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

基本信息

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
项目类别：
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

项目摘要

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.

Description (Description by Applicant): The ARYL HYDROCARBON RECEPTOR (AHR) is a ligand-activated transcription factor that MediaTES THE TOXIC AND Ogenic Effects of Numerous Environmental Contaminants, Inclouding DIOXIN-LIKE INDUSTRIAL WASTE COMPOUNDS and AROMATIC HYDROCARBONS IND AND AND AND AND AND AND和。如ASELY使用的基本模型，毒物模型与人类具有两个AHR（AHR1α和AR1β）的不同之处。或将单个祖先蛋白的多种作用分为重复的旁系同源物。 RT-PCR测量胚胎和成人组织中AHR1α和AHR1β的相对表达，定量验证其差异表达。一个或两个AHR ，在AIM 3中，我们将使用Talens产生缺乏AHR1α和 /或AHR1β的突变青蛙，以测量XLK-WG细胞中鉴定出的转录物的表达变化，用于胚胎的胚胎和t骨的the剂（例如，脊柱缺陷，脊柱缺陷）以及包括肝脏在内的特定组织的组织学变化。对众多地下科学家进行研究培训经验的测试。