Traditional African remedies induce hemolysis in a glucose-6-phopshate dehydrogenase deficient zebrafish model

Abstract

Traditional remedies are widely used throughout Africa in routine care for infants. However, such remedies could have detrimental effects. Acute bilirubin encephalopathy (ABE) and kernicterus spectrum disorder (KSD) are common newborn health conditions in the developing world, contributing to substantial neonatal mortality and morbidity. They frequently occur in children with glucose-6-phopshate dehydrogenase (G6PD) deficiency. Using our established zebrafish model of G6PD deficiency, we tested the effects of three traditional compounds used in the care of the newborn umbilical cord: eucalyptus oil, methylated spirits, and Yoruba herbal tea. We found that eucalyptus oil induced a 13.4% increase in a hemolytic phenotype versus control, while methylated spirits showed a 39.7% increase in affected phenotype. Yoruba herbal tea exposure showed no effect. While methylated spirits are already a known pro-oxidant, these data indicate that eucalyptus oil may also be a hemolytic trigger in those with G6PD deficiency. Discovering which agents may contribute to the pathophysiology of G6PD deficiency is critical to eliminate ABE and KSD, especially in countries with a high prevalence of G6PD deficiency. The next step in elucidating the role of these agents is to determine the clinical correlation between the use of these agents and ABE/KSD.

Figure 1

Compound tested in the G6PD-deficient zebrafish model. (A) Eucalyptus oil. (B) A variety of roots, bark, herbs to brew Yoruba tea (right panel). (C) Methylated spirits.

Figure 2

Hemolysis in a G6PD-deficient zebrafish model. Top micrograph shows a zebrafish at 72 hpf with intact red blood cells. Bottom micrograph shows an edematous G6PD-deficient zebrafish at 72 hpf after 48 h of methylated spirit exposure. Both animals were subject to o-dianisidine staining to show hemoglobin containing red cells. Zebrafish were imaged using a Leica DFC340FX fluorescent microscope with PlanAPO 1.6×/0.05 NA objective. Image capture was performed with Leica Application Suite X 3.6.0.2010 (https://www.leica-microsystems.com/products/microscope-software/p/leica-application-suite/).

Figure 3

Enumeration of the hemolytic phenotype in G6PD-deficient zebrafish after compound exposure. (A) Impact of eucalyptus oil exposure and regular fishwater on zebrafish in the following groups: G6PD MO, and random MO, uninjected control group. (B) Impact of methylated spirits exposure versus regular fishwater on zebrafish in the following groups: G6PD MO, and random MO, uninjected control group. (C) Impact of Yoruba tea exposure versus regular fishwater on zebrafish in the following groups: G6PD MO, and random MO, uninjected control group.

Figure 4

Treatment effect of compounds on G6PD-deficient zebrafish. Tallies were normalized to phenotype data produced in the unexposed embryos to produce a treatment effect. Random MO and G6PD MO injected animals were compared using multivariate analysis was used to separate effects from morpholino exposure versus compound exposure.