2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptosis in central nervous system: Correlation between local circulation failure and apoptosis in the dorsal mibdrain of zebradish embryos

Apoptotic cell death caused by TCDD (2,3,7,8-tetrachlorodibenzo-’-dioxin) was investigated in early embryos of zebrafish (Danio rerio), which is an excellent model for study of environmental toxicology. Embryos were exposed to TCDD from 24hours post fertilization (hpf) until observation in petri dishes at 28.5degC. As shown by terminal transferase-mediated nick-end-labeling (TUNEL) staining, TCDD exposure caused a significant increase in the number of TUNEL-positive cells, especially in the dorsal midbrain (optic tectum). The ultrastructures of these TUNEL-positive cells showed characteristics of apoptosis such as condensation and cleavage of nuclear chromatin. The effects of TCDD on TUNEL-positive cells were abolished by a caspase inhibitor. During TCDD exposure, the percentages of TUNEL-positive cells reached maximum at 60 hpf and decreased to the basal level by 96 hpf. In order to examine the effects of TCDD on circulation, time-lapse recordings were made using a digital video camera attached to a light microscope. It was found that TCDD decreased the blood flow in the mesencephalic vein of the dorsal midbrain at about 50 hpf, but the blood flow recovered to the control level by 60 hpf with a similar concentration dependency to that of the induction of apoptosis, whereas the blood flow in the trunk did not significantly change until 72 hpf. Induction of cell apoptosis and the blood flow retardation by TCDD were mimicked by bata-naphthoflavone, an agonist of the arylhydrocarbon receptor (AhR) and almost abolished by alpha-naphthoflavone, an antagonist of AhR. Both effects by TCDD were similarly inhibited by cytochrome P450 inhibitors and antioxidants. The same range of TCDD greatly enhanced mRNA expressions and immunoreactivity of cytochrome P450 1A in endothelial cells of the vessels in the brain, including the mesencephalic vein. These results suggested that TCDD induces apoptosis in the central nervous system of zebrafish embryos through local circulation failure, which could be caused by AhR activation, induction of P450 1A or oxidative stress