Developmental Hazard of Environmentally Persistent Free Radicals and Protective Effect of TEMPOL in Zebrafish Model

Abstract: Environmentally persistent free radicals (EPFRs) can be detected in ambient PM2.5, ciga-rette smoke, and soils and are formed through combustion and thermal processing of organic ma-terials. The hazards of EPFRs are largely unknown. In this study, we assess the developmental tox-icity of EPFRs and the ability of TEMPOL (4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl) to pro-tect against such hazards using zebrafish embryos. Particles containing EPFRs were acquired by dosing dichlorobenzene (DCB) vapor on the Cab-o-sil/5% CuO particles at 230 °C in vacuo (referred to as DCB-230). The particles were suspended in ultrapure water to make 1 mg/mL of stock solution from which series dilution was undertaken to obtain 10, 20, 30, 40, 50, 60, 80, and 100 μg/mL final test solutions, which were then placed in individual wells with a 4 h postfertilization (hpf) zebrafish embryo. Plates were run in duplicate to obtain a sample size of 24 animals per concentration; 12 embryos were exposed per concentration per plate. Statistical analysis of the morphology endpoints was performed. We investigated overt toxicity responses to DCB-230 in a 22-endpoint battery that included developing zebrafish from 24–120 hpf. Exposure to concentrations greater than 60 μg/mL of DCB-230 induced high mortality in the developmental zebrafish model. Exposure to EPFRs in-duced developmental hazards that were closely related to the concentrations of free radicals and EPFRs. The potential protective effects of TEMPOL against EPFRs’ toxicity in zebrafish were inves-tigated. Exposure to EPFRs plus TEMPOL shifted the concentration to an induced 50% adverse ef-fect (EC50), from 23.6 to 30.8 μg/mL, which verifies TEMPOL’s protective effect against EPFRs in the early phase of zebrafish development.


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