In vivoassessment of respiratory burst inhibition by xenobiotic exposure using larval zebrafish

Abstract

Currently, assessment of the potential immunotoxicity of a given agent involves a tiered approach for hazard identification and mechanistic studies, including observational studies, evaluation of immune function, and measurement of susceptibility to infectious and neoplastic diseases. These studies generally use costly low-throughput mammalian models. Zebrafish, however, offer an excellent alternative due to their rapid development, ease of maintenance, and homology to mammalian immune system function and development. Larval zebrafish also are a convenient model to study the innate immune system with no interference from the adaptive immune system. In this study, a respiratory burst assay (RBA) was utilized to measure reactive oxygen species (ROS) production after developmental xenobiotic exposure. Embryos were exposed to non-teratogenic doses of chemicals and at 96 h post-fertilization, the ability to produce ROS was measured. Using the RBA, 12 compounds with varying immune-suppressive properties were screened. Seven compounds neither suppressed nor enhanced the respiratory burst; five reproducibly suppressed global ROS production, but with varying potencies: benzo[a]pyrene, 17b-estradiol, lead acetate, methoxychlor, and phenanthrene. These five compounds have all previously been reported as immunosuppressive in mammalian innate immunity assays. To evaluate whether the suppression of ROS by these compounds was a result of decreased immune cell numbers, flow cytometry with transgenic zebrafish larvae was used to count the numbers of neutrophils and macrophages after chemical exposure. With this assay, benzo[a]pyrene was found to be the only chemical that induced a change in the number of immune cells by increasing macrophage but not neutrophil numbers. Taken together, this work demonstrates the utility of zebrafish larvae as a vertebrate model for identifying compounds that impact innate immune function at non-teratogenic levels and validates measuring ROS production and phagocyte numbers as metrics for monitoring how xenobiotic exposure alters the innate immune system.

Acute Aquatic Toxicity to Zebrafish and Bioaccumulation in Marine Mussels of Antimony Tin Oxide Nanoparticles

“…Small size, low cost-maintenance, reduced housing requirements, quick development, and high genetic and physiologic similarity with humans [ 32 , 33 ] are some of the attractive features that make zebrafish ( Danio rerio ) a consensually accepted and used animal model to evaluate the potential risk of different xenobiotics present in water [ 34 , 35 , 36 ]. Following the 3Rs (replacement, reduction, and refinement) principle and ethically accepted by the European legislation [ 37 ], the Fish Embryo Acute Toxicity (FET) test [ 38 ] has been implemented as a reliable assay in the environmental toxicology field.…”