Modulation by dioxin of ovarian estrogen synthesis

描述（由申请人提供）：目前的问题涉及卵巢 2，3，7，8-四氯二苯并对二恶英（TCDD）在调节中的作用位点 女性生殖功能。TCDD被认为是毒性最大的 一种人类已知的物质，主要作为废物的副产品产生 焚化除了作为致畸剂胚胎毒素或肿瘤 进展，TCDD是实验室物种中已知的内分泌干扰物， 野生动物，以及有限的数据，人类。我们已经证明了 卵巢雌激素的分泌，以及卵巢mRNA表达的改变， 雌激素和二恶英或芳香烃受体（ER和AHR）， 对怀孕母鼠体内注射四氯二苯并对二恶英后的围青春期幼鼠。 证据强烈支持卵巢作为TCDD作用的主要靶点。 由于卵巢（包括颗粒细胞（GC））具有AHR， 独特地研究TCDD对人卵巢雌激素的作用 使用体外培养范例进行生物合成。我们假设TCDD 通过结合AHR和减少卵巢功能来发挥抗生殖作用 在类固醇生物合成途径中的一个或多个基因座处的类固醇生成，例如，通过 改变P450侧链裂解（SCC，CYP 11 A）或芳香酶（CYP 19）的水平 mRNA、蛋白质和活性;对雌激素代谢酶无影响 （CYP1A1，1B1）。目标（1a）。确定低剂量（环境） 相关）TCDD对类固醇生成终点的影响。我们将对小学生进行TCDD 成年女性的卵巢GC培养物;并表征对 SCC mRNA水平采用定量RT-PCR，活性采用生化方法， 和酶蛋白使用西方免疫印迹;（1b）由于我们的数据在大鼠 也显示P450芳香酶的调节，我们将评估终点 在上述（1a）中对于这种酶;（2）意想不到但可以想到的替代物， 雌激素合成减少是卵巢通过细胞色素代谢雌激素 P450。我们将评估卵巢CYP 1A 1和1B 1的mRNA水平， 优先催化雌激素（雌二醇和雌酮）转化为2-OH-， 4-OH代谢物;并通过色谱法分离任何产物， 为了评估是否会同时发生合成减少和催化分解， 同时在卵巢中。总的来说，这些机制分子研究 将指出卵巢是TCDD作用的主要靶点，并将帮助我们 确定二恶英导致生殖功能障碍的原因， 动物和人类。该地区将作为“种子赠款”， 初步数据，并协助我们决定采取行动，在一个 R 01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.