Pro-Oxididant Induce Hemolysis in a Zebrafish Model of Glucose-6-Phosphate Dehydrogenase Deficiency.

Abstract 2078

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common genetic defect and enzymopathy worldwide with an estimated 400 million persons affected. G6PD deficiency causes acute hemolysis in children exposed to pro-oxidants. Mouse models have suffered from their inaccuracy recapitulating the disease because of early death or lack of significant hemolysis in response to oxidative challenge. The zebrafish is an expanding model for many hematopoietic diseases. It's ex utero development and optical clarity offer advantages not obtained using mouse models. We targeted morpholinos to the 5-prime coding region of zebrafish g6pd. The g6pd morphants showed a significant reduction in G6pd protein expression and G6pd enzyme activity. We found a dose-dependant phenotype in zebrafish g6pd morphants. Low dose morpholino, 1.2 pmols, embryos displayed no overt phenotype, while high dose, 1.5 pmols, g6pd morphants displayed over a 50% reduction in levels of hemoglobin as determined by o-dianisidine staining. This hemolysis resulted in a phenotype displaying severe pericardial edema, which could be rescued by injection of zebrafish g6pd mRNA. Embryos injected with random morpholinos displayed no abnormalities. Common pro-oxidants include menthol, as a therapeutic ointment used in West Africa; naphthalene, an active ingredient in mothballs; and more commonly antimalaria drugs such as primaquine. We next tested whether an oxidative challenge on the low dose morpholino injected embryos by treatment with menthol or naphthol (the active form of naphthalene). We found these compounds significantly increased free radical production using a 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate fluorescence-based assay as well as an increase in hydroethidine staining, another indicator of free radical production. When low dose morphants were exposed to either compound, they displayed massive cardiac edema and significantly reduced hemoglobin levels due to hemolysis. Hemolysis was verified by performing similar experiments in fish with labeled erythrocytes, gata1:DsRed where we found a 60% reduction in gata1 positive cells by flow cytometry. Imagestream flow cytometry revealed that remaining erythrocytes stained positive for annexin V indicating active hemolysis. We were also able to show hemolysis and massive pericardial edema also occurred with exposure to the prototypical antimalaria drug, primaquine, which displayed increased reactive oxygen species at day 6 –7 post fertilization. In conclusion, we have faithfully developed a vertebrate model of G6PD deficiency in which in vivo hemolytic crisis occurs after exposure to pro-oxidants similar to what occurs in children. These compounds pose significant health risks to infants and children with G6PD deficiency. Utilizing this model, we will learn new information about novel cell processes and metabolic pathways that are disrupted in G6PD deficiency driven hemolysis. In addition, we will be able to screen potential anti-oxidants for their ability to reduce hemolysis. With the ultimate goal to attenuate the morbidity caused by the severe hemolytic crises occurring in children with G6PD deficiency.