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

Environmentally persistent free radicals (EPFRs) can be detected in ambient PM_2.5, cigarette smoke, and soils and are formed through combustion and thermal processing of organic materials. The hazards of EPFRs are largely unknown. In this study, we assess the developmental toxicity of EPFRs and the ability of TEMPOL (4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl) to protect 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 induced 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 investigated. Exposure to EPFRs plus TEMPOL shifted the concentration to an induced 50% adverse effect (EC50), from 23.6 to 30.8 µg/mL, which verifies TEMPOL's protective effect against EPFRs in the early phase of zebrafish development.