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

1)对非洲爪哇(Xenopus laevis(Xenopus))进行了4万个克隆的显微图像分析，研究了2，3，7，8-四氯二苯并二恶英(Todd)的作用机理。Todd在受精后27小时诱导1个Done，减少5个克隆，7 d后诱导41个克隆，诱导14个克隆，减少42个克隆。然而，除了细胞色素P4501A(CYP1A6，7)外，我们没有发现任何Done，这被Northern印迹分析所证实。XAHR已在1dpf中表达，并在TCDD诱导细胞色素P1A6和7的表达的同时，增加了7dpf。3)注射AHR2吗啉(AHR2-MO)或细胞色素P1a吗啉(CyP1a-MO)可阻断血管内皮细胞中细胞色素P1a蛋白的诱导，并对…中已观察到的TCDD发育毒性的所有终点具有保护作用更多的包括循环衰竭和下颌骨发育障碍。在所有观察中，CYP1A-MO对较高的TCDD浓度比AHR2-MO更有效。这些结果表明，AHR2介导的斑马鱼胚胎血管内皮细胞细胞色素P1a的诱导与TCDD引起的多种毒性有关。4)选择性COX2抑制剂可明显抑制TCDD诱导的中脑循环衰竭，但选择性COX1抑制剂和选择性5-脂氧合酶(5-LOX)抑制剂不能。抗COX2的吗啡反义寡核苷酸(COX2-MO)也能恢复TCDD所致的中脑循环衰竭。此外，COX2抑制剂和COX2-MO均可阻断TCDD诱导的背侧中脑细胞凋亡。在包括颈动脉在内的部分血管中检测到COX2的转录本，而TCDD对其表达没有影响。这些结果提示COX2参与了TCDD诱导的中脑循环衰竭斑马鱼的发育。5)斑马鱼发育下巴表达Sonic Hedgehog(Shh)、Tiggy Winkle Hedgehog(TWhh)及其受体及其转录因子。Shh功能缺陷突变体或正常幼虫用刺猬信号抑制剂环丙胺处理后，颌骨生长明显减慢。TCDD处理的幼虫也表现出颌骨发育迟缓，但其受体和转录因子的转录水平几乎没有受到影响，但在发育中的下颌骨中Shh和TWhh的表达明显减少。AHR2-MO抑制Hhedgehog表达的减少，并抑制TCDD毒性的典型宏观终点。这些结果表明，TCDD暴露引起的Shh减少发生在AHR途径激活后，可能是对颌骨发育的不利影响的贡献。较少

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