Mechanisms of Aryl-Hydrocarbon-Induced Ovotoxicity

说明书(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主要原因是香烟烟雾。多环芳烃导致早产的证据 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。这些化学物质特别耐人寻味，因为有一种 PAN的胞内结合蛋白称为芳烃受体 (AHR)。AHR是一种基本的螺旋-环-螺旋转录因子，调节 配体相互作用和核转位后的基因表达。在.期间 在这笔赠款的上一个资助周期中，P1完成了 验证PAN介导的AHR在体内激活的中心假设 卵母细胞通过增加编码bax、a基因的表达来诱导细胞凋亡 Bcl2基因家族中促凋亡的成员。然而，几个新的问题 在这些研究过程中产生了。首先，AHR的功能是否设置了“细胞凋亡 细胞转录调控对女性生殖细胞易感性的影响 除BAX外，还有死亡调控基因？其次是二恶英的无能为力， 一种已知的AHR配体，与AHR反应的差异有关，可损害卵巢 靶细胞死亡调控基因中的元件(AHRE)侧翼序列 (例如，Bax)传递多环芳烃-AHR与二恶英-AHR相互作用的特异性？ 第三，在卵母细胞中诱导Bax基因表达是否需要 允许AHR-bax基因启动子相互作用的AHR相互作用蛋白(S)？ 根据这里提供的初步数据，PT假设 PAH-AHR。但不是二恶英-AHR，相互作用转录设定细胞死亡 女性生殖细胞中的易感性“变阻器”有利于细胞凋亡。这是 通过在卵母细胞中表达的辅助激活因子(S)完成，该辅助激活因子促进 泛激活的AHR与细胞死亡调控的功能相互作用 基因启动子拥有啊。为了检验这一假设，以下是具体的 提出了继续这些研究的目标：1)确定 暴露于辐射的卵母细胞促凋亡基因bak的表达增加 PAN，如果需要PAN-AHR相互作用才能增加转录活性 如果需要内源性bak基因的表达 PAN的卵毒作用；2)检测A基因的表达模式 已知在卵母细胞中表达的关键凋亡调控基因的数量包括 被AHR缺乏和/或由PAH驱动的AHR激活所改变；3)产生 AHR在卵母细胞中启动细胞死亡信号的基因表达谱 一种新近发展起来的用于小型生物的微型基因芯片技术 样本；4)描述“核心”两侧的核苷酸序列的作用 Bax基因启动子中的五个核苷酸在指定对 PAN和二恶英激活的AHR；以及，5)鉴定AHR与AHR的相互作用 卵母细胞中的蛋白质，使用基质辅助激光解吸和 电离/飞行时间质谱仪(MALDI/TOF-MS)，这可能是 参与特定细胞系选择性诱导Bax基因 由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.