Aryl hydrocarbon receptor (AHR) deficiency 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（AHR）缺陷

基本信息

批准号：
10652101
负责人：
WADE H POWELL
金额：
$ 34.78万
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 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10652101
关键词：
Acute Adult Adverse effects African Agonist Amphibia Animals Apoptosis Aromatic Polycyclic Hydrocarbons Aryl Hydrocarbon Receptor Biological Assay Biological Metamorphosis Cardiovascular system Cell Cycle Cell Cycle Regulation Cell Differentiation process Cell Line Cell physiology Cells Chemical Exposure Chemicals Cultured Cells Data Defect Deformity Development Dioxins Diploidy Disease Edema Embryo Embryonic Development Environment Environmental Pollutants Exhibits Fetal Development Flow Cytometry Frequencies Future Gene Expression Alteration Gene Proteins Gene Targeting Generations Genes Genomics Head Health Hepatic Histology Human Immune Individual Industrial Waste Knock-out Larva Length Life Ligands Measures Mediating Modeling Molecular Morphology Mus Mutation Null Lymphocytes Oocytes Organogenesis Ovarian Pathway interactions Persons Petroleum Phenotype Physicians Play Ploidies Process Proliferating Propidium Diiodide Protein Deficiency Proteins Rana Regimen Risk Assessment Role Sampling Scientist Severities Stains Standardization Tadpoles Testing Tetrachlorodibenzodioxin Thyroid Gland Tissues Toxic effect Toxicology Training Variant Weather Western Blotting Work Xenopus Xenopus laevis cigarette smoke comparative developmental disease developmental toxicity embryo/fetus gastrulation genome editing human disease improved insight liver development mature animal mutant paralogous gene pollutant protein function protein structure receptor teratogenesis thyroid disruption transcription factor transcriptome sequencing undergraduate student

项目摘要

Project Summary. The Aryl Hydrocarbon Receptor protein (AHR) underlies multiple human disease states. AHR is a ligand-activated transcription factor that mediates toxicity of numerous environmental contaminants, including dioxin-like compounds such as 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) from industrial waste and polynuclear aromatic hydrocarbons in weathered crude oil and cigarette smoke. AHR also plays roles in ordinary development of liver, cardiovascular system, and oocytes, disruption of which by toxic agonists or mutation can cause disease in adults. African clawed frogs, pseudotetraploid Xenopus laevis and diploid X. tropicalis, are widely studied aquatic models of vertebrate development. Xenopus is also used in developmental toxicity screens at two life stages. FETAX (Frog Embryo Teratogenesis Assay-Xenopus) detects acute developmental toxicity resulting from chemical exposure at early life stages. The Amphibian Metamorphosis Assay (AMA) models late fetal development in humans, measuring chemical disruption of remodeling of a tadpole to a froglet. The human-health relevance of AHR function in developmental toxicity is poorly understood in frogs, limiting the validity of these assays for human risk assessment. An understanding of the fundamental developmental and cellular functions of frog AHRs is essential for the use of this model in toxicological studies. The overall objective is to use genome-edited, AHR-deficient Xenopus models, including ahr-null X. tropicalis animals and X. laevis cell lines, to identify specific functions of AHRs in developing frogs and cultured cells. Studies under Aim 1 will identify functions of AHR in X. tropicalis development. We will characterize phenotypes displayed in ahr-null frogs, using the FETAX regimen to compare frequency and severity of typical developmental defects in ahr-null and wild-type embryos, including survival, length, axial and head deformities, and edemas. We will also test the hypothesis that ahr-null frogs display hepatic and ovarian phenotypes typical of AHR-/- mice, comparing morphology and histology. Finally, we will use a suite of assays to determine how loss of ahr alters tissue remodeling during metamorphosis. We will couple morphological endpoints with RNAseq studies to determine gene expression alterations underlying phentoypes. In Aim 2, we will use X. laevis cell lines lacking one of the two ahr paralogs in this species, testing the hypothesis that individual AHR paralogs play distinct role in cell cycle regulation. We will compare slow-growing ahr1a-null mutants with ahr1b-null and wild-type XLK-WG cells by flow-cytometry, establishing cell cycle distribution of each. We will also test the hypothesis that ahr1a-null cells are prone to apoptosis. Preliminary data suggest that AHR1a and AHR1b regulate distinct sets of gene targets. We will test this hypothesis in RNAseq studies. This AREA project will provide comparative insight into the pathways of AHR-mediated developmental toxicity shared among vertebrate models. It will also lend mechanistic underpinning to frog developmental toxicity screens such as FETAX and AMA, improving their validity for human risk assessment.

项目摘要。芳基烃受体蛋白 (AHR) 是多种人类疾病状态的基础。 AHR 是一种配体激活的转录因子，可介导多种环境污染物的毒性，包括工业废物中的二恶英类化合物，例如 2,3,7,8 四氯二苯并-对二恶英 (TCDD) 风化原油和香烟烟雾中的多核芳烃。 AHR 还发挥着以下作用：肝脏、心血管系统和卵母细胞的正常发育，被有毒激动剂或药物破坏突变可导致成人患病。非洲爪蛙、伪四倍体非洲爪蟾和二倍体 X. 热带生物是被广泛研究的脊椎动物发育的水生模型。爪蟾也用于两个生命阶段的发育毒性筛查。 FETAX（青蛙胚胎致畸试验-非洲爪蟾）检测生命早期阶段因接触化学品而导致的急性发育毒性。两栖动物变态试验 (AMA) 模拟人类晚期胎儿发育，测量化学破坏将蝌蚪改造为青蛙。 AHR 功能在发育毒性中与人类健康的相关性是对青蛙的了解甚少，限制了这些检测对人类风险评估的有效性。一种理解青蛙 AHR 的基本发育和细胞功能对于该模型的使用至关重要毒理学研究。总体目标是使用基因组编辑的、AHR 缺陷的非洲爪蟾模型，包括 ahr-null X.tropicalis 动物和 X.laevis 细胞系，以确定 AHR 在发育青蛙中的特定功能和培养的细胞。目标 1 下的研究将确定 AHR 在热带 X. 发育中的功能。我们将使用 FETAX 方案比较频率和 ahr-null 青蛙中显示的表型特征 ahr 缺失和野生型胚胎典型发育缺陷的严重程度，包括存活率、长度、轴向和头部畸形和水肿。我们还将检验 ahr 缺失青蛙显示肝脏和卵巢的假设 AHR-/- 小鼠的典型表型，比较形态学和组织学。最后，我们将使用一套分析方法以确定 ahr 的损失如何改变变态过程中的组织重塑。我们将结合形态学 RNAseq 研究的终点以确定表型背后的基因表达改变。在目标 2 中，我们将使用缺乏该物种中两个 ahr 旁系同源物之一的 X. laevis 细胞系，测试以下假设：各个 AHR 旁系同源物在细胞周期调节中发挥着独特的作用。我们将比较生长缓慢的 ahr1a-null 通过流式细胞术分析具有 ahr1b-null 和野生型 XLK-WG 细胞的突变体，建立细胞周期分布每个。我们还将检验 ahr1a-null 细胞容易凋亡的假设。初步数据表明 AHR1a 和 AHR1b 调节不同的基因靶标集。我们将在 RNAseq 研究中检验这一假设。该 AREA 项目将为 AHR 介导的发育毒性途径提供比较见解脊椎动物模型之间共享。它还将为青蛙发育毒性提供机制基础 FETAX 和 AMA 等屏幕，提高了人类风险评估的有效性。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

ARNT gene multiplicity in amphibians: characterization of ARNT2 from the frog Xenopus laevis.

DOI：
10.1002/jez.b.45
发表时间：
2003-12
期刊：
Journal of experimental zoology. Part B, Molecular and developmental evolution
影响因子：
0
作者：
A. J. Rowatt;John J. DePowell;W. Powell
通讯作者：
A. J. Rowatt;John J. DePowell;W. Powell

Developmental differences in elimination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during Xenopus laevis development.

非洲爪蟾发育过程中消除 2,3,7,8-四氯二苯并-对二恶英 (TCDD) 的发育差异。