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

亲电体或氧化应激诱导2相解毒酶的基因表达。在这项研究中，我们试图确定这种诱导的分子基础，通过筛选突变斑马鱼，有缺陷的亲电反应。我们用乙基亚硝基脲（ENU）作为诱变剂在成年雄鱼中引入随机突变。从ENU诱变的鱼衍生的F1后代和随后的F2家庭进行了饲养。使用F3幼虫进行突变鱼的筛选。用马来酸二乙酯（DEM）、金诺芬（AUR）或对照溶剂二甲基亚砜处理受精后5 d的F3幼虫6 h，通过整体原位杂交分析这些幼虫中gstp表达的诱导。我们寻找突变株系，其中包括幼虫显示没有或弱诱导的gstp后，处理的DEM或AUR。我们筛选了125个F2家系，并分离出了一些在第二阶段诱导中有缺陷的突变系。我们还分离出70行显示缺陷的形态发生。另一方面，为了提高我们的突变鱼筛选，我们试图产生转基因鱼表达GFP亲电处理后。为了这个目的，我们利用gstp的基因调控区，因为在我们测试的2期基因中，它在DEM处理后显示出最强的诱导。通过GFP-报告基因分析，我们证明了gstp转录起始位点上游3 kb的区域足以被Nrf 2反式激活。因此，我们试图建立在其染色体中含有该gstp-GFP报告基因构建体的稳定转基因株系。结果，分离出三个转基因株系。此外，我们分析了gstp调控区中Nrf 2反式激活的关键顺式调控元件，最终证明位于转录起始位点上游50 bp的帕雷序列对Nrf 2的反式激活和结合都是必需的。

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.