Figure 3

Induction of ROS and DNA fragmentation in erythrocytes and the endocardium by β-lapachone treatment. A: Embryos at 24 hours post-fertilization (hpf) were treated with DMSO or β-lapachone for 4 h and fixed at 30 and 48 hpf for either hbae1 hybridization or o-dianisidine (ODA) staining. B: DMSO- and β-lapachone-treated embryos were incubated with CM-H₂DCFDA for 1 h at 29 hpf, and both bright-field (a, c) and fluorescent (b, d-f) images under a green fluorescent protein (GFP) filter were recorded. Atrial boundary is depicted by white dotted lines. Arrows indicate flowing erythrocytes with green fluorescence from the common cardinal vein to the atrium of the heart (d-f) of β-lapachone-treated embryos. C: Embryos were treated with DMSO or β-lapachone at 48 hpf for 4 h, fixed at 52 hpf, and stained with ODA. After paraffin sectioning, TUNEL reactions were conducted, and fluorescein-dUTP-labeled erythrocytes were detected in β-lapachone-treated embryos (f, h). DIC images are shown (a, e), and the inset figure in panel a shows the position of sectioning. Tail border is illustrated by white dotted lines. Arrows in panels a and e indicate ODA-stained erythrocytes. D: Embryos were treated with DMSO or β-lapachone at 48 hpf for 4 h and fixed at 52 hpf. After paraffin sectioning, TUNEL reactions were conducted, and fluorescein-dUTP-labeled cells located in the endocardium were detected in β-lapachone-treated embryos (f, h, j, l). In addition, fluorescein-dUTP-labeled erythrocytes were detected in the yolk near the heart (f, j). Red dotted lines indicate borders of head and yolk while white dotted lines illustrate ventricle boundaries. * indicates erythrocytes. V, ventricle. Scale bars represent 100 μm.