High-Content Screening Assay for Identification of Chemicals Impacting Cardiovascular Function in Zebrafish Embryos
- Authors
- Yozzo, K.L., Isales, G.M., Raftery, T.D., and Volz, D.C.
- ID
- ZDB-PUB-130918-7
- Date
- 2013
- Source
- Environmental science & technology 47(19): 11302-11310 (Journal)
- Registered Authors
- Keywords
- none
- MeSH Terms
-
- Aminobenzoates/pharmacology
- Anesthetics/pharmacology
- Animals
- Body Size/drug effects
- Cardiotoxins/toxicity*
- Cardiovascular System/drug effects*
- Coronary Circulation/drug effects
- Embryo, Nonmammalian/drug effects*
- Embryo, Nonmammalian/physiology
- Heart Rate/drug effects
- High-Throughput Screening Assays*
- Zebrafish
- PubMed
- 24015875 Full text @ Env. Sci. Tech.
Targeted assays are needed to better evaluate effects of chemicals on organogenesis and begin classification of chemicals by toxicologically relevant modes-of-action. Using transgenic zebrafish (fli1:egfp) that stably express eGFP within vascular endothelial cells, we have developed and optimized a 384-well-based high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular function at sub-lethal, non-teratogenic concentrations. Following static exposure of one embryo per well from 5-72 hours post-fertilization (hpf), automated image acquisition procedures and custom image analysis protocols are used to quantify body length, circulation, heart rate, pericardial area (a biomarker for cardiac looping defects), and intersegmental vessel area within freshly hatched live embryos. After optimizing 72-hpf anesthetization procedures, we evaluated each endpoint across four independent control plates containing 384 initial embryos per plate. Survival and imaging success rates across these plates ranged from 93-99% and 42-74%, respectively. Criteria were then defined for assay success and analysis of treatments, and 10 chemicals were screened for targeted effects on cardiovascular function. Compared to existing zebrafish-based assays, this method provides a comprehensive discovery platform with 1) increased sample sizes; 2) broad concentration-response format; and 3) the ability to identify chemicals that target cardiovascular function at non-teratogenic concentrations.