Elucidation of molecular mechanism of the phase 2 xenobiotic induction using zebrafish system

亲电或氧化应激诱导第二相解毒酶的基因表达。在这项研究中，我们试图通过筛选对亲电物质有缺陷的突变斑马鱼来确定这种诱导的分子基础。我们使用乙基亚硝胺(ENU)作为诱变剂，在成年雄鱼中引入了随机突变。从ENU诱变的鱼类和后续的F2家系中培育出F1后代。以F3代仔鱼为材料进行了突变鱼的筛选。用顺丁烯二酸二乙酯(DEM)、金诺芬(Auranofin)或对照溶剂二甲基亚砜处理受精后5d的F3幼虫6h，用整体原位杂交法分析GSTP在这些幼虫中的表达。我们寻找了突变株系，其中包括经DEM或AuR处理后没有或弱诱导GSTP的幼虫。我们筛选了125个F2家系，分离出了一些在第二阶段诱导中存在缺陷的突变株系。我们还分离了70个在形态发生上存在缺陷的株系。另一方面，为了提高我们的突变体鱼的筛选水平，我们试图通过亲电处理获得表达绿色荧光蛋白的转基因鱼。为此，我们利用了GSTP的基因调控区，因为在我们测试的第二阶段基因中，它在DEM处理后显示出最强的诱导作用。通过GFP-Report分析，我们证明GSTP转录起始点上游的3kb区域足以被Nrf2反式激活。因此，我们试图建立染色体上含有GSTP-GFP报告结构的稳定转基因株系。结果共分离到3个转基因株系。此外，我们分析了GSTP调控区中Nrf2反式激活的关键顺式调控元件，最后证明了位于转录起始点上游50bp的pare序列对于反式激活和Nrf2结合都是必不可少的。

Electrophiles or oxidative stresses induce gene expression of phase 2 detoxification enzymes. In this study, we tried to identify molecular basis of this induction by screening mutant zebrafish that has defects in response to electrophiles. We introduced random mutations in adult male fish using ethylnitrosourea (ENU) as a mutagen. F1 progenies derived from ENU mutagenized fish and subsequent F2 families were raised. Screening of mutant fish was carried out using F3 larvae. F3 larvae at 5 days after fertilization were treated with diethylmaleate (DEM), auranofin (AUR), or control solvent dimethylsulfoxide for 6 hours, and induction of gstp expression in these larvae were analyzed by whole-mount in situ hybridization. We searched for mutant lines which included larvae displaying no or weak induction of gstp after treatment of DEM or AUR. We have screened 125 F2 families and isolated some candidates of mutant lines which had defects in phase 2 induction. We also isolated 70 lines displaying defects in morphogenesis. On the other hands, in order to improve our mutant fish screening, we tried to generate transgenic fish which expresses GFP after treatment of electrophiles. For this purpose, we utilized the gene regulatory region of gstp since it showed the strongest induction after treatment of DEM among phase 2 genes we had tested. By GFP-reporter analysis, we demonstrated that 3 kb region upstream of transcriptional initiation site of gstp was enough for transactivation by Nrf2. We therefore tried to establish stable transgenic lines of containing this gstp-GFP reporter construct in their chromosomes. As a result, three transgenic lines were isolated. Furthermore, we analyzed critical cis-regulatory elements for Nrf2 transactivation in the gstp regulatory region, and finally demonstrated that PARE sequence which was located just 50 bp upstream of transcriptional initiation site was essential for both the transactivation and Nrf2 binding.