MECHANISMS OF ARYL HYDROCARBON INDUCED OVOTOXICITY

来自流行病学和动物研究的数据表明 多环芳烃(PAHs)引起卵泡 然而，多环芳烃破坏的机制 女性生殖细胞是未知的。最近的研究表明 证明存在一种细胞内结合蛋白 多环芳烃被称为芳烃受体复合体(AHRC)。 由于AHRC作为基因的转录调节因子发挥作用 在配体结合后，多环芳烃的作用可能直接 通过基因表达的改变来调节。此外，多环芳烃 被包括卵巢在内的许多细胞代谢成反应性的 可以破坏细胞氧化状态的中间体。AS 这种多环芳烃还可能通过以下方式间接造成卵泡破坏 与氧化应激相关的途径。最近的数据支持 细胞凋亡的基础作用，以及由几个 最近在正常的自然损耗中发现了“细胞死亡基因”。 卵巢生殖细胞和卵泡。特别值得关注的是 基因，Bax，它编码了一种死亡易感因子， 是卵巢细胞死亡所必需的。Bax基因启动子 包含由氧化激活的转录反应元件 应激，以及公认的芳烃反应元件 (AHRE)，表明多环芳烃可诱导Bax的表达 直接(AHRE)和间接(氧化应激)机制。 因此，假设多环芳烃诱导的生殖细胞损失是 通过细胞凋亡，而这种细胞凋亡是通过 直接激活(PAH-AHRC通过AHRE起作用)和 间接(多环芳烃代谢和氧化应激)途径 以增强Bax的表达。为了解决这一假设， 提出了以下具体目标：1)表征剂量-- 多环芳烃对生殖细胞凋亡的时间依赖性影响 胎鼠和生后小鼠卵巢的卵泡颗粒细胞； 2)确定bax基因在生殖细胞中的表达是否增加 以及暴露于多环芳烃的颗粒细胞，如果这种反应和 诱导细胞凋亡依赖于PAH-AHRC 相互作用；3)评估多环芳烃是否导致氧化应激 代谢参与了多环芳烃诱导的卵毒性；4)使用一种 用BAX功能丧失的小鼠模型来确定BAX功能是否正常 多环芳烃的卵毒作用需要蛋白质；以及5) 评估Bax拮抗剂(BAX受体的产物)是否过表达 BCL-2生存基因)在小鼠卵母细胞中传递保护作用 PAH诱导细胞凋亡。使用最先进的分子 生物学方法，这些研究的长期目标是 破译细胞内的效应器通路 多环芳烃对卵巢生殖细胞(卵母细胞)的有害作用 颗粒细胞作为一种识别新的干预部位的手段 预防毒物引起的组织损伤。

Data from epidemiological and animal studies suggest that polycyclic aromatic hydrocarbons (PAHs) cause ovarian follicular degeneration; however, the mechanisms by which PAHs destroy female germ cells are unknown. Recent studies have demonstrated the existence of an intracellular binding protein for PAHs termed the aryl hydrocarbon receptor complex (AHRC). Since the AHRC functions as a transcriptional regulator of genes following ligand binding, the actions of PAHs may be directly mediated via altered gene expression. Additionally, PAHs are metabolized by many cells including those of the ovary, to reactive intermediates that can disrupt the oxidative state of the cell. As such, PAHs may also cause follicular destruction indirectly by pathways associated with oxidative stress. Recent data support a fundamental role for apoptosis, and proteins encoded by several recently characterized "cell death genes," in the normal attrition of ovarian germ cells and follicles. Of particular interest is the gene, bax, which encodes a death-susceptibility factor that appears to be required for ovarian cells to die. The bax gene promoter contains transcriptional response elements activated by oxidative stress, as well as a consensus aryl hydrocarbon response element (AHRE), suggesting that PAHs can induce bax expression by both direct (AHRE) and indirect (oxidative stress) mechanisms. Therefore, it is hypothesized that PAH-induced germ cell loss is mediated via apoptosis, and that this apoptosis occurs via activation of direct (PAH-AHRC acting via the AHRE) and indirect (PAH metabolism and oxidative strees) pathways leading to enhanced bax expression. To address this hypothesis, the following specific aims are proposed: 1) to characterize the dose- and time-dependent effects of PAHs on apoptosis in germ cells and follicular granulosa cells of fetal and post-natal mouse ovaries; 2) to determine if bax gene expression is increased in germ cells and granulosa cells exposed to PAHs, and if this response and the induction of apoptosis are dependent upon PAH-AHRC interaction; 3) to evaluate if oxidative stress resulting from PAH metabolism is involved in PAH-induced ovotoxicity; 4) to use a BAX loss-of-function mouse model to determine if functional BAX protein is required for the ovotoxic effects of PAHs; and 5) to evaluate if overexpression of BAX antagonist (the product of the bcl-2 survival gene) in mouse oocytes conveys protection from PAH-induced apoptosis. Using state-of-the-art molecular biological approaches, the long-range goal of these studies is to decipher the intracellular effector pathways that mediate the deleterious actions of PAHs in ovarian germ cells (oocytes) and granulosa cells as a means to identify novel sites of intervention for precenting toxicant-induced tissue damage.