CYP Genes and the Generation of Oxidative Stress

CYP 基因与氧化应激的产生

• 批准号: 7576787
• 负责人: JOHN J STEGEMAN
• 金额: $ 63.47万
• 依托单位: WOODS HOLE OCEANOGRAPHIC INSTITUTION
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-10 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7576787
• 关键词: 2-tert-butylhydroquinone; Address; Agonist; Aromatic Hydrocarbons; Aromatic Polycyclic Hydrocarbons; Aryl Hydrocarbon Receptor; Benzo(a)pyrene; Binding; CYP1A1 gene; Cell Line; Cells; Chemicals; Confocal Microscopy; Cytochrome P450; DNA Damage; Defect; Development; Dose; Embryo; Endothelial Cells; Enzymes; Family member; Fishes; Gene Expression; Gene Expression Profiling; Gene Family; Generations; Genes; Global Change; Hepatocyte; Mammals; Measures; Mitochondria; Modeling; Nucleic Acid Regulatory Sequences; Oligonucleotides; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxidoreductase; Pathology; Pathway interactions; Pattern; Predisposition; Process; Production; Proteins; Reaction Time; Reactive Oxygen Species; Regulation; Relative (related person); Reporter; Research Personnel; Response Elements; Retrotransposon; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Source; System; Technology; Testing; Tetrachlorodibenzodioxin; Toxic effect; Transcript; Translations; Xenobiotics; Zebrafish; biological adaptation to stress; in vivo; knock-down; morpholine; novel; oxidative DNA damage; oxidative damage; programs; research study; toxicant

The overall objective of these studies is to employ the zebrafish (Danio rerio) model to understand the relative role of multiple cytochrome P450 1 (CYP1) and aldo-keto reductase (AKR) enzymes in chemically dependent oxidative stress and DNA damage during development. Oxidative stress may be a key mechanistic point at which pathways leading to toxicity converge. Xenobiotic metabolizing enzymes may contribute to formation of reactive oxygen species (ROS) by toxic/carcinogenic halogenated and polynuclear aromatic hydrocarbons that are aryl hydrocarbon receptor agonists. These compounds elicit severe defects during development, and recent studies suggest a role for CYP1A in some of those effects via uncoupling of the CYP catalytic cycle, or through formation of metabolites that undergo redox cycling resulting in ROS generation. The hypothesis to be addressed is that the multiple CYP1 gene family members, including CYP1A, CYP1B and the novel CYP1C genes, as well as AKR, are involved in generation of ROS leading to oxidative damage and further effects during development in zebrafish. We will test this hypothesis by evaluating the sources and consequences of ROS generation. In the Specific Aims we will: 1) Evaluate changes in expression of CYP1s and AKRs and ROS generation, in relation to global changes in gene expression, the oxidative stress response and DNA damage in embryos exposed to tert-butylhydroquinone or the protoxicants (2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene). 2) Establish the temporal and cellular patterns of expression of CYP1 A, CYP1B and novel zebrafish CYP1C and AKR genes and their regulation by chemicals during development. 3) Determine the roles of cloned and heterologously expressed zebrafish CYP and AKR in generation of ROS. 4) Establish that CYP1s and AKR contribute significantly to ROSformation in intact cells. 5) Employ morpholino technology to knock down the expression of the CYP1A, CYP1B, CYP1C and AKR genes in embryos, and determine the effect of gene elimination on toxicant-induced formation of ROS in vivo. Knock-down fish will be examined for a) ROS formation, b) altered gene expression measured with microarrays and by PCR analysis of expression of targeted selected genes including a novel zebrafish retrotransposon potentially regulated by oxidative stress, and c) the occurrence of DNA damage.

这些研究的总体目的是采用斑马鱼（Danio Rerio）模型来了解 多个细胞色素P450 1（CYP1）和Aldo-Keto还原酶（AKR）酶的相对作用 在发育过程中，化学依赖的氧化应激和DNA损伤。氧化应激可能是 导致毒性融合的途径的关键机械点。异生物代谢酶 可能有助于通过有毒/致癌的卤代形成活性氧（ROS），并且 芳基烃受体激动剂的多核芳族烃。这些化合物引起 开发过程中的严重缺陷，最近的研究表明CYP1A在其中一些中的作用 通过切开CYP催化循环的效果，或形成经历氧化还原的代谢产物 骑自行车导致ROS产生。要解决的假设是多个CYP1基因 包括CYP1A，CYP1B和新型CYP1C基因在内的家庭成员以及AKR参与 ROS产生导致斑马鱼发育过程中氧化损伤和进一步的影响。我们 将通过评估ROS产生的来源和后果来检验这一假设。在具体中 目的我们将：1）评估CYP1和AKRS和ROS生成的表达变化 基因表达的全球变化，氧化应激反应和暴露胚胎中的DNA损伤 到TERT叔丁基氢醌或蛋白毒剂（2,3,7,8-四氯迪本佐-P-二恶英和苯并[A] pyrene）。 2）建立CYP1 A，CYP1B和新型斑马鱼的表达的时间和细胞模式 CYP1C和AKR基因及其在发育过程中对化学物质的调节。 3）确定角色 克隆和异源表达的斑马鱼CYP和AKR产生ROS。 4）确定 CYP1和AKR在完整细胞中有显着贡献。 5）雇用吗啡 拆除CYP1A，CYP1B，CYP1C和AKR基因的表达的技术， 并确定消除基因对毒物诱导的体内ROS形成的影响。击倒 将检查鱼类形成的鱼类，b）用微阵列测量和 通过PCR分析靶向选定基因的表达，包括新型斑马鱼逆转录盆地 可能受氧化应激调节，c）DNA损伤的发生。