Xenoestrogens: Genomic and Non-genomic Mechanisms

描述（由申请人提供）：双酚A（BPA）和辛基酚（OP）在环境中含量丰富，与雌激素受体（ER）结合，并作为部分雌激素激动剂。尽管越来越多的证据表明其对生殖道的多重影响，但对其在神经内分泌轴内的作用谱，特别是对雌激素敏感的垂体催乳素的作用谱知之甚少。我们以前报道，这些化合物诱导催乳细胞增殖，增加催乳素（PRL）基因的表达和释放，改变垂体ER α和ER β的表达，并影响女性生殖道的生长和形态。而这些影响需要几个小时到几天的时间发生，涉及基因转录和蛋白质合成，我们最近发现，低纳摩尔浓度的雌激素/异种雌激素诱导非常快速（10分钟内）激活MAP激酶（MAPK）系统和刺激PRL释放培养的垂体前叶细胞。我们的主要目的是比较基因组与非基因组的作用，雌激素/外源性雌激素在垂体和确定细胞内钙，一氧化氮（NO）的释放，MAPK激活和核ER反式激活，导致增加激素释放，基因激活和催乳细胞增殖之间的相互作用。具体目标1将测试雌激素通过与钙依赖性胞吐作用相关的膜ER快速刺激PRL释放并且还可能涉及一氧化氮（NO）释放的假设。游离和结合雌激素/异种雌激素将用于测定：a）在不存在和存在ER拮抗剂的情况下，从分散的垂体前叶细胞或GH 3催乳细胞快速刺激PRL释放，B）通过钙、NO或MAPK的阻断剂逆转该作用，c）响应雌激素的细胞内钙浓度或NO释放的变化，d）在多巴胺存在下雌激素增加PRL释放的能力，和e）对雌激素的PRL分泌反应是否经历受体脱敏。具体目标2将检验以下假设：短时间暴露于雌激素/异种雌激素足以启动MAPK和ER信号通路的顺序激活，导致诱导选定的靶基因和改变的细胞增殖/凋亡。在存在或不存在MAPK和ER抑制剂的情况下，将用游离和结合雌激素脉冲追踪催乳素。在此后的不同时间，我们将确定：a）诱导PRL和血管内皮生长因子（VEGF）基因表达，B）改变细胞对雌激素敏感性的ER亚型的表达，和c）增加的催乳素细胞增殖。具体目标3将使用基因阵列方法来检验以下假设：雌激素敏感的Fischer 344（F344）和雌激素不敏感的Sprague道利（SD）大鼠之间，响应于雌二醇和BPA而上调/下调的垂体基因谱不同。这些研究的结果应提供急需的实验基础，评估的脆弱性，脑垂体的侮辱内分泌干扰物。

DESCRIPTION (provided by applicant): Bisphenol A (BPA) and octylphenol (OP) are abundant in the environment, bind to the estrogen receptor (ER) and act as partial estrogen agonists. In spite of accumulating evidence on their multiple effects on the reproductive tract, little is known about their spectrum of actions within the neuroendocrine axis, especially on the estrogen-sensitive pituitary lactotroph. We previously reported that these compounds induced lactotroph proliferation, increased prolactin (PRL) gene expression and release, altered pituitary ERalpha and ERbeta expression and affected the growth and morphology of the female reproductive tract. Whereas these effects take hours to days to occur and involve gene transcription and protein synthesis, we recently found that low nanomolar concentrations of estrogens/xenoestrogens induced very rapid (within 10 min) activation of the MAP kinase (MAPK) system and stimulated PRL release from cultured anterior pituitary cells. Our main objective is to compare genomic vs. non-genomic actions of estrogens/ xenoestrogens in the pituitary and determine interactions between intracellular calcium, nitric oxide (NO) release, MAPK activation and nuclear ER transactivation which result in increased hormone release, gene activation and lactotroph cell proliferation. Specific aim 1 will test the hypothesis that estrogens rapidly stimulate PRL release via a membrane ER that is linked to calcium-dependent exocytosis and may also involve nitric oxide (NO) release. Free and conjugated estrogens/xenoestrogens will be used to determine: a) rapid stimulation of PRL release from dispersed anterior pituitary cells or GH3 lactotrophs in the absence and presence of ER antagonists, b) reversal of this effect by blockers of calcium, NO or MAPK, c) changes in intracellular calcium concentrations or NO release in response to estrogens, d) the ability of estrogens to increase PRL release in the presence of dopamine, and e) whether the PRL secretory response to estrogens is subjected to receptor desensitization. Specific aim 2 will test the hypothesis that a short exposure to estrogens/xenoestrogens is sufficient to initiate sequential activation of the MAPK and ER signaling pathways, resulting in the induction of selected target genes and altered cell proliferation/apoptosis. Lactotrophs will be pulse-chased with free and conjugated estrogens in the presence or absence of MAPK and ER inhibitors. At various times thereafter we will determine: a) induction PRL and vascular endothelial growth factor (VEGF) gene expression, b) expression of ER isoforms that alter cellular sensitivity to estrogens, and c) increased lactotroph proliferation. Specific aim 3 will use a gene array approach to test the hypothesis that the profile of pituitary genes that are upregulated/downregulated in response to estradiol and BPA differs between the estrogensensitive Fischer 344 (F344) and the estrogen-insensitive Sprague Dawley (SD) rat. The results of these studies should provide much needed experimental foundation for assessing the vulnerability of the pituitary gland to insults by endocrine disruptors.