Modulation by dioxin of ovarian estrogen synthesis

描述(申请人提供)：目前的问题是卵巢癌 2，3，7，8-四氯二苯并对二恶英(TCDD)的作用部位 女性的生殖功能。TCDD被认为是毒性最大的 人类已知的物质，主要作为废物的副产品而产生 焚烧。除了充当致畸剂、胚胎毒素或肿瘤外 在实验室物种中，TCDD是一种已知的内分泌干扰物， 野生动物，从有限的数据来看，人类。我们已经证明了衰减 卵巢雌激素的分泌和卵巢mRNAs表达的改变 雌激素和二恶英或芳香烃受体(ER和AHR) 妊娠母鼠体内注射TCDD后的青春期大鼠仔鼠。 证据有力地支持卵巢是TCDD行动的主要目标。 由于卵巢(包括颗粒细胞)具有AHR，我们现在建议 唯一研究TCDD对人卵巢雌激素作用的方法 使用体外培养范例进行生物合成。我们的假设是TCDD 通过结合AHR和减少卵巢而发挥抗生殖作用 类固醇生物合成途径中一个或多个位点的类固醇合成，例如通过 修饰P450侧链裂解(SCC，CYP11A)或芳香酶(CYP19)的水平 对雌激素代谢酶无影响。 (CYP1A1、1B1)。目标(1a)。要确定低剂量(环境)的影响 相关)TCDD对类固醇激素生成终点的影响。我们将把TCDD管理到小学 成年女性卵巢GC培养；并表征对卵巢GC的影响 定量RT-PCR法测定SCC mRNA水平，生化方法测定活性， 和酶蛋白的免疫印迹；(1b)由于我们在大鼠中的数据 也显示P450芳香酶的调节，我们将评估终点 在上述(1a)中用于该酶；(2)意想不到但可以想到的替代品 雌激素合成减少是卵巢中雌激素通过细胞色素的代谢 P450。我们将评估卵巢细胞色素P1A1和1B1的mRNAs水平，这是 优先催化雌激素(雌二醇和雌酮)生成其2-OH-和 4-OH代谢物；并用层析分离任何产物， 为了评估是否会同时发生合成减少和分解代谢 同时在卵巢中。总的来说，这些机械论的分子研究 将指出卵巢是TCDD行动的主要目标，并将帮助我们 用二恶英观察到的生殖功能障碍的基础 动物和人类。这片土地将作为“种子基金”用于生产 初步数据，并协助我们决定在 R01应用程序。

DESCRIPTION (provided by applicant): The current problem regards an ovarian site of action for 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in modulation of female reproductive function. TCDD has been referred to as the most toxic substance known to man and is produced primarily as a by-product of waste incineration. Aside from acting as a teratogen, embryotoxin, or tumor progressor, TCDD is a known endocrine disrupter in laboratory species, wildlife, and from limited data, humans. We have demonstrated attenuated secretion of ovarian estrogen, and altered expression of ovarian mRNAs for estrogen- and dioxin- or aromatic hydrocarbon receptor (ER and AHR) in peri-pubertal rat pups after in-vivo administration of TCDD to pregnant dams. The evidence strongly supports the ovary as a major target of TCDD action. Because ovary (including granulose cells (GC) possesses AHR, we now propose uniquely to investigate the action of TCDD on human ovarian estrogen biosynthesis using an in-vitro culture paradigm. Our hypothesis is that TCDD exerts an anti-reproductive effect by binding AHR and diminishing ovarian steroidogenesis at one or more loci in steroid biosynthetic pathway, e.g., by modifying levels of P450 side-chain cleavage (SCC, CYP11A) or aromatase (CYP19) mRNA, protein and activity; and has no effect on estrogen-metabolizing enzymes (CYP1A1, 1B1). Aim (1a). To determine effects of low-dose (environmentally relevant) TCDD on steroidogenic endpoints. We will administer TCDD to primary ovarian GC cultures from adult women; and characterize the effects on the levels of SCC mRNA using quantitative RT-PCR, activity by biochemical methods, and enzyme protein using Western immunoblotting; (1b) since our data in rat also show modulation of P450 aromatase enzyme, we will evaluate the endpoints in (1a) above for this enzyme; (2) unexpected but conceivable alternative to reduced estrogen synthesis is ovarian metabolism of estrogen via cytochromes P450. We will evaluate levels of mRNAs for ovarian CYP1A1 and 1B1, which preferentially catalyze estrogens (estradiol and estrone) to their 2-OH- and 4-OH metabolites, respectively; and separate any products chromatographically, in order to assess whether both reduced synthesis and catabolism might occur simultaneously in the ovary. Collectively, these mechanistic molecular studies will point to the ovary as a major target of TCDD action, and will aid us in determining the basis for the reproductive dysfunction observed with dioxin in animals and humans. This AREA will serve as a "seed grant" to produce preliminary data and assist us in deciding the course of action to pursue in a R01 application.