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.

Modulating glutathione thiol status alters pancreatic β-cell morphogenesis in the developing zebrafish (Danio rerio) embryo

“…3 ; Table 1 ), indicating Nrf2a stabilization ameliorates oxidative stress. Both 77.6 and 776 μM tBOOH are sublethal concentrations with no overt toxic or growth effects in the zebrafish embryo [ 17 , 26 ]. A 24-h pretreatment with 100 μM NAC, which bolsters cellular GSH pools, prevented the altered Nrf2a expression effects seen with both 77.6 μM and 776 μM tBOOH in 100% of the fish, indicating that GSH can protect against Nrf2a stabilization in pancreatic β-cells ( Fig.…”

“…Disruptions of the GSH system during organogenesis are known to induce adverse outcomes such as cell migration defects, premature differentiation, apoptosis and organ agenesis [ 12 , 15 , 16 ]. In the zebrafish ( Danio rerio) , GSH levels in the developing GI tract and pancreas are especially susceptible to redox modulators when compared to other developing organ systems [ 17 ]. Between 24 and 48 h post fertilization (hpf), the zebrafish go through the pharyngula stage – a period of rapid organogenesis [ 18 ].…”

“…1 ) [ 20 ]. Given the specific, spatiotemporal changes in GSH levels of the developing embryo [ 17 , 19 ], coupled with intricate developmental processes that dictate pancreatogenesis, we hypothesized that there is differential sensitivity of the pancreas to redox disruptions at distinct developmental timepoints.

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“…A multitude of redox stressors – endogenous metabolic byproduct ROS and exogenous xenobiotics – can deplete GSH, stabilize Nrf2 protein, and thus activate Nrf2 nuclear translocation and target gene expression [ 25 , 29 ]. Since the developing GI tract, including the pancreas, is susceptible to GSH depletion [ 17 ], it is a sensitive target of redox stress. Moreover, redox dysfunction is implicated as a mechanism of pancreatic β-cell dysfunction underlying diabetes, with Nrf2 activation protecting against the onset of diabetes [ [30] , [31] , [32] ].…”

“…Moreover, pancreatic function and development is conserved among zebrafish and humans, and the glutathione redox trajectory during concordant developmental stages between the mouse and zebrafish are also similar [ 25 ]. The zebrafish Nrf2 co-ortholog to human Nrf2 is Nrf2a [ 17 , 27 , 33 ]. We identify distinct temporal windows of redox susceptibility governing pancreatic organogenesis in vivo and demonstrate the lowered antioxidant defenses of the endocrine pancreas in situ .…”