High-Throughput Video Processing of Heart Rate Responses in Multiple Wild-type Embryonic Zebrafish per Imaging Field.

Heart rate assays in wild-type zebrafish embryos have been limited to analysis of one embryo per video/imaging field. Here we present for the first time a platform for high-throughput derivation of heart rate from multiple zebrafish (Danio rerio) embryos per imaging field, which is capable of quickly processing thousands of videos and ideal for multi-well platforms with multiple fish/well. This approach relies on use of 2-day post fertilization wild-type embryos, and uses only bright-field imaging, circumventing requirement for anesthesia or restraint, costly software/hardware, or fluorescently-labeled animals. Our original scripts (1) locate the heart and record pixel intensity fluctuations generated by each cardiac cycle using a robust image processing routine, and (2) process intensity data to derive heart rate. To demonstrate assay utility, we exposed embryos to the drugs epinephrine and clonidine, which increased or decreased heart rate, respectively. Exposure to organic extracts of air pollution-derived particulate matter, including diesel or biodiesel exhausts, or wood smoke, all complex environmental mixtures, decreased heart rate to varying degrees. Comparison against an established lower-throughput method indicated robust assay fidelity. As all code and executable files are publicly available, this approach may expedite cardiotoxicity screening of compounds as diverse as small molecule drugs and complex chemical mixtures.

Zebrafish Locomotor Responses Reveal Irritant Effects of Fine Particulate Matter Extracts and a Role for TRPA1.

Exposure to fine particulate matter (PM) air pollution causes adverse cardiopulmonary outcomes. Yet, the limited capacity to readily identify contributing PM sources and associated PM constituents in any given ambient air shed impedes risk assessment efforts. The health effects of PM have been attributed in part to its capacity to elicit irritant responses. A variety of chemicals trigger irritant behavior responses in zebrafish that can be easily measured. The purposes of this study were to examine the utility of zebrafish locomotor responses in the toxicity assessment of fine PM and its chemical fractions and uncover mechanisms of action. Locomotor responses were recorded in 6-day-old zebrafish exposed for 60 min in the dark at 26 °C to the extractable organic matter of a compressor-generated diesel exhaust PM (C-DEP) and 4 of its fractions (F1-F4) containing varying chemical classes of increasing polarity. The role of the transient receptor potential (TRP) cation channel TRPA1, a chemical sensor in mammals and zebrafish, in locomotor responses to C-DEP, was also examined. Acrolein, an environmental irritant and known activator of TRPA1, and all extracts induced concentration-dependent locomotor responses whose potencies ranked as follows: polar F3 > weakly polar F2 > C-DEP > highly polar F4 > nonpolar F1, indicating that polar and weakly polar fractions that included nitro- and oxy-polyaromatic hydrocarbons (PAHs), drove C-DEP responses. Irritant potencies in fish positively correlated with mutagenic potencies of the same extracts in strains of Salmonella sensitive to nitro- and oxy-PAHs, further implicating these chemical classes in the zebrafish responses to C-DEP. Pharmacologic inhibition of TRPA1 blocked locomotor responses to acrolein and the extracts. Taken together, these data indicate that the zebrafish locomotor assay may help expedite toxicity screening of fine PM sources, identify causal chemical classes, and uncover plausible biological mechanisms.