Blood vessels are primary targets for 2,3,7,8-tetrachlorodibenzo-p-dioxin in pre-cardiac edema formation in larval zebrafish.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has adverse effects on the development and function of the heart in zebrafish eleutheroembryos (embryos and larvae). We previously reported that TCDD reduced blood flow in the mesencephalic vein of zebrafish eleutheroembryos long before inducing pericardial edema. In the present study, we compared early edema (pre-cardiac edema), reduction of deduced cardiac output and reduction of blood flow in the dorsal aorta and cardinal vein caused by TCDD. In the same group of eleutheroembryos, TCDD (1.0 ppb) caused pre-cardiac edema and circulation failure at the cardinal vein in the central trunk region with the similar time courses from 42 to 54 h post fertilization (hpf), while the same concentration of TCDD did not significantly affect aortic circulation in the central trunk region or cardiac output. The dependence of pre-cardiac edema on TCDD concentration (0-2.0 ppb) at 55 hpf correlated well with the dependence of blood flow through the cardinal vein on TCDD concentration. Several treatments that markedly inhibited TCDD-induced pre-cardiac edema such as knockdown of aryl hydrocarbon receptor nuclear translocator-1 (ARNT1) and treatment with ascorbic acid, an antioxidant, did not significantly prevent the reduction of cardiac output at 55 hpf caused by 2.0 ppb TCDD. TCDD caused hemorrhage and extravasation of Evans blue that was intravascularly injected with bovine serum albumin, suggesting an increase in endothelium permeability to serum protein induced by TCDD. The results suggest that the blood vessels are primary targets of TCDD in edema formation in larval zebrafish.

Usefulness of zebrafish in evaluating drug-induced teratogenicity in cardiovascular system.

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.

Protective effect of prostacyclin against pre-cardiac edema caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin and a thromboxane receptor agonist in developing zebrafish.

The role of prostaglandin pathways has been suggested in some toxicological responses to dioxins. Cyclooxygenase type 2b (COX2b), thromboxane synthase, and the thromboxane receptor (TP) pathway have been implicated in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced pre-cardiac edema in developing zebrafish at 55 h post fertilization (hpf). Pre-cardiac edema refers to edema located in a small cavity between the heart and body wall of zebrafish eleutheroembryos. In the present study, we assessed the role of prostacyclin, which counteracts some biological effects of thromboxane, in TCDD-induced pre-cardiac edema. Pre-cardiac edema induced by TCDD exposure (0.5 and 1 ppb) beginning at 24 hpf was markedly inhibited by exposure to beraprost (5 and 10 µM), a prostacyclin receptor (IP) agonist, beginning at 33 hpf. The preventive effect of beraprost was reduced by exposure to CAY10441 (10 µM), an IP antagonist starting at 33 hpf. Knockdowns of the IP receptor (IP-KD) with two different morpholinos caused edema by themselves and enhanced pre-cardiac edema caused by the low concentration of TCDD (0.5 ppb). On the other hand, short exposure beginning at 48 hpf to U46619 (7.5-30 µM), a thromboxane receptor agonist caused pre-cardiac edema, which was inhibited by exposure beginning at 48 hpf to both ICI-192,605 (24 µM), a TP antagonist, and beraprost. Expression of prostacyclin synthase was increased from fertilization, plateaued by 48 hpf, and was maintained until at least 96 hpf. Overall, the results demonstrate a preventive effect of prostacyclin on TCDD-induced pre-cardiac edema in developing zebrafish.

Involvement of COX2-thromboxane pathway in TCDD-induced precardiac edema in developing zebrafish.

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 canceled 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.