283 - Mapping glutathione utilization in the developing zebrafish embryo

Abstract

Glutathione (GSH), the most abundant endogenous redox buffer in vertebrates, plays a key role in organogenesis and embryonic development. Toxic insults during development are commonly neutralized by glutathionylation and excretion. Despite its importance, scant information exists regarding organ-specific GSH utilization during development. Monochlorobimane (MCB) is a dye that is conjugated with GSH by glutathione-s-transferase (GST) to form a fluorescent adduct. Here, we use MCB staining to visualize organ-specific location of GSH utilization in live developing zebrafish embryos at developmental timepoints commonly used in the OECD fish embryo acute toxicity test. At 24 hours post-fertilization (hpf), GSH conjugation with MCB was highest in the heart, brain and yolk, increasing in the gut and otolith at 48 hpf. By 72 hpf, MCB staining was higher in the endoderm-derived lineage, and at 96hpf it was brightest in the liver, gut, and gill arches. We then tested the sensitivity and specificity of MCB staining with known GSH modulators . A 10-minute treatment at 48 hpf with the prooxidant tert-butyl hydroperoxide, caused organ-specific reductions in MCB staining, with the heart and brain losing 30% fluorescence, and, the gut and myotome losing 50%. A 24 hour treatment from 24-48 hpf with the GSH precursor N-Acetylcysteine resulted in significantly increased fluorescence, with the brain showing a 640% increase and the heart showing a 350% increase. The gut and myotome showed a 100% increase. Ethacrynic acid, a highly specific GST inhibitor caused decreased fluorescence; the heart, brain and myotome lost 30% fluorescence and the gut lost 50%. 2-AAPA, an inhibitor of Glutathione Reductase, caused similar organ specific losses in fluorescence with the gut being more susceptible than the heart and brain. Our data indicate highly specific spatio-temporal GSH utilization, with different organs having differing resilience against GSH system disruptions during embryonic development.