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)模型来理解 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的氧化应激和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。异源生物代谢酶 可能通过有毒/致癌的卤化和 属于芳烃受体激动剂的多核芳香烃。这些化合物引发了 发育过程中的严重缺陷，最近的研究表明，CYP1A在其中一些 通过解偶联CYP催化循环或通过形成经历氧化还原的代谢物而产生的影响 骑车会导致ROS的产生。需要解决的假设是，多个CYP1基因 家族成员，包括细胞色素P1a、细胞色素P1B和新的细胞色素P1C基因，以及AKR都参与了这一过程。 ROS的产生导致斑马鱼发育过程中的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。在具体的 目的我们将：1)评估细胞色素P450受体和AKRs的表达变化和ROS产生与以下方面的关系 暴露于胚胎中的基因表达、氧化应激反应和DNA损伤的全球变化 对叔丁基对苯二酚或致毒物质(2，3，7，8-四氯二苯并-对二恶英和苯并[a]芘)。 2)建立了新斑马鱼细胞色素P41A、细胞色素P1B和新斑马鱼基因表达的时间和细胞模式 细胞色素P1C和AKR基因及其在发育过程中的化学调控。3)确定 克隆并异源表达斑马鱼CYP和AKR，生成ROS。4)建立 细胞色素P450 1和AKR对完整细胞中的玫瑰花环形成有重要作用。5)使用吗啡 降低胚胎中细胞色素P1a、细胞色素P1B、细胞色素P1C和AKR基因表达的技术， 并在体内检测基因消除对毒物诱导的ROS形成的影响。拆卸 FISH将被检查a)ROS的形成，b)用微阵列测量的基因表达变化，以及 通过对包括一个新的斑马鱼反转录转座子在内的靶基因的表达进行PCR分析 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.