The cardiovascular system is one of the most characteristic and important targets for developmental toxicity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in fish larvae. However, knowledge of the mechanism of TCDD-induced edema after heterodimerization of aryl hydrocarbon receptor type 2 (AHR2) and AHR nuclear translocator type 1 (ARNT1) is still limited. In the present study, microscopic analysis with a high-speed camera revealed that TCDD increased the size of a small cavity between the heart and body wall in early eleutheroembryos, a toxic effect that we designate as precardiac edema. A concentration-response curve for precardiac edema at 2 days post fertilization (dpf) showed close similarity to that for conventional pericardial edema at 3 dpf. Precardiac edema caused by TCDD was reduced by morpholino knockdown of AHR2 and ARNT1, as well as by an antioxidant (ascorbic acid). A selective inhibitor of cyclooxygenase type 2 (COX2), NS398, also markedly inhibited TCDD-induced precardiac edema. A thromboxane receptor (TP) antagonist, ICI-192,605 almost abolished TCDD-induced precardiac edema and this effect was cancelled by U46619, a TP agonist, which was not influential in the action of TCDD by itself. Knockdown of COX2b and thromboxane A synthase 1 (TBXS), but not COX2a, strongly reduced TCDD-induced precardiac edema. Knockdown of COX2b was without effect on mesencephalic circulation failure caused by TCDD. The edema by TCDD was also inhibited by knockdown of c-mpl, a thrombopoietin receptor necessary for thromobocyte production. Finally, induction of COX2b, but not COX2a, by TCDD was seen in eleutheroembryos at 3 dpf. These results suggest a role of the COX2b-thromboxane pathway in precardiac edema formation following TCDD exposure in developing zebrafish.

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To confirm the usefulness of zebrafish for evaluating the teratogenic potential of drug candidates, the effect of O-ethylhydroxylamine hydrochloride (OHY), which induces mutagenesis by methylation, was evaluated in teratogenicity studies in rats and zebrafish. In the rat teratogenicity study, OHY-induced cardiovascular malformations such as increased abnormal vascular structures and ventricular septal defects. In the teratogenicity study using zebrafish-injected microspheres and green fluorescent protein-expressing Tg zebrafish (flk1:EGFP), OHY exposure was associated with the loss or malformation of the mandibular arch, opercular artery, and fourth branchial arch. These results suggested that OHY-induced external malformations in zebrafish eleutheroembryos adequately reflect OHY's teratogenicity in rat fetuses. Moreover, the zebrafish teratogenicity study incorporating vascular morphological examinations, including those of blood vessels in the heart, head and trunk, is an easy and reliable screening method to detect potential drug-induced teratogenicity and phenotypic characteristics.