Aryl hydrocarbon receptor multiplicity in a frog model of dioxin toxicity

二恶英毒性青蛙模型中芳基碳氢化合物受体的多样性

• 批准号: 8687034
• 负责人: WADE H POWELL
• 金额: $ 30.26万
• 依托单位: KENYON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2018-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8687034
• 关键词: AHR gene; Adult; Affinity; African; Animals; Aromatic Hydrocarbons; Aromatic Polycyclic Hydrocarbons; Aryl Hydrocarbon Receptor; Automobile Driving; Binding; Biological Assay; Biological Metamorphosis; Biological Models; Brain; Breeding; CYP1A1 gene; CYP1A6 gene; Carbazoles; Cardiovascular system; Cell Differentiation process; Cell Line; Cells; Communities; Complex; Defect; Development; Developmental Process; Dioxins; Edema; Embryo; Environment; Environmental Pollution; Enzymes; Event; Exhibits; Eye; Future; Gene Expression; Gene Targeting; Generations; Genes; Genome; Genomics; Genotype; Goals; Grant; Growth; Health; Histology; Human; Human Development; Immune; In Situ Hybridization; Industrial Waste; Knock-out; Ligands; Liver; Mammals; Measures; Mediating; Messenger RNA; Modeling; Molecular; Nervous system structure; Organ; Organogenesis; Pattern; Petroleum; Phenotype; Physicians; Physiology; Play; Proteins; Rana; Receptor Signaling; Relative (related person); Reporter; Research; Research Training; Reverse Transcriptase Polymerase Chain Reaction; Risk Assessment; Role; Sampling; Scientist; Signal Transduction; Spinal; Staging; Structure; T cell differentiation; Tadpoles; Testing; Tetrachlorodibenzodioxin; Tissues; Toxic effect; Toxicity Tests; Toxicology; Transcript; Transcription Coactivator; Transcriptional Regulation; Work; Xenobiotics; Xenopus; Xenopus laevis; Xenopus sp.; activating transcription factor; aryl hydrocarbon receptor ligand; carbazole; cigarette smoking; comparative; developmental toxicology; duplicate genes; environmental chemical; experience; human AHR protein; human disease; in vivo; infancy; innovative technologies; loss of function mutation; mRNA Expression; mature animal; member; mutant; nuclease; null mutation; paralogous gene; receptor function; research study; tissue regeneration

DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic and carcinogenic effects of numerous environmental contaminants, including dioxin-like industrial waste compounds and aromatic hydrocarbons in petroleum and cigarette smoke. AHR also plays essential roles in normal developmental processes, including liver development, cardiovascular development and T-cell differentiation. Alterations in AHR activity thus underlie multiple human disease states. Humans have one Ahr gene. The mechanisms by which the single AHR carries out such diverse, seemingly unrelated functions are poorly understood. The African clawed frog, Xenopus laevis, is a widely used model of both basic vertebrate development and developmental toxicology. The frog model differs from humans in having two AHRs (AHR1α and AHR1β), which resulted from a taxonomically unique genome duplication ~40 mya. Their transcripts exhibit distinct expression patterns in adult frog, raising the possibility of subfunctionalization, or partitioningof multiple roles of a single ancestral protein into duplicate paralogs. This AREA grant will support an undergraduate lab group to test the hypothesis that AHR1α and AHR1β have non-redundant functions in frog development, transcriptional regulation, and/or toxicity. The proposed project has three Specific Aims. In Aim 1, we will use in situ hybridization and quantitative RT-PCR to measure the relative expression of AHR1α and AHR1β mRNAs in embryonic and adult tissues, quantitatively verifying their differential expression. Studies under specific Aim 2 will test the hypothesis that AHR1α and AHR1β regulate distinct sets of target genes. Using transcription activator-like effector nucleases (TALENs) we will edit the genome of X. laevis cell line XLK-WG to knock out expression of one or both AHRs. Resulting mutant cell lines will be treated with the potent xenobiotic AHR ligand TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) or the endogenous AHR ligand FICZ (6-formylindolo[3,2b]carbazole), and resulting changes in mRNA expression will be measured and characterized on the genomic scale by RNAseq. Finally, in Aim 3 we will employ TALENs to generate mutant frogs lacking AHR1α and/or AHR1β. Knockout tadpoles will be used to test the hypothesis that each AHR paralog plays a distinct role in frog development or toxicity. In addition to measuring expression changes in transcripts identified in XLK-WG cells, we will examine TCDD and FICZ-treated embryos and tadpoles for common gross morphological defects (e.g. edema, spinal defects) as well as histological changes in specific tissues, including liver. Our comparative approach capitalizes on the opportunity presented by the two X. laevis AHRs to dissect the multiple, complex functions of the single human protein. Understanding the differences between frog and human AHR signaling will also aid toxicological risk assessment involving FETAX and similar developmental toxicity tests employing frog embryos. Finally, this AREA project will provide a technologically sophisticated, long-term research training experience for numerous undergraduate scientists.

描述（由申请人提供）：芳烃受体（AHR）是一种配体激活的转录因子，介导许多环境污染物的毒性和致癌作用，包括二恶英样工业废物化合物和石油和香烟烟雾中的芳烃。AHR在正常发育过程中也起着重要作用，包括肝脏发育、心血管发育和t细胞分化。因此，AHR活性的改变是多种人类疾病状态的基础。人类有一个Ahr基因。单个AHR执行如此多样、看似不相关的功能的机制尚不清楚。非洲爪蟾（Xenopus laevis）是基础脊椎动物发育和发育毒理学的一个广泛使用的模型。蛙模型与人类的不同之处在于具有两个ahr (AHR1α和AHR1β)，这是由分类上独特的基因组重复造成的~ 40mya。它们的转录本在成年青蛙中表现出不同的表达模式，这提高了亚功能化的可能性，或者单个祖先蛋白的多个角色划分为重复的相似物。该区域基金将支持一个本科实验室小组测试AHR1α和AHR1β在青蛙发育、转录调节和/或毒性中具有非冗余功能的假设。拟议的项目有三个具体目标。在Aim 1中，我们将使用原位杂交和定量RT-PCR来测量AHR1α和AHR1β mrna在胚胎和成人组织中的相对表达，定量验证它们的差异表达。特定Aim 2下的研究将检验AHR1α和AHR1β调控不同靶基因的假设。利用转录激活因子样效应核酸酶（TALENs）对X. laevis细胞系XLK-WG基因组进行编辑，敲除其中一个或两个ahr的表达。由此产生的突变细胞系将用强效外源AHR配体TCDD（2,3,7,8-四氯二苯并-对二恶英）或内源AHR配体FICZ（6-甲酰基林多洛[3,2b]咔唑）处理，并通过RNAseq在基因组尺度上测量和表征由此产生的mRNA表达变化。最后，在Aim 3中，我们将使用TALENs产生缺乏AHR1α和/或AHR1β的突变青蛙。基因敲除的蝌蚪将被用来检验假设，即每个AHR平行体在青蛙的发育或毒性中起着不同的作用。除了测量XLK-WG细胞中转录本的表达变化外，我们还将检查TCDD和ficz处理的胚胎和蝌蚪中常见的大体形态缺陷（如水肿、脊柱缺陷）以及特定组织（包括肝脏）的组织学变化。我们的比较方法利用了两个X. laevis AHRs提供的机会来剖析单个人类蛋白质的多种复杂功能。了解青蛙和人类AHR信号之间的差异也将有助于涉及FETAX的毒理学风险评估和使用青蛙胚胎的类似发育毒性试验。最后，这个AREA项目将为众多本科科学家提供技术成熟的长期研究培训经验。