Mapping glutathione utilization in the developing zebrafish (Danio rerio) embryo

Abstract

Glutathione (GSH), the most abundant vertebrate endogenous redox buffer, plays key roles in organogenesis and embryonic development, however, organ-specific GSH utilization during development remains understudied. Monochlorobimane (MCB), a dye conjugated with GSH by glutathione-s-transferase (GST) to form a fluorescent adduct, was used to visualize organ-specific GSH utilization in live developing zebrafish (Danio rerio) embryos. Embryos were incubated in 20 μM MCB for 1 h and imaged on an epifluorescence microscope. GSH conjugation with MCB was high during early organogenesis, decreasing as embryos aged. The heart had fluorescence 21-fold above autofluorescence at 24 hpf, dropping to 8.5-fold by 48 hpf; this increased again by 72 hpf to 23.5-fold, and stayed high till 96 hpf (18-fold). The brain had lower fluorescence (10-fold) at 24 and 48 hpf, steadily increasing to 30-fold by 96 hpf. The sensitivity and specificity of MCB staining was then tested with known GSH modulators. A 10-min treatment at 48 hpf with 750 μM tert-butylhydroperoxide, caused organ-specific reductions in staining, with the heart losing 30% fluorescence, and, the brain ventricle losing 47% fluorescence. A 24 h treatment from 24-48 hpf with 100 μM of N-Acetylcysteine (NAC) resulted in significantly increased fluorescence, with the brain ventricle and heart showing 312% and 240% increases respectively, these were abolished upon co-treatment with 5 μM BSO, an inhibitor of the enzyme that utilizes NAC to synthesize GSH. A 60 min 100 μM treatment with ethacrynic acid, a specific GST inhibitor, caused 30% reduction in fluorescence across all measured structures. MCB staining was then applied to test for GSH disruptions caused by the toxicants perfluorooctanesulfonic acid and mono-(2-ethyl-hexyl)phthalate; MCB fluorescence responded in a dose, structure and age-dependent manner. MCB staining is a robust, sensitive method to detect spatiotemporal changes in GSH utilization, and, can be applied to identify sensitive target tissues of toxicants.