Study of the mechanism of developmental toxicology by dioxin with cDNA microarray analysis

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1)We carried out the extensive macmarray analysis with forty thousand clones for Xenopus laevis (Xenopus) to investigated the mechanism of 2,3,7,8-tetrachlomdibenzo-gdioxin (TODD). TODD caused induction of 1 done and reduction of 5 clones at 27 hours post fertilization (hpf), induction of 41 clones at 7 dpf and induction of 14 clones and reduction of 42 clones. However, we mould not find any done, which was confirmed with northern blot analysis except cytochmme P4501A(CYP1A6, 7).2)We originally cloned a cDNA of arylhydrocarbon receptor from Xenopus (xAHR). xAHR was already expressed from ldpf and was increased by 7dpf with concomitant induction of CYP1A6 and 7 by TCDD. While CYP1B was induced, Arnt and glutathion transferase was not affected by TCDD treatment.3)Injection with either AHR2 morpholino (AHR2-MO) or CYP1A morpholino (CYP1A-MO) blocked induction of CYP1A protein in the vascular endothelium and protected against all endpoints of TCDD developmental toxicity already observed in … More cluding circulation failure and impairement of lower jaw growth. For all observations, CYP1A-MO was more effective against the higher TCDD concentrations than AHR2-MO. These suggest that AHR2-mediated CYP1A induction in the vascular endothelium of the zebrafish embryo is involved in various toxicity elicited by TCDD.4)TCDD-inducecl mesencephalic circulation failure was markedly inhibited by selective COX2 inhibitors but not by selective COX1 inhibitor and a selective 5-lipoxygenase (5-LOX) inhibitor. Morpholino antisense oligos against COX2 (COX2-MO) also recovered mesencephalic circulation failure by TCDD. Furthermore, TCDD-induced apoptosis in dorsal midbrain was also abolished by both COX2 inhibitor and COX2-MO. COX2 transcripts were detected in some vessels including carotid artery, while the expression was not affected by TCDD. These results suggest the involvement of COX2 in TCDD-induced mesencephalic circulation failure in developing zebrafish.5)Zebrafish developing lower jaw expresses Sonic hedgehog (Shh), Tiggy"winkle hedgehog (Twhh) and their receptors as well as their transcription factors. Shh function-defective mutant or normal larva treated with cyclopamine, an inhibitor for hedgehog signaling showed marked retardation of jaw growth. Larvae treated with TCDD also exhibiting jaw retardation showed a marked reduction of Shh and Twhh expression in the developing lower jaw, although transcripts of their receptors and transcriptional factors were hardly affected. AHR2-MO inhibited the reduction of hedgehog expression as well as the typical macroscopic endpoints of TCDD toxicity. These results suggest a reduction in Shh by TCDD exposure occurs postactivation of the AHR pathway and possible contribution to the adverse effects on the development of the jaw. Less