Developmental toxicity of environmental chemicals

描述（由申请人提供）：拟议研究的目标是研究双酚A （BPA）在前列腺和精囊发育过程中介导细胞信号系统破坏的机制。BPA从塑料中浸出，导致胎儿大量暴露在人体中。胎儿小鼠暴露于低剂量双酚a会导致前列腺数量、总体大小和雄激素受体（AR）的永久性增加，而精囊大小则会永久性减少；初步证据表明，这是由于双氢睾酮（DHT）形成所需的5a还原酶减少所致。我们的第一个假设是，低剂量的环境相关BPA（和低剂量的二乙基雌酚，DES，作为阳性对照）对胎鼠前列腺的影响是通过与雌激素受体α (ERa)结合，诱导EGF，随后也诱导IGF-1基因活性（以及这些生长因子的合成），导致AR基因表达和AR蛋白的永久性增加。我们的第二个假设是，高剂量的DES（而不是BPA）将导致相反的结果，并通过与DHT竞争与AR结合来干扰前列腺发育。在精囊中，我们预测EGF和IGF-1将出现剂量依赖性抑制，导致与剂量相关的持久的a-还原酶活性下调。为了验证这些假设，我们的第一个具体目标是进行体内研究，对怀孕的CD-1小鼠进行环境相关剂量的BPA（也包括DES）。我们的第二个具体目标是进行高剂量BPA和DES的体内研究，直到最大耐受剂量。我们的第三个具体目标涉及去除胎儿泌尿生殖窦和沃尔夫管，用于原代培养研究，通过给予极低至亚致死剂量的DES和BPA来回答特定的机制问题。我们还将确定EGF和igf - 1是否模拟DES和BPA的作用，以及是否对这些蛋白质进行抗体抑制作用。第四个具体目标是确定高剂量的DES（而非双酚a）是否会与DHT竞争与AR结合，从而产生抗雄激素作用。在这些研究中，前列腺和精囊将在妊娠第18天、出生后第3天和成年后代中进行检查。我们将首先关注AR， ERa， ERB， 5a-还原酶，EGF和IGF-1，并通过RT-PCR和western blot分析测量mRNA水平和蛋白水平。器官形态将通过组织切片的3-D计算机重建来确定，再加上对上述mrna和相应蛋白的原位杂交和免疫细胞化学，包括细胞类型、增殖和凋亡的标记。5a-还原酶活性将通过放射测定法测定。

DESCRIPTION (provided by applicant): The goal of the proposed research is to investigate mechanisms mediating disruption by bisphenol A (BPA) of cellular signalling systems during prostate and seminal vesicle development. BPA leaches from plastic resulting in significant fetal exposure in humans. Exposure of fetal mice to low doses of BPA results in a permanent increase in prostate gland number, overall size and androgen receptors (AR), while a permanent decrease in seminal vesicle size occurs; preliminary evidence suggests this is due to a decrease in the enzyme 5a-reductase, required for dihydrotestosterone (DHT) formation. Our first hypothesis is that the effects of low, environmentally relevant doses of BPA (and low doses of diethylstilbestrol, DES, as a positive control) in the fetal mouse prostate occur via binding to estrogen receptor alpha (ERa), induction of EGF and, subsequently, also IGF-1 gene activity (and synthesis of these growth factors), leading to a permanent increase in AR gene expression and AR protein. Our second hypothesis is that much higher doses of DES, but not BPA, will result in the opposite outcome and interfere with prostate development via competing with DHT for binding to AR. In the seminal vesicles, we predict that there will be a dose-dependent inhibition of EGF and IGF-1, resulting in a permanent dose-related down-regulation of 5a-reductase activity. To test these hypotheses our first specific aim is to conduct in vivo studies in which pregnant CD-1 mice are administered environmentally relevant doses of BPA (and also DES). Our second specific aim is to conduct in vivo studies with high doses of BPA and DES, up to the maximum tolerated dose. Our third specific aim involves removing the fetal urogenital sinus and Wolffian ducts for studies in primary culture to answer specific mechanistic questions by administering very low through sub-lethal doses of DES and BPA. We will also determine whether EGF and IGF-l mimic effects of DES and BPA, and if administering antibodies to these proteins inhibits effects. The fourth specific aim is to determine whether high doses of DES, but not BPA, compete with DHT for binding to AR, thus producing an antiandrogenic effect. In these studies the prostate and seminal vesicles will be examined on gestation day 18, postnatal day 3 and in adult offspring. We will initially focus on AR, ERa, ERB, 5a-reductase, EGF and IGF-1, and measure both mRNA levels by RT-PCR and protein levels by western blot analysis. Organ morphology will be determined by 3-D computer reconstruction from histological sections, coupled with in situ hybridization and immunocytochemistry for the above mRNAs and corresponding proteins, including markers for cell types, proliferation and apoptosis. 5a-reductase activity will be determined by radiometric assay.