CYP Genes and the Generation of Oxidative Stress

这些研究的总体目标是利用斑马鱼（Danio rerio）模型来了解 多种细胞色素 P450 1 (CYP1) 和醛酮还原酶 (AKR) 的相对作用 发育过程中化学依赖性氧化应激和 DNA 损伤。氧化应激可能是 导致毒性的途径汇聚的关键机制点。异生物质代谢酶 有毒/致癌的卤化物可能会导致活性氧 (ROS) 的形成 多核芳香烃是芳基烃受体激动剂。这些化合物引起 发育过程中的严重缺陷，最近的研究表明 CYP1A 在其中一些缺陷中发挥作用 通过 CYP 催化循环的解偶联或通过氧化还原代谢物的形成产生影响 循环导致ROS的产生。要解决的假设是多个 CYP1 基因 家族成员，包括 CYP1A、CYP1B 和新的 CYP1C 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 生成的来源和后果来检验这一假设。在具体 我们的目标是： 1) 评估 CYP1 和 AKR 表达以及 ROS 生成的变化，与 暴露的胚胎中基因表达、氧化应激反应和 DNA 损伤的整体变化 叔丁基氢醌或前毒剂（2,3,7,8-四氯二苯并-对二恶英和苯并[a]芘）。 2) 建立CYP1A、CYP1B和新型斑马鱼的时间和细胞表达模式 CYP1C 和 AKR 基因及其在发育过程中受化学物质的调节。 3）确定角色 在ROS的产生中克隆并异源表达斑马鱼CYP和AKR。 4）确定 CYP1 和 AKR 对完整细胞中 ROS 的形成有显着贡献。 5) 使用吗啉 降低胚胎中 CYP1A、CYP1B、CYP1C 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。击倒 将检查鱼的 a) ROS 形成，b) 使用微阵列测量的基因表达改变，以及 通过 PCR 分析选定的目标基因（包括新型斑马鱼反转录转座子）的表达 可能受到氧化应激的调节，以及 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.