CYP Genes and the Generation of Oxidative Stress

这些研究的总体目标是采用斑马鱼（Danio rerio）模型来理解 多种细胞色素P450 1（CYP 1）和醛酮还原酶（AKR）在 化学依赖性氧化应激和DNA损伤。氧化应激可能是一种 导致毒性的途径汇聚的关键机制点。异生物质代谢酶 可能有助于通过有毒/致癌的卤代和 多核芳烃是芳烃受体激动剂。这些化合物引起 最近的研究表明，CYP 1A在其中一些发育过程中的作用， 通过解偶联的氧化还原催化循环，或通过形成代谢物， 循环导致ROS产生。要解决的假设是，多个CYP 1基因 家族成员，包括CYP 1A、CYP 1B和新的CYP 1C基因，以及AKR，参与 ROS的产生导致斑马鱼发育过程中的氧化损伤和进一步影响。我们 将通过评估ROS产生的来源和后果来验证这一假设。在具体 我们的目的是：1）评价CYP 1 s和AKR表达的变化以及ROS的产生， 胚胎暴露后基因表达、氧化应激反应和DNA损伤的总体变化 转化为叔丁基对苯二酚或原氧化剂（2，3，7，8-四氯二苯并对二恶英和苯并[a]芘）。 2)建立CYP 1A、CYP 1B和新斑马鱼的时间和细胞表达模式 CYP 1C和AKR基因及其在发育过程中的化学调控。3)确定的角色 克隆并异源表达了斑马鱼ROS产生的抑制剂和抑制剂AKR。4)证明 CYP 1 s和AKR对完整细胞ROS的形成有重要作用。5)使用吗啉代 敲低胚胎中CYP 1A、CYP 1B、CYP 1C和AKR基因表达的技术， 并确定基因消除对体内毒物诱导的ROS形成的影响。敲低 将检查鱼的a）ROS形成，B）用微阵列测量的改变的基因表达， 通过PCR分析靶向选择基因的表达，包括一种新的斑马鱼反转录转座子 可能受氧化应激调节，和c）DNA损伤的发生。

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.