2,3,7,8-Tetrachlorodibenzo-p-dioxin Toxicity in the Zebrafish Embryo: Local Circulation Failure in the Dorsal Midbrain Is Associated with Increased Apoptosis

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on local circulation and apoptosis in the midbrain were investigated in zebrafish (Danio rerio) embryos during early development. Embryos were exposed to TCDD from 24 h post fertilization (hpf) until observation, in water maintained at 28.5 degrees C. TCDD decreased blood flow in the mesencephalic vein, the only vessel perfusing the dorsal midbrain of the embryo. At 50 hpf, blood flow was maximally reduced in this vessel and gradually returned to the control level at 60 hpf. In contrast, blood flows in the trunk and in other vessels of the head of the embryo did not significantly change until 72 hpf. Furthermore, TCDD exposure caused apoptosis in the midbrain at 60 hpf, and the TCDD dose response relationship for this effect was similar to that for reduced blood flow in the mesencephalic vein at 50 hpf. The effects of TCDD on apoptosis in the midbrain, but not on blood flow, were abolished by Z-VAD-FMK, a general caspase inhibitor. TCDD effects on both endpoints were mimicked by beta-naphthoflavone (BNF), an aryl hydrocarbon receptor (AHR) agonist, and almost abolished by concomitant exposure to TCDD and alpha-naphthoflavone (ANF), an AHR antagonist. Concomitant exposure to TCDD and either an inhibitor of cytochrome P450 (CYP) (SKF525A or miconazole) or an antioxidant (N-acetylcysteine or ascorbic acid) inhibited these effects of TCDD. The incidence of apoptosis in the midbrain was inversely related to blood flow in this brain region following these various treatments and graded TCDD exposure concentrations (r = -0.91). The same range of TCDD exposure concentrations that reduced blood flow and increased apoptosis in the midbrain greatly enhanced CYP1A mRNA expression and immunoreactivity at 50 hpf in endothelial cells of blood vessels including the mesencephalic vein and the heart, but not the brain parenchyma. Taken together, these results suggest that TCDD induces apoptosis in the midbrain of the zebrafish embryo secondary to local circulation failure, which could be related to AHR activation, induction of CYP1A, and oxidative stress.