in data content, but less cost-efficient). However, zebrafish assays are only likely to be useful if they can be shown to have high predictive power. We examined this issue by assaying 60 water-soluble compounds representing a range of chemical classes and toxicological mechanisms.

Methodology/principal findings: Over 20,000 wild-type zebrafish embryos (including controls) were cultured individually in defined buffer in 96-well plates. Embryos were exposed for a 96 hour period starting at 24 hours post fertilization. A logarithmic concentration series was used for range-finding, followed by a narrower geometric series for LC(50) determination. Zebrafish embryo LC(50) (log mmol/L), and published data on rodent LD(50) (log mmol/kg), were found to be strongly correlated (using Kendall's rank correlation tau and Pearson's product-moment correlation). The slope of the regression line for the full set of compounds was 0.73403. However, we found that the slope was strongly influenced by compound class. Thus, while most compounds had a similar toxicity level in both species, some compounds were markedly more toxic in zebrafish than in rodents, or vice versa.

Conclusions: For the substances examined here, in aggregate, the zebrafish embryo model has good predictivity for toxicity in rodents. However, the correlation between zebrafish and rodent toxicity varies considerably between individual compounds and compound class. We discuss the strengths and limitations of the zebrafish model in light of these findings.

Figure 1. Cumulative mortality and infertility of zebrafish in buffer or egg water.

Embryos were kept in 92 mm Petri dishes with 40 ml of either buffer or egg water, 60 eggs per dish. Each error bar represents ±SEM of N = 420 embryos each for buffer and egg water. A, cumulative infertility and early mortality in buffer. B, the same, in egg water. There is no significant difference between the two media in terms of survival and fertilization percentage.

Figure 2. Rate of evaporation from 96-well plates at 28.0°C.

Buffer was dispensed in four different 96-well plates. A, cumulative average percentage buffer loss per plate. All wells were initially filled with 250 µL buffer. B, percentage buffer loss after 96 h, per well, as a function of well position. The letters A–H and the numbers 1–12 correspond to the standard coordinates embossed into 96-well plates. All wells were initially filled with 250 µL buffer. Only the wells with grey columns were measured.

Figure 3. Correlation between zebrafish embryo Log LC₅₀ and rodent Log LD₅₀ for the 60 compounds tested in this study.

Zebrafish embryo LC₅₀ was determined based on cumulative mortality after 96 h exposure of compounds from three independent experiments and rodent LD₅₀ was taken from the literature. Key: blue, regression line; solid black lines, 0.25 and 0.75 quartiles; dashed line, perfect correlation line. The slope of the regression line (blue) is 0.73403.

Figure 4. Linear regression model: rodent log LD₅₀ and zebrafish embryo log LC₅₀.

The effect of the different compounds on the slope and intercept of the ANCOVA model. Although we must consider the effect of the unknown error in the rodent LD₅₀ values, the different compound classes seem to cluster in different regions in the graph.