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

Abstract

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.

A critical evaluation of TRPA1-mediated locomotor behavior in zebrafish as a screening tool for novel anti-nociceptive drug discovery

“…Several behavior models of neurological and neuropsychiatric-like behavior have been created in zebrafish that mimic those established for rodents, such as conditioned place preference 13 and anxiety-like behavior 14 . Increased zebrafish locomotor behavior has also been previously observed by both thermal and chemical activation of TRPA1 channels 15,16 . Fortunately, TRPA1 channels are relatively conserved across species ranging from planarians to humans 17 , and the peripheral and central nociceptive systems of zebrafish are similar to many vertebrates such as mice and humans 18–20 .…”

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

“…The slope of the linear regression derived from the concentration-responses of each extract was used to determine the potency as described previously 27,30 (Fig. 6a).…”

“…All studies were carried out in accordance with the guidelines of, and approved by, the Institutional Animal Care and Use Committee at the U.S. EPA’s National Health and Environmental Effects Research Laboratory. The fish were housed in an Association for Assessment and Accreditation of Laboratory Animal Care-approved animal facility with a 14:10 h light:dark cycle with lights on at 08:30 h. Embryos were reared and bred as described previously 30 . Briefly, breeding utilized an in-house strain of zebrafish ( D. rerio ; undefined, outbred stock originally obtained from Aquatic Research Organisms, Hampton, NH and EkkWill Waterlife Resources, Ruskin, FL) that were bred annually to wild-type fish to increase genetic diversity.…”

Evaluation of Developmental Toxicity, Developmental Neurotoxicity, and Tissue Dose in Zebrafish Exposed to GenX and Other PFAS

“…Finally, analysis of larval behavior data is not standardized. Here, we opted to use a repeated measures ANOVA and parametric mixed model analysis to account for the complex data structure that contains multiple, repeated locomotor activity values for each animal over the 40-min testing period (Catron et al 2019a;Irons et al 2013;Phelps et al 2017;Stevens et al 2018). However, zebrafish behavior data are not normally distributed and may often be below the LOD.…”

“…If a test for linear trend was significant (p < 0:05), with developmental toxicity observed at the highest concentration tested, nonlinear regression was performed with a Hill slope curve fitting for half maximal effective concentration (EC 50 ) value determinations. For DNT assay results shown in Figures 3, 6, and 8, a repeated measures ANOVA analysis was used to detect differences in swimming behavior between exposed and control larvae (Catron et al 2019b;Irons et al 2013;Phelps et al 2017;Stevens et al 2018). These analyses were performed using SAS (version 9.4; SAS Institute Inc.).…”