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

1)对4万份非洲爪蟾（Xenopus laevis）克隆进行了广泛的macmarray分析，探讨了2,3,7,8-四氯二苯并-二恶英（TODD）的作用机制。在受精后27小时（hpf）诱导1个克隆、5个克隆减少，在受精后7小时（dpf）诱导41个克隆，诱导14个克隆、42个克隆减少。然而，除了cytochme P4501A（cyp1a6,7）外，我们没有发现任何其他基因，这与northern blot分析证实的结果一致。2)我们最初从爪蟾（xAHR）中克隆了一个芳烃受体cDNA。xAHR已经从ldpf中表达，TCDD同时诱导CYP1A6和7，xAHR增加7dpf。在诱导CYP1B的同时，TCDD不影响Arnt和谷胱甘肽转移酶。3)注射AHR2 morpholino （AHR2- mo）或CYP1A morpholino （CYP1A- mo）阻断了血管内皮中CYP1A蛋白的诱导，并保护了TCDD发育毒性的所有终点，包括循环衰竭和下颌生长障碍。在所有观察中，CYP1A-MO比AHR2-MO更有效地对抗较高浓度的TCDD。这表明，ahr2介导的CYP1A在斑马鱼胚胎血管内皮的诱导参与了tcdd引起的各种毒性。4)选择性COX2抑制剂显著抑制tcdd诱导的中脑循环衰竭，而选择性COX1抑制剂和选择性5-脂氧合酶（5-LOX）抑制剂则不抑制tcdd诱导的中脑循环衰竭。抗COX2的Morpholino反义寡核苷酸（COX2- mo）也能通过TCDD恢复中脑循环衰竭。此外，COX2抑制剂和COX2- mo均可消除tcdd诱导的中脑背侧细胞凋亡。在颈动脉等部分血管中检测到COX2转录本，TCDD不影响其表达。这些结果提示COX2参与了发育中的斑马鱼tcdd诱导的中脑循环衰竭。5)斑马鱼发育中的下颌骨表达Sonic hedgehog （Shh）、Tiggy"winkle hedgehog （thwhh）及其受体和转录因子。Shh功能缺陷突变体或正常幼虫用环巴胺（一种刺猬信号抑制剂）处理后，显示出明显的颌骨生长迟缓。经TCDD处理的幼虫也表现出下颌发育迟缓，尽管其受体和转录因子的转录几乎没有受到影响，但发育中的下颌Shh和thwhh的表达明显减少。AHR2-MO抑制了hedgehog基因表达的降低以及TCDD毒性的典型宏观终点。这些结果表明，暴露于TCDD后，Shh的减少发生在AHR通路激活后，并可能对颌骨发育产生不利影响。少

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