High-Content Screening Assay for Identification of Chemicals Impacting Cardiovascular Function in Zebrafish Embryos

Abstract

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 sublethal, nonteratogenic concentrations. Following static exposure of one embryo per well from 5 to 72 h postfertilization (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 end point across four independent control plates containing 384 initial embryos per plate. Survival and imaging success rates across these plates ranged from 93 to 99% and 42 to 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 nonteratogenic concentrations.

High-Content Screening in Zebrafish Embryos Identifies Butafenacil as a Potent Inducer of Anemia

“…Due to transparency, small size, and rapid development ex utero , zebrafish are highly amenable to whole-organism imaging during embryogenesis and, as a result, high-content screening (HCS) assays for identifying and prioritizing hazardous compounds for further testing. To this end, using transgenic zebrafish ( fli1:egfp ) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well HCS assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations [8]. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization (hpf).…”

“…Therefore, chemicals within the EPA's ToxCast Phase-I chemical library were ranked from most to least acutely toxic based on available data for zebrafish survival and gross malformations present at 6 days post fertilization (dpf) following an 8-hpf to 5-dpf exposure [12]. Using protocols described in Yozzo et al [8], 26 of the most acutely toxic chemicals within this library (Table S1) were first screened in concentration-response format (0.05–50 µM) to evaluate the ability of this assay to detect cardiovascular toxicity at non-teratogenic concentrations. Based on results from this pilot screen, we then conducted initial hit validation studies to (1) determine whether exposure to chemicals with a common site of action resulted in similar effects on cardiovascular development and (2) begin characterizing the potential mechanism leading to cardiovascular toxicity.…”

Automatic Zebrafish Heartbeat Detection and Analysis for Zebrafish Embryos

“…Up to date, an assessment of the heart rate is often practiced manually by counting heartbeats from slow motion replay of videotape recordings, what is neither practical for large numbers of hearts to be analyzed nor is it accurate when determining beat-to-beat intervals as needed for detection of arrhythmias. 7 Existing software for semiautomated analysis of heartbeats [7][8][9][10][11][12][13] has several limitations. One is that the embryos have to be anesthetized by adding Tricaine 11,14 or MS-222 12 to avoid movement of zebrafish embryos during videoing.…”

“…7 Existing software for semiautomated analysis of heartbeats [7][8][9][10][11][12][13] has several limitations. One is that the embryos have to be anesthetized by adding Tricaine 11,14 or MS-222 12 to avoid movement of zebrafish embryos during videoing. Adding compounds that prevent embryo movement such as anesthetics, sedatives, or neuromuscular junction blockers can alter the heart rate.…”

Recent progress in the use of zebrafish for novel cardiac drug discovery

“…This assay allows the analysis of automatically acquired images and the evaluation of the heart rate and blood circulation. Furthermore, the impact of chemical compounds on morphological parameters such as body length and intersegmental vessel development can be assessed using this transgenic zebrafish line [45]. Only very recently, Lin et al developed a pseudodynamic three-dimensional imaging system that determines a variety of cardiac function parameters: heart rate, ventricular stroke volume, ejection fraction, cardiac output, diastolic filling, and ventricular mass in Tg(BMP4:eGFP) zebrafish larvae up to 6 dpf [46].…”