Characterizing sources of variability in zebrafish embryo screening protocols

Abstract

There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Literature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure media (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.

Keywords: Non-mammalian species; developmental toxicity; high-throughput screening.

Fig. 1:. Zebrafish embryos in a 384-well tissue culture plate

Photographs of transgenic fluorescent zebrafish embryos taken under a microscope using transmitted light in the top panel (A) and using fluorescence capture in the bottom panel (B). Images are captured simultaneously. One zebrafish embryo is immersed in 50 μL of embryo media in each 3X3 mm well of the 384-well tissue culture plate. The embryos in this image were placed in the well at 5 hpf and the image was taken at 72 hpf.

Fig. 2:. Zebrafish embryos in a 96-well tissue culture plate

Photograph of zebrafish embryos in a 96-well tissue culture plate, taken under a microscope using transmitted light.

Fig. 3:. Distribution of active chemicals at EPA and Oregon State University in comparison to logP

ToxCast chemicals that were determined to be active at both EPA and Oregon State University (OSU) were plotted based on their logP. The top box plot (grey box) is the logP distribution of all the chemicals in ToxCast Phase I and II. The middle box plot (red box) is the distribution of the actives reported by the EPA laboratory using chorionated embryos and semi-static dosing. The lower box (blue box) is the distribution of the actives reported by OSU using dechorionated embryos and static dosing. Results demonstrate that active chemicals at both laboratories share similar distribution of lipophilicity.

Fig. 4:. Total numbers of active chemicals as determined by EPA and Oregon State University

All overlapping ToxCast chemicals run at both EPA and Oregon State University (OSU) were plotted against logP along with the numbers of active chemicals at each institution. Results demonstrate that a greater number of chemicals were active when using chorionated embryos and semistatic dosing conditions (i.e., EPA protocol).

Fig. 5:. General structure of the zebrafish egg

The zebrafish egg is approximately 0.7mm in diameter. The chorion (outer layer) has a thickness of 3.5μM and is fenestrated with 0.5 μM diameter pores allowing passage of water, ions, and chemicals. The fluid-filled perivitelline space overlays the vitelline membrane, which surrounds the yolk and the blastoderm, which will become the developing embryo. Figure adapted from (Jones et al., 1978).