Mechanisms of Aryl-Hydrocarbon-Induced Ovotoxicity

描述（由申请人提供）：多环芳烃（PAH）是 通过化石燃料燃烧释放到环境中。另一条主要路线 人类接触多环芳烃的最主要原因是香烟烟雾。PAH导致早产的证据 流行病学和动物研究都提供了卵巢衰竭的证据。 然而，PAH损害卵巢的机制仍然不清楚， 几十年这些化学物质特别有趣，因为存在一种 PAN的细胞内结合蛋白，称为芳香烃受体 （AHR）。AHR是一种碱性螺旋-环-螺旋转录因子， 配体相互作用和核转位后的基因表达。期间 在该赠款的上一个资助周期，P1完成了实验， 验证PAN介导的AHR激活的中心假设， 卵母细胞通过增加编码Bax的基因的表达诱导凋亡， bcl-2基因家族的促凋亡成员。然而，几个新的问题 在这些研究中出现。首先，AHR是否具有设置“细胞凋亡”的功能， 通过细胞转录调控雌性生殖细胞的“易感性” 死亡调控基因除了bax？第二，是二恶英不能， 一种已知的AHR配体，损害与AHR反应差异相关的卵巢 “靶”细胞死亡调控基因中的AHRE侧翼序列 (e.g., bax），传达特异性的PAH-AHR与二恶英-AHR相互作用？ 第三，卵母细胞中bax基因表达的诱导是否需要 允许AHR-bax基因启动子相互作用的AHR相互作用蛋白？ 根据本文提供的初步数据，PT假设， PAH-AHR。而不是二恶英-AHR，相互作用转录设置细胞死亡 易感性“变阻器”在女性生殖细胞有利于凋亡。这是 通过在卵母细胞中表达的共激活因子实现， PAN激活的AHR和细胞死亡调节之间的功能相互作用 具有AHRE的基因启动子。为了验证这一假设，以下具体 建议继续进行这些研究的目的是：1）确定是否 促凋亡巴克基因的表达在暴露于 PAN，如果PAN-AHR相互作用是增加转录活性所必需的 巴克基因的表达，如果需要内源性巴克基因的表达， PAN的卵毒性作用; 2）检查是否表达模式， 已知在卵母细胞中表达的一些关键凋亡调控基因， 通过AHR缺陷和/或PAH驱动的AHR激活改变; 3）产生 AHR启动的卵母细胞死亡信号的基因表达谱， 最近开发的用于小生物的微型基因阵列技术， 4）描绘“核心”侧翼核苷酸序列的作用 bax基因启动子中的五核苷酸AHRE在指定对 PAN与二恶英激活的AHR;以及，5）识别AHR相互作用 卵母细胞中的蛋白质，使用基质辅助激光解吸， 电离/飞行时间质谱（MALDI/TOF-MS），其可以是 参与特异性细胞系选择性诱导bax基因 通过PAN激活的AHR转录。

DESCRIPTION (provided by applicant): Polycyclic aromatic hydrocarbons (PAH) are released into the environment by fossil fuel combustion. Another primary route of human exposure to PAH is cigarette smoke. Evidence that PAH cause premature ovarian failure has been provided by both epidemiological and animal studies. However, the mechanisms by which PAH damage the ovary have remained obscure for decades. These chemicals are particularly intriguing as there exists an intracellular binding protein for PAN termed the aryl hydrocarbon receptor (AHR). The AHR is a basic helix-loop-helix transcription factor that regulates gene expression following ligand interaction and nuclear translocation. During the previous funding cycle of this grant, the P1 completed experiments to validate the central hypothesis that PAN-mediated activation of the AHR in oocytes induces apoptosis by increasing expression of the gene encoding Bax, a pro-apoptotic member of the bcl-2 gene family. However, several new questions arose during these studies. First, does the AHR function to set the "apoptosis susceptibility" of female germ cells by transcriptional regulation of cell death regulatory genes in addition to bax? Second, is the inability of dioxin, a known AHR ligand, to damage the ovary related to differences in AHR response element (AHRE) flanking sequences in "target" cell death regulatory genes (e.g., bax) that convey specificity for PAH-AHR versus dioxin-AHR interactions? Third, does the induction of bax gene expression in oocytes require an AHR-interacting protein(s) that permits AHR-bax gene promoter interaction? Based on preliminary data presented herein, the PT has hypothesized that PAH-AHR. but not dioxin-AHR, interaction transcriptionally sets the cell death susceptibility "rheostat" in female germ cells to favor apoptosis. This is accomplished via a coactivator(s) expressed in oocytes that facilitates functional interaction between the PAN-activated AHR and cell death regulatory gene promoters possessing AHRE. To test this hypothesis, the following Specific Aims are proposed for continuation of these studies: 1) to determine if expression of the pro-apoptotic bak gene is increased in oocytes exposed to PAN, if PAN-AHR interaction is required for increased transcriptional activity of the bak gene, and if expression of the endogenous bak gene is required for the ovotoxic effects of PAN; 2) to examine if the expression patterns of a number of key apoptosis regulatory genes, known to be expressed in oocytes, are altered by AHR deficiency and/or by PAH-driven AHR activation; 3) to produce a gene expression profile for AHR initiated cell death signaling in oocytes using a microscale gene array technology recently developed for small biological samples; 4) to delineate the role of nucleotide sequences flanking the "core" five-nucleotide AHRE in the bax gene promoter in specifying responses to the AHR activated by PAN versus dioxin; and, 5) to identify AHR-interacting proteins in oocytes, using matrix-assisted laser desorption and ionization/time-of-flight mass spectrometry (MALDI/TOF-MS), that may be involved in specifying cell lineage-selective induction of bax gene transcription by the PAN-activated AHR.